Biochem Handout

Ross University School of Medicine
Course Handout
Biochemistry and Genetics
Department of Biochemistry
Drs Blanchetot, Beevers, Buxbaum, Grogan, James, Larsen,
LaVille, Meisenberg, Sands, Smolanoff and Mrs Lambert
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Contents
Welcome xix
I. Semester one, Mini I 1
1. Introduction to Biomolecules 3
1.1. Elements and molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2. Covalent Bonds And Non-covalent Interactions . . . . . . . . . . . . . . . . 3
1.2.1. Covalent bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2. Non-covalent interactions . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3. Bonds in biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4. Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.5. Acids and bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6. Fats and carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.7. Objectives in summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.7.1. Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2. Energy changes and rates of chemical reactions 15

2.1. Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2. Reaction kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2.1. Order of Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.2. The principle of Le Chatelier . . . . . . . . . . . . . . . . . . . . 21
2.3. Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.4. Example questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3. Amino acids and proteins 27

3.1. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1.1. General structure of amino acids . . . . . . . . . . . . . . . . . . . . 27
3.1.2. The 22 amino acids in proteins . . . . . . . . . . . . . . . . . . . . . 28
3.1.3. The pI-value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.1.4. The one-letter code . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.1. The peptide bond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
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3.2.2. Protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.2.3. Proteins in the laboratory . . . . . . . . . . . . . . . . . . . . . . . . 66
3.3. Protein folding diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.3.1. Spongiform encephalopathies . . . . . . . . . . . . . . . . . . . . . . 77
3.3.2. Morbus Alzheimer . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.3.3. Morbus Parkinson . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.4. Chorea Huntington . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.5. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4. DNA and Gene Expression 91

4.1. DNA Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.1.1. Bases, Nucleosides and Nucleotides . . . . . . . . . . . . . . . . . . . 91
4.1.2. The DNA Double-Helix. . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.1.3. Chemical stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.1.4. Supercoiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.2. DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.2.1. Semi-conservative replication . . . . . . . . . . . . . . . . . . . . . . 94
4.2.2. DNA polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.2.3. Steps in bacterial DNA replication . . . . . . . . . . . . . . . . . . . 95
4.3. RNA and Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.3.1. RNA Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.3.2. Types of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.3.3. Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.3.4. Post-transcriptional processing . . . . . . . . . . . . . . . . . . . . . 97
4.4. Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.4.1. The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.4.2. tRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.4.3. Ribosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.4.4. Steps in translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.4.5. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.5. Regulation of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.6. Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.6.1. Virus Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.6.2. The Lytic Cycle of Bacteriophage T4 . . . . . . . . . . . . . . . . . . 102
4.6.3. The Lysogenic Cycle of λ phage . . . . . . . . . . . . . . . . . . . . . 103
4.6.4. Animal Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.6.5. RNA Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.6.6. Retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.6.7. Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.7. Genetic Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
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4.8. Types of Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

4.8.1. Parasexual Processes in Bacteria . . . . . . . . . . . . . . . . . . . . 105
4.9. Objectives in Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5. The Human Genome and Mutations 109

5.1. The Human Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.2. Chromatin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.2.1. Histones and Nucleosomes . . . . . . . . . . . . . . . . . . . . . . . . 110
5.2.2. Repetitive DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.2.3. Mobile DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
5.2.4. Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
5.2.5. Telomeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.2.6. DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.3. Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.3.1. Types of Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.3.2. Causes of Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.3.3. Mutagenesis Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.4. DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.4.1. Repair Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
5.5. Eukaryotic Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.5.1. Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.5.2. mRNA Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.5.3. Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.6. Regulation of Eukaryotic Gene Expression . . . . . . . . . . . . . . . . . . . 117
6. Chromosome Aberrations 121

6.1. The Human Karyotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.2. Sex Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.3. Types Of Chromosome Aberrations . . . . . . . . . . . . . . . . . . . . . . . 123
6.4. Autosomal Trisomies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.4.1. Trisomy 21 (Down syndrome) . . . . . . . . . . . . . . . . . . . . . 124
6.4.2. Trisomy 18 (Edward syndrome) . . . . . . . . . . . . . . . . . . . . 126
6.4.3. Trisomy 13 (Patau syndrome) . . . . . . . . . . . . . . . . . . . . . 126
6.5. Sex Chromosome Aberrations: Male . . . . . . . . . . . . . . . . . . . . . . 127
6.5.1. Klinefelter syndrome . . . . . . . . . . . . . . . . . . . . . . . . . 127
6.5.2. XYY constitution (“murderer chromosome”) . . . . . . . . . . . . . . 127
6.5.3. XX Males . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
6.6. Sex Chromosome Aberrations: Female . . . . . . . . . . . . . . . . . . . . . 128
6.6.1. Turner’s syndrome (gonadal dysgenesis) . . . . . . . . . . . . . . . 128
6.6.2. Triple-X (47, XXX, “superfemale”) . . . . . . . . . . . . . . . . . . . 128
6.6.3. XY females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
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6.7. Abnormal Sexual Development With Normal Chromosomes . . . . . . . . . 129

6.7.1. True hermaphroditism . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.7.2. Mixed Gonadal Dysgenesis . . . . . . . . . . . . . . . . . . . . . . . 129
6.7.3. Female Pseudohermaphroditism. . . . . . . . . . . . . . . . . . . . . 129
6.7.4. Male Pseudohermaphroditism . . . . . . . . . . . . . . . . . . . . . . 130
6.7.5. Chromosomal Rearrangements . . . . . . . . . . . . . . . . . . . . . 131
7. Enzymes 137
7.1. History of enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
7.2. Classification of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
7.2.1. Systematic name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
7.2.2. Enzyme classes and EC codes . . . . . . . . . . . . . . . . . . . . . . 138
7.3. Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.3.1. The Henri-Michaelis-Menten (HMM)-equation . . . . . . . . . . . . . 140
7.3.2. Catalytic perfection . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
7.3.3. Environmental influences on enzyme activity . . . . . . . . . . . . . 145
7.3.4. Cooperativity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
7.4. Enzyme inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
7.4.1. Competitive inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . 150
7.4.2. Uncompetitive inhibition . . . . . . . . . . . . . . . . . . . . . . . . . 154
7.4.3. Noncompetitive inhibition . . . . . . . . . . . . . . . . . . . . . . . . 154
7.4.4. Mixed inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
7.5. Enzyme inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
7.6. Enzymes with multiple substrates or products . . . . . . . . . . . . . . . . . 161
7.7. How do enzymes do it? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.7.1. Protease reaction mechanism . . . . . . . . . . . . . . . . . . . . . . 163
7.8. Coenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
7.8.1. Adenosine Triphosphate (ATP) . . . . . . . . . . . . . . . . . . . . . 165
7.8.2. Redox Coenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
7.8.3. Other Coenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.9. Enzymes in clinical diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . 169
7.10. Membrane Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7.10.1. Passive transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7.10.2. Active transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
7.11. Homeostasis of the Intracellular Environment . . . . . . . . . . . . . . . . . 173
7.11.1. Ion concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
7.12. Useful web resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
7.14. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
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8. Methods in Molecular Medicine 181

8.1. Restriction Endonucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.2. Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.3. Southern Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.4. The Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . 183
8.5. DNA Sequencing and Deduced Functions . . . . . . . . . . . . . . . . . . . 185
8.6. Gene Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
8.6.1. Fluorescent in-situ Hybridization (FISH) . . . . . . . . . . . . . . . . 186
8.6.2. Deletion Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
8.6.3. Linkage Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
8.6.4. Candidate Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
8.7. Cloning and Genomic Libraries . . . . . . . . . . . . . . . . . . . . . . . . . 187
8.8. cDNA Cloning and Expression Cloning . . . . . . . . . . . . . . . . . . . . . 189
8.9. Site-Directed Mutagenesis and Protein Engineering . . . . . . . . . . . . . . 190
8.10. Use of DNA Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
8.10.1. Southern Blotting with Allele-Specific Probes . . . . . . . . . . . . 191
8.11. Use of PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
8.12. Dot-Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
8.13. OBJECTIVES IN SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . 194
II. Semester one, Mini II 197
9. Glycolysis: Splitting glucose in half 199

9.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
9.2. Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
9.2.1. Reactions of glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . 200
9.2.2. Regulation of glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . 200
9.2.3. Inhibition of glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . 201
9.2.4. Anaerobic glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
10.Plasma Proteins 203

10.1. Plasma Proteins: Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
10.1.1. Functions of plasma proteins . . . . . . . . . . . . . . . . . . . . . . 203
10.2. Separation of Plasma Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 204
10.2.1. Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
10.2.2. Transport Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
10.2.3. Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
10.3. Plasma Proteins in Disease States . . . . . . . . . . . . . . . . . . . . . . . . 206
10.3.1. Plasma Components for Clinical Use . . . . . . . . . . . . . . . . . . 207
10.4. Clinical Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
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11.Blood Coagulation 213

11.1. The Biochemistry of Blood Coagulation . . . . . . . . . . . . . . . . . . . . 213
11.1.1. Primary hemostasis (platelet-plug formation) . . . . . . . . . . . . . 213
11.1.2. Secondary hemostasis (fibrin clot formation) . . . . . . . . . . . . . . 213
11.1.3. Clinical Correlates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
11.1.4. Laboratory Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
11.1.5. Features of Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . 215
11.1.6. Genetics of Blood Coagulation . . . . . . . . . . . . . . . . . . . . . 216
11.2. Objectives in Brief . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
12.Blood 219
12.1. Blood Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
12.1.1. The ABO system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
12.1.2. The Rhesus System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
12.1.3. Other blood group systems . . . . . . . . . . . . . . . . . . . . . . . 221
12.2. Structure of Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
12.2.1. Chemical Inactivation of Hemoglobin . . . . . . . . . . . . . . . . . . 221
12.2.2. Allosteric Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
12.2.3. The Hemoglobinopathies . . . . . . . . . . . . . . . . . . . . . . . . . 225
12.3.1. Blood Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
12.3.2. Hemoglobin and Myoglobin Biochemistry . . . . . . . . . . . . . . . 228
12.3.3. Hemoglobinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
III. Semester one, Mini III 231
13.Mendelian Inheritance 233

13.1. The Patterns of Mendelian Inheritance . . . . . . . . . . . . . . . . . . . . 233
13.2. Variations of gene transmission and expression . . . . . . . . . . . . . . . . 234
13.3. Functional classification of mutations . . . . . . . . . . . . . . . . . . . . . . 235
13.3.1. Loss of function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
13.3.2. Gain of function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
13.4. Linked Markers for Genotype Prediction . . . . . . . . . . . . . . . . . . . . 237
13.5. Pedigree Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
13.5.1. X-linked recessive inheritance . . . . . . . . . . . . . . . . . . . . . . 239
13.6. Modified Risk: Bayes Theorem . . . . . . . . . . . . . . . . . . . . . . . . . 240
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14.Krebs- (TCA-) cycle: Burning the carbon skeleton 243

14.1. The Pyruvate Dehydrogenase Reaction . . . . . . . . . . . . . . . . . . . . . 243
14.1.1. The pyruvate dehydrogenase complex . . . . . . . . . . . . . . . . . 243
14.1.2. Overall reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
14.1.3. Functional impairment . . . . . . . . . . . . . . . . . . . . . . . . . . 243
14.2. The TCA Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
14.2.1. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
14.2.2. Products of the TCA cycle . . . . . . . . . . . . . . . . . . . . . . . 244
14.2.3. Regulation of pyruvate dehydrogenase and TCA cycle . . . . . . . . 244
14.3. Inhibition of the TCA cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
14.3.1. Other reactions of TCA cycle intermediates . . . . . . . . . . . . . . 245
14.4. Shuttles Across the Inner Mitochondrial Membrane . . . . . . . . . . . . . . 246
14.5. Shuttles for electrons from cytoplasmic NADH + H+ . . . . . . . . . . . . . 246
14.5.1. Other substrates and products . . . . . . . . . . . . . . . . . . . . . 247
14.6. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
15.Respiratory Chain, Oxidative Phosphorylation and Reactive Oxygen Species 249

15.1. Respiratory Chain: Burning Hydrogen . . . . . . . . . . . . . . . . . . . . . 249
15.1.1. Overall reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
15.1.2. The redox potential . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
15.1.3. Components of the electron transport chain . . . . . . . . . . . . . . 250
15.2. Making ATP from electricity . . . . . . . . . . . . . . . . . . . . . . . . . . 251
15.2.1. Phosphorylation sites . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
15.2.2. Mechanism of oxidative phosphorylation . . . . . . . . . . . . . . . . 252
15.2.3. Energy yield from glucose . . . . . . . . . . . . . . . . . . . . . . . . 252
15.2.4. Regulation of electron flow and phosphorylation . . . . . . . . . . . . 252
15.2.5. Inhibition of oxidative phosphorylation . . . . . . . . . . . . . . . . . 252
15.3. Reactive Oxygen Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
15.3.1. Protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 254
15.4. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
16.Single-Gene Disorders and Traits 257

16.1. Skeletal and Connective Tissue Diseases . . . . . . . . . . . . . . . . . . . . 257
16.2. Skeletal Dysplasias and Dysostoses . . . . . . . . . . . . . . . . . . . . . . . 258
16.3. Diseases Of Muscles and Peripheral Nerves . . . . . . . . . . . . . . . . . . . 259
16.3.1. Other Muscular Dystrophies . . . . . . . . . . . . . . . . . . . . . . . 259
16.3.2. Myotonic Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
16.3.3. Peripheral Neuropathies . . . . . . . . . . . . . . . . . . . . . . . . . 260
16.4. CNS Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
16.4.1. Hereditary ataxias . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
16.4.2. Mental Retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
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16.5. Blood Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

16.5.1. Clotting Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
16.5.2. Structural defects of RBCs . . . . . . . . . . . . . . . . . . . . . . . 263
16.6. Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
16.6.1. Occulocutaneous albinism . . . . . . . . . . . . . . . . . . . . . . . . 263
16.6.2. Epidermolysis bullosa (EB) . . . . . . . . . . . . . . . . . . . . . . . 264
16.7. Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
16.8. Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
16.9. Blindness and Deafness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
16.10.“Harmless” Mendelian Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
16.11.Tuberous Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
16.12.Phenylketonuria (PKU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
16.13.Hemochromatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
16.14.Diseases Caused by Unusual Mutations . . . . . . . . . . . . . . . . . . . . . 268
16.14.1.Imprinting-related Syndromes . . . . . . . . . . . . . . . . . . . . . . 268
16.14.2.Lepore Hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
16.15.Objectives in Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
17.Hormone Biochemistry 271

17.1. Types of Extracellular Messenger . . . . . . . . . . . . . . . . . . . . . . . . 271
17.2. Hormone Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
17.3. Types of Hormone Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
17.4. Receptors Coupled to G-Protein . . . . . . . . . . . . . . . . . . . . . . . . . 273
17.5. Cyclic AMP (cAMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
17.6. Calcium and Phosphatidylinositol . . . . . . . . . . . . . . . . . . . . . . . . 275
17.7. Cyclic GMP (cGMP). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
17.8. Desensitization of Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
IV. Semester two, Mini I 279
18.Vitamins and minerals 281

18.1. Nutrient doses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
18.1.1. The US Recommended Daily Allowance . . . . . . . . . . . . . . . . 283
18.1.2. Assessing nutrient stores and needs . . . . . . . . . . . . . . . . . . . 283
18.2. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
18.2.1. Fat soluble vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
18.2.2. Water soluble vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . 299
18.3. Minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
18.3.1. Mass elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
18.3.2. Trace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
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18.3.3. Ultratrace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

19.Carbohydrate Metabolism 349

19.1. Gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
19.1.1. The first bypass: From pyruvate to phosphoenolpyruvate (PEP) . . . 349
19.1.2. The second and third bypasses . . . . . . . . . . . . . . . . . . . . . 349
19.1.3. Substrates of gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . 350
19.1.4. Energy balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
19.1.5. Regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . 350
19.2. Glycogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
19.2.1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
19.2.2. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
19.2.3. Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
19.2.4. Difference between liver and muscle . . . . . . . . . . . . . . . . . . . 352
19.2.5. Regulation of glycogen metabolism . . . . . . . . . . . . . . . . . . . 352
19.2.6. Glycogen storage diseases . . . . . . . . . . . . . . . . . . . . . . . . 353
19.3. Dietary Fructose and Galactose . . . . . . . . . . . . . . . . . . . . . . . . . 354
19.3.1. Fructose metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
19.3.2. Galactose metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . 355
19.4. The Pentose Phosphate Pathway . . . . . . . . . . . . . . . . . . . . . . . . 355
19.4.1. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
19.4.2. Products and regulation . . . . . . . . . . . . . . . . . . . . . . . . . 356
19.4.3. Physiological role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
19.4.4. Glucose-6-phosphate dehydrogenase deficiency . . . . . . . . . . . . . 357
19.5. The ‘Minor’ Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
19.5.1. The Polyol Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
19.5.2. Synthesis of Amino Sugars . . . . . . . . . . . . . . . . . . . . . . . . 358
19.5.3. The uronic acid pathway. . . . . . . . . . . . . . . . . . . . . . . . . 358
19.6. Practice Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
20.Lipid Metabolism 361

20.1. Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
20.2. Utilization of Dietary Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
20.3. Adipose tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
20.3.1. Triglyceride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
20.4. Fatty Acid Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
20.5. Ketogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
20.6. Aberrations Of Fatty Acid Metabolism . . . . . . . . . . . . . . . . . . . . . 367
20.7. From Carbohydrate To Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
20.8. Phosphoglyceride Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . 368
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20.9. Sphingolipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

20.10.Cholesterol And Bile Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
20.11.Lipoprotein Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
20.12.Lipoprotein Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
20.13.Lipoproteins and Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . 374
20.14.Lipoprotein Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
20.15.Practice Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
20.16.Objectives In Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
V. Semester two, Mini II 379
21.Nutritional Management of Disease 381

21.1. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
21.2. Weight Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
21.3. Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
21.3.1. Types of diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . 383
21.3.2. Management of Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . 385
21.4. Coronary Heart Disease (CHD) . . . . . . . . . . . . . . . . . . . . . . . . . 387
21.4.1. Dietary Guidelines for Reducing Blood Cholesterol . . . . . . . . . . 388
21.5. Prevention of Coronary Heart Disease (CHD) . . . . . . . . . . . . . . . . . 391
21.6. Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
21.7. The Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
21.7.1. Disease of the Biliary System . . . . . . . . . . . . . . . . . . . . . . 397
21.7.2. Parenchymal Liver Disease . . . . . . . . . . . . . . . . . . . . . . . 398
21.8. The Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
21.8.1. Glomerulonephritis and Nephrotic Syndrome . . . . . . . . . . . . . 400
21.8.2. End-Stage Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . 401
21.9. Nutritional Therapy In Surgery And Injury . . . . . . . . . . . . . . . . . . 402
22.Amino Acid Metabolism 405

22.1. Protein metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
22.1.1. Biological value of proteins . . . . . . . . . . . . . . . . . . . . . . . 406
22.2. Nitrogen metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
22.2.1. Nitrogen transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
22.2.2. Urea-cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
22.3. Catabolism of the carbon backbone . . . . . . . . . . . . . . . . . . . . . . . 414
22.3.1. Ala and Ser enter glycolysis . . . . . . . . . . . . . . . . . . . . . . . 415
22.3.2. Glu, Gln, Asp and Asn enter the Krebs-cycle . . . . . . . . . . . . . 418
22.3.3. Gly, Thr and Ser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
22.3.4. The sulphur-containing amino acids: Met and Cys . . . . . . . . . . 422
22.3.5. Branched-chain amino acids: Val, Ile, Leu . . . . . . . . . . . . . . . 422
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22.3.6. Phe and Tyr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

22.3.7. Trp and Lys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
22.3.8. Pro, Arg, His and ornithine . . . . . . . . . . . . . . . . . . . . . . . 429
22.4. Compounds derived from amino acids . . . . . . . . . . . . . . . . . . . . . 431
22.4.1. Carnitine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
22.4.2. Creatine and creatinine . . . . . . . . . . . . . . . . . . . . . . . . . 432
22.4.3. Polyamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
22.5. Physiology of amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
22.5.1. Metabolism of amino acids . . . . . . . . . . . . . . . . . . . . . . . 435
22.5.2. Inherited amino acid transporter deficiencies . . . . . . . . . . . . . . 435
22.6. Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
22.7. Objectives in Summary: Amino acid metabolism . . . . . . . . . . . . . . . 437
23.Biochemistry of Digestion 439

23.1. Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
23.1.1. Digestive secretions in man . . . . . . . . . . . . . . . . . . . . . . . 439
23.1.2. Undigestible materials . . . . . . . . . . . . . . . . . . . . . . . . . . 445
23.1.3. Lactose intolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
23.1.4. Zymogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
23.2. Intermediary metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
23.3. Carbohydrate metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
23.4. Fat metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
23.5. Metabolism of amino acids and protein . . . . . . . . . . . . . . . . . . . . . 452
23.6. Alcohol Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
23.7. Intermediary metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
23.8. Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
23.9. Digestion: Objectives in summary . . . . . . . . . . . . . . . . . . . . . . . . 455
24.Heme, Purines and Pyrimidines 457

24.1. Heme Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
24.1.1. Disorders of Heme Biosynthesis . . . . . . . . . . . . . . . . . . . . . 457
24.2. Heme Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
24.2.1. Hyperbilirubinemia and Jaundice . . . . . . . . . . . . . . . . . . . . 461
24.3. Purines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
24.3.1. Purine Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
24.3.2. Purine Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
24.4. Pyrimidine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
24.5. Salvage pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
24.6. Deoxyribonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
24.7. Anti-neoplastic and anti-bacterial drugs acting on nucleotide metabolism . . 468
24.8. Hyperuricemia and Gout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
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25.Nutrition During the Life Cycle 473

25.1. Nutrition in Pregnancy and Lactation . . . . . . . . . . . . . . . . . . . . . 473
25.2. Breastfeeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
25.2.1. Composition of Human Milk . . . . . . . . . . . . . . . . . . . . . . 476
25.2.2. Dietary recommendations for lactating mothers . . . . . . . . . . . . 477
25.3. Feeding the Weaning Age Group . . . . . . . . . . . . . . . . . . . . . . . . 478
25.4. Feeding the School Child . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
25.5. Feeding Adolescents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
25.6. Nutrition in the Elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
26.Cell Cycle Control and Cancer 485

26.1. Cell Cycle Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
26.2. Normal Cells in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
26.3. Cyclins and the Retinoblastoma Protein . . . . . . . . . . . . . . . . . . . . 486
26.4. p53 and the Damage Response . . . . . . . . . . . . . . . . . . . . . . . . . 487
26.5. Growth Control by External Stimuli . . . . . . . . . . . . . . . . . . . . . . 488
26.6. Mitogenic Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
26.7. Principles of Malignant Transformation . . . . . . . . . . . . . . . . . . . . . 490
26.8. Cellular Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
26.9. Nuclear Proteins in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
26.10.Virally-Induced Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
26.11.Inherited Cancer Susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . 493
26.12.Objectives in Brief . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
27.Immunoglobulins and Immunogenetics 497

27.1. Antibody Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
27.1.1. Structure of Immunoglobulin G (IgG) . . . . . . . . . . . . . . . . . 497
27.1.2. Heterogeneity of Immunoglobulins . . . . . . . . . . . . . . . . . . . 499
27.1.3. Other Ig Domain Proteins . . . . . . . . . . . . . . . . . . . . . . . . 500
27.2. Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
27.2.1. The Major Histocompatibility Locus (MHC) . . . . . . . . . . . . . 501
27.2.2. Immunoglobulin Gene Structure . . . . . . . . . . . . . . . . . . . . 506
27.3. MHC (HLA) and Clinical Risk of Disease . . . . . . . . . . . . . . . . . . . 508
27.3.1. MHC Polymorphisms and Disease Risk . . . . . . . . . . . . . . . . . 508
27.3.2. Familial Immune Disorders . . . . . . . . . . . . . . . . . . . . . . . 508
28.Inherited diseases of metabolism 511

28.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
28.1.1. Significance of inherited diseases of metabolism . . . . . . . . . . . . 511
28.1.2. Mechanism of IDoM . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
xiv
Contents
28.1.3. Newborn screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

28.2. Cytosolic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
28.2.1. G6PDH deficiency – Favism . . . . . . . . . . . . . . . . . . . . . . . 516
28.2.2. Galactosemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
28.2.3. Errors of fructose metabolism . . . . . . . . . . . . . . . . . . . . . . 518
28.2.4. Lactase persistence/restriction . . . . . . . . . . . . . . . . . . . . . 518
28.2.5. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
28.3. Glycogen storage diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
28.4. Lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
28.4.1. I-cell disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
28.4.2. Mucopolysaccharidoses and sphingolipidoses . . . . . . . . . . . . . . 522
28.5. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
28.5.1. Pyruvate metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
28.5.2. β-oxidation of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . 532
28.6. Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
28.7. Endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
28.7.1. Biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
28.8. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
VI. Semester two, Mini III 545
29.Advanced DNA technology 547

29.1. Germline Gene Manipulations (analysis of gene function) . . . . . . . . . . . 547
29.1.1. Cre/LoxP system for recombination . . . . . . . . . . . . . . . . . . 549
29.2. RNAi (inhibitory RNA, Knock-down) . . . . . . . . . . . . . . . . . . . . . 550
29.3. Microarray Technology (DNA Chips) . . . . . . . . . . . . . . . . . . . . . . 550
29.4. Somatic Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
29.5. Proteomics Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
29.6. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
30.Population Genetics and Genetic Counseling 555

30.1. Genotype Frequencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
30.1.1. The Hardy-Weinberg equation . . . . . . . . . . . . . . . . . . . . 555
30.2. Inbreeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
30.3. Mutation and Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
30.4. Genetic Counseling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
30.5. Diagnostic Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
30.6. Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
30.7. Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
30.8. Screening Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
30.9. Assisted Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
xv
Contents
30.10.Objectives in Brief . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
31.Integration of Metabolism 569

31.1. Regulation of Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . 569
31.1.1. Hormonal Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
31.1.2. Metabolic role of organs . . . . . . . . . . . . . . . . . . . . . . . . . 570
31.2. The respiratory quotient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
31.3. The Starve-Feed Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
31.3.1. Phases of the starve-feed cycle . . . . . . . . . . . . . . . . . . . . . 574
31.3.2. Role of organs during starve-feed cycles . . . . . . . . . . . . . . . . 576
31.3.3. Unbalanced meals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
31.4. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
31.4.1. Adipose tissue in obesity . . . . . . . . . . . . . . . . . . . . . . . . . 584
31.4.2. Appetite control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
31.4.3. Role of gut flora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
31.4.4. Beneficial effects of dietary restriction . . . . . . . . . . . . . . . . . 589
31.4.5. Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
31.4.6. Systems biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
31.4.7. Metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
31.5. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
32.Multifactorial Inheritance 607

32.1. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
32.2. Quantitative Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
32.3. Common Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
32.4. Congenital Malformations (“Birth Defects”) . . . . . . . . . . . . . . . . . . 611
32.4.1. Nongenetic Causes Of Congenital Malformations . . . . . . . . . . . 612
32.5. Quantitative Trait Loci (QTLs) . . . . . . . . . . . . . . . . . . . . . . . . . 615
32.6. Examples Of Susceptibility Genes . . . . . . . . . . . . . . . . . . . . . . . . 616
VII. Appendix 619
33.Answers to the example questions 621

33.1. Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
33.2. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
33.3. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
33.4. Amino acid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
33.5. Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
xvi
Contents
33.6. Integration of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
34.Tables 629
34.1. Conversion from non-metric to metric units . . . . . . . . . . . . . . . . . . 630
34.2. Symbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
34.3. Greek alphabet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
34.4. The genetic code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
34.5. Periodic system of the elements . . . . . . . . . . . . . . . . . . . . . . . . . 634
35.Acronyms 635
xvii
Welcome
The faculty of the Biochemistry Department at Ross University welcome you to the bio-
chemistry course. The following handouts are provided to supplement lectures and reading
material in your text to complement the required textbooks:
G. Meisenberg & W.H. Simmons: Principles of Medical Biochemistry, 2nd ed., Philadel-
phia (Mosby) 2006
L.B. Jorde, J.C. Carey, M.J. Bamshad & R.L. White: Medical Genetics, 3rd updated
ed., Philadelphia (Mosby) 2006
You should look at the appropriate chapters of both the handout and your textbooks before
a lecture, this will enhance your learning success.
Please understand that this handout set is in no way a sure indication of what you will
be required to know for exams. This material can be viewed as the minimum information
students are expected to know. Additional reading material for this course can be found
in the library. Reserve materials, texts and journals in this subject area are also available.
We encourage you to use these materials in addition to your assigned materials throughout
the semester. Enhancing and updating your knowledge of biochemistry requires you to
take the long view, and develop habits which foster lifetime learning. Discussion of medical
biochemistry topics with faculty and other students is helpful for integrating this core
knowledge with your other subjects.
The following web-sites are a small sampling of additional material which can be accessed
online. We encourage your use of internet resources to enhance your integration of course
material.
General Biochemistry Courses:

http://www.kumc.edu/biochemistry/bioc800/biocindx.htm
http://web.indstate.edu/thcme/mwking/
http://tutor.lcsf.ucsb.edu/instdev/sears/biochemistry/
Metabolism:
http://www.gwu.edu/ mpb/
Clinical Biochemistry:
http://www.qub.ac.uk/cm/cb/text/studgide/index.htm
xix
Database searching for journal articles, and biochemical information:

http://www3.ncbi.nlm.nih.gov/Entrez/index.html
Enjoy your learning experience in this course! And remember, the faculty are here to help
you succeed.
Sincerely yours,
The Biochemistry Faculty
xx
Part I.
Semester one, Mini I

1. Introduction to Biomolecules
1.1. Elements and molecules
Biomolecules contain only a few of the 92 elements that exist in nature: carbon hydrogen,
oxygen (all), nitrogen (proteins, nucleic acids, vitamins), sulfur (proteins, vitamins, inter-
mediates), phosphate (nucleic acids, some proteins, many metabolic intermediates). Some
elements occur in charged form: sodium, potassium, calcium, magnesium, chloride.
If you know the composition of a molecule, you know its molecular weight. Example: Glucose
(C6 H12 O6 ) has a molecular weight (MW) of 180. One mol is the molecular weight in grams
(g). 1 mol of glucose is 180 g. Concentrations of dissolved molecules are often given in mol
per liter (mol/L, or M), millimole per liter (mmol/L), or milligrams per 100 ml (90 mg/dl).
Can you calculate its molar concentration?
1.2. Covalent Bonds And Non-covalent Interactions
1.2.1. Covalent bonds
The covalent bonds that connect the atoms in molecules are formed by binding electron
pairs which orbit both atoms. Formation and breakage of covalent bonds requires chemical
reactions which, in the body, are catalyzed by enzymes.
Table 1.1.: Important elements

Element biol. abund. terrest. abund. atomic mass
(% w/w) (% w/w) (Da)
Carbon (C) 18.5 0.030 12
Hydrogen (H) 9.5 0.140 1
Oxygen (O) 65.0 65.0 16
Nitrogen (N) 3.3 0.005 14
Sulfur (S) 0.3 0.050 32
Phosphorus (P) 0.2 0.120 31
3
1.2.2 Biochemistry and Genetics
Table 1.2.: Composition of the human body

Class of Molecule Content (%) MW (Da)
Water 60.0 18
Inorganic salt, soluble 0.7 = atomic weight
Inorganic salt, insoluble 5.5 = atomic weight
Protein 16.0 5 × 103 –1 × 106
Triglyceride (fat) 13.0 ≈ 800
Membrane lipids 2.5 400–1000
Carbohydrates 1.5 >1000 (polysaccharides)
Nucleic acids 1.2 2 × 104 –2 × 108
Electronegativity is the tendency of an atom to attract electrons. The order of electroneg-

ativities of atoms in biomolecules is:
O>N>S>C≈H
1.2.2. Non-covalent interactions
The binding electron pair of a covalent bond is displaced towards the more electronegative
atom. This creates a dipole, with partial positive and negative charges at opposite ends.
Opposite charges attract each other, therefore dipole-dipole interactions can form between
different polarized bonds. Hydrogen bonds are a type of dipole-dipole interaction involv-
ing a hydrogen bound to an electronegative atom (O or N). The water molecule is a dipole
with a partial negative charge on the oxygen and partial positive charge on the hydrogens:
The unusual physical properties of water (for example its high boiling point) are caused by
hydrogen bonds between water molecules.
δ+ δ+
H H
O
δ-
Ions carry either a positive charge (cations) or a negative charge (anions). They form ion-
dipole interactions with water. That is why many salts are water soluble: the interactions
with water can overcome the electrostatic interaction (“salt bond”) in the salt crystal.
Organic molecules that contain positive and negative charges interact strongly with water,
and most of them are water soluble. Also molecules that form hydrogen bonds with water
are water soluble. Molecules that have mostly nonpolar bonds, (especially the hydrocarbons
which consist of carbon and hydrogen) are insoluble in water.
4
Bonds in biomolecules 1.3
H2N NH2 H2N CH3 H3C CH3 H3C CH3

C C C C
> > > H2
O O O
Urea Acetamide Acetone Propane
Polar interactions (ion-ion, ion-dipole, dipole-dipole) are also important for interactions
within and between biomolecules. Hydrophobic interactions are formed between nonpolar
groups (usually hydrocarbon) in the molecules.
Nonpolar groups occupy space between the water molecules preventing their interactions.
Therefore, hydrophobic groups aggregate to minimize their interface with water. Van-der-
Waals interactions are weak non-covalent forces between neighboring molecules. All non-
covalent bonds are weak. They form and break spontaneously, therefore all non-covalent
bonding is reversible.
1.3. Bonds in biomolecules
Functional groups determine chemical reactivity and physical properties (solubility, melting
point, boiling point) of the molecule. Most important functional groups (R= “residue”, the
remainder of the molecule):
Alkyl groups R−CH3 (methyl), R−CH2−CH3 (ethyl) etc. Nonpolar (hydrophobic) groups.
Most common in lipids.
Hydroxy group R−OH Non-ionizable, forms dipole-dipole interactions. Occurs in carbo-

hydrates, ethanol, some amino acids.
Carboxy group This is the most important acidic group in biomolecules. In fatty acids,
amino acids, and many metabolic intermediates. The carboxy group carries a negative
charge at pH = 7.
_
O O O O O O
C C C
R R R
5
1.3 Biochemistry and Genetics
Carbonyl group Non-ionizable, forms dipole-dipole interactions. Occurs in monosaccha-

rides.
O H O R
C C
R R
aldehyde ketone
Amino group These are the most important basic groups in biomolecules. In amino acids
and biogenic amines. The nitrogen of aliphatic amines carries a positive charge at pH = 7.
H H R" R"
+
R N| R N| R N| R N R'"
H R' R' R'
primary amine secondary amine tertiary amine quarternary ammonium base
Sulphydryl group R−SH Weakly acidic. In cysteine, and coenzyme A.
Hemiacetal Non-ionizable, in the ring forms of monosaccharides.

OH
R C H
OR'
Hemiacetal
Most biomolecules are large molecules (macromolecules) which are formed when functional
groups in their building blocks react with the formation of water. These reactions are called
condensation reactions. The reversal of a condensation reaction is called hydrolysis.
Condensation reactions are endergonic (energy-requiring), while hydrolysis reactions are
exergonic (energy-releasing):
Carboxylic ester From carboxy group and hydroxy group. Example: Triglycerides (fat)
O O
R C + HO R" R C O R" + H-O-H
OH
Phosphate ester From hydroxy group and phosphate. Example: Many intermediates of
carbohydrate metabolism. All esters can be cleaved by acid and, especially, alkaline hydrol-
ysis.
O O
O P OH + HO R O P O R + H-O-H
O O
6
Bonds in biomolecules 1.3
Phosphodiester From hydroxy group and phosphate ester. Examples: Nucleic acids, phos-
pholipids.
O O
R O P OH + HO R' R O P O R' + H-O-H
O O
Mixed anhydride From two different acids (e.g., carboxylic acid and inorganic phosphate).
In some metabolic intermediates.
O O O
O
O P OH + C R O P O C R + H-O-H
HO
O O
Phosphoanhydride From phosphate and phosphate (-ester). Example: Energy-rich bonds

in ATP.
O O O O
O P OH + HO P O R O P O P O R + H-O-H
O O O O
Ether From two hydroxy groups. In some O-methylated and hydroxy group.
R OH + HO R' R O R' + H-O-H
Acetal From hemiacetal and hydroxy group. In Carbohydrate, where they are known as
glycosidic bonds.
R' O R' O
R" C OH + HO R R" C O R + H-O-H
H H
Thioesters From sulfhydryl group and carboxy group. In coenzyme A thioesters (acetyl-
CoA, fatty acyl-CoA).
O
O
R C + HS R' R C S R' + H-O-H
OH
Amides From (carboxylic) acid and ammonia or amine. Example: Peptide bonds in pro-
teins.
7
O O
R C + NH3 R C + H-O-H
OH NH2
O O
R C + H2N R' R C + H-O-H
OH N R'
H
The formation of anhydride bonds and thioester bonds requires more energy than the
formation of the other bonds. These bonds are called energy-rich bonds.
1.4. Isomers
Isomers are chemically different molecules of identical composition. There are three different
types:
Positional isomers differ in the positions of atoms or functional groups within the molecule.
H
H O
C H C OH
H C OH C O
H C OH H C OH
H H
Glyceraldehyde Dihydroxyacetone
Geometric isomers differ in the relative geometric position of different parts of the mole-
cule, for example cis-trans isomers at double bonds. Substituents at double bonds are
planar, and they don’t rotate:
R' H R' H
R H H R
cis-isomer trans-isomer
Optical isomers arise when a carbon has four different substituents making it an asymmet-
ric carbon. If the molecule has only one asymmetric carbon, the isomers are mirror
images or enantiomers. Also disastereomers differ only in the position of some groups
in space, but they are not mirror images. Unlike positional isomers and diastereomers,
enantiomers have identical physicochemical properties. But they rotate the plane of
8
Acids and bases 1.5
polarized light in opposite directions. Optical isomerism is also called chirality. Ex-
ample:
O O O O
C C
+ +
H C N H3 H3N C H
R R
D-amino acid L-amino acid
Alternative positional, geometric and optical isomers are not equivalent biologically. En-
zymes can distinguish between the isomers.
1.5. Acids and bases
A proton can be transferred from one water molecule to another in a spontaneous, reversible
reaction 2H2 O *
) H3 O+ + OH−
In distilled water, [H3 O+ ] = [OH− ] = 1 × 10−7 M, and [H3 O+ ] × [OH− ] = 10 × 10−14 M2 .
This means that any rise in [H3 O+ ] must be compensated by a decline in [OH− ] and vice
versa. The concentration of [H3 O+ ] is usually written as the “proton concentration” [H+ ],
although free protons are uncommon in water. The proton concentration is expressed as
the pH value. The pH value is the negative logarithm of [H+ ].
pH = 7 neutral ([H+ ] = 1 × 10−7 M)

pH < 7 acidic ([H+ ] > 1 × 10−7 M)
pH > 7 alkaline ([H+ ] < 1 × 10−7 M)
The pH of cells and body fluids has to be kept constant. Normal blood pH: 7.4.
Acids are substances which donate protons to water. They increase [H+ ] and decrease the
pH. Bases are substances which accept protons from water: They decrease [H+ ] and increase
the pH.
Many biomolecules contain acidic and basic groups which undergo reversible protona-
tion/deprotonation reactions. The protonation state of such groups depends on the sol-
vent pH. This can be understood in terms of mass action: H+ is a reactant, therefore its
concentration drives protonation and deprotonation reactions.
The major acidic group in biomolecules is the carboxy group, which undergoes the following
ionization reaction:
O O
R C + H2O R C + H3O+
OH O
9
This is a nonenzymatic, instantaneous, freely reversible equilibrium reaction. Note that

the carboxylate anion (R − COO− ) is itself a base, ready to accept a proton and reform
the carboxylic acid in the reverse reaction. The carboxylate anion is the conjugate base of
the carboxylic acid. Acids are uncharged in the protonated form and negatively charged
(anionic) in the deprotonated form.
The major basic group in biomolecules is the primary amino group:

+
R NH2 + H3O+ R N H3 + H2O
The ammonium salt (R − N+ H3 ) is itself an acid, ready to release a proton and reform the
primary amino group in the reverse reaction. The ammonium salt is the conjugate acid of
the amine. Bases are positively charged (cationic) in the protonated form and uncharged
in the deprotonated form.
The protonation/deprotonation reaction

O O
R C + H2O R C + H3O+
OH O
can be described by its dissociation constant Ka :
[R−COO− ] × [H + ] [R−COOH]
Ka = or [H + ] = Ka × (1.1)
[R−COOH] [R−COO− ]
We can put this whole equation into the negative logarithm:
[R−COOH] [R−COO− ]
pH = pK a − log( ) = pK a + log( ) (1.2)
[R−COO− ] [R−COOH]
The pKa value is an intrinsic property of the ionizable group. If the pH equals the pKa , the
group is half-protonated; if pH > pKa it is mostly deprotonated; if pH < pKa , it is mostly
protonated.
Important ionizable groups in biomolecules:
Acidic groups, deprotonated at pH 7: Carboxy group, phosphate ester, phosphodiester.
Acidic groups, protonated at pH 7: Sulfhydryl group, phenolic hydroxy group.
Basic groups, protonated at pH 7: Aliphatic amino groups.
Basic groups, deprotonated at pH 7: Aromatic amines.
10
Fats and carbohydrates 1.6
1.6. Fats and carbohydrates
Triglycerides (“fat”) are esters formed from glycerol and fatty acids
H2C OH
HO CH
C OH
Glycerol H2
H2 H2 H2 H2 H2 H2 H2
C C C C C C C COOH
H3C C C C C C C C
H2 H2 H2 H2 H2 H2 H2
Fatty acid (Palmitic acid)
Palmitic acid is a saturated fatty acid. Monounsaturated fatty acids have a single C=C
double bond, and polyunsaturated fatty acids have more than one C=C double bond.
2
O H O
O H
H2C C C
Triglycerides are not water-soluble. They are used as energy stores in adipose tissue. Non-
polar molecules like the triglycerides are called lipids. Lipids other than triglycerides occur
in biological membranes. They include the phospholipids, glycolipids and cholesterol.
Carbohydrates are made from monosaccharides. These are polyalcohols containing an

aldehyde group (aldoses) or keto group (ketoses). Monosaccharides may have:
3 carbons Trioses 6 carbons Hexoses
4 carbons Tetroses 7 carbons Heptoses
5 carbons Pentoses etc.
Examples:
O O O O
CH CH CH CH2OH CH
HC OH HC OH HO CH O C HC OH
HO CH HO CH HO CH HO CH HC OH
HO CH HC OH HC OH HC OH HC OH
HC OH HC OH HC OH HC OH CH2OH
CH2OH CH2OH CH2OH CH2OH
D-Galactose D-Glucose D-Mannose D-Fructose D-Ribose

Gal Glu Man Fru Rib
Only D-isomers are shown as these are prevalent in nature.
Gal, Glc and Man are not enantiomers but diastereomers because they are not mirror
11
images. Monosaccharides that are distinguished by the orientation of substituents around

a single asymmetric carbon are called epimeres.
Most monosaccharides form ring structures by a hemiacetal or hemiketal bond between the
carbonyl group and one of the hydroxy groups:
O CH2OH
O OH
HO OH
OH
OH β-D-glucose
HO
OH OH
CH2OH
OH O α-D-glucose
D-glucose OH
OH OH
OH
Glucose spends 99.9975 % of its time in the ring form. C-1 in the ring form is asymmetric.
It gives rise to α and β isomers which are called anomers. Because the ring can open
and close spontaneously, α and β equilibrate until 34 % is α and 66 % is β. This is called
mutarotation.
Disaccharides (from 2 monosaccharides), (from “a few” monosaccharides) and (from

“many” monosaccharides) are formed by . These are acetal or ketal bonds that involve at
least one anomeric (aldehyde or keto carbon). Glycosidic bonds involving the anomeric
carbon of a carbohydrate can also be formed with non-carbohydrate components.
Important disaccharides:
Maltose Glucose+ Glucose (α-1,4 bond)
Lactose Galactose + Glucose (β-1,4 bond)
Sucrose Glucose + Fructose (α, β-1,2 bond)
Important polysaccharides
Starch Glucose, with α-1,4 bonds and some α-1,6 bonds.
Glycogen Like starch, but with more α-1,6 bonds
Cellulose Glucose, with β-1,4 bonds
Monosaccharides and disaccharides are water soluble. Polysaccharides are hydrated, but not
all are water soluble. Monosaccharides have reducing properties because of the presence of
the carbonyl carbon.
12
Molecular Structure 1.7.1
1.7. Objectives in summary
1.7.1. Molecular Structure
1. Recognize in chemical formulas the important functional groups and bond types,
including hydroxy, carbonyl, carboxy, sulfhydryl and amino groups, and ester, ether,
glycosidic, thioester and amide bonds.
2. Know the relative electronegativities of O,N,S,C and H. State that chemical bond
formation is usually endergonic while hydrolytic bond cleavage is exergonic.
3. Define the term “energy rich bond”.
4. Describe the principal types of non-covalent interaction between biomolecules, in-
cluding hydrogen bonds, salt bonds, van der Waals interactions and hydrophobic
interactions.
5. Describe structural features of biomolecules, which tend to increase or decrease water
solubility, including the effect of pH and charge pattern.
6. Know the definition of terms, such as, redox reaction, anion, cation, zwitterions, pH
value, isoelectric point and buffering capacity.
7. Application of the Henderson-Hasselbalch equation to problems in which either
the pH the pKa or the ratio of protonated/unprotonated form has to be calculated.
8. Recognition of asymmetric carbons in chemical structures and predict existence of
optical isomers.
9. Recognize the general structures of glycerol, fatty acids, and triglycerides in the chem-
ical formula.
10. Describe the properties of fatty acids and triglycerides with respect to water solubility,
chemical stability of the ester bond and effects of pH changes on protonation state
water solubility.
11. Recognition of the general structure of monosaccharides both in open -chain form and
the Hayworth projection.
12. Define the terms aldose, ketose, triose, pentose, hexose, hemiacetal, hemiketal, and
glycosidic bond.
13. Name the chemical building blocks and bond types in nucleic acids.
13
2. Energy changes and rates of chemical
reactions
Thermodynamic properties are those features of a chemical reaction that are related
to its energy balance and equilibrium. Kinetic properties are those related to the rate
(velocity) of the reaction. Only the kinetic properties of reactions are changed by enzymes.
2.1. Thermodynamics
The following definitions of important terms are oversimplified, but sufficient for the purpose
of medical biochemistry. It is important that you know them as they will be frequently used
to describe reactions that go on in our body.
Temperature is the random movement of molecules, and is measured in K. 0 K is the lowest
theoretically possible temperature, where not only all molecules, but even the electrons of
their atoms, would be at rest. In practice, this temperature can never be reached. Note that
temperatures are measured in Kelvin (K), but temperature differences in °. There is no
such thing as °K. Alternatively, temperatures may also be measured in degrees Celsius °C
(confusing, isn’t it?). You convert from °C to K by adding 273.16. In formulas, the capital
T represents temperature. In the US the unit °F was used in the past, but you should avoid
any non-SI units, they make a lot of additional work and are a source of calculation error.
Heat (q) flows between bodies of different temperature, with q = C × ∆T . C is the heat
capacity of the bodies (amount of energy you need to increase the temperature by a given
amount), and ∆T the difference in temperature between them. The Greek letter ∆ (Delta)
always symbolizes differences. In formulas, the heat leaving a body is denoted with a nega-
tive, heat entering a body with a positive sign. For an example see fig. 2.1. Heat is measured
in Joule J.
Work w is anything that “somehow” can be converted to the lifting of weights. When work
is transferred across system boundaries then work done on a system gets a positive, system
done by a system a negative sign. The unit for work is the Joule (J).
Energy is the sum of heat and work:
E = q + w = const (2.1)
15
Figure 2.1.: Heat flowing between bodies of different temperatures. For explanations see
text.
40 °C 20 °C
60 °C 0 °C 30 °C 30 °C
In other words, energy can not be created nor destroyed. This statement is called the first
law of thermodynamics. Since both heat and work are measured in J, energy of course
has that unit too.
A system is some part of the universe, which is separated from the rest (see fig. 2.2). We
distinguish:
Open systems can exchange both matter and energy with the rest of the environment.
Example: an open test tube.
Closed systems can exchange energy, but not matter with the environment. A stoppered
test tube would be an example, matter can no longer leave or enter, but we may still
heat its content.
Insulated systems (also called “adiabatic” systems) can exchange neither matter nor en-
ergy with the environment. If we placed our stoppered test tube into a Dewar-vessel,
energy exchange would be prevented. Such vessels have a double-layer construction,
with vacuum as insulator between the inner and outer glass bottle to minimize con-
duction and convection. The bottles have a silver coating on the vacuum side, to limit
radiation. You may have used such vessels to keep your coffee hot.
Standard conditions: Some thermodynamic properties of a system depend on the condi-
tions that system is in. Chemists have agreed to reference their measurement to a pressure of
1014 hPa (standard atmospheric pressure), a temperature of 25 °C (= 298 K) and a concen-
tration of 1 M for all reactants. The latter point is an inconvenience for biochemists, because
if [H+ ] = 1 M, then pH= 0 (see later). Very few biochemical reactions occur at such low a
16
Thermodynamics 2.1
Figure 2.2.: From the left to the right: open, closed and insulated (adiabatic) systems.
open closed insulated
pH, so biochemical standard conditions are pH 7. Biochemists also define standard values
for ionic strength (250 mM) and [Mg2+ ] (3 mM), since these influence the activity of many
enzymes. The values chosen simply reflect those that we find in living cells. If a parameter
X is measured under chemical standard conditions, we write X 0 , if it was measured un-
der biological standard conditions we write X 00 . In older books you may find X 0 ’ instead,
because IUPAC changed the nomenclature several years ago.
Enthalpy (H) is the heat of a chemical reaction. It is expressed in Joule/mol (J/mol).
Enthalpy itself can not be measured, but changes in enthalpy (∆H) can. To do these
measurements, we have to assign the value 0 J/mol to some arbitrary state. By convention,
this value is assigned to pure elements in their stable state under standard conditions.
If heat is produced by a reaction, we give ∆H a negative sign and call the reaction exother-
mic. If enthalpy is consumed during a reaction, we give ∆H a positive sign, such reactions
are called endothermic.
The change in enthalpy is given by:
∆H = ∆E + P × ∆V (2.2)
where P is the pressure (in Pa) and ∆V the change in volume. The product P × ∆V is the
mechanical work done during a reaction. Because biochemical reactions usually happen in
dilute aqueous solutions, ∆V ≈ 0 and hence ∆H ≈ ∆E. This is a useful result because it is
17
difficult to measure a change in energy, but much easier in to measure changes in enthalpy.
For biochemical reactions we can thus use ∆H as an approximation for ∆E, which is the
much more important parameter.
Entropy (S) is a measure of chaos. If you look at fig. 2.1, you see two bodies of different
temperature. If you bring them close together, heat could theoretically flow from the cold
body to the hot, making the cold body even colder and the hot body even hotter. The first
law of thermodynamics would allow that, because the energy lost by the cold body exactly
balances the energy gained by the hot. However, everyday experience shows us that this is
not what will happen. Instead, heat will flow from the hot body to the cold, until both have
the same temperature. This is an example for a general principle called the second law of
thermodynamics: The direction of a reaction is the one where the entropy (disorder) of
the universe is maximized, that is ∆S ≥ 0.
Note: The second law of thermodynamics allows for a local reduction of entropy as long
as the global entropy increases. Life does exactly that: Our sun sends out energy in form
of photons, constantly increasing universal entropy. Plants catch this orderly stream of
photons and dissipate it, again increasing the entropy of the universe. However, part of
this entropy difference is not given up to the universe, but saved in the form of organic
compounds like sugar, which animals then consume.
There is a third law of thermodynamics which states that the entropy of an ideal crystal
at a temperature of 0 K is 0 J mol−1 K−1 . This law allows us to measure entropies, but is of
no other consequence to biochemistry. You may safely assign it to passive knowledge.
Water below 100 °C is liquid, above that temperature it is gaseous, but we have to expend
energy to convert water of 100 °C into steam of 100 °C (the heat of evaporation). Gases, of
course, have a much higher entropy than liquids. Thus energy, temperature and entropy of
a reaction must be linked. This linkage is provided by the Gibbs free energy ∆G through
the Gibbs-Helmholtz-equation:
∆G = ∆H − T ∆S (2.3)
∆G is a very important parameter, which we will encounter again and again in this course.
It determines the direction of a chemical reaction:
∆G < 0: reaction proceeds from left to right (v+ > v− )
∆G = 0: reaction is at equilibrium (v+ = v− )
∆G > 0: reaction proceeds from right to left (v+ < v− )
By convention, chemical reactions are always written in the direction where ∆G is nega-
tive.
∆G is that part of ∆H of a reaction that can be used to do useful work. T ∆S is the part
of ∆H which is lost as heat. Therefore ∆G determines the energy efficiency of a reaction.
18
Reaction kinetics 2.2
This is, of course, a key driving force in evolution since any energy not lost as heat may
be used for growth and reproduction. Thus it is not surprising that the energy efficiency
of living organisms is close to the thermodynamically allowed maximum, and often much
higher than that of man-made devices.
Physical processes in which chemical bond energies don’t change (∆H = 0, for example
diffusion across a membrane) are also driven by ∆G via an increase in entropy (i.e. a
negative value for [T × ∆S]). The body has to use chemical bond energy to combat the
tendency for increasing entropy. That’s what metabolic energy is good for!
Summary:
Exothermic reaction: ∆H < 0, heat is released.
Endothermic reaction: ∆H > 0, heat is absorbed
Exergonic reaction: ∆G < 0, reaction can proceed
Endergonic reaction: ∆G > 0, reaction cannot proceed.
Equilibrium: ∆G = 0 concentration of reactants doesn’t change over time, as the rates for forward and
2.2. Reaction kinetics
In theory, all chemical reactions are reversible. If only non-covalent interactions are in-
volved, as in protonation-deprotonation, antigen-antibody binding and hormone-receptor
binding, the reactions are always found at equilibrium concentrations. Chemical reac-
tions in which covalent bonds are formed and broken are also reversible, but there is an
energy barrier to the reaction. Therefore the reactions are not always found at equilibrium
concentrations. The equilibrium is described by the equilibrium constant (Keq ). For the
reaction
k+1
GGGGGGGB
aA + bB + ... F GG zZ + yY + ... (2.4)
k−1
we get:
order of forward reaction: a + b + ...
order of backward reaction z + y + ...
velocity of forward reaction v+ = k+1 × [A]a × [B]b × ...
velocity of backward reaction v− = k−1 × [Z]z × [Y ]y × ...
[Z]z ×[Y ]y ×... k+1

equilibrium constant Keq = [A]a ×[B]b ×...
= k−1 (law of mass action)
19
Although the equilibrium constant is a thermodynamic characteristic, it is related to the

rate constants of the forward (k+1 ) and reverse reactions (k−1 ), since at equilibrium the
rates of the forward reaction and the reverse reaction are equal:
k+1 × [A]a × [B]b × ... = k−1 × [Z]z × [Y ]y × ... (2.5)

[Z]z × [Y ]y × ... k
a b
= +1 = Keq (2.6)
[A] × [B] × ... k−1
The actual free energy change (∆G) for the reaction is:
[Z]z × [Y ]y × ...

∆G = ∆G00 + RT × ln (2.7)
[A]a × [B]b × ...
with R = universal gas constant (8.314 472(15) J mol−1 K−1 ). Note that here the actual,
not the equilibrium concentrations are used.
At equilibrium, ∆G = 0, therefore there is a logarithmic relationship between Keq and

∆G00 :
Keq ∆G00 (kJ/mol)
1 × 10−5 28.5
1 × 10−4 22.8
1 × 10−3 17.1
1 × 10−2 11.4
1 × 10−1 5.7
1 0.0
1 × 101 -5.7
1 × 102 -11.4
1 × 10 3 -17.1
1 × 104 -22.8
1 × 105 -28.5
Note: By convention, capital K denotes equilibrium constants, small k rate constants. It

is important that you do not mix these up!
2.2.1. Order of Reactions
Zero-order reaction: The reaction rate v is independent of the substrate concentration.

Zero-order reactions are observed only in catalyzed reactions when the catalyst is saturated
with substrate. The decrease of the substrate concentration is linear. Formula (k = rate
constant, units are mol/s):
v=k (2.8)
20
The principle of Le Chatelier 2.3
First-order reaction: The reaction rate is proportional to the substrate concentration

(units of k are s−1 ):
v = k × [A] (2.9)
The decrease of the substrate concentration is asymptotic, and its half-life can be deter-
mined.
Second-order reaction: The reaction rate depends on the concentrations of 2 substrates:
v = k × [A] × [B] (2.10)
The unit of k is M−1 s−1 .
Pseudo-first-order reaction: There are 2 substrates, but one is present in large excess and
is not rate-limiting. kap describes the apparent rate constant for these conditions. Example:
Hydrolysis-reactions: A + H2 O → B + C, the water concentration is 1000 g/l / 18 g/mol
= 55.5 mol/l, almost independent of the concentration of the other reactants. Thus the
reaction rate will depend only on [A]:
v = kap × [A] (2.11)
2.2.2. The principle of Le Chatelier
If the conditions of a reaction are changed, the equilibrium will change in such
a way as to counteract the change.
This is an extremely important rule, both in chemical technology and in biochemistry. It

allows the yield of a chemical reaction to be influenced. Examples:
• If you dissolve salts in water, the solution becomes cold. If you increase the tempera-
ture, more salt can be dissolved.
• If you remove one of the products of a reaction, more reactants are turned into prod-
ucts to maintain the equilibrium constant. Thus by constantly removing the products,
you can drive an equilibrium reaction to completion.
• If you add one of the reactants in large excess, more of the other reactants is converted
to product. This allows you to use an expensive reactant more completely, at the
expense of the other (cheaper) ones.
21
2.3. Catalysis
All “true” chemical reactions, catalyzed or uncatalyzed, go through a short-lived unstable

transition state (‡):
without catalyst
a
with catalyst
The transition state has a higher free energy content than the substrate and the product,
creating an energy barrier between S and P. This energy barrier, known as the energy of
activation (∆Ea ), is the difference in the free energy contents of S and ‡. A condition
that is stable kinetically, but is not the most favorable state thermodynamically, is called
metastable. The human body is metastable.
Catalysts increase the rate of the reaction by decreasing the free energy of activation. The
transition state of the catalyzed reaction is different from that of the uncatalyzed reaction.
It has a lower free energy content. The equilibrium of the reaction remains unchanged, since
only ∆Ea , but not ∆G, is changed. The catalyst is not consumed during the reaction, it
may undergo a bond with the substrate temporarily, but is regenerated before the reaction
is complete.
2.4. Example questions
1) Entropy, driving force of a reaction The heat of evaporation of water is 40.7 kJ/mol,
its boiling point 100 °C. The change in entropy ∆S during evaporation is approximately
A 0.109 J mol−1 K−1
B 109 J mol−1 K−1
C 407 J mol−1 K−1
D −109 J mol−1 K−1
22
Example questions 2.4
E −407 J mol−1 K−1
2) Ligand binding to receptor The plasma membrane of a cell contains 10 000 receptors
for the hormone X, and 1000 need to have hormone bound to stimulate the maximal re-
sponse in the cell.The dissociation constant Kd = 1 nM. How high does the concentration
of the hormone need to be to get maximal response of the cell? Carefull: there is a trick in
this question!
A 0.05 nM
B 0.11 nM
C 0.25 nM
D 0.50 nM
E 0.73 nM
3) Virus capsid stability Virus have a capsid that is made of many copies of one or a few
proteins. These assemble in a regular pattern, where they are held together by non-covalent
bonds. In the host cell, where the virus is manufactured, the cytosolic concentration of the
capsid protein(s) is high, and the law of mass action favors virus assembly. However, once
the virus is released from the cell the concentration of capsid protein(s) drops to essentially
zero, and the capsid should disintegrate. The very existence of viral diseases proves that
this is not the case. Why?
A The process is controlled thermodynamically, by a negative free energy ∆G.
B Disassembly would lower the entropy ∆S.
C The process is controlled kinetically by a high activation energy ∆E a
D The process is controlled by a high heat of reaction ∆H.
E The law of mass action does not apply to living systems.
4) Elimination of drugs from the body When a drug enters the body, it can distribute
only through part of it. For example, hydrophilic drugs will dissolve in cytosol and intersti-
tial fluid, but not in bones or body fat. The part of the volume of the body available to a
drug is called its distribution volume, measured in L. Elimination of a drug from the body
is often, but not always, a first order process.
A drug (molecular mass 1000 Da) has a distribution volume of 50 L, and is eliminated with
first-order kinetics and a half life period of 2 h. 6 h after injection a blood sample is taken
and found to contain 12.5 nM. How much was injected?
23
A) 1 mg B) 2 mg C) 5 mg D) 10 mg E) 20 mg
5) Effect of pKa on membrane diffusion The depicted compounds are used as local
anesthetics for small surgical procedures (dentistry, stitching of wounds). They act in the
protonated form (BH+ ) by binding to and blocking the sodium channel from the cytosolic
side of the membrane of nerve cells. From the blood stream they enter the cells by passive
diffusion, that is, in their unprotonated (B) form. Which of these compounds acts fastest?
O
H H
N N N
N N O
O O
H2N
Lidocain, pKa = 7.7 Bupivacaine, pKa = 8.1 Procaine, pKa = 8.9
A Lidocaine
B Bupivacaine
C Procaine
D no difference
6) Follow-up: A.B. Drofnats, MD treats a patient with an abscess. Before draining the
abscess he applies topical lidocain creme to prevent pain. Upon starting the procedure the
good doctor gets a nose-job from his patient. Why?
7) pH-dependent drug trapping Aspirin® (acetylsalicylate) is an over-the-counter pain

killer, which is also used to prevent stroke as it reduces blood clotting. The drug has a pKa -
value of 3.4. What will be the approximate ratio of the drugs concentration in blood (pH
= 7.4) over that in stomach (pH = 1.4)? Assume that there is a concentration equilibrium,
that no transport proteins for aspirin exist and that stomach juice and plasma are separated
by a simple biomembrane as the diffusion barrier.
A 0.0001
B 0.01
C 1
D 100
E 10000
24
Objectives 2.5
2.5. Objectives
Students should be able to

• define the terms system, energy, enthalpy, entropy, work, heat, temperature, free en-
ergy, activation energy, reaction order
• Define the equilibrium and dissociation constant of chemical reactions
• Describe in qualitative terms the relationship between free energy change and equi-
librium constant.
• Describe the difference between thermodynamic and kinetic properties of reactions.
• use fundamental thermodynamic and kinetic equations to characterize a reaction.
• discuss how le Chatelier’s principle can be used to change the equilibrium of a
reaction
• State the importance of the transition state in chemical reactions, its relative free
energy content, and the effect of enzymes on the transition state.
• define the term catalyst and state the consequences of its presence on a chemical
reaction.
• Recognize the difference between zero-order and first-order reaction, and state the
conditions leading to zero of first order kinetics in catalyzed reactions.
25
3. Amino acids and proteins
... everything that living things do can be understood in terms of the jigglings
and wigglings of atoms.
(R. Feynman: Lectures in Physics)
3.1. Amino acids
3.1.1. General structure of amino acids
Amino acids contain a carboxy group, an amino group, a hydrogen atom and a variable
side chain R (“residue”). These four groups are bonded to a central, asymmetric (chiral)
carbon called the alpha-carbon. Only L-amino acids are found in proteins. However,
D-amino acids are found in the bacterial cell wall and in several antibiotics. In humans,
D-Ser is produced by astrocytes to regulate the response of NMDA-receptors and long-term
potentiation. The carboxy group has a pKa close to 2 while the amino group pKa ranges
from 9 to 10. Thus, amino acids can exist in-
different protonation states:
-
COOH COO COO
+ +
H3N CH H3N CH H2N CH
R R R
pH = 1 pH = 7 pH = 11
At pH = 7, amino acids exist as zwitterions - molecules that possess both a positive and
a negative charge.
27
Figure 3.1.: Structure of amino acids. Top left: Amino acids can form zwitterions (Zwitter
(Ger.) = hermaphrodite) with a positive and a negative charge. Top right:
Because the α-carbon bears 4 different substituents, it is chiral (exception:
glycine where R = H). Bottom: Naming convention of carbon atoms in an
amino acid. Figure taken from [Buxbaum, 2007].
R
O OH O O
C C
+
H2N C H H3N C H
R R CO H N
NH2 ,
ε δ γ
α O β
H2N C C C C C C
H2 H2 H2 H2 H OH
3.1.2. The 22 amino acids in proteins
Also some of the amino acid side chains are ionizable1 :

Group Amino Acid pKa
Carboxy Glutamate, aspartate 4.0
Sulfhydryl Cysteine 8.0
Selenol Selenocysteine 5.2
Phenolic OH Tyrosine 10.0
Guanidino Arginine 12.5
Amino Lysine 10.8
Imidazole Histidine 6.0
The number and pKa values of ionizable groups in a molecule can be determined experi-
mentally by a titration curve, which plots solution pH as a function of increasing quantity
of added base (or acid). Each titration plateau indicates an ionizable group and its position
shows the pKa to that group. Ionizable groups “buffer” the pH of a solution because they
release or absorb protons at pH values close to their pKa .
You have to know the (approximate) structures of the 22 amino acids (see fig. 3.2). The
amino acid side chains can engage in a number of non-covalent interactions and covalent
bonds:
1
pKa values depend on the solvent environment, and vary widely in active sites of enzymes and unique
protein environments.
28
The 22 amino acids in proteins 3.1.2
Figure 3.2.: Structures of the 22 amino acids encoded by genes. Acidic groups are marked
red, basic blue. Post-translational modifications can significantly change the
properties of amino acids in proteins. Sec and Pyl are rare amino acids encoded
by alternatively used stop-codons (see the genetic code in fig. 34.4 on page 632).
Picture taken from [Buxbaum, 2007].
O O
C
+
H 3N CH
H
Glycine (Gly, G)
O O O O O O O O
C C C C
+ +
H 3N
+
CH H 3N
+
CH H3N CH H3N CH
C H C OH C SH C Se
H2 H2 H2 H2
Alanine (Ala, A) Serine (Ser, S) Cysteine (Cys, C) Selenocystein (Sec, U)
O O O O
O O O O O O C C
C C C +
+ + H3N CH
+
H3N
+
CH H 3N CH H3N CH
H3N CH
CH2 CH2
HC CH3 HC OH CH2
C C S CH3
CH3 CH3 C H2
O O H2N O
Valine (Val, V) Threonine (Thr, T) Aspartic acid (Asp, D) Asparagine, (Asn, N) Methionine (Met, M)
O O O O O O O O O
O C
C C C C
+ +
H3N
+
H3N CH H 3N
+
CH H 3N CH H 3N
+
CH CH
CH2 HC CH3 CH2 CH2 CH2
HC CH3 CH2 CH2 CH2 CH2
CH3 CH3 C C CH2
O O O NH2
NH
C
Leucine (Leu, L) Isoleucine (Ile, I) Glutamic acid (Glu, E) Glutamine (Gln, Q) +
H2N N H2
Arginine (Arg, R)
O O O O
C C
+ +
H 3N CH H3N CH
CH2 CH2
CH2 CH2
CH2 CH2
CH2 CH2
+
N H3 NH
Lysine (Lys, K) C O
N CH3
O O O O O O
C C C
+ + + Pyrrolysine (Pyl, O)
H3N CH H 3N CH H 3N CH
CH2 CH2 CH2
NH
O
Phenylalanine (Phe, F) Tyrosine (Tyr, Y) Tryptophan (Trp, W)
O O O O
C C
H
+ C H3N
+
CH
H2N
CH2
NH
+
Proline (Pro, P) N
H Histidine (His, H)
29
Amino Acid Hydrophobic Hydrogen Salt bond Covalent

interaction bond bond
Glycine (Gly, G) (+) - - -
Alanine (Ala, A) + - - -
Valine (Val, V) ++ - - -
Leucine (Leu, L) +++ - - -
Isoleucine (Ile, I) +++ - - -
Serine (Ser, S) + + - Ph., CHO
Threonine (Thr, T) ++ + - Ph., CHO
Cysteine (Cys, C) + (+) + Disulfide
Selenocysteine (Sec, U) + ++ + -
Methionine (Met, M) ++ - - -
Phenylalanine (Phe, F) +++ - - -
Tyrosine (Tyr, T) ++ + - Ph.
Tryptophan (Trp, W) ++ + - -
Aspartate (Asp, D) - ++ +++ -
Asparagine (Asn, N) - +++ - CHO
Glutamate (Glu, E) + ++ +++ -
Glutamine (Gln, Q) + ++ - -
Lysine (Lys, K) ++ +++ - -
Pyrrolysine (Pyl, O) +++ + - -
Arginine (Arg, R) + +++ +++ -
Histidine (His, H) - +++ + -
Proline (Pro, P) ++ - - -
Ph. = phosphate ester; CHO = glycosidic bond.
3.1.3. The pI-value
At some pH an amino acid has the same number of positive and negative charges. This
pH is called the isoelectric point (pI) of that amino acid. While pKa is a property of an
individual ionizable group, pI is a property of an entire molecule. The pI is calculated from
the pKa -values on both sides of the form of the amino acid which bears no net charge.
30
Table 3.1.: Properties of the amino acids found in proteins vary widely. This allows amino acids to fulfill different roles
in a protein.
Amino acid 3-letter 1-letter MW pK1 pK2 pK3 pI Hydropathy Helix Surface Volume Abund.
2 3
(Da) (−COOH) (NH+3 ) (R) (kJ/mol) propensity (Å ) (Å ) (%)
Alanine Ala A 89 2.34 9.69 - 6.01 +1.8 0.00 115 67 9.0
Arginine Arg R 174 2.17 9.04 12.48 10.76 -4.5 0.21 225 167 4.7
Asparagine Asn N 132 2.02 8.08 - 5.41 -3.5 0.65 160 148 4.4
Aspartic acid Asp D 133 1.88 9.60 3.65 2.77 -3.5 0.43 150 67 5.5
Cysteine Cys C 121 1.96 8.18 10.28 5.07 +2.5 0.68 135 86 2.8
Glutamic acid Glu E 147 2.19 9.67 4.25 3.22 -3.5 0.39 180 114 6.2
Glutamine Gln Q 146 2.17 9.13 - 5.65 -3.5 0.16 190 109 3.9
Glycine Gly G 75 2.34 9.60 - 5.97 -0.4 1.00 75 48 7.7
Histidine His H 155 1.82 9.17 6.00 7.59 -3.2 0.56 195 118 2.1
Isoleucine Ile I 131 2.36 9.68 - 6.02 +4.5 0.41 175 124 4.6
The pI-value
Leucine Leu L 131 2.36 9.60 - 5.98 +3.8 0.21 170 124 7.5
Lysine Lys K 146 2.18 8.95 10.53 9.74 -3.9 0.26 200 135 7.0
Methionine Met M 149 2.28 9.21 - 5.74 +1.9 0.24 185 124 1.7
Phenylalanine Phe F 165 1.83 9.13 - 5.48 +2.8 0.54 210 135 3.5
Proline Pro P 115 1.99 10.96 - 6.48 -1.6 3.16 145 90 4.6
Selenocysteine Sec U 168 2.16 9.40 5.20 3.68 rare
Serine Ser S 105 2.21 9.15 13.60 5.68 -0.8 0.50 115 73 7.1
Threonine Thr T 119 2.11 9.62 13.60 5.87 -0.7 0.66 140 93 6.0
Tryptophan Trp W 204 2.38 9.39 - 5.89 -0.9 0.53 255 163 1.1
Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 -1.3 0.49 230 141 3.5
Valine Val V 117 2.32 9.62 - 5.97 +4.2 0.61 155 105 6.9
3.1.3
31
pI = 5.97
_ _
HO O pK1 = 2.34 O O pK2 = 9.60 O O
+ C + C C
H3N CH2 H3N CH2 H2N CH2
pI = 5.97
glycine
12
_ _
HO O pK1 = 2.34 O O pK2 = 9.60 O O
+10 C + C C
H3N CH2 H3N CH2 H2N CH2
8
glycine
12
pH
10
2
pH
0
0 0.5 1 1.5 2
4 OH-Equivalents (mol/mol)
If the amino acid contains only the Carboxy-group C0 and the amino group on Cα , the
2
pI = 3.22
isoelectric point is simply the average of their pKa s:
0
_ _ _
HO O O 0.5 O O
× (pK1 + pK+O2 ) C=O1/2 × (2.34 O
(3.1)
0 1.5 1 2
+
C pI += C1/2 OH-Equivalents (mol/mol) + 9.60)
C = 5.97
H3N CH pK1 = 2.19 H3N CH pKR = 4.25 H3N CH pK2 = 9.67 H2N CH
CH2
pI = CH
3.22
2 CH2 CH2
CH2 CH2 CH2 CH2
_ _ _
HO C O O C O O C O O C O
H+O C O H+O C O _+O C O _O C O
H3N CH pK1 = 2.19 H3N CH pK = 4.25 H3N CH pK = 9.67 H2N CH
R Glutamate 2
CH2 12 CH2 CH2 CH2
CH2 CH2 CH2 CH2
10
C C C C
HO O HO O _O O _O O
8
Glutamate
12
pH
10
2
pH
0
0 0.5 1 1.5 2 2.5 3
2
pI = 7.59
_ _ _
HO O 0 O O O O O O
C 0 0.5 C 1 1.5 2 C 2.5 3 C
+ + OH-Equivalents (mol/mol) +
H3N CH pK1 = 1.82 H3N CH pK R = 6.00 H3N CH pK2 = 9.17 H2N CH
If, however,
CH2 the amino acidCHhas 2
a negatively CH charged
2
R-group CH (like
2 Glu), we have to identify
H H H H
the formC N with
CH
no net charge.
_ C N This
CH
is the_ second
pI =C7.59
N from
CH
the_ left,
C N which
CH
has one positive and
one negative
HO C O
C
N
charge. TheCHpK + aH-values on +either
O C ON O C O
C
N
side are 2.19HC and 4.25, and the pI is the
O C O N
+ H + H + H
average,
H3N CH3.22.pK1 = 1.82 H N CH pKR = 6.00
3 H3N CH pK2 = 9.17 H N CH
2
Histidine
CH2 12 CH2 CH2 CH2
H H H H
C N C N C N C N
CH 10 CH CH CH
C N C N C N C N
H + H H + H H H
32 8
Histidine
12
pH
10
2
pH
0
0 0.5 1 1.5 2 2.5 3
4
0
0 0.5 1 1.5
The one-letter
2 2.5
code3 3.1.4
OH-Equivalents (mol/mol)
pI = 7.59
_ _ _
HO O O O O O O O
C C C C
+ + +
H3N CH pK1 = 1.82 H3N CH pKR = 6.00 H3N CH pK2 = 9.17 H2N CH
CH2 CH2 CH2 CH2
H H H H
C N C N C N C N
CH CH CH CH
C N C N C N C N
H + H H + H H H
Histidine
12
10
8
pH
0
0 0.5 1 1.5 2 2.5 3
OH-Equivalents (mol/mol)
For amino acids with a positively charged R-group the rational is similar: The third form
from the left has one positive and one negative charge. The pKa -values on either side are
6.00 and 9.17, and the pI is the average, 7.59.
3.1.4. The one-letter code
Amino acids are abbreviated with the first three letters of their name. However, this takes
three bytes in computer data bases. The 26 letters of the roman alphabet are sufficient
to code for 22 amino acids, which is a more economical use of computer memory. Since,
however, several amino acids start with the same letter (e.g. Ala, Arg, Asp, Asn), we can
not simply use the first letter. The following list should help you to remember single letter
codes:
• Amino acids with a unique first letter: Cys, His, Ile, Met, Ser, Val
• Where several amino acids start with the same letter, common amino acids are given
preference: Ala, Gly, Leu, Pro, Thr
• Letters other than the 1st letter are used for Asn (asparagiN), Arg (aRginine), Tyr
(tYrosine)
• Similar sounding names: Asp (asparDic acid), Glu (glutEmate), Gln (Qtamine), Phe
(Fenylalanine)
• The remaining amino acids have letters that do not occur in their name: Lys (K close
to L), Trp (W reminds of double ring), Sec (U), Pyl (O)
33
• X for any aa
3.2. Proteins
3.2.1. The peptide bond
A peptide bond is formed by the condensation of two amino acids under elimination of
water:
Carboxy-terminal O
end + O
O H3N C
O C O O C
C R´ H2O
O C O R" C R"
C
+ R´ C HN
H3N
NH C O
+ O C R´ C
C R NH
O +
H3N H2O O C
C R
O C Amino-terminal C R
+
end +
H3N H3N
Aminoacids Dipeptide Tripeptide
Polypeptide Oligopeptide
(> 20) (< 20)
Protein: Polypeptide with biol. function
The product is a dipeptide. Addition of further amino acids to the chain leads to tripeptides,
tetrapeptides and so on. Chains of up to 20 amino acids are called oligopeptides (oligo
= few), and longer ones polypeptides (poly = many). Proteins are polypeptides with a
biological function.
Polypeptides range in size from a few amino acids to hundreds or even thousands. Pro-
teins consist either of a single polypeptide chain, or they are formed from separate polypep-
tide chains called subunits. Some proteins contain other components, so-called prosthetic
groups.
Peptide bonds are formed between the carboxy and α-amino groups of two amino acids.
Each peptide has an amino terminus, conventionally written on the left, and a carboxy
terminus, written on the right side.
The peptide bond is rigid. Because of mesomery with the C0 =O-bond it has “partial double
bond character” (see fig. 3.4). Like the C=C bond, it is planar and cannot rotate. The H and
34
The peptide bond 3.2.1
Figure 3.3.: Aspartam is a peptide used as an artificial sweetener in the food industry.
O O
C CH3
O O
C N CH
+ H
H3N CH CH2
CH2
-
COO
aspartyl-phenylalanine-1-methyl ester
(Aspartam®)
Figure 3.4.: Left: The definition of the dihedral angle of the bond between the atoms B
and C. The dihedral angle is then the angle between the bonds A-B and C-D.
Right: The geometry of the peptide bond. For details see text. Picture taken
from [Buxbaum, 2007].
α
+α
ω ψ
α
τ
γ
φ
35
Figure 3.5.: Left: Ramachandran plot of the frequency of φ, ψ-values in proteins. Certain
pairs of angles do not occur since they lead to clashes between the atoms of
neighboring amino acids. Right: At a certain φ, ψ C’=O of the preceding amino
acid clashes with the N-H of the following one, we define this pair of dihedral
angles as 0°,0°. Figure taken from [Buxbaum, 2007].
all amino acids except Gly and Pro
2000
1800
2000 1600
1400
1200
1000
1500
800
600
freq 400
1000 200
0
500
180
135
90
0 45
-180
-135 0
-90 -45 ϕ
-45
0 -90
45 -135
φ 90
135
180-180
O of the peptide bond are in the trans-configuration. Formally, we express the same idea by
saying that the dihedral angle of the peptide bond ω is fixed to 180°. The other two bonds
in the polypeptide backbone can rotate. Therefore the polypeptide can fold, bend and twist
itself into a variety of shapes. In particular, the dihedral angles of the C0 −Cα -bond ψ and
of the Cα −N-bond φ are flexible. However, not all angles are allowed, because some lead
to clashes between the atoms of neighboring amino acids. If we look at the frequency of
amino acids with given φ, ψ-values (Ramachandran-plot, see fig. 3.5) we see that certain
values are not represented at all (forbidden values). The angles φ, ψ which results in a clash
between C0n =O and Nn+1 −H is defined as 0°, 0°.
3.2.2. Protein structure
Primary structure
The sequence of amino acids in the polypeptide chain of a protein is called its primary
structure. By convention, the sequence is read from the N- to the C-terminus, i.e. in the
direction in which proteins are synthesized in the cell.
36
Protein structure 3.2.2
Figure 3.6.: Subtilisin (PDB-code 1gcl, left) and Chymotrypsin (PDB-code 1oxg, right) are
both Ser-proteases, that use the classical catalytic triade (Ser, His, Asp, shown
as wire diagram) in the catalytic center to cleave proteins. These amino acids
are far apart in the sequence, but close to each other in the folded protein. Both
proteins, however, have completely different sequence and secondary structure.
This is an example of convergent evolution (“re-inventing the wheel”). The
proteins are called iso-enzymes (iso = the same).
Because each position in the primary structure can be occupied by any of the 20 common
amino acids, the possible number of combinations is huge. For example, a protein with
100 amino acids has 20100 = 1.3 × 10130 possible sequences. Given that our universe is
about 13.7 × 109 a ≈ 4.32 × 1017 s old, creationists have argued that proteins can not have
been created by a process of random mutation and selection. This argument is fallacious,
however, since it makes the (unspoken!) assumption that the function of a protein can only
be met by one particular amino acid sequence. The existence of isoenzymes, proteins with
different primary structure but the same function, proves this assumption wrong.
Secondary structure
The secondary structure of a protein is any regular, repetitive folding pattern in the mole-
cule. Only a few secondary structures are energetically possible:
37
Figure 3.7.: Different ways to represent the structure of a protein. For proteins with more
than just a few amino acids the space-filling (a) or even the wire diagram (b)
become unreadable. If only the backbone of the protein is drawn (c), this can be
avoided. However, disulphide bonds then dangle in free space (d). To increase
readability, the disulphide bonds are drawn to the backbone instead (e, which
of course is chemically wrong!). For larger proteins even backbone diagrams are
too crowded, and structural elements are presented in schematic form instead
(f). Figure taken from [Buxbaum, 2007].
(a) spacefilling (b) wire diagram
(c) wire + backbone (d) dangling disulphide bond
(e) disulphide connected (f) schematic (PDB 1m40)
38
Figure 3.8.: Signal-peptide for import into mitochondria. Most mitochondrial proteins are
encoded in the nucleus, they are synthesized in the cytosol and them imported
via a transport system that spans both mitochondrial membranes. An am-
phipatic α-helix serves as the recognition signal for binding of the nascent
protein to the transporter. Note that the helical wheel projection is viewed
from the N-terminus. Figures taken from [Buxbaum, 2007].
(a) side-view (b) helical wheel projection
The α-helix In the α-helix the polypeptide is wound in a counterclockwise spiral around
an imaginary axis. Such a spiral is called righthanded, because if you hold your right hand
with the thumb pointing from N- towards C-terminus the fingers curl counter-clockwise.
There are 3.6 amino acids per turn, each turn is 5.4 Å long with a pitch of 1.5 Å per residue.
φ, ψ = −57°, −47°, and the R-groups stick outward. This compact, rod-like structure is
maintained by hydrogen bonds between the components of the peptide bonds: Each peptide
bond C=O forms a hydrogen bond with the peptide bond N−H four amino acid residues
ahead of it. Because all N-termini point in the same direction, an α-helix has a dipole
moment and can bind to charged molecules. Proline and glycine don’t fit well into the
α-helix. The α-helix is the most common secondary structure in proteins. It occurs both
in many fibrous (long, stretched-out) proteins (such as myosin and keratin), and in many
globular (compact-shaped) proteins. Often α-helices have a polar side (facing the outside
of a protein) and an non-polar one which is buried in the interior (amphipatic helix).
Function of α-helices:
• An α-helix of 22 amino acids is long enough to span a double membrane. The part
of the helix that is inside the membrane consists of hydrophobic amino acids that
39
Figure 3.9.: Keratin is made from coiled-coils of α-helices. Figure taken from [Nelson et al.,
2008].
Figure 3.10.: In heptad-repeats (Leu-zipper) (here tropomyosin, PDB-code 1lc2) every 7th
aa is Leu → hydrophobic interactions. This leads to specific associations of α-
helices by hydrophobic interactions. This is a stereo-diagram, if you look at the
images cross-eyed, you will see 3 figures, the middle of which is 3-dimensional
(this takes some practice).
40
can interact with the lipid tails of the membrane. Hydrophilic amino acids on both
ends interact with the cytosol and the interstitial fluid, respectively. On the cytosolic
end you find more positively charged amino acids, but on the extracellular end more
negatively charged ones, because the potential of a cell is negative inside (−70 mV).
At the interface between the membrane and the aqueous environment one finds pre-
dominantly aromatic amino acids and Lys.
• Amphipatic α-helices at the N-terminus of a protein serve as recognition sites for the
import into mitochondria. Every 4th or 5th amino acid is positively charged, so that
all positive charges are in the same quadrant of the helix (see fig. 3.8).
• Two α-helices wound around each other form a coiled coil. Keratin consists of such
coiled-coils (see fig. 3.9). These are held together by disulphide bonds. Breaking these
with thioglycolic acid is the basis of the permanent wave.
• Heptad-repeats (Leu-zippers) are α-helices where every 7th amino acid is leucine.
Such helices associate because of hydrophobic interactions between the Leu-residues,
allowing for specific dimerization of proteins. Some DNA-binding proteins have this
structure.
The β-sheet In the β-pleated sheet, the polypeptide backbone is stretched out. Different
portions of the protein are aligned in a parallel or antiparallel fashion, forming hydrogen
bonds between a N−H group of one strand and a C=O-group in a neighboring strand. This
gives rise to large, blanket-like structure. The main difference between α-helix and β-pleated
sheet is that in the α-helix hydrogen bonds occur between residues of the same helix, while
in a β-pleated sheet they occur between residues of neighboring strands. Nevertheless, a
single β-strand is stable since the amino acids in this extended structure have plenty of
“wiggling” space without running into steric hindrance (look up the coordinates in the
Ramachandran plot!), resulting in entropic stabilization. The R-groups poind up- and
downwards in turn, making amphipatic sheets with polar and non-polar or positive and
negative faces possible. The entire sheet is rarely flat, but has a right-handed twist, in
extreme cases forming a β-barrel.
In an anti-parallel β-sheet the stands change direction, going in turn from N → C and
vice versa. They are usually joined together by β-turns (see later). φ, ψ = −138°, 137°.
In a parallel β-sheet the N-termini of all strands point in the same direction, φ, ψ =
−116°, 111°. The hydrogen bonds are oblique to the strand direction, hence the parallel β-
sheet is less stable than the anti-parallel. The strands in a parallel β-sheet are often joined
by α-helices, which form the “return-leg”.
Silk-protein is an important example for the use of β-sheets in biologically important
structures. Since the amino-acids within a β-strand are already in an extend conformation,
silk shows little elasticity and has an extremely high tensile strength, as any extension
41
Figure 3.11.: Anti-parallel β-pleated sheet (here PDB-code 1qjp). Neighboring strands have
different directions, and are joined by β-turns. Hydrogen bonds holding the
sheet together run vertically to the strands.
Figure 3.12.: Parallel β-pleated sheet (here PDB-code 2v9s). All strands have the same di-
rection, the “return-legs” are either α-helices or coils. Hydrogen bonds holding
the sheet together run obliquely to the strands.
42
would require breaking covalent bonds. On the other hand, the strands are held together
by hydrogen bonds only, giving silk cloth this wonderful soft flow.
The PII (syn.: poly-Pro or polypeptide II) helix is a left-handed helix with three residues
per turn and φ, ψ = −70°, 140°. Like the single β-strand, it is stabilized by entropy, not
by hydrogen bonds. Pro frequently occurs in this structure, but not all PII helices contain
Pro.
Table 3.2.: Collagen-related inherited diseases. FACIT = fibrillar associated col-

lagens with interrupted triple helices. OMIM (Online Mendelian
inheritance in man) is a catalogue of inherited diseases at
http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim, which you should
bookmark.
Type composition function OMIM Gene Location diseases
I α1(I)2 α2(I) skin, tendon, +120150 COL1A1 17q21.3-q22 OI, EDS, osteo-
bones porosis
*120160 COL1A2 7q21.3-q22.1 OI, EDS, Marfan,
osteoporosis
II α1(II)3 cartilage, vitre- +120140 COL2A1 12q12-q13.2 chondrodysplasia
ous body
III α1(III)3 skin, muscle, ves- *120180 COL3A1 2q31-q32.3 EDS
sels, fetus
IV α1(IV )...α6(IV ) basal lamina *120130 COL4A1 13q34 porencephaly,
brain small vessel
disease
*120090 COL4A2 13q34
source of tumsta- *120070 COL4A3 2q36-q37 Alport syn-
tin drome, benign
familial hema-
turia
*120131 COL4A4 2q35-q37 Alport syn-
drome, benign
familial hema-
turia
*303630 COL4A5 X Alport syn-
drome
*303631 COL4A6 Xq22 diffuse leiomy-
omatosis
V α1(V ), α2(V ), α3(V ) interstitium, pla- 120215 COL5A1 9q34.2-q34.3 EDS
centa
*120190 COL5A2 2q14-q32 EDS
*120216 COL5A3 19p13.2
43

VI α1(V I), α2(V I), α3(V I) interstitium, *120220 COL6A1 21q22.3 Bethlem my-
intima opathy, Ullrich
congenital mus-
cular dystrophy
*120240 COL6A2 21q22.3 Bethlem my-
opathy, Ullrich
congenital mus-
cular dystrophy
*120250 COL6A3 2q37 Bethlem my-
opathy, Ullrich
congenital mus-
cular dystrophy
VII α1(V II)3 below basal lam- *120120 COL7A1 3 Epidermolysis
ina of skin bullosa, toenail
dystrophy
VIII α1(V III), α2(V III) hemidesmosomes *120251 COL8A1 3q11.1-q13.2
in skin
*120252 COL8A2 1p34.2-p32.3 Fuchs endothe-
lial corneal dys-
trophy
IX α1(IX), α2(IX), α3(IX) cartilage, vit- 120210 COL9A1 6q12-q14 Stickler Syn-
reous body drome, Multiple
(FACIT) epiphyseal dys-
plasia
*120260 COL9A2 1p33-p32.2 Multiple epiphy-
seal dysplasia
*120270 COL9A3 20q13.3 Multiple epiphy-
seal dysplasia, In-
tervertebral disc
disease
X α1(X)3 hypertrophic 120110 COL10A1 6q21-q22
+ mineralizing
cartilage
XI α1(XI), α2(XI) cartilage 120280 COL11A1 1p21 Collagenopathy
type I+II
120290 COL11A2 6p21.3
XII α1(XII)3 FACIT *120320 COL12A1 6q12-q13
XIII α1(XIII)3 transmembrane *120350 COL13A1 10q22
protein
XIV α1(XIV )3 FACIT *120324 COL14A1 8q23
XV α1(XV )3 proteoglycans in *120325 COL15A1 9q21-q22
basal lamina
XVI α1(XV I)3 FACIT *120326 COL16A1 1p35-p34
XVII α1(XV II)3 hemidesmosomes +113811 COL17A1 10q24.3 Epidermolysis
in skin bullosa
XVIII α1(XV III)3 source of endosta- *120328 COL18A1 21q22.3 Knobloch syn-
tin drome
XIX α1(XIX)3 FACIT *120165 COL19A1 6q12-q13
XX α1(XX)3 COL20A1 20q13.33
44
Figure 3.13.: Collagen (PDB-code 1cag). To make the tight association between the three
strands clearer, one each is drawn space-filling, as wire diagram and as carbon-
backbone. Note the repeating Gly-X-Pro (yellow, green, brown; with X often
hydroxy-Pro) sequence. Marked in blue is a Gly→Ala mutation that prevents
a close fit and destabilize the molecule. Such mutations cause for example
Ehlers-Danlos-syndrome.

XXI α1(XXI)3 FACIT *610002 COL21A1 6p12.3-p11.2
XXII α1(XXII)3 cell adhesion in *610026 COL22A1 8q24.3
the lung?
XXIII α1(XXIII)3 610043 COL23A1 5q35.3
XXIV α1(XXIV )3 embryonic bone 610025 COL24A1 1p22.3-p22.2
formation
XXV α1(XXV )3 cell membranes in *610004 COL25A1 4q25 Alzheimer
brain
XXVI α1(XXV I)3 embryonal tissues *608927 EMID2 7q22.1
XXVII α1(XXV II)3 all tissues *608461 COL27A1 9q33.1
XXVIII α1(XXV III)3 neural and con- 609996 COL28A1 7p21.3
nective tissue
The most important example for the PII -helix are the collagens, which consist of 3 PII -
helices wound around each other (hetero- or homo-oligomer). In the human genome there
are 42 collagen genes, which encode for 28 known collagen types. Of these types I, II and III
are the most important. Each of the three molecules in collagen has 1050 amino acids, with
the sequence Gly-X-Pro. The sharp angle of the Pro peptide bond allows the sharp turn in
the molecule, and the small R-residue of Gly (only a H) allows the 3 protein molecules to
wrap around each other. If only a single of the Gly-residues in one of the collagen chains
is mutated, wrapping is no longer possible, leading to osteogenesis imperfecta (brittle
bone disease, collagen I), to Ehlers-Danlos-syndrome (collagen I, III or V) with too
brittle or too elastic ligaments and death by vascular or organ rupture, epidermolysis
45
Figure 3.14.: β-turn (here in PDB-code 1qiv) are most common between the strands of an
anti-parallel β-sheet.
bullosa (blistering of skin, collagen XVII), or to Alport-syndrome (collagen IV, kidney

and hearing defects).
Heating turns collagen into gelatine. The dissociation temperature of collagen is influenced
by the hydroxylation of proline. Vitamin C (ascorbic acid) is required for the correct
function of Pro-hydroxylase. In scurvy, the dissociation temperature of collagen drops
below the body temperature of 37 °C, explaining the connective tissue weakness typical for
this condition.
Apart from collagen, PII -helices also occur in SH3-domains, which occur in proteins
involved in signal transduction (more about this in the course on hormone effects).
The hairpin turn Hairpin turns allow the protein to fold back onto itself in a 180° angle.
This is required, for example, in anti-parallel β-sheets. Since the C=O- and N−H-groups of
a turn are not all involved in hydrogen bond formation within the protein, they are often
surface-exposed and interact with water. They may also occur in the catalytic center of
enzymes, where they are involved in substrate binding. Because of its small size, Gly is
often found in turns. There are two types of hairpin turn; the β-turn with 4 amino acids
(frequent, φ, ψ = −56°, 137°.), and the rare γ-turn with 3 amino acids.
The coil A coil is basically any secondary structure except those mentioned above. It is
important to realize that all the amino acids in a coil have defined positions, making the
term “random coil”, often found in textbooks, false. Coils give the protein flexibility, which
allows for conformational changes. Since their peptide bonds are not involved in intra-
protein hydrogen bonding, they are often exposed to interact with water, small ligands
or other proteins. Coils tend to tolerate mutations better than other structures and are
46
therefore hot-spots for evolution. In the chapter on immunoproteins you will learn that it
is coils that give antibodies their specific binding properties.
Tertiary structure
describes how the elements of secondary structure of a protein are organized in 3D-space.
Tertiary structures are formed by
hydrophobic interactions of amino acid side chains. Typical globular proteins have a core
of hydrophobic side chains, while hydrophilic side chains are on the surface where
they interact with water or with other proteins. If hydrophobic residues were exposed
to water, the water would have to form an ordered cage around them, which would
decrease the entropy of the system.
van der Waals-interactions, which are fluctuating dipole interactions with a bond en-
ergy of 4–17 kJ/mol. The bond length is ≤ 4 Å.
hydrogen bonds, which are interaction between permanent partial charges. The bond length
is about 3 Å, the bond energy is 2–6 kJ/mol if both partners are partially charged and
up to 21 kJ/mol if one partner is fully charged. If the distance between the partners is
too large, an indirect hydrogen bond may be formed where water acts as a bridge.
salt bridges, which are interactions between fully charged groups. The bond length is 2.8 Å
and the bond energy normally 10–30 kJ/mol, but can be significantly higher if both
groups are buried in a hydrophobic environment.
Disulfide bonds, which are formed by an oxidative reaction between two Cys residues after
folding of the protein into its higher-order structure. They may occur between two
Cys residues in the same polypeptide (intra-chain), or between different polypeptides
(inter-chain). Disulfide bonds are uncommon in cytosolic proteins, but may be formed
in the oxidizing environment of the endoplasmic reticulum (ER). They are therefore
present in secreted and plasma membrane proteins. Bond length is 2.2Å and bond
energy 167 kJ/mol.
Coordination around cofactors Several amino acids in a protein are involved in the co-
ordination of metal ions (Ca, Zn, Fe, Mg, Na, K) or other cofactors, such as heme or
FAD.
Tertiary structure and evolution Domains are independently folding parts of a protein,
which can often be isolated by gentle proteolysis under retention of function. Evolutionary
they are often parts of eukaryotic proteins which originated from the fusion of a prokaryotic
operon into a single protein. Most domains are between 50 and 250 amino acids long, shorter
domains would be devoid of function and longer once may be unable to fold properly.
47
Figure 3.15.: If you have done the logical thing and plotted the φ, ψ-values for the various
secondary structures into the Ramachandran-plot you should have gotten
something like this. At the top of the diagram are the extended structures
(parallel and antiparallel β-sheets, PII -helix and turn). The big peak below is
at the coordinates of the α-helix, the small peak to the right is the left-handed
αl -helix, a rare structure that proteins can adopt only for a few amino acids.
48
Motives are the folding patterns of proteins. Evolutionary they are often, but not necessar-
ily, the result of common ancestry. Folding patterns are much more stable during evolution
than amino acid sequences; we can recognize common ancestry in cases where there is no
longer a statistically recognizable sequence homology. This is understandable: a change of
an amino acid may not result in much functional change, but a change in protein folding
certainly would.
Because the folding patterns are so illuminating, efforts have been made to categorize
proteins by tertiary structure. The most important one is structural classification of
proteins (SCOP). Lesser known attempts like class architecture topology homology
(CATH) and families of structurally similar proteins (FSSP) give largely similar
results. SCOP recognizes the following classifications:
Class relative content of α-helix and β-strand.
Fold Major structural similarity, identical structural elements and topological connection.
Superfamily Domains with common folding pattern and similar function. Probable com-
mon evolutionary origin despite low sequence similarity.
Family Proteins have high structural and sequence similarity. They clearly have a common
ancestor.
For our purposes, only the class is important:
All α proteins contain only α-helices, or their content of β-strands is insignificant. Example:
hemoglobin (1hga)
All β proteins contain only β-strands, or their content of α-helices is insignificant. Example:
Immunoglobulins (ifc2)
α/β Proteins with alternating α-helices and β-strands → parallel β-sheets. Example: Thiore-
doxin (2trx)
α + β Proteins with segregated α-helices and β-strands → anti-parallel β-sheets. Example:

Lysozyme (132l)
Multi-domain proteins have domains belonging to different classes. Example: DnaK (1dkz)
Membrane and cell surface proteins (excluding the immune system). Often the trans-
membrane domains are α-helices (e.g. bacteriorhodopsin, 1m0k), but β-barrels (e.g.
OmpA, 1qjp) may also occur.
Small proteins usually dominated by metal ligands, heme, disulphide bridges. Example:
insulin (1mso)
Coiled coil proteins α-helices wound around each other. Example: Fibrinogen (1m1j)
49
Figure 3.16.: Intrinsically disordered proteins exist in 3 states (“the trinity”): folded, molten
globule and disordered. Each of these may have its own biological function.
ordered
(solid-like)
molten globule extended

(fluid-like) (gas-like)
Intrinsically disordered proteins contain relatively long coil segments, possibly inter-
spersed with short segments of other secondary structures. Their tertiary structure is either
an extended strand or a molten globule. When these disordered segments interact with
other proteins, they bind under re-folding to a different secondary and tertiary structure
with the partner protein serving as a folding template. We do not yet fully understand these
sequences, it appears that stretches of many identically charged amino acids keep that part
on the surface of the protein as an extended coil, preventing the hydrophobic interactions
required for folding. A large number of hydrophobic amino acids (more than 5 in a row)
result in considerable entropy gains upon binding to a partner, these segments often form
molten globules.
In enzymes, intrinsic disorder is relatively rare (although examples exist, e.g., chorismate
mutase). It is an important feature of many regulatory proteins, however. These regulating
factors may bind to many regulated proteins, and their structure is different with each
partner. On the other hand, a regulated protein may bind different regulating ones, forc-
ing each of them into similar structures. Thus the intrinsically disordered segments allow
one-to-many and many-to-one relationships between regulating and regulated proteins and
effectively dissociate specificity and affinity: folding upon binding results in loss of entropy,
this reduces ∆G and hence increases Kd . As a result, binding is readily reversible, allow-
ing quick termination of a signalling event. The surface area available for binding is larger
2
(70 Å per residue), at the same time the flexibility to accommodate binding is increased.
Binding is often regulated by post-translational modification of proteins like phosphoryla-
50
tion (see later), this can change Kd by several orders of magnitude and generates molecular
switches.
Disordered sequences are very sensitive to proteolytic attack. This creates technical prob-
lems for the researcher and is certainly one of the reasons why the significance of these
segments are only now more fully appreciated. It also leads to rapid degradation of the
protein inside the cell, an important feature for regulatory function.
The interaction between partners is robustly encoded in the domains and maintained during
evolutionary events which modify, rather than abolish, an interaction. Experiments indicate
that early enzymes probably were more disordered and hence flexible, by becoming more
structured enzymes specialized on a particular function. Because of their importance in
regulation, intrinsically disordered proteins are more frequent in eukaryotes (more than 500
have been identified) than in prokaryotes. An exception to that rule are the Apicomplexa,
who have unusually many intrinsically disordered proteins. Important pathogens, like the
malaria parasite, belong into that clade. How pathogenicity depends on protein disorder is
a topic of current research.
Amyloid-formation – a process causing various debilitating diseases – can be understood in
similar terms (see later).
Quaternary structure
Quaternary structure describes how several polypeptide chains (“subunits”) come together
to form a single, functional protein. The subunits are held together by the same forces that
we have discussed for tertiary structure. Depending on the number of subunits in a protein,
we speak of mono-, di-,...,oligo- or polymers. If all subunits are identical, we precede this by
homo- and else by hetero-. Sometimes different subunits come together to form a protomer,
and several of these then form the functional protein. An example would be hemoglobin,
which is a diprotomer, each protomer is a heterodimer composed of an α- and a β-subunit.
Determination of protein structures
Astounding progress has been made in the determination of protein structures over the
last few decades. The protein data base (PDB) started in 1971 with only 7 entries.
As of April 2010 almost 65 900 structures have been submitted, and of those, about 3900
were non-redundant. Unfortunately, only 234 membrane protein structures are available,
even though membrane proteins make 70 % of the 500 or so drug targets. Difficulties in
membrane protein structure determination seriously impedes drug development.
Several methods can be used to determine the structure of a protein, each with its own
advantages and disadvantages:
51
X-ray crystallography Soluble proteins are crystallized, then investigated by X-ray diffrac-
tion. No size limit (whole virus).
Nuclear magnetic resonance Atomic distances within a molecule are measured in solution.
Size limit ≈ 20 kDa.
Electron microscopy uses 2D-crystals or molecules sorted by orientation to calculate “av-
erage” picture. Resolution limited to 10–20 Å, but works on insoluble proteins.
All these methods require large amounts (≈ 100 mg) of pure protein.
Since primary structure determines folding (Anfinsen-hypothesis), folding of a protein
with a known amino acid sequence should be predictable. People have tried to identify
which combinations of amino acids tend to occur in which secondary structure, and then
predict the structure of an unknown protein. In practice, success is still very limited because
protein structure is not only determined by short-range interactions between neighboring
amino acids, but also by interactions between amino acids which are far apart in the primary
structure, but come close together in the final tertiary structure. If the structure of a
similar protein is available, “threading” is more promising. In theory, one should be able to
calculate the structure by taking all possible bond energies into account and minimizing the
free energy ∆G of the protein in a computer. However, these “ab initio” (Lat.: from first
principles) calculations require way more computational power than even modern super-
computers have (for computing experts: the problem is NP-complete).
Protein folding and denaturation
Protein chemists work from the assumption that all the information coding for the native
3D-structure of a protein is contained in its amino acid sequence, and that no extraneous
information is required to direct folding. This is known as the Anfinsen-hypothesis (N.P.
1972).
Levinthal’s paradox: Assume a protein with 100 peptide bonds, each of which can
assume 6 stable conformations (α-helix, β↑↑ -sheet, β↑↓ -sheet, PII -helix, turn, coil).
Since each of these states is characterized by a φ, ψ-angle pair, this results in 26 = 64
possible angles per peptide bond and 10064 = 10128 for the entire protein (note that this is
an underestimate!).
Rotation around a σ-bond takes about 10−13 s, thus folding by random testing of all possible
angles would take 10128 × 10−13 s = 10128−13 s = 10115 s. Our best estimate for the age of
the universe is 13.7 × 109 a ≈ 4.32 × 1017 s. Proteins therefore should never fold.
In reality, folding is a rapid process; in E. coli at 37 °C a 100 amino acid protein folds in
about 5 s. During folding hydrophobic residues are buried in the interior and hydrophilic
52
Figure 3.17.: Protein folding reduces free energy (G). The native structure is the one with
the lowest free energy. However, proteins may get kinetically trapped in local
minima of the energy landscape. At the same time the entropy (S) is also
reduced, symbolized by the width of the funnel.
residues appear on the outside of the protein, resulting in a compact “molten globule”
structure. This brings amino acids so close to each other that the formation of hydrogen
bonds between peptide bonds gives rise to secondary structure.
Energetics and kinetics of protein folding Amino acids undergoing folding have a choice
of undergoing interactions with either
P other amino acids or with water. Thus only the dif-
ference ∆Gfolding = ∆Ga−a − ∆Ga−w is available to stabilize the native structure of
P
proteins. Although folding decreases the enthalpy (H) (which stabilizes native structure),
it also decreases the entropy (S), which tends to destabilize native structure. Thus protein
folding is a compromise between forces, and the actual stabilization energy is only about
20–40 kJ/mol, about 10× the thermal energy at room temperature (2.5 kJ/mol). This mar-
ginal stability of proteins has a good side however: it allows protein flexibility required
for ligand binding and enzymatic activity.
Although protein folding is a spontaneous process driven by thermodynamic forces and

hence, given enough time, all proteins will eventually arrive at their native structure, kinet-
ically the process can be trapped in local minima in the energy landscape. Such metastable
intermediates would expose hydrophobic patches on their surface, which leads to protein ag-
53
Figure 3.18.: Reaction cycle of adenylate kinase, an enzyme that catalyzes the reaction 2
ADP * ) ATP + AMP. Substrate binding and product release are accompanied
by considerable movement of the protein, with coils serving as hinges. Such
movement is possible only because the stabilization energy of protein folding
is relatively low and can be overcome by the binding energy for the substrates.
Film taken from [Vonrhein et al., 1995].
54
gregation. Note that proteins in the cytosol are very closely packed at about 300–400 mg/ml.
The average distance of protein molecules is just one protein diameter, and the space be-
tween them is filled with water, salts and metabolites. As opposed to quaternary structure,
such aggregates have no reproducible structure and no biological function. On the contrary,
they may interfere with cellular function (see later).
Molecular chaperons and chaperonins Cells have two lines of defences against misfolded
proteins:
Molecular chaperons bind to unfolded proteins and prevent their aggregation until these
proteins can achieve folding. Binding/unbinding cycles of chaperons may or may not
require the hydrolysis of ATP. Examples: Hsp70, Hsc70, crystallins.
Molecular chaperonins use the energy of ATP-hydrolysis to actively unfold misfolded pro-
teins, giving them a second chance to arrive at the proper fold. Example: GroES/GroEL.
Unfolding occurs in a “beaker” formed by the chaperonin, in which the client protein
can try to refold without disturbance from other proteins (at “infinite dilution”). The
beaker has a diameter of about 45 Å, enough to contain proteins (or protein-domains)
of up to 60 kDa.
Note: Neither chaperons nor chaperonins actively fold proteins, they merely protect them
against aggregation during the folding process.
Protein denaturation The normal interactions that maintain the higher-order structures
of proteins are weak and can be disrupted easily. Heat denaturation occurs when the protein
is heated to more than 40–70 °C. This results in loss of biological activity and precipitation.
Renaturation is sometimes possible with small proteins (ribonuclease, lysozyme) under lab-
oratory conditions, but denaturation is irreversible in the real world (example: boiled egg).
Once a few bonds within the protein are broken by the increasing movement of the protein
chain the protein is destabilized and further bonds are broken more and more easily. Hence
heat denaturation of proteins is a highly cooperative process. Humans die if their body
temperature exceeds 42 °C because key proteins are denatured.
Also other insults can denature proteins:
Strong acids and bases denature proteins by disrupting ionic interactions.
Organic Solvents can denature proteins by disrupting hydrophobic interactions. Proteins

are not soluble in organic solvents. More water soluble solvents (e.g. ethanol or ace-
tone) bind water and thus reduce the concentration of water available to the protein.
Detergents also disrupt hydrophobic interactions. They can denature proteins without
precipitating them.
55
Figure 3.19.: The pathway from genome to phenome is studied at different levels with dif-
ferent methods. The resulting complex data can only be handled by advanced
computer techniques. Figure from [Buxbaum, in press].
Small hydrophilic substances , such as urea can denature proteins when they are present
in very high concentration.
Salts precipitate proteins because they reduce the concentration of water available to main-
tain protein structure.
Heavy metal ions (lead, mercury) bind to carboxylate or sulfhydryl groups of proteins.
That’s why they are toxic!
The covalent bonds in proteins are more robust, but peptide bonds are hydrolyzed by
heating in strong acids and bases, and by proteolytic enzymes. Disulfide bonds are cleaved
by reducing and oxidizing agents.
Post-translational modification of proteins
The human genome contains ≈ 30 000 genes. mRNA-processing (alternative splicing, mRNA
editing etc.) results in ≈ 3 mRNAs per gene. Post-translational modification of the pro-
teins produced from them creates ≈ 10 different protein species from each mRNA. Thus
the human proteome consists of ≈ 106 proteins, with different function, regulation, destruc-
tion...
Glycosylation is the process of enzymatic transfer of oligosaccharide (sugar) trees to the

proteins. They are affixed either to the OH-groups of Ser or Thr (O-linked) or to the acid
amide group of Asn (N-linked). Other amino acids (Arg, Tyr, Trp, Hyl, Hyp) are involved
much less frequently, e.g., collagen. Addition occurs in the ER and the Golgi-apparatus to
56
the extracellular domain of membrane proteins and to secreted proteins. Cytosolic proteins
are rarely glycosylated. In bacteria, glycosylation occurs in the periplasm.
Glycosylation is required for proper protein folding. Glycosylation inhibitors are used as
antiviral drugs (e.g. nojirimycin or desoxynojirimycin). Sugar-trees are also required as
“address labels” in the intracellular transport of proteins between compartments. For exam-
ple, in I-cell disease enzymes of the lysosome can not be transported into this organelle
because the enzyme which transfers the sugar mannose-6-phosphate to them is defective.
They are excreted into the blood stream instead and, as a consequence, the lysosomes are
non-functional.
On the cell surface, the sugar-trees of membrane proteins serve as recognition sites for cell-
cell-interactions, as immunological determinants (blood group antigens, see fig. 3.20) and –
since everything has to have a downside too – as docking sites for bacteria and virus.
Clostridium ssp. infection Glycosylation of Rho GTPases by bacterial enzyme results in

loss of a nucleotide binding site
Type II diabetes glutamine:Fru-6-P amidotransferase stimulation leads to increased [GlcN Ac],

transfer of GlcNAc to regulatory proteins involved in insulin resistance
Congenital disorders of glycosylation failure to produce dolicholpyrophosphate-sugar tree,

no N-linked glycosylation → neurological defects, failure of maturation of N-linked
glycoproteins
Leucocyte adhesion deficiency GDP-Fuc is not produced or not imported into Golgi, no
fucosylation of EGF-motives occurs.
Paroxysmal nocturnal hemoglobinuria The GPI-anchors of proteins in granulocytes and

B lymphocytes are missing.
Glucosylation This is one of those points where you really have to watch your mouth:
Although glucosylation and glycosylation are spelled only with one different letter, they
denote completely different processes. Glycosylation is an enzymatic process and carefully
orchestrated by the cell. Glucosylation is a spontaneous process that does not require any
enzymes.
The velocity of this reaction depends on the concentration of glucose in the blood. This
has a direct medical application: In diabetics, the concentration of glucated hemoglobin
(HbA1c ) depends on the average blood glucose concentration during the lifespan of an
erythrocyte (about 3 mo).
The formation of advanced glycation end-products (AGE) from glucosylated proteins

is thought to be involved in aging and in long-term diabetic damage.
57
Figure 3.20.: The AB0 system of blood group antigens. Sugar trees are added to both
proteins (O-linked) and lipids. All people can make the 0-antigen. Transfer of
an additional GlcNAc-residue creates the A-, of an additional Gal-residue the
B-antigen. People who have the GlcNAc-transferase only have the blood group
A, while those who have the Gal-transferase only have blood group B. People
with both enzymes have blood group AB, those with neither have blood group
0. Picture taken from [Buxbaum, 2007].
58
Figure 3.21.: Glucosylation of proteins by the aldehyde group of glucose proceeds via an un-
stable Schiff-base and Amadori-rearrangement to a stable ketosamine. Dur-
ing roasting, this is converted into caramels via the Maillard-reaction. These
are responsible for the taste of cooked food. Ketosamine may also be converted
to advanced glycation end-products (AGE) by Strecker-degradation.
+ H2O
COOH OH COOH H H COOH
O
HC + H2N CH HC N CH HC N CH
+
H
HC OH R' HC OH R HC OH R'
R R R
Schiff base Immonium
CH2OH COOH
O COOH HC N CH
H + H
N CH H C OH R'
OH R' R
OH
Amadori-Rearrangement
Glucosylamine OH
H2O
COOH
O
C H2C N CH
H Strecker degradation
H2C N C C O R'
H
C O R' !
R
ketosamine
R
Maillard-Reaction
Reductone (Caramel)
Figure 3.22.: The tripeptide glutathione serves as a redox-coupler in our cells. Figure taken
COOH
H2N CH
O OH
CH2 C
O
CH2 C N CH2
H
C N CH
O H
CH2
SH
*OXWDWKLRQH*6+
( -Glu-Cys-Gly)
59
Disulphide bonds Reduction of the SH-groups of two cysteine residues leads to the forma-
tion of a covalent bond. The cell uses the tripeptide glutathione (see fig. 3.22) as reducing
agent: −SH + HS− + GSSG * ) −S−S− + 2GSH. Disulphide bond formation does not hap-
pen in the reducing environment of the cytosol, but in the ER (or the bacterial periplasm)
which is oxidizing. Special enzymes, protein disulphide isomerase (PDI), make sure
that the right Cys residues undergo disulphide bond formation.
When cytosolic proteins are used in the laboratory one has to make sure that their SH-
groups are not oxidized by air oxygen, which would lead to inactivation. The buffers there-
fore usually contain an antioxidant like β-mercaptoethanol or dithiotreitol.
Some mucolytic pharmaceuticals, like N-acetylcysteine (ACC), work by breaking S−S-

bonds in mucus proteins, decreasing the viscosity of mucus and making it easier to clear
from the airways.
Bacterially expressed eukaryotic proteins are often miss-folded and precipitate as inclusion
bodies because bacteria are less active in disulphide formation than eukaryotes. However,
bacteria do have an enzyme operon (Dsb, short for disulphide bond) for formation and
isomerization of protein disulphide bonds in their periplasm.
Proteolysis Proteolysis is involved in the activation of proenzymes, for example in the

digestive system. Digestive enzymes (e.g. trypsin, chymotrypsin) are produced as inactive
precursors, so that the can not harm the cells secreting them. Once released into the intes-
tine, they are activated by cleaving off a part of the enzyme that was blocking the active
site. Pro-hormones (e.g. insulin) are activated in a similar manner. On the other hand,
proteins no longer needed can be inactivated by proteolysis (e.g. cyclins in cell cycle).
Proteolysis may also be used to remove signal peptides. For example, proteins destined for
the intermembrane space of mitochondria carry a signal protein for mitochondrial import
(see fig. 3.8) which leads to their import into the mitochondrial matrix. There the signal
peptide is cleaved off by matrix protease, exposing a second signal directing the proteins
export into the intermembrane space through a different transporter.
A special form of proteolysis is protein splicing. This reaction is carried out by a protease
within the protein itself, the intein. This protease cuts itself out of the protein and rejoins
the flanking segments (exteins), and all this without requiring any external proteins, co-
factors or sources of energy (like ATP)! Inteins are now used as self-cleaving affinity-tags
to make protein pharmaceuticals.
Hydroxylation Protein hydroxylation occurs on Pro and Lys residues, for example in
collagen (see section on collagen above for further discussion).
60
Another protein regulated by Pro-hydroxylation are the hypoxia induced transcription

factors (HIF). These consist of two subunits, α and β. In the presence of oxygen, the
α-subunit is hydroxylated on P402 and P564 by HIF-prolylhydroxylases (PDH-1, -2 and
-3), leading to their proteasomal destruction. In the absence of oxygen, the α-subunits
accumulate and form a complex with β, which binds to hypoxia response elements in
the cellular DNA. As a consequence, oxygen consumption of the cell is down-regulated, it
can survive low oxygen supply for a longer time. This mechanism may one day be exploited
to increase the survival time of organs in infarct or transplantation, e.g., with inhibitors of
PDH-1 (currently available inhibitors produces too many side effects due to concomitant
inhibition of PHD-2 and -3).
Phosphorylation/dephosphorylation The transfer of phosphoryl groups from ATP to the

hydroxy groups of Ser, Thr and Tyr (rarely onto His, Asp and Glu) is important for the
reversible regulation of enzyme activity. The transfer is catalyzed by protein kinases,
the removal by protein phosphatases. Thus the reaction is rapidly reversible at minimal
expense for the cell (a single high energy phosphate bond). You will study many examples
for this type of regulation in the next couple of months, 1/3 of all proteins in the cell
undergo regulatory phosphorylation/dephosphorylation cycles.
Acetylation/deacetylation Transfer of acetyl-groups from acetyl-CoA onto the -amino

group of Lys by protein acetylases, and their removal by protein deacetylases, are
also used for regulation of enzymatic activity. We know much less about acetylation than
about phosphorylation. Many DNA-binding proteins are regulated by acetylation, because
the acetylated Lys is much less likely to be protonated, hence less likely to bind to the
negative charges of DNA. Three classes of deacetylases are known: class I and II hydrolyze
the bond with water, while class III deacetylases (sirtuins) use NAD+ , thus their activity
depends on the nutritional status of the cell. This is probably the mechanism behind the
observation that caloric restriction prolongs the life expectancy of lab animals.
Methylation/demethylation Transfer of methyl-groups from S-adenosyl methionine (SAM)

onto proteins may also serve regulatory purposes, but we know very little about it. Transfer
can be to
carboxyl groups, forming methyl esters. This reaction is used to mark damaged proteins
for destruction, but also in signal cascades of unknown function.
amino groups, forming methyl amines. The function is unknown. There are no N-demethylases,
so the modification is permanent.
thiol groups, forming thioesters. The function is unknown.
Unlike phosphorylation, methylation has never been observed to occur on hydroxyl-groups.
61
Figure 3.23.: ADP-ribosylation of proteins. Figure taken from [Buxbaum, 2007].

O NH2
H2N N N
Protein Arg NH2 + N

O O
N N
O P P O
O O O
O O
HO OH HO OH
NAD+
Cholera
toxin
NH2
N N
O
H
Protein Arg N N N
O O + NH2
O P P O
O O O N
O O
H
HO OH HO OH
Addition/removal of hydrophobic tails Addition of
palmityl- (fatty acid) groups to internal Cys or Ser
myristoyl- (fatty acid) groups to N-terminal Gly
farnesyl- or geranylgeranyl (isoprenoids) groups to C-terminal Cys
converts cytosolic enzymes to membrane-bound (cytosolic leaflet). Since this is required for
the activation of some enzymes, the transferases make possible drug targets (e.g. anti-cancer
drugs).
S-Nitrosylation occurs on Cys-residues: R−SH + NO ) * R−S−Ṅ−OH. The Cys must be

positioned between a basic and an acidic residue (either in primary, tertiary or quaternary
structure) because of acid-base catalysis. Nitrosylation serves as an additional pathway
for NO regulation besides cGMP-dependent kinases. This pathway too has not been fully
explored.
62
ADP-ribosylation ADP-ribosylation is used by some bacterial toxins (Vibrio cholerae,

Bordetella pertussis) to inactivate cellular proteins. This is the starting point of the patho-
mechanism of the diseases associated with these bacteria (cholera and whooping cough,
respectively).
Deamidation Removal of the acid amide group from Gln or Asn, forming Glu and Asp,
respectively. May be followed by racemization (formation of D-amino acids).
• catalytic removal by bacterial pathogenicity factors (cytotoxic necrotizing factors)

on heterotrimeric G-proteins and small GTPases → GTPase activity is inhibited, the
protein can not go from the active GTP-bound to the inactive GDP-bound form.
• spontaneous (Asn faster than Gln): age determination in
forensic science long lived, low turnover proteins (bone, teeth)
archaeology rate constant depends on temperature, humidity, soil pH etc. Hence

better suited for determination of relative age within a series of finds.
AMPylation (adenylation) is performed from ATP onto critical Thr hydroxygroups of

Rho GTPases and other important regulatory proteins by Vibrio parahaemolyticus Vibrio
outer protein S (VopS). This results in depolymerisation of the actin cytoskeleton,
affected cells round up. Like in ADP-ribosylation the bacterial toxin uses a readily available
energy-rich substrate to disable critical host proteins. Like the ADP-ribosylation from NAD
or glucosylation from UDP-glucose AMPylation from ATP is performed by a bacterial
A/B toxin to interfere with host defences. The toxins use readily available, energy-rich
co-substrates to modify and hence inactivate key proteins. AMPylation however is (at least
in bacteria) also used for control of metabolism: The glutamine synthetase of E. coli is
controlled in part by AMPylation of Tyr-397, the adenylyl transferase responsible in turn
is controlled by uridilylation.
Transfer of peptides Ubiquitin is transferred onto proteins no longer needed, the pro-
teins are then degraded in the proteasome. Ubiquitin is transferred to the -amino-group
of lysine, forming an isopeptide bond. There is a whole family of small ubiquitin-like
modifiers (SUMO) which are transferred in a similar manner, but about whose function
we have little knowledge.
63
The relationship between protein structure and function: Green fluorescent protein
GFP is produced by the coelenterate Aequorea victoria. It accepts energy from a chemilu-
minescent protein (which would otherwise produce blue light) and translates it into green
light by bioluminescence resonance energy transfer (BRET). The function of bio-
luminescence in these animals is unknown. Because of its intensive green fluorescence GFP
has become a favorite marker in molecular biology. The genetic information for GFP is
attached to the gene for the protein under investigation, so that a fusion product is gener-
ated. Thus both the amount of target protein produced and its subcellular localization can
then be studied by fluorescence video microscopy.
Production of the fluorophore of GFP from 3 neighboring amino acids This process
can proceed in the absence of other proteins and cofactors except oxygen. Thus GFP can
be used as marker in any aerobic cell.
Ser-65 Tyr-66 Gly-67
OH OH OH
238 aa precursor
folding cyclisation
O CH2O O CH2 CH2
H τ = 10 min
N C C N C C N C C C O C O
H H H H H2 HN H C O HN H C O
CH2 H H
N C C N C C N C C N C C
OH H H2 H H2
CH O
2 CH OH
2
OH OH
τ = 3 min
dehydration
H2O
OH OH
Aequorea victoria GFP H2O2 O2

CH CH2
λex = 395 + 488 nm, λem = 508 nm
C O C O
N OC N H C O
H oxidation H
N C C N C C N C C N C C
τ~1h H H2
H H2
CH2 CH2
OH OH
The reaction increases the system of conjugated double bonds (π-system) compared to Tyr
and shifts the absorbtion maximum from 280 to 395 nm.
Stereo representation of the crystal structure of GFP (PDB-code 1ema) The pro-
tein consists mainly of antiparallel β-strands, which together form a β-barrel. An α-helix
with the fluorophore runs in the center of the barrel, where the fluorophore is protected
from collisions with water and, in particular, oxygen. Any such collision would prevent flu-
orescence by taking away energy from the excited fluorophore. This accounts for the high
64
quantum efficiency (green photons produced per blue photons absorbed) of GFP.
The amino acids that interact with the fluorophore in the active center of GFP
GFP has 2 absorption maxima, that of the non-ionized fluorophore (phenol) at 395 nm
(UV) and that of the ionized (phenolate) at 488 nm (blue).
In the native protein Ser-65 donates a hydrogen bond to Glu-222 which makes deprotonation
of Glu-222 easier. The negative charge on this residue then prevents deprotonation of the
phenyl-group of the fluorophore. If Ser-65 is mutated to Ala, ionization of the fluorophore
becomes easier, the absorbtion maximum at 395 nm is reduced and that at 488 nm becomes
stronger. The phenolate ion forms a hydrogen bond with Thr-203, if this is mutated to Ile
the fluorophore is stabilized in the phenol-form and the absorbtion maximum at 395 nm
becomes stronger at the expense of that at 488. If Thr-203 is mutated to an aromatic
amino acid like Tyr stacking of the π-systems leads to a red-shift of both absorbtion and
emission maxima by 20 nm because of the reduced exited state energy (yellow fluorescent
protein). If Tyr-66 is replaced by Trp or His, the maxima are blue-shifted to 436/476 nm
(cyan fluorescent protein) and 390/450 nm (blue fluorescent protein), respectively.
65
Figure 3.24.: Biuret reaction of proteins. The Cu2+ is reduced by the protein in alkaline
solution to Cu+ , which forms a purple complex with the protein. The Cu+ can
undergo further reactions, which are the basis of the BCA and Lowry-tests.
Figure taken from [Buxbaum, in press].
R O R O R O
H H
C C C C C C
N N N
H
-
OH
H2O
R O R O R O
_ H
C C C C C C
N N N
H
H
N N N
Cu
2+
+ C C
H
C C
H
C C
O R O R O R
R O R O R O
H
C C C C C C
N N N
H +
Cu
H
N N N
C C C C C C
H H
O R O R O R
purple complex
λmax = 540 nm
3.2.3. Proteins in the laboratory
Determination of protein concentration
Absorbance Proteins do not absorb visible light (380–760 nm) and are uncolored unless
they contain a colored prosthetic group (flavins, heme, Cu, Fe...). However, almost
all proteins contain tyrosine and/or tryptophan. The aromatic rings of these amino
acids absorb UV-light, with a maximum at 280 nm.
The biuret assay In alkaline solution, copper salts (Cu2+ ) are reduced by the protein to
Cu+ , which forms a violet complex with substances containing two or more peptide
bonds (see fig. 3.24
The Lowry Method Similar to biuret, but more sensitive. The Cu+ produced in the biuret
reaction and the tyrosine residues in the protein react with molybdophosphoric acid
66
Proteins in the laboratory 3.2.3
Figure 3.25.: Reaction of amino acids and other primary amines with ninhydrin. Note that
Pro is a secondary amine and gives a different reaction, turning yellow instead
of purple.
O COO
-
OH H3N
+
CH
2 +
OH R
O
Ninhydrin
O O
O
N + CH + CO2 + H3O+
R
O O
Ruhemann's purple (λmax = 570 nm)
(Mo6+ ), forming molybdenum blue (Mo4+ and Mo5+ ). Very commonly used, but time-
consuming. Color yield depends on the Tyr-content of the protein. Interference by
complex forming and reducing agents, detergents and many other common chemicals.
BCA-reaction The Cu+ formed in the biuret reaction forms an intensively purple com-
plex with bichinchonic acid (BCA). Interference by complex-forming and reducing
chemicals, but not by detergents.
The ninhydrin reaction Ninhydrin reacts with primary amino groups to yield a purple
product. Used for free amino acids (see fig. 3.25), also proteins after hydrolysis.
Fluorescent amine reagents like OPA and fluoram react with primary amino groups in
proteins. Since most labs don’t have a fluorimeter this type of assay is rarely used.
Fluorescent yield depends on aa composition of the protein.
Bradford-assay This method is based on the fact that proteins bind hydrophobic dyes
like CBB-G250, which have different colors in aqueous and hydrophobic environment.
Color yield depends on the properties of the protein.
67
Figure 3.26.: Proteins are easily damaged when not handled with care. Just as in medicine
the first rule is “Firstly, do no harm”. Figure taken from the Pierce-catalogue.
Separation and purification of proteins
The purification of proteins is the first step in elucidating their properties, since in a mixture
you could never tell which protein is responsible for an observed effect. Purification is usually
not possible with a single technique, but requires the judicious combination of several steps.
Since we can not really predict the behavior of a protein in the various techniques, protein
purification still is more art than science and requires experienced operators.
Crude separation
Precipitation Proteins can be precipitated without denaturing them by
Adjustment of pH Protein solubility is minimal at the isoelectric point.
Adjustment of salt concentration A modest salt content enhances solubility because

intermolecular salt bonds are disrupted. High salt concentrations (> 10 %) reduce
the solubility because the salt competes with the protein for water.
Organic solvents The addition of water-miscible organic solvents (ethanol , acetone

) at low temperature precipitates proteins, usually without denaturing them.
Dialysis This method uses a porous cellophane membrane to separate molecules by size:
salt and small molecules are removed from the protein solution.
68
Figure 3.27.: Globulins are, as the name implies, spherical molecules with an even charge
distribution. Such proteins are soluble in distilled water and low concentration
salt solutions, they are precipitated by high salt concentrations. Albumins on
the other hand have an asymmetric shape and charge distribution, they are
held together by ionic bonds. As a consequence they are not soluble in distilled
water, low salt concentrations are required to break these bonds (salting in).
High concentrations of salt precipitate the protein again. Figure taken from
[Buxbaum, 2007].
Globulins Albumins
_ + _ _ _ _
_+ +_ + _ + _ + _
+ + +
+_ + _ _ _
_ + + +
+_ _+ + _ + _ + _
+ + _ + _ + _
_ _
+ _ + _
+ +
_ _
_ + _ _ + _ + +
_+ +_ +_ _ _
_+ + +
_ + _ + _
+_ + +_ + _ _
_ _ + _ + _ + _
+ + +
+_ _+ +_ _+
+ + _ _ _
+ + +
+ _ + _ + _
+ _ + _ + _
Figure 3.28.: Principle of chromatography. For details see text. Figure taken from
[Buxbaum, 2007].
6ROYHQW
0DWUL[
7HVWWXEH
ZLWKIUDFWLRQ
WLPH

ion exchange affinity, HIC gel filtration
69
Chromatography Chromatography is a widely used technique to separate mixtures of

different compounds either on an analytical or a preparative scale. The basic principle
is that compounds partition between a stationary (usually solid, sometimes liquid) and
a mobile (fluid or gas) phase. The partitioning coefficient Kp = [A]m /[A]s determines
how fast the substance A moves. From the various chromatographic formats only column
chromatography is used to separate proteins.
Gel filtration (size exclusion chromatography (SEC)) This method separates proteins (and
other macromolecules) by molecular size using small porous beads of a cross-linked
gel in a column chromatographic procedure. Big molecules come out first.
Ion exchange chromatography (IEC) The stationary phase contains charged groups which
interact with oppositely charged groups on the protein. Proteins are eluted by increas-
ing the salt concentration and/or changing the pH.
Affinity chromatography A ligand (substrate, antibody, inhibitor, etc.) is covalently linked
to the stationary phase. The target protein binds and is then eluted with free ligand.
Reversed phase Chromatography (RPC) A lab work-horse for the separation of drug-
molecules, amino acids, peptides and small proteins. This method normally separates
based on the selective retention of analytes on a hydrophobic ligand (usually C18 ).
An organic solvent like acetonitrile is used to chase off the retained proteins one at
a time. This method would denature proteins, so much less hydrophobic residues
are used (butyl- or phenyl-): hydrophobic interaction chromatography (HIC).
Proteins are loaded onto the column at high salt concentrations to maximize their
interactions with the hydrophobic groups, elution is with a gradient of decreasing salt
concentrations.
Electrophoresis Proteins are charged if the pH6= pI. Thus they can be separated in an
electric field, depending on the method used separation can be on size+shape, charge or pI.
Since electrical currents produce heat, which denatures proteins, electrophoretic methods
are usually used for small-scale (analytical) separations, where the large surface area /
volume ratio makes cooling easier.
Electrophoresis At a pH above its isoelectric point, a protein migrates to the anode, below
the isoelectric point to the cathode. Electrophoresis can be done in gels, on thin layer,
cellulose acetate foils, etc. It is used in clinical laboratories to separate plasma proteins
or diagnostically important isoenzymes. It separates molecules by their charge/mass
ratio. However, in the presence of a charged detergent like sodium dodecylsulphate
(SDS) or cetyl trimetylammonium bromide (CTAB) proteins bind a roughly constant
amount of detergent (1 detergent molecule per 3 amino acids), the charge of the
bound detergent is then much larger than that of the protein itself. Hence all proteins
have the same charge/mass ratio and hence experience the same acceleration in an
70
Figure 3.29.: Separation of proteins by size in polyacrylamide gel electrophorese

(PAGE). Figure taken from [Buxbaum, 2007].
Mw (kDa)

Myosin (200)

200

Galactosidase (116.3)
100 Phosphorylase b (97.4)

Bovine serum albumine (66.2)

50 Ovalbumine (45)

Carbonic anhydrase (31)

Trypsin inhibitor (21.5)

20
Lysozyme (14.4)

10
relative migration distance

Gel add sample separate
Figure 3.30.: isoelectric focussing (IEF) separates proteins by pI. Figure taken from
[Buxbaum, 2007]
71
Figure 3.31.: 2D-electrophoresis of proteins. For details see text. Figure taken from
[Buxbaum, 2007].
pH 3 pH 10
200 kDa
14 kDa
mount IEF gel and load molecular mass marker after running 2nd dimension
electric field. However, their retention by a network of cross-linked gel-molecules is

size dependent.
Isoelectric focusing IEF a pH gradient is set up in a gel. In the electrical field, proteins
stop migrating when the local pH equals their pI.
2D-electrophoresis Proteins from a sample are first separated by pI using IEF, then in a
second run by size using SDS-PAGE. If this is done carefully, about 10 000 protein
spots can be resolved from a cell extract. This is a key method of proteomics, where
the protein content of healthy and diseased cells are compared to identify possible
drug targets.
Membrane proteins Membrane proteins are more difficult to purify than soluble ones,
because you first have to get them out of the membrane. Membrane attached proteins can
be washed of the membrane by high salt concentrations or high pH, but proteins with
transmembrane segments have to be solubilized with detergents.
Detergents are molecules with a hydrophilic and a hydrophobic end, that is, they are am-
phophilic. For this reason they can mediate between aqueous and lipophilic phases. The
use of detergents for cleaning makes use of this effect: The hydrophobic tails of the de-
tergent insert into an oil droplet, the hydrophilic head groups allow the complex to stay
in the aqueous phase. In addition, many detergents bear a charge on their head groups,
Coulombic repulsion between the head groups cause the oil droplets to disperse.
72
Figure 3.32.: Left: Detergent look a little bit like phospholipids, with a hydrophobic tail and
a hydrophilic head group. The head group may be uncharged, or it may bear
positive or negative charges. Right: Detergents tend to aggregate in aqueous
solution into micelles, where the hydrophobic tails point into the interior and
are shielded from the water. Figures taken from [Buxbaum, 2007].
O
H2
O C
O CH O CH3
+
C O P O C C N CH3
O H2 H2 H2
Phosphatidylcholine O CH3
-
SO3
+
Sodium dodecyl sulfate (SDS) Na
65 365 34 34

CH3 Br
+
N CH3
CH3

65 365 43 1/43 12 0/ 0/

/0/ .- 0/ .-
Cetyltrimethylammonium bromide (CTAB)
.- ,+ .- ,+

*) *)
H2

HO C
'(' ('
O
O C
H OH
!#! #$ %'#$ &% (' &% *) (' *)
OH
%&% &%
"#" $# $#
C C
H H H
Octyl-β-glucoside C C
H
OH
Figure 3.33.: Solubilization of membrane proteins by detergents. The detergents form tor-
roidal micelles around the transmembrane section of the proteins. In the mi-
celles the hydrophobic tails of the detergent interacts with the hydrophobic
amino acid side chains, the hydrophilic head groups keep the micelle in solu-
tion. Each micelle contains only a single protein molecule, making purification
possible. Once the protein is pure, it can be re-inserted into an artificial lipid
membrane, a liposome for further study. Figure taken from [Buxbaum, 2007].
11
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11
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11
00
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11
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00
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1111
73
Protein structural analysis
Amino acids sequences are known for many proteins. Steps in primary structural analysis
include:
Amino acid analysis Complete acid hydrolysis of the protein, followed by HPLC separation
of the amino acids for quantitative analysis.
Fragmentation of the protein into shorter peptides usually with proteolytic enzymes. The
fragments may be analyzed by mass spectrometry.
Amino-terminus determination using chemicals which selectively react with the terminal
amino group.
Sequencing using Edman degradation chemistry. Nowadays proteins sequences are often
determined indirectly by sequencing their genes, which is a lot less time consuming.
Secondary structure analysis is usually done using biophysical methods like circular dichro-
ism or infrared spectroscopy, which measure the α-helix and β-sheet content.
Tertiary and quaternary structure analysis are now done using X-ray crystal structure
determination and nuclear magnetic resonance (NMR) spectroscopy.
Verification of structures at least of peptides and small proteins is done by synthesizing
them from amino acids by the Merrifield-method.
3.3. Protein folding diseases
So far we have looked upon protein folding as driven by the reduction of ∆G to one stable,
called native, structure. However, there are proteins that have a metastable native structure
and that can fold into one or even several more stable structures with different biological
properties. Often, these proteins contain large segments of coil, with little or no other
secondary structure (intrinsically disordered proteins). When these segments come
into contact with a template protein they refold to fit that template, with the free energy of
binding driving the process. We have discussed above how this can be useful for regulatory
proteins.
The problem is that contact of some proteins with their alternative conformation results in
their autocatalytic conversion into the alternative conformation, which is not only devoid
of the biological function but also has a tendency to form aggregates, called amyloid.
Surprisingly all amyloid seem to have basically the same structure, fibres made of β-helices.
Many proteins that tend to form amyloid tend to have more Gln and Asn, aromatic and
β-branched chain and fewer polar amino acids than average proteins, however, this is not
a universal feature.
74
Protein folding diseases 3.3
Figure 3.34.: Edman-degradation of proteins. The amino acids are cleaved of one by one
from the N-terminus and analyzed. After each cycle the amino group of the
next amino acid is exposed for cleavage. About 50 amino acids can be se-
quenced this way, longer proteins must be cut into segments first. Figure
taken from [Buxbaum, 2007].
H
NH2 N C S
N C S
R CH NH
C O Phenylisothiocyanate R CH
NH C O
R' CH NH
C O R' CH
C O
Protein
Phenylthiourea
HCl
NH2
R' CH
N +
O C C S C O
H C NH
R
Phenylthiohydantoin Protein
amino acid (with N-terminal
aa removed)
75
Figure 3.35.: Merrifield-synthesis of peptides. An amino acid, with its amino-group pro-
tected by a Fmoc-group, is bound to a solid support via it’s carboxy-group.
The protective Fmoc-group is cleaved away so that it can react with a second
amino acid. The amino-group of that amino acid is protected by Fmoc, the
carboxy-group activated by DCCD to make the reaction with the exposed
amino-group of the first amino acid easier. This cycle of deprotection and
coupling is repeated until the peptide is fully synthesized. Then the peptide
is cleaved of the support and purified. Proteins of up to 100 amino acids can
be synthesized this way. Important: Unlike biological protein synthesis, which
goes from the N- to the C-terminus, chemical synthesis goes from C- to N-.
You need to keep that in mind when talking about sequences to a chemist!
Figure taken from [Buxbaum, in press].
O OH
C
H
N CH fmoc
fmoc HCl
R1 H2C Polystyrene H2C Polystyrene
O O O O
+ H
C C
N CH mild organic base H2N CH
fmoc
R1 R1
Cl C Polystyrene
H2
O DCCD
C
H
N CH
fmoc
R1
DCU
O
C
NH
fmoc =
O
DCCD =
N H
C DCU = C O
H2C Polystyrene
CH2
N NH
O O
C
H
O N CH
C R1
H
N CH
fmoc
R1
mild organic base

F C Polystyrene
H2 H2C Polystyrene fmoc
O O
O OH HF C
C H H2C Polystyrene
H O N CH
O O
O N CH C C
R1
C O H
R1 N CH
C N CH
O H O
C NH
CH
R1 C R1
N CH
N CH R1 H H2N CH
H R1
R1 R1
76
Spongiform encephalopathies 3.3.1
Figure 3.36.: Structure of amyloid formed by a yeast prion protein (HET-s fragment 218-
289, PDB-code 2rnm), determined by solid state (magic angle spinning) NMR.
Each protein forms a β-helix of 2 turns with 3 strands each. The helices
come together from different proteins (represented by different colors) to form
extended parallel β-sheet.
In yeasts there are several such proteins; the amyloid states are transferred to the daughter
cells in mitosis and meiosis (dominant), inheritance is in a non-Mendelian, cytosolic man-
ner. Some of these amyloids can be cured by growing cells in the presence of guanidinium
hydrochloride (GuHCl), a compound which unfolds proteins. Low temperature also pre-
vent the autocatalytic amyloid formation and can even break up amyloid fibrils; apparently
amyloid formation is driven mainly by entropy.
Amyloids are involved in – many say: the cause of – several severe human diseases. It is
however unclear whether the pathogenesis is really caused by the large amyloid aggregates
or rather by soluble, oligomeric intermediates. The latter have been shown to solubilize
phospholipid vesicles. In addition, trapping of other proteins in the aggregate – which then
can not perform their normal function – may also play a role.
Because the conversion of proteins into amyloid is a slow process amyloidoses tend to
manifest in the brain, where cell turnover is low, at advanced age and in the form of a
slow degeneration. In our aging society this puts severe strain on public health budgets
and the families of the affected. Because disease onset is past the usual age of reproduction
there is little evolutionary pressure against these diseases.
Detection of amyloid in histologic sections often relies on the metachromatic shift of color
or fluorescence of dyes bound to them, examples include Congo Red and Thioflavin T. The
effect is enhanced when the sample is viewed between crossed polarizers.
3.3.1. Spongiform encephalopathies
The first spongiform encephalopathy known was scrapie in sheep, first described in the 18th
century. Other diseases affect humans:
77
Figure 3.37.: Left: X-ray structure of part of the normal PrPc . Middle: Hypothetical model
of PrPsc . Figure taken from [Doenecke et al., 2005]. Right: Vacuolization in
the brain of a patient with CJD. Figure curtesy of Dr. Yakubovskyy, RUSM.
Gerstmann-Stäussler-Scheinker-disease (GSS) Patients have difficulty to coordinate

their movement (ataxia, nystagmus, tremor), loose their speech and finally control over
body functions. Disease is protracted (1–11 a). Familiar cases have been described with
F198S or P102L in the prion protein.
Creutzfeldt-Jakob-disease (CJD) Patients suffer from myoclonus, ataxia, hallucina-

tions, loss of memory, change of personality and dementia, in the final stages aki-
netic mutism (decerebrate rigidity). Death usually occurs within 4–6 mo after first
symptoms. In familial cases the patients are younger than in sporadic (40 vs 65 a),
mutations E200K and V210I in prion protein have been reported.
fatal familial insomnia (FFI) At age 40–50 patients suddenly fail to sleep. Sleeping pills
show no effect. Secondary to sleep deprivation dysautonomia develops: myosis, ele-
vated blood pressure, heart rate and temperature, profuse sweating, myoclonus, impo-
tency. As the disease progresses, patients loose the ability to walk, keep their balance,
control their sphincter, speak. Hallucinations, panick attacks, agitation and phobias
develop, but unlike the other prion diseases patients retain their mental capacity un-
til almost the end. Death occurs between 0.5 and 3 a after onset. On autopsy one
finds neuronal degradation and reactive astrocytosis in the anterior and dorsomedial
thalamic nuclei, but without spongiosis. The disease is usually familial with D178N of
the prion protein. However, FFI occurs only if amino acid 129 is Met, if it is Val, the
D178N mutation leads to CJD. Spontaneous cases of FFI have also been reported.
78
Spongiform encephalopathies 3.3.1
Kuru is a disease spread by endo-cannibalism (ritual eating of deceased family members)

in the Fore-people in Papua-New Guinea. Although this custom has been stomped
out by the colonial power (Australia) in the 1950s, occasional new cases of kuru are
reported in elderly patients due to the extremely long incubation period of the disease.
Patients giggle and tremble uncontrollably (kuru = the laughing death), then loose
control over body and mind and finally die after about one year.
variant Creutzfeldt-Jakob-disease (vCJD) or “mad cow disease”, similar to kuru, is
spread by ingestion of infected meat, but the meat of British cows that during the
1980s had been fed with offal from scrapie-infected sheep without adequate precau-
tions. Prions which have crossed the species-barrier once are apparently much more
likely to do so again, so unlike scrapie vCJD can be spread to humans. The agent
crosses into the gut associated lymphatics and from there moves up the neural tissues
via spinal cord into the brain. Distinction between classical and variant CJD can be
made by tonsil biopsy, only in vCJD will it contain prions. Compared to classical
CJD patients are younger (median 29 instead of 65 a), the course of the disease is
more protracted (14 vs 4.5 mo) and the first symptoms are usually psychiatric (de-
pression, aggression and loss of memory). About 170 patients have been reported, 152
in Britain alone.
Of particular concern are iatrogenic transmissions of spongiform encephalopathy by surgical
instruments, neural tissue (Dura mater or corneas), or pharmaceuticals made from brain
(e.g. growth hormone, gonadotropin). No transmissions by blood transfusion have been
described so far, but it is considered a probable route.
All these diseases have in common the development of a spongiform encephalopathy,
where vacuoles form in the brain at disease-specific sites (see fig. 3.37, right).
The diseases can occur sporadically or familial, but they can also be transmitted by ingestion
of tissue from affected individuals. Unusual in this context is the extreme stability of the
pathogenic agent against decomposition, heat, radiation or disinfectants. The brain of a
sheep with scrapie, when fed to mice, proved infective after burial for 3 a! T. Alper and
her co-workers noticed in the 1960s that the agent is not destroyed by UV-light of 250 nm
or by nucleases, she therefore concluded that the agent may consist of proteins only.
S. Prusiner has vigorously followed up on this heretic idea and managed to identify the
protein involved (N.P. 1997), called prion protein (PrP). Prion stands for “proteinaceous
infectious agent”.
PrP is a membrane protein of unknown function (knock-out mice are phenotypically normal,
except that they do not get spongiform encephalopathy after ingesting scrapie-infected
brains). In normal brain PrP has a secondary structure consisting mostly of α-helices (PrPc ,
c = cellular, see fig. 3.37, left). In spongiform encephalopathy this protein changes its
secondary structure into a β-pleated sheet, this leads to protein aggregation (PrPsc , sc =
scrapie (see fig. 3.37, middle). The aggregates form long fibrils (see fig. 3.36). It is not known
79
whether the aggregates themselves are cytotoxic, or whether they are inert and the damage
is caused by a soluble intermediate.
The key point for understanding of prion diseases however is the realization that the con-
version from PrPc to PrPsc is auto-catalytic, in other words: a molecule of PrPc that comes
into contact with PrPsc changes its conformation into PrPsc . It gets even weirder than that:
There exists not only one alternative conformation of PrP, but the different spongiform
encephalopathies are caused by different strains of PrPsc , which have different conforma-
tions. These different conformations can be differentiated by their sensitivity to proteases
or chemicals that break up protein secondary structure like guanidinium hydrochloride or
urea.
3.3.2. Morbus Alzheimer
(dementia) is named after Alois Alzheimer (German psychiatrist and neuropathologist,

1864–1915), who first described it in 1906. The disease is clinically characterized by the loss
of (short term) memory, excitation, apathy, paranoia, depression, aggression with possible
violence, progressing over loss of language, immobility, incontinence to finally death. Usual
age of onset is ≥ 65 a, this disease is spontaneous and age related. About 2 % of the popu-
lation at age 65 a is affected, by age 80 a prevalence increases to 20 %. Beyond age 85 a the
prevalence decreases again, as the patients rarely reach that age. With the growing number
of elderly in advanced societies these patients already put a considerable strain on their
families and the public health system, this is likely to increase in future. WHO estimates
that there are currently about 29 × 106 patients living with Alzheimer’s disease, and that
this number will increase to 106 × 106 in 2050 (when the incidence will be 1:85).
There is also an inherited form of the disease characterized by an early onset (≤ 60 a).
Strictly speaking, the case described by Alzheimer was early-onset (pre-senile dementia),
the late onset form should be called “senile dementia of the Alzheimer type (SDAT)”, but
this distinction is rarely made.
In Alzheimer’s disease the β-amyloid precursor protein (APP) is proteolytically cleaved

extracellularly by β-secretase, then within the membrane by γ-secretase. The extracel-
lular fragment resulting from the latter cleavage is the β-amyloid, which forms neuritic
plaques. If instead by β-secretase the APP is first cleaved by α-secretase then the prod-
uct resulting from γ-secretase cleavage can not form plaques. The cytosolic part of APP
left over after cleavage is called APP intracellular domain (AICD). The role of AICD in
Alzheimer’s disease is controversial, as it appears short-lived. However, it interacts with
about 20 identified partner proteins. Amongst those is the histone acetyltransferase TIP60
(affects DNA/histone interactions and hence gene transcription) and the adapter FE65 (in-
creases stability of AICD). The AICD/FE65/TIP60 complex (“AFT”) can be found in the
nucleus, where it may act as transcription factor. In addition to the extracellular neuritic
80
Morbus Alzheimer 3.3.2
Figure 3.38.: Amyloid formation in Alzheimer’s disease.
soluble APP
NH 2 Amyloid aggregate
amyloid precursor protein
Amyloid
Secretase Secretase Secretase Secretase
extracellular
membrane
intracellular
AICD
COOH
Figure 3.39.: positron emission tomography (PET) scan of the brains of left: normal 20 a
old, middle: normal 80 a old, right: 80 a old with Alzheimer’s disease. Col-
ors denote metabolic activity in the brain (red = high to blue = low). Figure
© Alzheimer’s Disease Education and Referral Center, National Institute on
Aging
plaques, histology of brains from patients with Alzheimer’s disease also reveals neurofibril-
lary tangles inside the cells. These consist of hyperphosphorylated τ-protein (component of
the cell skeleton). One of the potential target genes identified for AFT is glycogen synthase
kinase 3β, which is one of the kinases responsible for τ-phosphorylation. If these results
could be confirmed, then AFT would connect plaque and tangle formation.
Further investigation into the biochemistry of Alzheimer-brains reveals dysfunctional mi-

tochondria which produce high amounts of reactive oxygen species (ROS) (more about
ROS in the lecture on oxidative phosphorylation, see chapter 15.3 on page 253). ROS are
very toxic to cells. It is currently unclear what the patho-mechanistic relationship between
plaques, tangles and ROS is.
In early onset (familial) Alzheimer’s disease several mutations have been found. The
genes PSEN1 (AD3, on chromosome 14) and PSEN2 (AD4, on chromosome 1) code for
81
Figure 3.40.: Left: neuritic plaques (amyloid) and neurofibrillary tangle (hyper-
phosphorylated τ-protein) in the brain of a patient with Alzheimer’s disease.
Right: amyloid angiopathy, resulting in brain hemorrhage. Pictures curtesy
of Dr. Yakubovskyy, Dept. of Pathology, RUSM.
presenilin 1 and 2, respectively, which are components of γ-secretase. Mutations in APP

(AD1, on chromosome 21q) have also been found. There is also significant association
between Alzheimer’s disease and the 4 allele of the ApoE protein (AD2, on chromosome
19), which is involved in the transport of cholesterol in blood. In addition, mitochondrial
DNA-polymorphism and several other mutations have been described as associated with
Alzheimer’s disease. Patients with Down’s syndrome (trisomy 21) are at increased risk
for Alzheimer’s too, because of their general mental deficiency the onset of Alzheimer’s
is particularly difficult to diagnose in these patients.
Because the cause of Alzheimer’s disease is unknown, prevention is in infancy. The fol-
lowing recommendations however are widely agreed upon:
• control of blood pressure and [cholesterol]
• balanced diet with minerals and vitamins
• no smoking
• profession with high intellectual activity
• high physical activity
• limited TV consumption
Several authors have described a connection between Alzheimer’s disease and high alu-
minium ion intake (e.g. from cooking utensils), however, this is now considered a red herring.
Treatment of Alzheimer’s disease is possible with various pharmaceuticals that have come
onto the market in the last couple of years, but all of them only reduce the symptoms, they
do not slow down disease progression and do not change the final outcome.
82
Chorea Huntington 3.3.4
3.3.3. Morbus Parkinson
The first description of this disease in modern medical literature was by the English physi-
cian James Parkinson in 1817, but several ancient sources describe what appears to be
Parkinson’s disease, e.g. in China the Yellow Emperor’s Internal Classics from 425 BC
and the Ayurveda in India (≈ 1000 BC).
In Parkinson’s disease (shaking palsy) the protein α-synuclein aggregates in the sub-
stantia nigra of the brain, forming Lewy-bodies. This leads to a failure of ER → Golgi
transport, resulting in the death of extrapyramidal cells in the pars compacta of the
substantia nigra. These cells would normally produce dopamine which acts on basal
ganglia. This results in a characteristic trembling, in slow movement and finally the ces-
sation of movement. Patients show a characteristic, bend-forward posture when standing.
Hallucinations, depression and other psychiatric symptoms may be seen. The disease usually
strikes between 50 and 60 a of age, more often in ♂ than in ♀. Morbus Parkinson is usually
caused by gene duplication, but contact with toxic chemicals (pesticides, trichloroethylene)
can show similar results.
Precursors of dopamine (L-Dopa (Levodopa)), dopamine agonists or substances that in-

terfere with dopamine breakdown (MAO-B or COMT inhibitors) are used to treat the
disease. You will learn to understand their action in the next semester. It is fascinating
that the herbal remedies described in ancient Chinese sources contain substances that act
like modern drugs against this disease.
3.3.4. Chorea Huntington
Huntington’s disease is caused by an expansion of CAG-repeats (base triplet encoding for

Gln, see the genetic code in the appendix) from 10–35 in the protein huntingtin. Poly-Gln
> 40 amino acids form β-sheets which lead to aggregation. Inheritance is dominant autoso-
mal with complete penetrance, chromosome 4p16.3. Huntingtin is required for endocytosis
and hence for recycling of vesicle membranes after exocytosis. The death of brain cells in
basal ganglia (putamen + caudate nucleus) reduces indirect inhibition of globus pallidus
internus, resulting in activation of thalamus and cortex. Huntington’s disease is charac-
terized by jerky movements (choreoathetosis, choreia = Gr. dance), cognitive and behavioral
defects. There are several other trinucleotide expansion diseases in other proteins.
Not fully understood is the role of tissue transglutaminases in Huntington’s and also
Parkinson’s disease. Transglutaminases form isopeptide bonds between glutamine (R)
and lysine (R’) residues in proteins (R−CO−NH2 + H2 N−R0 → R−CO−NH−R0 + NH3 ), the
most well known transglutaminase is factor VIII of the blood clotting cascade (see section
11.1.2 on page 213). Tissue transglutaminase (tTG) is an enzyme found in all organs,
including brain. It has been found that intramolecular crosslinks in α-synuclein and tTG
83
binding to synuclein increase as the disease progresses, but this may actually be a protective
mechanism to reduce amyloid production by preventing the β-sheet formation.
1) Effect of pH on enzyme activity Lysozyme is an enzyme that occurs in tears. It

hydrolyzes the murein sacculus of bacteria and thus protects us from eye infection. It’s
catalytic center contains two essential, acidic amino acid residues, Glu-35 (pKa = 5.9) and
Asp-52 (pKa = 4.5). Their R-groups have to be in the correct state (protonated (−COOH) /
deprotonated (−COO− )) for the enzyme to work. The diagram shows measurements of the
enzymes activity at constant enzyme and substrate concentration, but different pH. What
is the required state of the two R-groups in the active site?
pH dependency of lysozyme activity
100
90
80
Activity (% of maximum)
70
60
50
40
30
20
10
0
2 3 4 5 6 7 8 9 10
pH
A Glu-35 protonated, Asp-52 deprotonated
B Glu-35 protonated, Asp-52 protonated
C Glu-35 deprotonated, Asp-52 deprotonated
D Glu-35 deprotonated, Asp-52 protonated
E it does not matter
84
2) Structure of alpha-amino acids, radioactivity Which of the following statements is

false? Alanine, labeled with 14 C (β-emitter, half life period = 5500 a) on the carboxy-
group,
A produces electrons by radioactive decay.
B when decarboxylated produces radioactive CO2 and non-radioactive ethylamine.
C may be produced by sparging a solution of non-radioactive alanine with 14 CO

2.
D has the same pI as non-radioactive alanine.
E can be used by cells to make proteins.
3) pI-value of amino acid Lysine has the pKa -values 2.18 (−COOH), 8.95 (α-amino) and
10.53 (-amino). What is the pI?
A 3.50
B 5.57
C 7.22
D 9.74
E 11.83
4) Properties of amino acids Sickle cell anemia, an inherited disease, is caused by the
mutation Glu6Val in the β-subunit of hemoglobin. Compared to the normal protein you
would expect the mutated protein to move in a native electrophoresis experiment (that is,
without SDS):
A) less to (+) at pH 8, same distance at pH 1
B) same at pH 8, more to the positive at pH 1
C) more to (+) pH 8, more to the (-) at pH 1
D) less to the (-) at pH 8, less to the (-) pH 1
E) same distance under all conditions
85
5) Functional replacement of amino acids You are working on a research project to

elucidate the reaction mechanism of an enzyme. You think that a particular serine residue in
the protein is required for catalytic activity. To test this hypothesis you want to genetically
replace this amino acid by another, and then test whether the enzyme is still active.
Which amino acid would you choose to replace the Ser?
A Threonine
B Alanine
C Tryptophan
D Glutamic acid
E Histidine
6) pH dependence of solubility of amino acids Which of the following tripeptides would

you expect to be the most soluble in 1 M NaOH:
A) Phe-Ala-Val
B) Glu-Gly-Asp
C) Gln-Gly-Asn
D) Lys-Arg-His
E) Trp-Lys-Asn
7) Determination of protein concentration from UV-absorption Blood serum contains

many different proteins at a fairly constant concentration (6.0–7.8 g/dl). However, in several
serious diseases protein concentration is lowered (e.g. liver cirrhosis) or elevated (e.g. mul-
tiple myeloma). A quick way to determine the serum protein concentration is to measure
the UV-absorbance at 280 nm.
A 1:100 dilution of serum gives an absorbance of 0.4 at 280 nm in a standard cuvette of 1 cm
path length. Assume a molar extinction coefficient of 4 × 104 l mol−1 cm−1 and an average
molecular weight of 65 kDa for blood proteins. The protein concentration is approximately
A 6.0 g/dl
B 6.5 g/dl
C 7.0 g/dl
D 7.5 g/dl
E 8.0 g/dl
86
8) Strange disease A.B., 45 a old ♂, visits you because he can not sleep. Hypnotics
are without effect. You notice the following signs: myosis, hypertension, tachycardia and
elevated body temperature with diaphoresis. Over the following months the patient develops
dream-like states, dysarthria, myoclonus and impotency. He looses the ability to walk, keep
his balance and control his sphincter. However, his ability to think and understand does
not diminish until after 15 m the patient falls into a coma and finally dies quite suddenly.
Autopsy shows neuronal degradation with reactive astrocytosis limited to the anterior and
dorsomedial thalamic nuclei without spongiosis or inflammation. What is the most likely
diagnosis?
A astrocytoma
B myasthenia gravis
C morbus Alzheimer
D fatal familial insomnia
E new variant Creutzfeld-Jakob disease
9) Collagen related inherited disease X.Y. has malformation of his limbs because his
epiphyses ossified from several discrete centers with a stippled appearance in X-ray, the
shafts of the long bones are thickened. He also has a congenital cataract of both eyes and
mental retardation. Genetic analysis shows a mutation in the gene for collagen α1(II). What
is the most likely diagnosis?
A chondrodysplasia
B Ehlers-Danlos syndrome
C osteogenesis imperfecta
D Alport syndrome
E epidermolysis bullosa
10) CAG-length variation A.B., 45 year old male, visits you in your office because of
involuntary movements in arms, legs and face. He is very worried because the (protracted
and finally fatal) disease of both his father and paternal grandfather had started with the
same symptoms. Upon molecular investigation you find that the repeat-length of a CAG-
stretch in the gene for a protein involved in endocytosis is 155 (normal up to 35).
The most likely diagnosis is:
A) Chorea Huntington
B) Alzheimer’s disease
87
C) Kuru
D) Fatal familial insomnia
E) Creutzfeldt-Jakob-disease
3.5. Objectives
Students should be able to
1. Recognize the structures of the 22 major amino acids.
2. explain how these different structures affect their biological function.
3. explain what pKa -values are and how to calculate the pI.
4. Name the non-covalent interactions that can be formed by the different amino acid
side chains.
5. Describe the structure of the peptide bond, including its partial double bond charac-
ter, planarity and ability to engage in hydrogen bonding.
6. define the terms primary, secondary, tertiary and quaternary structure, fibrous pro-
tein, globular protein, albumin and globulin.
7. describe the structure of the α-helix and the β-pleated sheet and the role of hydrogen
bonds in their formation.
8. explain on suitable examples how particular structures allow a protein to serve its
biological function.
9. name the components of glycoproteins, lipoproteins, nucleoproteins, phosphoproteins,

heme proteins, flavoproteins and metalloproteins and describe the interaction between
the polypeptide and prosthetic group in each case.
10. describe the process of heat denaturation of proteins and its biological consequences.
11. list various types of denaturing agents and specify the mechanism by which they cause
denaturation.
12. state the susceptibilities of peptide bonds and disulfide bonds to acids, bases, oxidizing
and reducing agents.
13. know the use of UV absorbance, the biuret and Lowry methods for the measurement
of protein concentrations and describe why the result of such measurement depends
on the method used.
88
Objectives 3.5
14. name the principles by which proteins are separated in different types of chromatog-
raphy and electrophoresis.
15. describe the post-translational modification of proteins and their interaction with
prosthetic groups.
16. explain, using suitable examples, why mutation of a single amino acid in a protein
may result in genetic disease.
17. critically discuss the mechanism of prion and other protein folding diseases
89
4. DNA and Gene Expression
4.1. DNA Structure
4.1.1. Bases, Nucleosides and Nucleotides
DNA contains the two purine bases adenine (A) and guanine (G), and the pyrimidine bases
cytosine (C) and thymine (T). RNA contains uracil (U) instead of thymine.
NH2 O
N N N N
N N H2N N N
H H
Adenine Guanine
O O NH2
CH3
HN HN N
O N O N O N
H H H
Thymine Uracil Cytosine
Nucleosides consist of a base linked by an N-glycosidic bond to the 1’ -carbon of ribose or

2’-deoxyribose. Examples:
Adenosine A + ribose
Guanosine G + ribose
Cytidine C + ribose
Uridine U + ribose
2’-deoxyadenosine A + 2’-deoxyribose
2’-deoxythymidine T + 2’-deoxyribose
Nucleotides are nucleoside derivatives carrying 1, 2 or 3 phosphate groups at the 5’-carbon

of ribose or 2-deoxyribose. They are named as derivatives of their corresponding nucleosides.
91
Examples:
A + ribose + 1 phosphate Adenosine monophosphate (AMP)
U + ribose + 3 phosphates Uridine triphosphate (UTP)
4.1.2. The DNA Double-Helix.
DNA is an unbranched polymer of 2-deoxyribonucleoside monophosphates. These nucleotides

are linked by phosphodiester bonds between the 3’ end of one 2-deoxyribose and the 5’ end
of the next 2-deoxyribose. The phosphate groups are negatively charged. The molecule has a
polarity, with a 5’ end and a 3’ end. Conventionally, the 5’ end is written left and the 3’ end
right. The primary structure of the DNA strand can be described by its base sequence.
The Watson-Crick double helix (= B-DNA) is the principal higher-order structure of DNA.
Important features:
• The two strands of the double helix are antiparallel.
• The bases face inward to the helix axis while the sugar-phosphate backbone forms
two ridges.
• There are a major groove and a minor groove which are lined by the edges of the bases.
Minor grove binding proteins bind to the sugar/phosphate-backbone (e.g. histones),
in the major grove proteins can access the bases (e.g. regulators of transcription).
• In each strand, the bases are stacked flat one on top of the other.
• The bases interact by hydrogen bonds, forming A-T and C-G base pairs
• The two strands form a right-handed helix with about 10 bp per turn.
This structure has several important implications:
• A large number of unique DNA sequences can be generated by permutations of the 4

bases.
• The edges of the bases are exposed in the major and minor grooves. Therefore the
base sequence of the DNA can be recognized by DNA-binding proteins (see fig. 4.1).
• The two strands can be separated easily.
• The base sequence of one strand predicts exactly the base sequence of the comple-
mentary strand.
92
Supercoiling 4.1.4
Figure 4.1.: Glucocorticoid receptor bound to a glucocorticoid response element DNA

(PDB-file 1r40). Major and minor groove of the DNA are clearly visible. The
receptor belongs into the class of Zn-finger DNA-binding proteins. It’s 4 Zn-ions
are visible, also shown are the 4 Cys-residues that keep each Zn-ion in place.
Note however that the Zn-ions do not make contact with the DNA, rather 2
α-helices bind into the major grove of the DNA. The Zn-ions however ensure
the correct secondary structure of the protein.
4.1.3. Chemical stability
The covalent structure of DNA is quite stable. Rigorous conditions (boiling in strong acid)
are required to break the covalent bonds. The double helix, however, is destroyed by heat-
denaturation (“melting” of DNA).
• Short DNAs melt more easily than long DNAs.
• A-T-rich DNA melts more easily than C-G-rich DNA.
• Low ionic strength and alkaline pH favor melting.
The viscosity of the DNA solution decreases and the UV- absorption (measured at 260 nm)
increases. Denatured DNA renatures when cooled slowly. This process is called annealing.
Short DNAs anneal much faster than long DNAs.
4.1.4. Supercoiling
DNA can be overwound, with less than the canonical 10 base-pairs/turn. This is called a
positive supertwist. Or it can be underwound, with more than 10 bp/turn. This is called a
negative supertwist. Over- and under-winding can occur when the DNA duplex is circular
93
(in prokaryotes), or when the ends of the duplex are firmly attached to structural proteins
(in eukaryotes). The supertwisting is regulated by topoisomerases. Usually, the DNA in
cells is moderately underwound. This facilitates strand separation for transcription and
DNA replication.
4.2. DNA Replication
4.2.1. Semi-conservative replication
Mechanism of DNA replication:

• The double helix unwinds. The point where unwinding occurs is called the replication
fork.
• New DNA is synthesized in the replication fork, using the old strand as a template.
• The old strand keeps unwinding until the whole DNA is replicated.
• The replicated DNA consists of one complete old strand and one complete new strand.
4.2.2. DNA polymerases
New DNA is synthesized by DNA polymerases. Important properties of bacterial DNA

polymerases.
• They require deoxyribonucleoside triphosphate as precursors. dATP, dGTP, dTTP.
Pyrophosphate is released during the polymerization reaction.
• They synthesize DNA from 5’ → 3’.
• They require a single-stranded DNA template. Therefore the parental DNA duplex
has to unwind first before the DNA polymerases can start.
• They read their template from 3’ → 5’. Therefore the template strand and the new
strand are antiparallel. All base-paired nucleic acids are antiparallel.
• They incorporate only nucleotides that make proper base pairing with the base in the
template strand.
• They possess a 3’ -exonuclease activity which is specific for mismatched bases and
which is used for proofreading.
• Many bacterial DNA polymerases have a 5’ -exonuclease activity which is required
for DNA repair.
94
Steps in bacterial DNA replication 4.3.1
• The DNA polymerases require a primer with a free 3’-hydroxy group.
Bacteria have three DNA polymerases:
Poly I Most important for DNA repair. It has a low binding affinity for its template, and
therefore it makes only short pieces of DNA at a time: Low processivity.
Poly II Unknown function
Poly III The essential enzyme for DNA replication. Very high processivity.
Thanks to the 3’-exonuclease activities of the DNA polymerases, the error rate is only about
one mis-incorporated base per 1 × 108 nucleotides.
4.2.3. Steps in bacterial DNA replication
1. E. coli has a circular chromosome (≈ 3 × 106 bp) with a single replication origin called
Ori-c. Initiator proteins associate with Ori-c to start unwinding.
2. A helicase unwinds the DNA. This requires ATP.
3. The topoisomerase gyrase relaxes positive supertwists and actively pumps negative
supertwists into DNA (ATP-dependent). This prevents overwinding ahead of the mov-
ing replication fork. The gyrase is not in the replication fork.
4. Single-strand DNA binding proteins (SSB proteins) bind the single-stranded DNA to
prevent annealing.
5. A specialized RNA polymerase called primase synthesizes a small RNA primer.
6. Poly III starts DNA synthesis at the 3’ end of the primer.
7. One of the new strands, the leading strand, is synthesized continuously.
8. The other strand, the lagging strand, is synthesized piecemeal. The pieces are called
Okazaki fragments.
9. The small RNA pieces at the 5’ ends of the Okazaki fragments are removed by Poly
I and replaced by DNA.
10. The fragments of the lagging strand are connected by DNA ligase.
95
4.3. RNA and Transcription
4.3.1. RNA Structure
RNA can form a double helix like DNA, but is usually single-stranded except in some virus.
Cellular RNAs contain both base-paired segments (“stems”) and unpaired segments (“loop”).
An RNA strand can also anneal with a complementary DNA strand. Annealing between
nucleic acids of different kinds or from different sources, for example DNA with RNA, or
human DNA with chimpanzee DNA, is called hybridization.
4.3.2. Types of RNA
There are three major types of cellular RNA, all of them synthesized as copies of DNA
sequences:
Messenger RNA (mRNA) carries the information of the linear DNA sequence to the ri-
bosomes where proteins are synthesized. In eukaryotes each mRNA is copied from
a single gene and encodes a single polypeptide chain (“monocistronic” mRNA). In
prokaryotes most mRNAs are copied from a string of genes and encode more than one
polypeptide (“polycistronic” mRNA). By definition, each gene codes for one polypep-
tide.
Ribosomal RNA (rRNA) is a structural component of the ribosome. There are 3 prokary-
otic and 4 eukaryotic rRNAs, each of them present in a single copy in the ribosome.
Transfer RNA (tRNA) carries amino acids to the ribosome for protein synthesis.
4.3.3. Transcription
RNA synthesis is by transcription from a DNA template. It requires the enzyme RNA
polymerase. Mechanism: like DNA synthesis, but
• The precursors are ATP, GTP, CTP and UTP.
• RNA polymerase does not need a primer
• RNA polymerase has no nuclease activities.
96
Post-transcriptional processing 4.4.1
Steps in transcription (E. coli )
1. RNA polymerase, sliding along the DNA, binds to the promoter. This requires the
σ (sigma) subunit of RNA polymerase. Promoters look different in different genes,
with consensus sequences at about −10 (TATAAT, “Pribnow box”) and −35 bp
(TTGACA) from the start of transcription.
2. RNA polymerase, separates a short stretch of DNA duplex.
3. RNA polymerase synthesizes the RNA in the 5’ → 3’ direction using the original
strand of the DNA strands as a template strand. The template strand is called the
coding strand. The DNA anneals behind the RNA polymerase.
4. There is a termination site at the end of the gene where the RNA polymerase falls off
its template. Most bacterial termination sequences contain a palindrome.
All bacterial RNAs are synthesized by the same RNA polymerase. The rate-limiting steps
in transcription are promoter recognition and strand separation. The error rate of RNA
synthesis about 1 in 10 000. The life span of mRNA is only a few minutes in bacteria but
several hours in eukaryotes. Only 3 % of the RNA in E. coli is mRNA.
4.3.4. Post-transcriptional processing
Modification of RNA after transcription:
• mRNA is usually not modified in prokaryotes but translated immediately. But exten-
sive processing occurs in eukaryotes.
• tRNA has many modified bases, both in prokaryotes and eukaryotes: methylated
bases, pseudouridine, inosine (nucleoside containing hypoxanthine), ribothymidine
(thymine bound to ribose) etc. Most tRNAs are processed from precursor transcripts
by nuclease cleavages.
• rRNA is processed by nucleases from a transcript that contains the sequences of all
major ribosomal RNAs (5 S, 16 S, 23 S in bacteria, 5.8 S, 18 S, 28 S in humans). There
are some methylated bases (prokaryotes) or ribose residues (eukaryotes) in rRNA.
97
4.4. Protein Synthesis
4.4.1. The Genetic Code
The genetic code (see fig. 34.4 on page 632) describes the relationship between the base se-
quence of the mRNA (and the DNA) and the amino acid sequence of the protein. Important
features:
• A base triplet called a codon on the mRNA specifies an amino acid.
• There are 61 amino acid coding codons. One of them (AUG, coding for formyl me-
thionine) is also used as a start codon. There are 3 stop codons: UAA, UAG and
UGA.
• The code is non-overlapping and comma-less. There is no overlap between codons and
no empty spaces in between.
• The code is co-linear. The codons on the mRNA are in the same sequence as the
encoded amino acids in the polypeptide.
• The code is unambiguous. Each codon specifies one and only one amino acid.
• The code is degenerate. Most amino acids are specified by more than one codon.
• The code is universal. There are only minor deviations, especially in the small genomes
of mitochondria and chloroplasts.
4.4.2. tRNA
tRNAs are small RNAs (80 nucleotides in length) which bring amino acids to the ribosome.
Typical “cloverleaf” structure. The 3’-terminus, ending in the sequence CCA, binds the
amino acid through an ester bond with one of the hydroxy groups of ribose. The anticodon
base-pairs with the codon of mRNA during translation. There is a certain freedom of pairing
between the third codon base and the first anticodon base (“wobble”). Therefore cells can
do with less than 61 tRNAs.
Aminoacyl-tRNA synthetase are cytoplasmic enzymes which transfer an amino acid to the
3’ end of the tRNA. ATP is hydrolyzed to AMP + pyrophosphate in this reaction. Each
aminoacyl-tRNA synthetase has a high specificity for “its” tRNA and amino acid. This
specificity is required for the fidelity of the genetic code.
98
Antibiotics 4.4.5
4.4.3. Ribosomes
Ribosomes consist of rRNA + protein. Subunits: 30 S + 50 S = 70 S in bacteria, 40 S + 60 S

= 80 S in eukaryotes. The 16 S rRNA (bacteria) or 18 S rRNA (eukaryotes) is in the small
subunit, the others in the large subunit. Ribosomes self-assemble from rRNA + ribosomal
proteins. The subunits are separate in the resting state and form the complete ribosome only
when they synthesize proteins. Magnesium is required for ribosome assembly. The ribosome
has 2 binding sites for tRNA: the P site (P = peptidyl) contains the peptidyl-tRNA during
the elongation phase, the A site (A = aminoacyl, or acceptor) accepts the newly incoming
aminoacyl-tRNA.
4.4.4. Steps in translation
1. The initiation complex is formed when the 30 S subunit binds to the 5’-terminal part
of the mRNA and to the initiator-tRNA that carries formyl-methionine (bacteria)
or methionine (eukaryotes). In bacteria (but not eukaryotes), the Shine-Delgarno
Sequence on the mRNA has to base-pair with a complementary sequence in the 16 S
ribosomal RNA. Then the 50 S subunit is added. The formation of the initiation
complex requires soluble cytoplasmic proteins called initiation factors, and one GTP
is hydrolyzed to GDP + phosphate.
2. The fMet-tRNA is in the P-site, base-paired with the start codon AUG. Elongation
starts when an aminoacyl-tRNA is placed into the A site by elongation factor EF-Tu.
This requires GTP hydrolysis.
3. Next the peptide bond formed by the peptidyl transferase activity of the large ribo-
somal subunit.
4. Translocation brings the newly formed peptidyl-tRNA from the A site to the P-site.
The mRNA moves 3 nucleotides along the ribosome. This requires GTP hydrolysis.
5. Termination of translation occurs when a stop codon is encountered in the mRNA

sequence. The polypeptide is hydrolyzed from the tRNA after the binding of protein
releasing factors to the stop colon.
4.4.5. Antibiotics
Many (not all) antibiotics inhibit protein synthesis:
Rifampicin An inhibitor of bacterial RNA polymerase
Actinomycin D Inhibits transcription by DNA-base intercalation.
99
Streptomycin Inhibits binding of fMet-tRNA and causes misreading of mRNA in prokary-

otes.
Tetracycline Inhibits the binding or aminoacyl-tRNA in prokaryotes.
Chloramphenicol Inhibits the peptidyl transferase of prokaryotes.
Cycloheximide Same as chloramphenicol, but in eukaryotes.
Erythromycin Inhibits translocation in prokaryotes.
Puromycin A structural analog of aminoacyl-tRNA. Causes premature polypeptide chain

termination.
Clinical note: Most bacteria can survive for considerable periods of time without protein
synthesis. Therefore most drugs inhibiting bacterial protein synthesis are only bacteriosta-
tic, not bactericidal.
4.5. Regulation of Gene Expression
Gene expression is regulated, most often at the level of transcription and sometimes by post-
transcriptional mechanisms. Proteins that are synthesized sometimes and sometimes not are
called inducible. Proteins that are synthesized at all times are called constitutive.
Examples in E. coli of gene regulation:
The lac operon contains genes for the catabolism of the disaccharide lactose. Lactose
is transported into the cell by the carrier lactose permease and then cleaved into glucose
and galactose by the enzyme β-galactosidase. These proteins, together with the enzyme
thiogalactoside transacetylase, are produced in the presence of lactose but not its absence.
The genes for these 3 proteins are clustered in the lac operon. They produce a polycistronic
message which is translated into separate polypeptides. Organization of the lac operon:
100
Regulation of Gene Expression 4.5
P I P O A B C
Repressor mRNA
repressor protein binds to operator
and blocks transcription of A, B, C genes
Protein
With inducer: Presence of inducer prevents

repressor binding to operator
A, B, and C are the genes for β-galactosidase, lactose permease and the transacetylase.
These are the structural genes that are expressed as polycistronic unit. P is the promoter,
where transcription begins. O is the operator. The operator is a binding site for a repressor
protein, next to (and overlapping) the promoter. The binding of the repressor prevents
transcription. I is the gene for the repressor. It is expressed constitutively. The repressor
binds to the operator in the absence of lactose. In the presence of lactose the repressor binds
allo-lactose (formed from lactose). Allo-lactose binding causes a conformational change in
the repressor which prevents its binding to the operator, thus allowing RNA polymerase to
transcribe the structural genes. The operon consists of a promoter, operator and structural
genes. The I gene may be next to the operon (as in the lac operon), but this is not always
the case.
Other Catabolic Operons are also induced by nutrients, usually by the removal of a
repressor protein from the operator.
The Tryptophan Operon contains 5 structural genes which encode enzymes for trypto-
phan biosynthesis. These genes are expressed in the absence but not the presence of external
tryptophan. In this case the repressor protein itself (“apo-repressor”) does not bind to the
operator, but the binding of tryptophan to the repressor induces a conformational change
which causes the repressor to bind to the operator: Tryptophan acts as a corepressor. Tryp-
tophan makes feedback inhibition of its own synthesis.
Catabolite Repression is a general regulatory mechanism for carbon metabolism in E.

coli. Because glucose is the favored tasty treat of bacteria (it enters glycolysis directly),
many catabolic operons in E. coli are transcribed only in the absence of glucose. The level
of cyclic AMP (cAMP) is low when glucose is present and high when glucose is absent.
cAMP binds to CAP (Catabolite Gene Activator Protein). The CAP-cAMP complex binds
101
to the promoter region of many catabolic operons and stimulates RNA polymerase binding
and transcription.
4.6. Virus
4.6.1. Virus Structure
Virus are obligatory intracellular parasites: they can replicate only by infecting a host cell.
Outside the cell, the virus exists as an inert particle called a virion. The virion consists
of:
Nucleic acid: DNA or RNA, single-stranded or double-stranded, but only one kind is
present. Virus have anywhere between 3 and 250 genes.
The capsid is a protein capsule that surrounds the nucleic acid. It is formed from a few
structural proteins which polymerize into a symmetrical shape. Nucleic acid + capsid
are called nucleocapsid.
An envelope is present only in some animal virus, not in bacteriophages (=bacteria-infecting
virus). It is a piece of membrane acquired from the host cell that contains viral pro-
teins called spike proteins.
4.6.2. The Lytic Cycle of Bacteriophage T4
Lytic infection by the DNA bacteriophage T4 and related phages proceeds in the following
sequence:
Adsorption: The tips of the tail fibers attach to a surface component of E. coli which
serves as a “phage receptor”. This determines the host range: bacteria without the
phage receptor cannot be infected.
Penetration: the tail sheath contracts, the phage DNA is injected into bacterium. The
capsid remains outside.
Synthesis of phage proteins: The phage DNA serves as a template for mRNA synthesis,
and viral proteins are synthesized on bacterial ribosomes. Phage proteins produced
include: - A DNase which degrades the host genome. Viral DNA is protected be-
cause it contains hydroxymethylcytosine instead of ordinary cytosine - Enzymes
for nucleotide biosynthesis - DNA polymerase, DNA ligase - Viral capsid proteins -
Lysozyme and phospholipase which destroy the bacterial cell envelope.
Transcription of viral genes occurs in a specific sequence. Immediate-early genes
are transcribed first, followed by delayed-early and late genes. RNA polymerase of
102
Animal Virus 4.6.5
the host is used for immediate-early transcription and later is modified chemically by
viral enzymes. This modified polymerase is specific for late transcripts.
Virion assembly: DNA and capsid proteins self-assemble into virus particles.
Lysis: The host cell is destroyed (lysed) after about 25 minutes. About 200 virions are
released.
4.6.3. The Lysogenic Cycle of λ phage
λ-phage has double-stranded DNA with mutually complementary single-stranded ends (co-
hesive ends, or “sticky ends”). After viral DNA had entered the host cell, the sticky ends
anneal and are joined by the bacterial DNA ligase. At this time the λ phage may proceed
along the lytic pathway or follow the lysogenic pathway. In the latter case, it inserts it-
self into the bacterial chromosome with the help of a virally-encoded integrase enzyme,
always between the gal and bio operons. The inserted viral genome is called lysogenic. In
the λ-prophage only the gene for the λ-repressor protein is transcribed and translated.
The λ-repressor protein prevents the transcription of the other prophage genes. When the
cellular level of this repressor protein drops too low, the prophage excises itself from the
bacterial chromosome to enter the lytic pathway. This usually happens when the bacterium
is exposed to damaging environmental influences such as radiation or heat-stress.
4.6.4. Animal Virus
Some typical differences between animal virus and bacteriophages:
• Some animal virus replicate in the nucleus and others in the cytoplasm.
• After adsorption to the cell surface the complete virus particle enters the cell, often
by endocytosis.
• Instead of lysing the cell, animal virus are most commonly shed by budding from the
plasma membrane, not all virions are released at the same time, and the cell may
recover.
• Some virus acquire an envelope while budding out of the nucleus or through the
plasma membrane.
• The immune system can fight viral infections either by forming antibodies to capsid
proteins or spike proteins (followed by phagocytosis of the antibody-coated virus), or
by destroying the virus-infected cells which display viral proteins on their surface.
103
4.6.5. RNA Virus
RNA virus can be double-stranded, (+) single-stranded or (-) single-stranded. (+) ss RNA
can serve as mRNA while (-) ss RNA is complementary to the mRNA. All RNA virus require
a virally-encoded RNA-dependent RNA polymerase (“RNA replicase”). (-) ss RNA virus
have to carry at least one copy of this enzyme in the virus particle. Viral RNA replicases
have no proofreading ability, therefore the mutation rates are very high.
4.6.6. Retrovirus
Retrovirus are enveloped virus containing 2 copies of their (+) -stranded RNA genome in
the virus particle together with the viral enzyme reverse transcriptase.
Retroviral Life Cycle
1. The nucleocapsid enters the host cell by simple fusion of the viral envelope with the
host cell membrane.
2. The reverse transcriptase copies the viral RNA into a double-stranded DNA (a cDNA)
which becomes inserted into the host cell genome with the help of a virally-encoded
integrase. The inserted viral cDNA contains the viral genes flanked by long terminal
repeats which contain the viral promoter.
3. After its insertion into the host cell DNA, the viral DNA directs the synthesis of viral
RNA and proteins. The viral RNA is used both for protein synthesis and as the new
genomic DNA.
4. New virus particles assemble from viral RNA + protein. These bud out of the cell
continuously.
Like the RNA replicases, the retroviral reverse transcriptases have a high error rate. Most
retrovirus (the AIDS virus is an exception) cannot infect non-dividing cells because they
cannot get across the nuclear envelope.
4.6.7. Plasmids
Plasmids are semi-independent genetic entities in bacteria, consisting of circular double-

stranded DNA carrying a few genes. The genes are not essential for bacterial survival un-
der ordinary conditions but confer special abilities: antibiotic resistance, toxin production,
ability to metabolize usual substrates, etc. They also have a replication origin and genes
104
Objectives in Summary 4.9
regulating their own replication. Most important: R-factors are plasmids that make the bac-
terium resistant to antibiotics. The plasmid-encoded enzyme β-lactamase (“penicillinase”)
destroys penicillin.
4.7. Genetic Recombination
4.8. Types of Recombination
The joining of different DNA molecules is called genetic recombination. There are 2 types:
Site-specific recombination requires a specific integrase enzyme which recognizes sequences
on one or both of the DNA molecules to be joined. The integration of λ-phage and of
the retroviral cDNA are examples.
General recombination (=homologous recombination) requires no specific sequences, but
sequence identity or similarity between the recombining DNAs: transformation in bac-
teria, crossing-over during meiosis.
4.8.1. Parasexual Processes in Bacteria
Bacteria don’t make “real” sex, but they can exchange genetic information through para-
sexual processes:
Transformation is the uptake of foreign DNA. This is random in some species, but other
species take up selectively DNA of their own species. The DNA integrates into the
chromosome by homologous recombination.
Transduction is the transfer of cellular DNA by a bacteriophage.
Conjugation is the transfer of DNA by a self-transmissible plasmid. The F-factor of E.
coli causes the formation of sex pili which can form a cytoplasmic bridge between the
F-factor carrying cell (F+ -cell) and a cell without F-factor (F− -cell). The F factor
sends a copy of itself into the F− -cell. Also some R-factors are self-transmissible.
4.9. Objectives in Summary
• Describe the covalent structures of DNA and RNA, including their constituent monomers,
bond types, functional groups and ionization state.
• Describe the structure of the Watson-Crick double helix, with approximate helix
parameters, relevant non-covalent interactions, and the rules of base pairing.
105
• Define the process of melting of DNA.
• Define the different types of nucleases.
• Name the different types of DNA polymerases of pro- and eukaryotes and identify
their functionally important properties.
• Define the semiconservative model of DNA replication.
• Identify the proteins involved in prokaryotic DNA replication, and state the sequence
of their actions.
• Know the functionally important characteristics of prokaryotic and eukaryotic RNA

polymerases.
• Define the terms “template strand” and “coding strand”.
• List the important properties of the genetic code.
• Describe the post-transcriptional processing of tRNA, rRNA, and mRNA in prokary-

otes and eukaryotes and the general structure of tRNA and the formation of aminoacyl-
tRNA.
• Identify the steps in ribosomal protein synthesis, their energy requirements and re-
quirements for soluble protein factors.
• Locate the sites of action for some common antibiotics on transcription or translation.
• Describe the operon model for the regulation of gene expression in prokaryotes.
• Define the principles of positive and negative control of transcription by DNA binding
proteins.
• Describe the principle events in the life cycles of DNA virus, RNA virus and retrovirus
and identify the required viral enzymes.
• Describe the properties of histones and their interaction with DNA in nucleosome
structure.
• Know the approximate proportions of protein-coding, non-coding, unique, moderately

repetitive and simple-sequence DNA in the human genome.
• Define the terms “promoter” and “enhancer”.
• Outline the importance of histones, chromatin structure, DNA methylation and sequence-
specific DNA-binding proteins for the regulation of eukaryotic gene expression.
• List the types of repetitive elements in the human genome, and describe the mecha-
nism for the mobility of Alu and LINE-1 sequences.
106
• Define the terms solitary gene, duplicated gene, gene family, pseudo-gene and processed
pseudo-gene.
• Describe the importance of telomere erosion and of telomerase for the mortality of
somatic cells.
• List the mechanisms by which transcription factors can be regulated.
• List the principal types of mutations, and predict their likely effects on the function-
ality of the encoded protein.
• Describe the causes of mutations, including replication errors, radiation and the major
types of chemical mutagens.
• Define the nature and functions of the major DNA repair systems, including the
3’-exonuclease activities of replication complexes, methyl-directed mismatch repair,
direct repair, base excision repair and nucleotide excision repair.
• Describe the major DNA repair defects, including chromosome breakage syndrome,
xeroderma pigmentosum, and Cockayne syndrome.
• State the importance of mutations for genetic diseases and carcinogenesis.
• Describe the diploid nature of the human genome and its implications for the expres-
sion of genetic disorders.
• Know the terms “homozygous”, “heterozygous”, “dominant” and “recessive”.
• Describe the molecular defect of sickle cell disease, its clinical expression, and the
relationship between the two.
• Define the terms “α-thalassemia”, “β-thalassemia”, thalassemia major and thalassemia
minor.
• Describe the clinical presentation in different types of thalassemia.
• State the reason for the beneficial effect of increased HbF-expression in patients
with sickle cell disease and β-thalassemia, and the beneficial effect of concurrent
α-thalassemia minor on the clinical source of sickle cell disease.
• List the treatment options for the major hemoglobinopathies.
107
5. The Human Genome and Mutations
5.1. The Human Genome
The human genome is the complete sequence of DNA bases in the human organism. The
finalized drafts of the complete human genome were published in 2001. The human genome
was completed after sequencing smaller genomes of model organisms. The entire genome se-
quences of about 1000 different virus and 100 microbes can be found in the Entrez Genome
Browser. The genomes represent both completed genomes and those for which sequenc-
ing is still in progress. The three domains of life - bacteria, archaea, and eukaryota - are
represented, as well as many virus and organelles.
Compared with model organisms the human genome is very large:
Organism Genome size (Mb) common name
E. coli 4.6 intestinal bacterium
S. cerevisiae 12.1 bakers yeast
A. thaliana 100 mouse-ear cress
D. melanogaster 140 fruit fly
M. musculus 3300 mouse
H. sapiens 3000 human
T. aestivum 17000 wheat
The human sequence and its variation is also vital in its importance in medicine. It provides
the basis for understanding human disease inheritance and susceptibility.
The genome is organized in chromosomes of differing size. Human diploid somatic cells
have 22 pairs of autosomes. In addition, females have two X chromosomes, males one X
and one Y. Mitochondria also contain DNA which makes up a small fraction of the entire
human genome.
The unit of DNA which encodes polypeptides is called the gene. In the human genome,
there are approximately 30 000 genes. As for most eukaryotes, human genes are not tightly
arranged with their nearest neighbors. Very long stretches of DNA occur between genes
and also within genes themselves. DNA in humans can be categorized as follows:
Unique DNA sequences Protein Coding Regions of Genes (Exons) 1.5 %
Non-coding Regions (Introns) 25.0 %
109
Other UNIQUE DNA sequences 12.0 %
Repetitive DNA sequences All repetitive DNA sequenced to date 53.0 %
Not sequenced 8.0 %
5.2. Chromatin Structure
5.2.1. Histones and Nucleosomes
Chromatin is about 50 % DNA and 50 % histones. Histones are small, basic proteins (lots
of Lys and Arg!) that come in 5 varieties: histones H1, H2A, H2B, H3 and H4. Histones are
well-conserved in all eukaryotes. Nucleosomes are formed from a core of histones (2 copies
each of histones H2A, H2B, H3 and H4), with about 140 bp of DNA wound around. About
60 bp are in the linker DNA between the nucleosomes. H1 sits on the linker DNA. Higher-
order structure: the nucleosomes coil up into a solenoid (diameter 30 nm). In the condensed
chromosomes, the 30 nm fibers are attached to scaffold proteins. The DNA in condensed
chromosomes consist of DNA:histones:non-histone proteins in 1:1:1 ratio. Euchromatin is
dispersed chromatin in which the genes can be transcribed. Heterochromatin is condensed
chromatin that cannot be transcribed. The mitochondrial DNA has no histones.
5.2.2. Repetitive DNA
There is about 3 × 109 bp of DNA in the human genome, with about 30 000 genes. Only
1.2 % of the DNA codes for proteins. The rest is junk DNA between the genes and in the
introns of the genes. Introns are sequences within the genes that are transcribed but not
translated. Some of the non-coding DNA is repetitive.
Tandemly repeated sequences are most abundant in the centromeric and telomeric
regions (“satellite DNA”). Simple-sequence DNA consists of short, tandemly repeated
sequences of between 2 and few dozen nucleotides in the repeat unit. Minisatellites and
microsatellites are tandem repeats outside the centromeres and telomerase.
Interspersed elements are sequences that occur, with variations, in different locations in
the genome. Alu sequences are about 300 bp long and are present in about 500 000 copies
in the genome (6–8 % of the total genome), with about 80 % sequence homology between
different copies. LINE-1 sequences are 6000 bp long. The complete sequence is present in
only a few thousand copies, but truncated LINE-1 sequences are very common.
110
Genes 5.2.4
Figure 5.1.: When hybridized with it’s mRNA, the DNA of adenovirus 2 forms loops. These
correspond to introns. Image from [Berget et al., 1977].
5.2.3. Mobile DNA
Alu sequences and LINE-1 sequences can shuffle their location. These sequences end in an
oligo-A tract, and they are framed by short direct repeats. They can jump to new locations
by reverse transcription:
1. The element is transcribed into an RNA by RNA polymerase III. This RNA becomes
polyadenylated, like an mRNA .
2. Reverse transcriptase makes a cDNA from this RNA. The complete LINE-1 elements
contain a gene for a reverse transcriptase.
3. The cDNA is inserted into a new genomic location.
These mobile elements are considered selfish DNA. They can cause mutations when they
jump into a gene. Another name for mobile element is transposon or less commonly
retroposon.
5.2.4. Genes
Genes occur mostly in one copy per haploid genome. Duplicated genes are present in 2 or
more copies which are close together on the chromosome. Gene families with similar, but
nonidentical functional genes are common, usually with closely-related members clustered
in the same chromosomal region. Example: globin genes. Pseudo-genes are nonfunctional
copies of functional genes. Usually they are close to their functional counterpart. Duplicated
genes, gene families and pseudo-genes arise by gene duplication, usually from crossing-over
(homologous recombination) between misaligned chromosomes during prophase of meiosis
I. Individual exons can duplicate (or be deleted) by the same mechanism.
111
Processed pseudo-genes are nonfunctional sequences that duplicate the exon sequences of
a functional gene. They are non-viral retroposons that are derived by the reverse tran-
scription of a cellular mRNA (or sometimes a partially processed hnRNA) and the insertion
of the resulting cDNA into the genome e.g., reverse transcribed by the Line-1 reverse tran-
scriptase. They are not necessarily close to their functional counterpart. Viral retroposons
are the remnants of retroviral genomes that have mutated into non-functionality.
5.2.5. Telomeres
Telomeres are the end pieces of the chromosomes. They consist of the repeat sequence
TTAGGG, which is repeated hundreds of times to a total length of several thousand bp.
They tend to shorten during repeated rounds of DNA replication because lagging strand
replication cannot go all the way to the end. Excessive shortening is prevented by the
enzyme telomerase, which adds to the repeat sequence by synthesizing new DNA on an
internal RNA template. Telomerase is active in the germline and in embryonic tissues, but
not in most adult tissues. Therefore the telomeres tend to shorten during a lifetime. This is
important for the mortality of cells, both in the body and in cell culture. Cancer cells have
telomerase, and they are immortal.
5.2.6. DNA Replication
There are many origins of replication in human DNA, about one every 100 000 bp. There
are several DNA polymerases:
Polymerase α and ζ replicate the lagging and the leading strand, respectively. Polymerase
β and are involved in DNA repair and/or recombination. Polymerase γ is in the mito-
chondria.
5.3. Mutations
Mutations are heritable changes in DNA structure. They arise as errors during DNA repli-
cation or are caused by DNA damage. The basal mutation rate is the mutation rate in the
absence of external mutagens. Mutations in the germline lead to genetic diseases. Muta-
tions in somatic cells cause cell dysfunction and, sometimes, cancer. All mutagens are also
carcinogens.
112
Causes of Mutations 5.3.2
5.3.1. Types of Mutation
Base substitutions are the most common type.
Transition A purine replaces another purine or a pyrimidine replaces another pyrim-

idine.
Transversion A purine replaces a pyrimidine or a pyrimidine replaces a purine.
Deletions One or several nucleotides, or a large piece with up to millions of nucleotides, is

lost.
Insertions One or several nucleotides, or a big chunk of DNA, is added.
Translocation A piece of DNA is transferred to another location in the genome.
Other Definitions
Point mutations are single-base substitutions. Silent mutations are base substitutions
that do not change the amino acid sequence because of the degeneracy of the genetic
code. Nonsense mutations change a codon for an amino acid to a stop-codon (UAA,
UAG, UGA) resulting in inappropriate termination of chain elongation during translation.
Missense mutations change a codon for an amino acid to a codon for a different amino
acid. Frameshift mutations are small insertions or deletions that change the reading
frame of the mRNA Splice-site mutations result in abnormal splicing. These lead to
anything from changes in protein length to frameshifts, which again often lead to truncated
proteins.
5.3.2. Causes of Mutations
1. The basal mutation rate is caused by spontaneous tautomeric shifts and hydrolytic re-
actions. Tautomeric shifts occur with thymine (keto ↔ enol shift) and adenine (amino
↔ imino shift). Spontaneous depurination is the most important type of hydrolytic
reaction.
2. UV radiation is a component of sunlight. It causes the formation of pyrimidine dimers

and other photoproducts. UV does not penetrate the skin, but it causes sunburn and
skin cancer.
3. Ionizing radiation (X-rays, radioactive radiation) is more energy-rich than UV ra-

diation. It causes many kinds of DNA damage, but especially double-strand beaks.
Ionizing radiation can penetrate the whole body.
113
4. Base analogs are “false” bases that are incorporated into DNA. Example: 5-bromouracil
can replace thymine.
5. Deaminating agents deaminate adenine to hypoxanthine and cytosine to uracil. Ex-
ample: nitrous acid.
6. Deamination of methylated cytosine leads to thymidine, which is not recognized as
an unnatural base in DNA, and therefore the repair of T::G mismatches often is
erroneous. This is the most common type of point mutation in the genome.
7. Alkylating agents alkylate N and O atoms in the bases. Examples: methyl bromide
and ethylene oxide.
8. Intercalating agents are flat, hydrophobic molecules that insert themselves between
stacked bases. Examples: acridine dyes and benzene. Ethidium bromide used to visu-
alize DNA in electrophoresis is another intercalating agent.
9. Virus may insert their own DNA into the host cell chromosome, either habitually
(retrovirus) or by accident (DNA virus).
Mutagens are most effective when they hit the cell immediately before or during DNA
replication (during S phase of the cell cycle), because there is no time for repair. Cancer
cells are more easily killed by radiation than normal cells because they divide more rapidly
and spend more time in S phase.
5.3.3. Mutagenesis Testing
The Ames test uses bacteria that are dependent on a particular nutrient as a result of a mu-
tation (auxotrophic bacteria). After exposure to the chemical, the bacteria are screened for
back-mutations which restore their ability to grow in the absence of the nutrient. Mutagens
increase the rate of back-mutations.
5.4. DNA Repair
All cellular organisms have repair systems for many different kinds of DNA damage. DNA
repair is possible if only one strand is damaged, therefore the undamaged second strand
can supply the sequence information for the repair enzymes.
Nucleotide Excision Repair Nucleotide excision repair removes bulky lesions including thymine
dimers, alkylations and bulky adducts. The mechanism of excision repair:
• A repair crew consisting of several specialized proteins scans the DNA.
• The repair complex binds to a bulky lesion.
114
Repair Defects 5.4.1
• Endonucleases make 2 incisions in the damaged strand, one 5’ and the other
3’ of the lesion.
• Helicases in the complex separate the two strands.
• The damaged DNA piece is removed.
• The resulting gap is filled by DNA polymerase.
• The last phosphodiester bond is formed by DNA ligase.
• Nucleotide excision repair reaches all parts of the genome, but there is a subsys-
tem for the preferential repair of transcribed genes.
Other Repair Systems Depurination (loss of a purine base by spontaneous hydrolysis) is

repaired by an AP endonuclease (AP = apurinic), followed by DNA poly-
merase and DNA ligase.
Deaminated bases are removed by base excision repair: hypoxanthine (formed from
adenine) and uracil (from cytosine) are clipped off by cleavage of the N-glycosidic
bond with D-ribose. The next steps are as in the repair of apurinic sites.
Post-replication mismatch repair corrects base mismatches and small insertions and
deletions after DNA replication. The repair enzymes can distinguish between the
strands because the old strand is methylated on specific sequences (GATC in E.
coli, CMG in eukaryotes). The repair system cuts the unmethylated new strand
either 5’ or 3’ of the mismatch. The damaged piece is removed by exonucleases
and replaced by DNA polymerase followed by DNA ligase.
5.4.1. Repair Defects
Xeroderma pigmentosum is a recessively inherited skin disease with premalignant and

malignant lesions on sun-exposed skin. There are 9 different types (“complementation
groups”), caused by deficiencies of different repair proteins.
Cockayne syndrome is also caused by defective nucleotide excision repair, but only
the preferential repair of transcribed genes is affected. No skin cancer, but poor growth,
neurological problems, and early senility.
Hereditary non-polyposis colon cancer (HNPCC) is an inherited cancer susceptibility

syndrome caused by defects in post-replication mismatch repair.
115
Chromosome breakage syndromes include Bloom syndrome, ataxia-telangiectasia,

and Fanconi anemia. These are multi-system disorders with an increased incidence of
chromosome breakage.
5.5. Eukaryotic Gene Expression
5.5.1. Transcription
There are 3 nuclear RNA polymerases in eukaryotes:
RNA polymerase I makes rRNA (in the nucleolus).
RNA polymerase II makes mRNA .
RNA polymerase III makes small RNAs: tRNA, 5 S rRNA.
α-amanitin is a mushroom poison (from Amanita phalloides, the “angel of death”-mushroom)

that inhibits RNA polymerase II.
5.5.2. mRNA Processing
Eukaryotic mRNA is processed in the nucleus before it is sent into the cytoplasm for trans-
lation. Steps:
1. Capping is the addition of a methylguanosine residue to the 5’ end of the mRNA (Fig-
ure 8.11 in the Meisenberg book). It occurs co-transcriptionally. The cap protects
the 5’ terminus from the action of nucleases.
2. Polyadenylation is the addition of a poly-A tail to the 3’ end of the transcript. Tran-
scription proceeds beyond the polyadenylation signal (consensus: AAUAAA). This is
followed by a nuclease cleavage and the enzymatic addition of the adenine nucleotides
(no template required!).
3. Splicing is the removal of intron sequences from the primary transcript. It requires
spliceosomes which consist of small nuclear ribonucleoproteins (snurps). The intron-
exon junctions have consensus sequences that are recognized by the spliceosome.
116
Regulation of Eukaryotic Gene Expression 5.6
5.5.3. Translation
The important steps are the same as in prokaryotes, but:
1. Eukaryotic proteins start with methionine, not formylmethionine.
2. There is no Shine-Delgarno sequence, but translational initiation depends on in-

teractions of mRNA with ribosomal proteins.
3. Eukaryotic mRNAs are not polycistronic, i.e., they normally encode only one protein.
This is normally positioned as the 5’ open reading frame in the mRNA .
Eukaryotic translation is inhibited by diphtheria toxin. This toxin modifies an elongation

factor covalently, thereby preventing translocation.
5.6. Regulation of Eukaryotic Gene Expression
1. Global Effects:
• Histones inhibit transcription non-selectively by tightly binding the DNA double-

helix. Histones are modified by acetylation, phosphorylation and methylation at
specific sites.
• DNA methylation inhibits transcription. Cytosine bases in the palindrome CG

are methylated on both strands, and methylation patterns are maintained during
DNA replication. Specific proteins interact with the methylated regions. Not all
CG palindromes are methylated: This is regulated.
2. Regulatory DNA Sequences:
• Promoter for RNA polymerase II extend to about -200 bp from the transcrip-
tional start site. Highly variable sequence, but most contain a TATA box 25–
30 bp upstream of the start site. Most promoter elements have to be on the
correct strand and in the correct 5’ → 3’ orientation.
• Enhancers can be present up to some thousand bp upstream or downstream of

the transcriptional start site. Enhancers increase the rate of transcription. They
need not be in correct 5’ → 3’ orientation to be effective.
• Silencers are like enhancers, but they reduce rather than enhance the rate of
transcription.
117
• Promoters, enhancers and silencers contain binding sites for regulatory proteins.
The individual binding sites are often called response elements. Note that in
eukaryotes the inhibitory effects of histones and DNA methylation have to be
overcome by sequence-specific DNA binding proteins that bind to promoter and
enhancer sequences.
3. Transcription Factors
• The regulatory proteins that bind to promoters and enhancers are called tran-
scription factors. General transcription factors are promoter-binding proteins
that are required for the transcription of all genes by a particular RNA poly-
merase. They are the functional equivalents of the bacterial σ-subunit. Others
affect the transcription of some genes but not others.
• Many transcriptional regulators bind DNA in a dimeric form, either as homod-
imers or as heterodimers of 2 slightly different polypeptides. Recognition occurs
in the major groove where the ’side-on’ steric features of base-sequences can be
recognized by proteins. Types:
Zinc-finger proteins (see fig 4.1 on page 93) contain between 2 and about a
dozen zinc fingers in their DNA-binding region: zinc complexed between 4
Cys or 2 Cys and 2 His residues, with an intervening loop.
Leucine-zipper proteins contain a DNA-binding basic domain, a dimerization
domain with leucine residues spaced 7 amino acids apart in an amphipatic
α-helix, and a transcriptional activator (or repressor) domain.
Helix-loop-helix proteins have a dimerization domain with 2 amphipatic α-
helices separated by a loop.
Helix-turn-helix proteins have 2 α-helices separated by a β-turn. One of the
α-helices fits into the major groove of the DNA.
• Regulation of Transcription Factors
a) The synthesis of transcription factors is controlled often in a cell type-specific
manner: most transcription factors (except the “general” transcription fac-
tors) are present only in certain cell types and at certain stages of cell dif-
ferentiation.
b) They can be controlled by the reversible binding of small molecules. Exam-
ple: receptors for steroid and thyroid hormones.
c) They are controlled by interactions with other proteins.
d) They are controlled by phosphorylation and dephosphorylation, frequently
in response to growth factors or the second messengers of hormones.
118
4. Post-transcriptional Controls
a) The use of alternative promoters or alternative polyadenylation signals can create
mRNAs which differ at the 5’ end or the 3’ end, respectively.
b) Alternative splicing of a single mRNA transcript can produce different polypep-
tides from the same gene.
c) RNA editing is the post-transcriptional chemical modification of a base in the
mRNA . The example, an amino acid coding codon can be modified into a stop
codon to produce a shorter protein.
d) mRNA stability varies in different cell types and under different conditions.
mRNA stability can be controlled by specific mRNA sequence binding-proteins
that impair the action of nucleases.
5. Translational Controls
a) Start-site variation occurs when alternative start codons can be used by the
ribosome. The resulting polypeptides differ at their N-terminus.
b) Initiation factor control works through the phosphorylation of the initiation fac-
tor eIF-2 which slows all translation. This occurs during the M phase of the cell
cycle, and when growth factors are not present.
c) Cap-binding proteins can inhibit translation by binding to the 5’ end of the
mRNA .
1. Know the proportion and characteristics of DNA sequence elements in the human
genome.
2. Describe the properties of histones and their interaction with DNA in nucleosome
structure.
3. Define the terms promoter, enhancer, and silencer.
4. Explain how histones, chromatin, DNA methylation, and transcription factors can
influence the rate of eukaryotic transcription.
5. Name and describe the principal types of mutations, and explain their effects on the
structure and function of proteins.
6. Describe the causes of mutations.
7. Name and describe the systems for repair of DNA lesions.
119
8. List inherited human diseases which result from faulty DNA repair systems.
9. Name the different types of eukaryotic DNA polymerases, and describe replication.
10. Know the different types of eukaryotic RNA polymerases, and accessory factors.
11. Describe the processes of post-transcriptional processing of tRNA, rRNA and mRNA
in eukaryotes.
120
6. Chromosome Aberrations
6.1. The Human Karyotype
Chromosomes become visible only during mitosis and meiosis. They are classified as:
Metacentric the centromere is in the middle.
Submetacentric intermediate between metacentric and acrocentric.
Acrocentric centromere near the end, forming a stubby short arm and a long arm. Only
the long arm has genes.
Telocentric the centromere is at the end; only one arm is present. Does not occur in
humans.
The short arm of a chromosome is designated by the letter p (petite, French for small, the
long arm by q. The acrocentric chromosomes have small satellites attached to their short
arms, which contain clusters of genes for ribosomal RNA; losing one or two of these clusters
will not affect the cell in any adverse way.
Diploid somatic cells have 22 pairs of autosomes. In addition, females have two X chro-
mosomes, males one X and one Y. The karyotype (= chromosome constitution) can be
examined during mitotic metaphase. Techniques:
• Bone marrow biopsy (no culture required)
• Leukocyte culture
• Fibroblast culture from the skin
• Amniotic cells, cultured like skin fibroblasts
Leukocyte culture is most important. Phytohemagglutinin (a lectin from beans) is used as
a mitogen, and colchicine is used to arrest mitosis in metaphase.
Human chromosomes can be grouped into:
Group A (1-3) large, metacentric to submetacentric.
Group B (4-5) submetacentric, smaller than group A.
Group C (6-12) submetacentric, smaller than group B.
121
Group D (13-15) acrocentric
Group E (19-18) submetacentric
Group F (19-20) small metacentric
Group G (21-22) small acrocentric
X chromosome like a C-group chromosome
Y chromosome small submetacentric
Banding techniques are staining procedures that distinguish between different types of
chromatin within the chromosomes: Q (quinacrine mustard) banding, G (Giemsa) banding,
and R (reverse) banding. They can distinguish different chromosomes within each group,
and they can detect structural abnormalities. Karyotype with banding costs about twice as
much as a regular karyotype. High-resolution banding with prophase chromosomes is not
often done in clinical routine, but is an important research technique.
Chromosome painting makes use of fluorescent-labeled probes that bind to one of the
chromosomes (as in FISH = fluorescent in-situ hybridization; FISH is discussed in the
section on recombinant DNA methods). Different-colored probes are used to distinguish
between chromosomes. Fluorescent probes can even be used to detect aneuploidy in the in-
terphase nucleus. This method tends to supplement or even replace classical karyotyping.
Heteromorphisms are variations in the karyotype that are in most cases not associated with
disease:
• Length variations of the long arm of the Y chromosome (Yq) are present in 10 % of
all males. Yq is mostly junk DNA, and most of these variants are innocuous.
• The satellites of the acrocentric chromosomes are variable. Also these heteromor-
phisms are generally asymptomatic.
• There are fragile sites on many chromosomes, which are visible as small constrictions.
Chromosome breakage at these sites can be induced by culturing in a folate-deficient
medium. Many fragile sites are variable, and most (but not all) of them are harmless.
• Centromeric heterochromatin, and also heterochromatic bands outside the centromeres,

can be variable.
Question: What should you do when you pick up a heteromorphism incidentally during
prenatal diagnosis?
122
Types Of Chromosome Aberrations 6.3
6.2. Sex Chromatin
In interphase nuclei, one of the two X chromosomes of a female is frequently visible as a

heterochromatic mass, the Barr body. Number of Barr bodies in diploid somatic cells
= number of X chromosomes minus 1: Normal females have one, normal males none. Sex
chromatin in buccal smear cells is an important screening test for sex chromosome aberra-
tions.
The Lyons hypothesis states that only one of the X chromosomes in a cell is in the dispersed,
genetically active form during interphase. Additional X chromosomes become heterochro-
matic and genetically inactive. This involves DNA methylation. Inactivation occurs about
15–16 days post-conception and is random. Inactivation can affect either the maternally-
derived or the paternally- derived X chromosome: normal females are mosaics of cell clones
in which either one or the other X chromosome is active. This is important for the ex-
pression of X-linked diseases in heterozygous females. The genes in the pseudo-autosomal
region on the short arm of the second X chromosome escape inactivation, and also some
other genes remain active. Therefore people with an abnormal number of X chromosomes
do have clinical abnormalities. Only the few genes in the pseudo-autosomal region have an
equivalent on the Y chromosome.
The chromatin of the Y chromosome can be demonstrated in the interphase nucleus by
Q-banding. This method can be used to distinguish X- and Y-bearing sperm cells. Most
of the long arm of the Y chromosome is heterochromatic and non-coding. The short arm
carries the male-determining SRY gene, which directs the development of the testis. It
codes for a transcription factor. There are also a few Y-linked genes that are required for
spermatogenesis, and mutations in these genes can lead to male infertility.
6.3. Types Of Chromosome Aberrations
Gross chromosomal aberrations are present in 1 out of every 160 live births and 35 % of
spontaneous abortions. More than half of all pre-implantation embryos are mosaics with
at least one abnormal cell line. These chromosome aberrations are a major reason why
even for young women the risk of pregnancy is never higher than 25 % to 39 % per month.
About 25 % of women with primary amenorrhea, 11 % of infertile males and 10 % of the
institutionalized mentally impaired have chromosomal abnormalities.
Numerical aberrations:
Aneuploidy: One chromosome is present in an abnormal copy number: trisomy, monosomy.
Polyploidy: The whole set is present in an abnormal number: triploidy (69 chromosomes
total), tetraploidy (92 chromosomes).
123
Consequences:
• Polyploidy results in fetal death.
• Monosomy for an autosome results in a nonviable embryo, trisomy is better tolerated.
• Trisomy of large autosomes causes death, only trisomies of some of the smaller auto-
somes are viable.
• Aneuploidy of sex chromosomes is well tolerated, but at least one X chromosome has
to be present.
• People with a Y chromosome are usually male, those without a Y chromosome are
usually female.
Aneuploidy results from non-disjunction during meiosis, or during mitosis in the germ
line. If it happened in a mitotic division of spermatogonia or oogonia, there is germline
mosaicism: presence of a whole tribe of abnormal cells in the gonad. This means there is
an increased recurrence risk after the birth of an affected child. Aberrations of chromosome
number generally are not inherited from an affected parent, but arise de novo.
Mosaicism is caused by non-disjunction during the first mitotic divisions after fertilization:
different somatic cells of the patient have different karyotypes.
Chromosomal rearrangements are aberrations of chromosome structure, such as large dele-
tions or translocations. They often arise de novo, but can also be inherited.
6.4. Autosomal Trisomies
Only 3 autosomal trisomies are observed in live-births:

Trisomy 21: Down syndrome About 1 in 800 births.
Trisomy 18: Edward syndrome About 1 in 8000 births
Trisomy 13: Patau syndrome. About 1 in 20 000 births.
6.4.1. Trisomy 21 (Down syndrome)
95 % of Down syndrome patients have trisomy 21, 3 % a Robertsonian translocation

(“translocation-type Down syndrome”), and 2 % are mosaics with trisomic and normal
cells. In 80 % of the cases non-disjunction occurred in the mother, usually in meiosis I.
About 60 % of trisomy 21 conceptuses abort spontaneously. Phenotype:
• Hypotonia (infants)
124
Trisomy 21 (Down syndrome) 6.4.2
• Short stature
• Short, stubby hands and feet
• Palpebral fissure, epicanthic fold
• Large, protruding tongue
• Small or malformed ears
• Flat occiput
• Short, broad neck
• Simian crease
• IQ typically between 25 and 50
• Infertility in males
There is an increased risk of:
Congenital heart defects (30–40 %): Endocardial cushion defect, atrial and ventricular
septal defects.
Childhood leukemia is 10–20 times more common than in euploid individuals. Both myel-
ogenous leukemia (infants) and lymphoblastic leukemia (older children) are increased.
Dementia in older patients. Life expectancy is reduced, but many patients live into their
50s and 60s. The neuropathologic changes of Alzheimer’s disease (plaques and tangles)
are present in patients dying at > 35 years. The β-amyloid precursor protein (APP)
gene encoding one of the components of Alzheimer plaques is located on chromosome
21.
Maternal Age frequency
Very young mother 1 in 1500
Mother 35 years 1 in 365
Paternal Age has only a very slight effect (if any, the effect is delayed 20 a relative to the
maternal age effect).
Prenatal diagnosis: Karyotype analysis after chorionic villus sampling or amniocentesis.

Also maternal serum α-fetoprotein (reduced in Down syndrome). The recurrence risk after
the birth of a trisomy 21 child to a young mother is about 1 % because of possible germline
mosaicism.
125
6.4.2. Trisomy 18 (Edward syndrome)
• 80 % of patients are female

• Birth-weight under 6 pounds
• Failure to thrive
• Hypertonicity, flexed fingers
• Prominent occiput
• Low-set, malformed ears
• Micrognathia
• Short sternum, small pelvis, rocker-bottom feet
• Cardiac and renal malformations
• 90 % die within a year
• Profound mental deficiency in survivors
6.4.3. Trisomy 13 (Patau syndrome)
• Apparent deafness
• Minor motor seizures
• Hypertonia/flexed fingers
• Sloping forehead, microcephaly
• Eye abnormalities
• Cleft lip, cleft palate
• Agenesis of the olfactory bulb, arhinencephaly, holoprosencephaly
• Cardiac/colon/uterine malformations
• Polydactyly
• Rocker-bottom feet
• 90 % die within a year
• Profound mental deficiency in survivors
126
XX Males 6.6.1
6.5. Sex Chromosome Aberrations: Male
6.5.1. Klinefelter syndrome
Incidence 1 in 1000 males, mild maternal age effect.
Chromosomes 80 % are 47, XXY. The others are mosaics (46, XY/XXY most common),
48, XXXY, or 49, XXXXY.
Phenotype (47, XXY): Small testes are the most consistent physical finding. Infertility.
Height is average or above average. Undervirilization, with poor growth of beard and
body hair, penis small or normal, 25 % develop gynecomastia. Testosterone is low,
estrogen variable, pituitary gonadotropins high. IQ is decreased by about 15 points.
More severe abnormalities (with mental deficiency) in 48,XXXY and 49, XXXXY.
Diagnosis Most patients present either with poor sexual development or infertility. Buccal
smear and/or karyotype is indicated in these situations. Klinefelter is the single most
important cause of severe male infertility. Treatment: Testosterone improves virility
but not fertility.
6.5.2. XYY constitution (“murderer chromosome”)
Incidence 1 in 1000 males. No parental age effect.
Phenotype No consistent abnormalities, except:
• Increased height
• IQ reduced by 10–15 points
• Increased risk of criminal conviction
XYY males are over represented in prisons (3 in 1000), institutions for the mentally defective
(3 in 1000) and institutions for mentally defective criminals (20 in 100), but most are never
diagnosed. Normal fertility; children are normal. Presence of the additional chromosome
does not constitute a legal defense.
6.5.3. XX Males
Rare (1 in 20 000). Usually caused by a small translocation of the testis-determining gene

to the X chromosome. Klinefelter-like phenotype.
127
6.6. Sex Chromosome Aberrations: Female
6.6.1. Turner’s syndrome (gonadal dysgenesis)
Incidence 1 in 5000
females Chromosomes 50–60 % are 45, XO. Others are mosaics (46, XX/45, XO; 47,
XXX/45, XO) or structural rearrangements. 99 % of XO fetuses abort spontaneously.
Gonads “Streak ovaries”, consisting of connective tissue without oogenesis. Degeneration of

the ovaries starts during fetal development.
At birth Peripheral lymphedema (first few days only, webbing of neck, 20 % have aortic
coarctation.
Later Short stature, final height is 140–1 50cm. Broad chest, widely-spaced nipples, cubitus
valgus, low posterior hairline.
At puberty There is no puberty.
Mental development is about normal.
Diagnosis Buccal smear, karyotype. Some are diagnosed before puberty because of slow
growth, others are diagnosed later during workup of primary amenorrhea. Treatment:
Estrogen after puberty, growth hormone in younger patients.
6.6.2. Triple-X (47, XXX, “superfemale”)
Incidence 1 in 1000 females, maternal age effect.
Phenotype No consistent abnormalities, but IQ is decreased 10–15 points, and some have
impaired fertility or menstrual problems. Their children are usually normal. 48, XXXXX
are rare, with mental deficiency and dysmorphic features.
6.6.3. XY females
Rare (1 in 20 000), caused by deletion of the testis-determining gene. Turner-like pheno-

type.
128
Female Pseudohermaphroditism. 6.7.3
6.7. Abnormal Sexual Development With Normal

Chromosomes
6.7.1. True hermaphroditism
Definition Presence of ovarian and testicular tissue, either as separate gonads or combined
in an ovotestis.
Incidence Very rare.
Phenotype A variable mix of male and female structures.
Chromosomes Most are 46, XX. Some are mosaics with a Y-containing cell line, some are
chimeras (created by the fusion of two embryos).
6.7.2. Mixed Gonadal Dysgenesis
Definition Presence of testis and streak ovary.
Incidence Rare
Phenotype Variable. Often virilization at puberty.
Chromosomes Most are mosaics 46, XY/45, XO.
6.7.3. Female Pseudohermaphroditism.
Definition Virilized phenotype in a female. Only ovaries are present.
Causes Prenatal androgen or progesterone treatment. Or a recessively inherited deficiency

of 11 — or 21 — hydroxylase in the adrenal cortex: congenital adrenal hyperplasia
(“adrenogenital syndrome”, incidence 1 in 10 000). The synthesis of corticosteroids is
blocked while androgen synthesis is possible.
129
Pituitary
ACTH
+
Cholesterol Progestins
Corticosteroids Androgens
The lack of corticosteroids causes excessive ACTH secretion and stimulation of the adrenal
cortex overproducing progestins and adrenal hyperplasia. With corticosteroids synthesis
blocked, the progestins are converted to androgens. These adrenal androgens make viril-
ization in females and precocious puberty in males. If the enzyme deficiency is complete,
there is dangerous hyponatremia and hyperkalemia because mineralocorticoids are missing.
Cortisol treatment permits normal development both in males and females. Early diagnosis
is important.
6.7.4. Male Pseudohermaphroditism
Definition Feminized phenotype in a genetic male.
Causes Defective androgen action. Androgen insensitivity (testicular feminization) is a rare

X-linked recessive disorder. The external phenotype is female. Female body propor-
tions, breast development, feminine psychosexual development. No uterus and fallop-
ian tubes, primary amenorrhea, no Wolffian duct structures, non-functional testes
are present in abdomen. Sometimes an “inguinal hernia” turns out to be a testis. Oth-
erwise after puberty when amenorrhea or infertility is the presenting problem. The
testes are prone to malignancies and should be removed. There is a whole spectrum
of incomplete androgen insensitivity, ranging from infertile male to phenotype female.
5α-reductase deficiency a rare autosomal recessive condition, prevents the formation of

dihydrotestosterone from testosterone. Dihydrotestosterone is the most potent andro-
gen. The external genitalia of a genetic male are mostly female at birth but internal
Wolffian duct structures are present. Virilization occurs at puberty.
130
Chromosomal Rearrangements 6.7.5
6.7.5. Chromosomal Rearrangements
Chromosomal rearrangements are caused by chromosome breakage, often followed by faulty

healing of the fragments. To be stable during mitosis, the rearranged chromosomes must
have one centromere and the telomeres.
Balanced rearrangements do not change the copy number of genes, therefore their phenotype
is normal. Genes can still be expressed, even on the wrong chromosome, as long as they
still have their promoter and enhancers.
Unbalanced rearrangements lead to a net excess or deficiency of genes, therefore the phe-
notype is abnormal.
Deletions
Both terminal and interstitial deletions occur, and they come in all sizes.
Turner syndrome Deletions in the short arm of the X chromosome (Xp-) make typical
Turner syndrome. Deletions of the long arm (Xq-) make only streak gonads and
primary amenorrhea.
Autosomal deletion syndromes Usually with a combination of physical abnormalities and

mental deficiency. Larger deletions (> 10 000 000 bp) can be seen in the banded kary-
otype, and smaller deletions can be diagnosed with FISH. Examples:
Cri-du-chat syndrome is caused by deletion of part of the short arm of chromosome

5 (5p-). Physical stigmata are mild, but there is severe to profound mental re-
tardation. Infants have a cat-like cry. 80 % of patients are female.
131
Williams syndrome is caused by an interstitial deletion in the long arm of chro-

mosome 7 (7q-). Patients are pixie-faced, with short stature, transient hypercal-
cemia, and mental retardation but spared verbal ability.
Ring chromosomes
This is a rare type of abnormal chromosome in which end pieces of the chromosome are
lost while the ring is formed:
Isochromosomes
Isochromosomes consist of two identical chromosome arms. They are formed by transverse
rather than lengthwise cleavage of the centromere during the metaphase to anaphase tran-
sition in meiosis II or mitosis.
IsoXq causes Turner’s syndrome. Isochromosomes of autosomes (other than acrocentrics)

are not compatible with life.
132
Chromosomal Rearrangements 6.7.5
Inversions
Inversions are either pericentric or paracentric. They are usually asymptomatic, but un-
balanced offspring can be produced if crossing-over occurs in the inverted segment during
meiosis.
Reciprocal Translocations
Breaks occur in two non-homologous chromosomes. The fragments unite to form two ab-
normal chromosomes:
133
Carriers of a reciprocal translocation are normal, but gametes with abnormal chromosomes
may be produced, resulting in unbalanced translocations in the offspring: High incidence of
stillbirths and spontaneous abortions.
Robertsonian Translocation (=centric fusion)
This is a fusion of the long arms of two acrocentric chromosomes after breakage near the
centromere.
The small fragment is lost. This consists of genetically inactive heterochromatin, as well
as rRNA genes. Normally, other rRNA loci are present and functional and therefore no
phenotype is expected.
134
The carriers of a Robertsonian translocation have only 45 chromosomes. They are phe-
notypically normal, but can produce abnormal offspring. Example:
Translocation-type Down syndrome is present in 3 % of unselected Down syndrome

patients. Typical features:
• Clinical severity is the same as trisomy 21
• There is no maternal age effect
• The cases are often familial
• The propositus has 46 chromosomes
• In many cases one of the parents (usually the mother) has 45 chromosomes.
Offspring derived from a gamete carrying both the translocation chromosome and a normal
chromosome 21 (but not the normal copy of the chromosome to which chromosome 21
is attached) will have translocation Down syndrome. In an asymptomatic carrier of a
Robertsonian t(21;21) translocation (isochromosome 21), all children will have Down
syndrome.
1. Define polyploid, aneuploid, monosomy, trisomy, deletion, translocation, balanced re-

arrangement, unbalanced rearrangement, ring chromosome and isochromosome.
2. Recall the approximate incidence values of chromosome aberrations.
3. Describe the causes of aneuploidy, mosaicism and chromosomal rearrangements.
4. Describe the normal function of the X and Y chromosomes in sex determination.
5. Provide examples of clinical situations in which sex chromatin determination should
be performed.
6. Describe methods for karyotyping and give examples of when a karyotype should be
performed.
7. List the phenotypic features associated with major chromosome aberrations and also
the more common trisomies and sex-chromosome ploidies.
8. Predict Down syndrome incidence based on maternal age.
9. Given a patient with a reciprocal (balanced) translocation, state the likely conse-
quences for the translocation carrier and future children.
135
10. Describe the clinical definitions of hermaphroditism, male/female pseudohermaphroditism,

mixed gonadal disgenesis.
11. Describe the pathogenesis in testicular feminization and adrenogenital syndrome.
12. Identify clinical signs and symptoms suggesting unbalanced chromosomal rearrange-
ments.
136
7. Enzymes
Life is an ordered sequence of enzymatic reactions

(R. Willstätter )
7.1. History of enzymology
Biotechnological use of enzymes in food production is a very ancient part of human culture.
The Codex Hamurabi (≈ 2100 BC) mentions the production of wine by fermentation, but
pottery used to drink wine or beer from has been found from much earlier dates. Homer
(≈ 600 BC) in the 5th song of his Iliad mentions the use of ficin (fig tree extract) in cheese
production, the first written record of the use of (semi-)purified enzymes.
Scientific work on enzymes started at the end of the 18th century with Reaumur and
Spalanzani, who noted that the stomach juice of buzzards and seagulls would digest meat
and soften bone, but would not act on plant material (enzymes are substrate specific)
and that the activity was lost on storage (enzymes are unstable).
The first to note the macromolecular character of proteins was J.F. Engelhart, who
in 1825 found the iron content of hemoglobin to be 0.334 % of the total mass, irrespective of
species. Since iron has an atomic mass 55.8 Da, the molecular mass of hemoglobin must be
n × 16.7 kDa, with n the number of iron atoms in a hemoglobin molecule (now known to be
4). This “hasty conclusion” drew a lot of ridicule from his contemporaries, who refused to
believe that any molecules could be that big. Nevertheless G.J. Mulder in 1838 coined the
term protein, which is derived from Greek and means “of prime importance”. M. Traube
suggested in 1877 that enzymes are proteins, but the idea that these “colloids” — with
which to work was below the dignity of any self-respecting chemist — could have such a
fundamental role was not accepted until J.B. Sumner crystallized pure urease in 1926.
Today we know that although most enzymes are indeed proteins, some RNAs also have
catalytic properties (“ribozymes”), a discovery made by T. Cech in 1980.
By the end of the 19th century it had become clear that both animals and plant produce
powerful enzymes, which allowed degradation of biological material in the test tube
under milder conditions (pH, temperature) than those required by a chemist. It was however
believed that the production of complex living matter could be performed only by the
organized ferments within a cell, which needed a special “vis vitalis” (Lat.: force of
137
life) to perform such reactions. This assumption is called vitalism. In 1897 Hans and
Eduard Buchner tried to preserve a cell-free juice pressed from yeast by addition of
household sugar, so they could market it for health purposes. Addition of sugar or salt
was a common preservation method before refrigerators became available. To their surprise
however the yeast juice was able to ferment the sugar under production of carbon dioxide,
a reaction that vitalists had claimed would be possible only in a living cell. Their chance
discovery opened the way for reductionism, the assumption that living organisms are
governed by the same laws of physics and chemistry that hold in a test tube. Reductionism
distinguishes modern medicine from shamanism and simple quackery. With this
discovery the distinction between ferments and enzymes lost its meaning, today we use both
terms synonymously.
7.2. Classification of enzymes
7.2.1. Systematic name
In the 19th century naming an enzyme was the privilege of its discoverer. This resulted in
names that had little systematic meaning, some of them we even use today as trivial names:
trypsin, diastase and so on. However, given that the average cell contains some 2000 different
enzymes this way of naming enzymes became rapidly untenable. Today we name enzymes
by taking the names of their substrate, adding the name of the reaction performed and the
ending “ase”. For example acetylcholine esterase is an enzyme that cleaves the ester bond in
the neurotransmitter acetylcholine, producing acetic acid and choline. Water as substrate
is not included in the name, thus it is acetylcholine esterase rather than acetylcholine:water
esterase.
7.2.2. Enzyme classes and EC codes
It turns out that enzymes perform only 6 classes of reactions:
1) Oxidoreductases catalyze the transfer of electrons, hydrid ions or hydrogen atoms be-
tween molecules:
NAD+: alcohol
H3C C OH + NAD
+
NADH + H
+
+ H3C C O
H
H2 oxidoreductase
ethanol ethanal (acetaldehyde)
138
Enzyme classes and EC codes 7.2.2
2) Transferases catalyze the transfer of functional groups between molecules

H2C OH 2-
H2C O PO3
O OH O OH
ATP : glucose
OH
+ ATP
phosphotransferase
OH + ADP
OH OH
D-glucose
OH OH
Glucose-6-phosphate
3) Hydrolases catalyze the transfer of functional groups to water

2-
H2C O PO3 H2C OH
O OH O OH
glucose-6-phosphatase
OH + H2O OH + HPO4
2-
OH OH
OH OH
glucose-6-phosphate D-glucose
4) Lyases form double bonds by removing functional groups from a molecule

- -
HC COO HC COO
aconitate
-
C COO + H2 O
hydratase
HC COO
-
- -
C COO HO C COO
H H
cis-aconitate iso-citrate
5) Isomerases transfer functional groups within a molecule

O OH
HC H2C
HC OH C O
glucose-6-phosphate
HO CH HO CH
isomerase
HC OH HC OH
HC OH HC OH
2- 2-
C O PO3 C O PO3
H2 H2
6) Ligases use the chemical energy of ATP-hydrolysis to form C-C, C-O, C-N or C-S bonds
- COO -
COO - COO pyruvate
C O
+ OH
+ ATP
carboxylase
C O + ADP + Pi
CH2
CH3
COO -
Pyruvate Bicarbonate Oxaloacetate
The Enzyme commission, responsible for naming of enzymes within the IUBMB/IUPAC
has introduced a 4-figure code number to uniquely identify each enzyme.
Example: ATP:glucose phosphotransferase 2.7.1.1 (glucokinase): the enzyme is a trans-

ferase (class 2), a phosphotransferase (subclass 7), transfer is to a hydroxy-group (1). The
fourth number has no systematic meaning, it simply allows the unique identification of each
enzyme.
139
Chemical and biological direction of a reaction We have learned in the thermodynamics

section of this course that all reaction are, at least in principle, reversible, and that catalysts
only accelerate the establishment of the equilibrium, without influencing the equilibrium
constant. Chemists by convention write a reaction so that when it proceeds from left to
right ∆G0 is negative, for example:
ATP + H2 O * ) ADP + Pi ∆G00 = −30.5 kJ/mol.
Within the mitochondria of a cell however the reaction runs in reverse direction, using
the energy liberated by oxidation of food to drive it. Thus the biological direction of the
reaction is (in this case) opposite to the chemical. Nevertheless, enzymes are named by
chemical direction, hence the enzyme is called systematically ATPase rather than ATP
synthase.
7.3. Kinetics
The first to develop a mathematical relationship between substrate concentration and re-
action velocity of an enzymatic reaction was the French physicochemist V. Henri in 1902.
Because the influence of the hydrogen ion concentration on enzyme activity was not known
at his time however, measurements did not follow the predicted values well. Once P.L.
Sørensen defined this role, introduced the pH-scale and the concept of buffering, L.
Michaelis and his postdoc M.L. Menten revisited the field and confirmed Henri’s re-
sult in 1913. In 1925 G.E. Briggs and J.B.S. Haldane came to the same result in a
more rigorous way.
7.3.1. The Henri-Michaelis-Menten (HMM)-equation
To derive this relationship, we start with the assumption of E. Fischer (1894) that enzymes
act by binding their substrate in a special pocket, called binding site, into which the
substrate fitted like a key into a lock:
k+1 k+2 k+3
E + SFGGGGGGGB GGGGGGGB
GG ES F GGGGGGGB
GG EP F GG E + P
k−1 k−2 k−3
Within the enzyme the substrate is turned over into product and finally released.
If we perform the reaction in the absence of product, then the release step becomes irre-
versible, giving us:
k+1 k+2 k+3
E + SFGGGGGGGB
GG ES G
FGGGGGGB
GG EP GGGGGGGAE + P
k−1 k−2
The conversion of S into P inside the enzyme is not accompanied by any binding or release
steps, it is therefore independent of the concentrations of the reactants and can not be
140
The Henri-Michaelis-Menten (HMM)-equation 7.3.1
Figure 7.1.: Phases of an enzymatic reaction. in the first phase (pre-steady state) the rate
of product formation is low because the ES-complex must be formed. In the
second, steady-state phase the rate of ES formation from E + S is equal to the
rate of its breakdown into E + P, [ES] is constant and [P] increases linearly
over time. In the last phase the substrate concentration becomes so low that
ES-formation slows down, leading in turn to a decrease in the rate of product
formation. Note the logarithmic time scale. Figure taken from [Buxbaum, 2007].
time-course of an enzymatic reaction
0.8
concentration (rel. units)
[S]
[E]
0.6 [ES]
[P]
0.4
0.2
0
1 10 100 1000 10000
time (rel. units)
non-steady state steady state depletion phase
investigated by kinetic methods. Hence one can further simplify the reaction scheme:
k+1 kcat
E + SFGGGGGGGB
GG ESGGGGGGGAE + P
k−1
If we mix enzyme and substrate, the ES-complex will begin to form. Initially, the rate of
formation of P will be zero, because there is no ES (law of mass action, see section 2.2 on
page 19). As [ES] increases, the rate of its breakdown into E + P will also increase, until
an equilibrium is established, where the rate of formation of ES from E + S is equal to
its rate of breakdown into E + P (see fig. 7.1). The Henri-Michaelis-Menten (HMM)-law of
enzyme kinetics, which we are about to derive, applies only in the steady-state phase of the
reaction.
The rate at which product is formed is, according to the law of mass action, given by
v = kcat × [ES] (7.1)
141
Figure 7.2.: Left: Plot of the Henri-Michaelis-Menten (HMM)-equation. Right: Rela-

tionship between the HMM-equation and a hyperbola. Figures taken from
[Buxbaum, 2007].
normalised Henri-Michaelis-Menten curve
y v y
0.75
v / Vmax
2
0.5
y2 = x - a2
0.25
x S
x
0
0 1 2 3 4 5 6 7 8 9 10
[S] / Km
In the absence of the second reaction step, [ES] would depend on [E], [S] and Kd =
k−1 /k+1 = 1/Keq :
[E] × [S]
[ES] = (7.2)
Kd
where Kd is the concentration of substrate where [ES] is half the total enzyme concentration
[E]t , in other words [E] = [ES].
Since however in an enzymatic reaction ES can break down not only into E + S, but also
into E + P, [ES] must be lower than predicted by equation 7.2. G.E. Briggs and J.B.S.
Haldane have shown in 1925 that equation 7.2 can still be used, however, that we have
to replace the dissociation constant Kd = k−1 /k+1 with the Michaelis-constant Km =
k−1 +kcat
k+1 . While Kd is the concentration of substrate where in the absence of turn-over half
the enzyme molecules have substrate bound, Km is the substrate concentration where the
rate of the enzymatic reaction is half maximal. If we replace [ES] in eqn. 7.1 with the
modified equation 7.2 and rearrange, we get the HMM-equation:
kcat × [E]t × [S] V × [S]

v= = max (7.3)
Km + [S] Km + [S]
This equation is graphed in fig. 7.2. We note that
• if [S] = ∞, all enzyme will be present as ES ([ES] = [E]t ), and the rate of product
formation will approach the maximum kcat × Et .
• if [S] Km , relative large changes in [S] will have only a small effect on v, in other
words, the reaction will be of zeroth order with respect to S. Example: If [S] = 10×Km ,
10Kd
then v = Vmax × 11K = Vmax × 0.91. If on the other hand [S] = 20 × Kd , then
d
142
The Henri-Michaelis-Menten (HMM)-equation 7.3.1
v = Vmax × 0.95. In other words, a doubling of the substrate concentration resulted

in a 4.4 % increase in v.
Vmax ×Km
• if the [S] = Km , then v = 2Km = Vmax /2
• if [S] Km , then the relationship between [S] and v can be approximated by a
straight line v ≈ [E] × [S].
The unit of the velocity of an enzyme reaction is the katal, 1 kat = 1 mol/s. In the older
literature you may still find the enzyme unit, 1 U = 1 µmol/s = 16.7 nkat.
Application: Forensic determination of blood alcohol concentration
In a considerable percentage of automobile accidents one or several of the drivers involved

are under the influence of intoxicating spirits. Because alcohol has a very detrimental effect
on the ability of motorists to control their vehicle, most countries have legal limits on
[alcohol] in the blood of motorists. Depending on jurisdiction, these tend to vary between
0.2–0.8 h, but some countries enforce a strict 0 h rule. In an “untrained” drinker 1.5 h
leads to unconsciousness, 4 h to death by respiratory suppression.
Ethanol is oxidized in our livers by alcohol dehydrogenase, which has a Km of 1 mM ≡
0.046 h. In other words, at legally relevant blood alcohol concentrations the enzyme works
near substrate saturation, the reaction is of zeroth order with about 0.15–0.2 h/h. There is
usually a considerable delay between an arrest for drunk driving and the taking of a blood
sample, but this relationship allows the back-calculation of the alcohol concentration at the
time of arrest or the time of an accident (which is the legally relevant one).
The Lineweaver-Burk-transformation
If you have experimentally determined the reaction rate v of an enzyme as function of the
substrate concentration [S], it is not always easy to determine Vmax , because the curve at
high substrate concentrations is so flat. This is especially true if your data points contain
experimental error (which they always do!).
Woolf had the idea to linearize the HMM-equation by using either one of three mathe-
matical transformations, but his work was largely ignored. Later these transformation were
re-discovered by other workers, whose name they now bear. The most important one is the
Lineweaver-Burk-transformation, which consists of taking reciprocals:
Vmax × [S] 1 1 K 1
v= ⇒ = + m × (7.4)
Km + [S] v Vmax Vmax [S]
This is the equation of a straight line (y = a + b × x), the y-intercept is 1/Vmax , the slope
is Km /Vmax and, as can be shown easily by setting to 0, the x-intercept is −1/Km .
143
Figure 7.3.: Lineweaver-Burk-transformation of v vs. [S] data results in a straight line,

but at the expense of a skewed standard deviation. Such plots can therefore not
be used to determine Km and Vmax by linear regression. Figures taken from
[Buxbaum, 2007].
original data space Lineweaver-Burk space
11
1 10
8
0.75
7
v / Vmax
Vmax / v
6
0.5 5
3
0.25
2
0 0
0 1 2 3 4 5 6 7 8 9 10 -1 0 1 2 3 4
[S] / Km Km / [S]
The other two linearizations are those of Hanes:

[S] [S] K
= + m (7.5)
v Vmax Vmax
and of Eadie-Hofstee:
v
v = Vmax − Km ∗ (7.6)
[S]
They are rarely used because they lead to a mixing of dependent and independent variables,
which causes problems when experimental results are evaluated by statistical methods. You
may safely place them into passive memory.
This is not to say that the Lineweaver-Burk-transformation were without such prob-
lems. On the contrary, taking of reciprocals transforms not only the data points, but also
their standard deviation, which becomes not only unsymmetrical, but extremely large for
low values of [S] (see fig. 7.3). The Lineweaver-Burk-transformation should therefore no
longer be used to estimate Km and Vmax from experimental data. We have better proce-
dures to do that by curve fitting directly to the original data on a computer (for experts:
Nelson/Mead-simplex and Marquardt/Levenberg algorithms). However, as we will
see later, the Lineweaver-Burk-transformation is still very useful for the presentation of
the results of kinetic experiments.
7.3.2. Catalytic perfection
k+1 , the rate of association between the enzyme and the substrate, is in aqueous solution
limited to ≈ 1 × 109 M−1 s−1 due to the viscosity of water (note that this is a second order
144
Environmental influences on enzyme activity 7.3.3
Table 7.1.: Some enzymes approaching catalytic perfection. Note that some enzymes achieve
a high efficiency by increasing kcat , others by decreasing Km . Data obtained from
BRENDA.
Enzyme Organism Substrate kcat Km kcat /Km
s−1 M M−1 s−1
β-lactamase E. coli ampicillin 1090 8.0 × 10−5 8.7 × 108
Carbonic anhydrase Mus musculus CO2 940 000 1.6 × 10−3 5.9 × 108
Catalase N. crassa H2 O2 125 000 2.5 × 10−4 5.0 × 108
AcChE H. sapiens Ac-S-choline 6500 4.6 × 10−5 1.4 × 108
Peroxidase Strep. faecalis NADH 83.3 2.0 × 10−6 4.2 × 107
Fumarase S. scrofa fumarate 364 5.0 × 10−6 7.3 × 107
Triose-P isomerase S. cerevisiae d-GA3P 16 700 1.1 × 10−3 5.6 × 107
rate constant). Indeed, if we increase the viscosity of the solution by addition of agarose or
other inert material, enzymatic reaction velocities are reduced.
In the linear part we can rewrite the HMM-equation in the following way:
Vmax [S] k
v= = cat [E][S] (7.7)
Km + [S] Km
where kcat /Km is called the efficiency constant of the enzyme. Note that it too, just like
k+1 is a second order rate constant.
k+1 is the rate at which substrate can bind to the enzyme, kcat /Km is the rate with which
the enzyme can convert the substrate into released product. If the efficiency constant is of
the same order of magnitude as k+1 , then the enzyme can handle the substrate as fast as
it can be delivered into its binding site, and the enzyme is called catalytically perfect.
Because the diffusion of the product of one enzyme to the substrate binding site of a
second enzyme can be the rate limiting step of a biochemical pathway, enzymes forming a
pathway may be arranged in complexes which directly pass intermediates between them.
This eliminates not only the time required for an intermediate to diffuse, but reduces the
risk of the intermediate undergoing other reactions, e.g. with water, before it can reach
the next enzyme. Elucidating the interactome of cells has become an important research
question.
7.3.3. Environmental influences on enzyme activity
You have already seen that enzyme structure depends on environmental factors like temper-
ature, pH, ionic strength or the presence of denaturing compounds. Of course the function
145
of enzyme depends on proper folding, so any of these factors will also affect the enzymatic
activity. In addition, the following points are relevant:
temperature higher temperatures mean more molecular motion and hence higher reaction
rates. However, beyond a certain temperature the enzyme gets inactivated, hence
enzymatic activity vs temperature is an optimum curve.
Osmolarity Enzymes need water as a “grease” for conformational changes. Osmolytes re-
duce the available water concentration and hence the enzymatic activity.
pH The protonation state of both the substrate and of amino acid R-groups in the catalytic
center of the enzyme are influenced by pH.
7.3.4. Cooperativity
As we have seen previously, interactions of substrates with many enzymes results in hyper-
bolic v vs. [S] curves. This is in particular the case when an enzyme has only one binding
site for a substrate. If however there are several binding sites, binding of substrate to one
site may change the conformation of the enzyme and hence its affinity for substrate at the
other binding sites. This effect is called cooperativity, it results in S-shaped (sigmoidal)
rather than hyperbolic binding curves.
In addition to binding sites for additional substrate molecules (“homotropic effect”) there
may also be binding sites for other molecules (“heterotropic effect”). Imagine a pathway
leading from some substrate A to a product Z via intermediates B, C...Y. In such cases
would it not be useful if the enzyme that catalyzes the conversion A *
) B (“Aase”) would be
turned off by high concentrations of the end product Z, and turned on by low concentra-
tions? This is called end product inhibition, and is achieved by heterotrophic allosteric
regulation of enzyme activity. Phosphofructokinase, which catalyzes the first commit-
ted step in glycolysis, the breakdown of food glucose, is an example for this. Binding of
a ligand to a receptor may also be regulated in such a fashion, e.g. oxygen binding to
hemoglobin, which is regulated by [O2 ] (homotropically) and by pH (Bohr-effect) and
2,3-BPG (heterotropically).
Cooperativity (using hemoglobin as a model) was first described by A. Hill in 1910. The
equation looks very much like the HMM-equation, except that the substrate occurs as a
power of h, the Hill-coefficient:
Vmax [S]h
v= (7.8)
Km + [S]h
The Hill-coefficient has the following meaning:
0 ≤ h < 1 signifies negative cooperativity, where binding of a ligand to one site impedes
the binding at other sites.
146
Cooperativity 7.3.4
Figure 7.4.: Top: Endproduct inhibition. In a biochemical pathway substrate A is turned

into intermediate B by the enzyme Aase, B is turned into C by Base and so
on. If the concentration of the final product Z is high, Z binds to Aase and
inhibits it allosterically. At low concentrations of Z, Z is released from Aase
and the activity of Aase increases. Bottom: This results in a allosteric effect
on Aase, which has S-shaped (“sigmoidal”) v vs. [S] curves, the shape of the
curve depends on [Z]. As a consequence the velocity of the reaction depends
not only on [A], but also on [Z]. Figure taken from [Buxbaum, 2007].
Allosteric regulation of enzyme activity (Km and Vmax constant)

100
80
60
v (% of Vmax)
nH = 0.50
nH = 1.00
nH = 1.75
40 nH = 2.50
nH = 3.50
20
0
0 1 2 3 4
[S]/Km
147
Figure 7.5.: Top: Changes in the Hill-coefficient h affect the shape of the v/S-curve. h = 1
results in hyperbolic curves (no cooperativity, HMM-law), if h > 1 (positive
cooperativity) then the curves are S-shaped (sigmoidal). If h < 1 (negative
cooperativity) the curves are flatter than with enzymes following the HMM-
law. Bottom: Binding of regulators often does not change h but either K0.5
(K-type) or Vmax (V-type, much rarer).
allosteric enzyme of K-type
h = 0.50
h = 1.00
h = 1.75
0.75 h = 2.50
h = 3.50
v / Vmax
0.5
0.25
0
0 0.5 1 1.5 2 2.5 3
[S] / Km
Oxygen binding to HbA: Effect of 2,3-BPG Allosteric enzyme of V-type

100
2
1.75
80
1.5
Oxygen Saturation (%)
allosteric inhibitor
no effector
allosteric activator
60 1.25
v / Vmax
40
0.0 mM
0.5 mM 0.75
1.0 mM
2.0 mM
6.0 mM 0.5
20
0.25
0 0
0 1 2 3 4 5 6 0 2 4 6 8 10
pO2 (kPa) [S] / Km
148
Cooperativity 7.3.4
Figure 7.6.: Sequential model of cooperativity according to Koshland et al. The enzyme
exists in a T-form (squares) with low and a R-form (circles) with high affinity
for substrate. Empty molecules are white, molecules with bound substrate cyan.
As more and more binding sites are filled, the probability of the protein to exist
in the R-form increases. In the sequential model of Monod et al. the entire
protein is either in the T- or in the R-form, i.e., only the outer two columns
exist.
all tense all relaxed
L L L L L
L L L L
L
L L L L L
L L
L L L
L L
L L L
L L L L L
L L
L L L
L L L
L L L
L L L L
L L L L L
L L L L L L L L L L
L
L L L L L
L L L L L L
149
h=1 means that there is no cooperation at all (binding follows HMM-kinetics)
1 < h < n positive cooperativity, binding of one ligand to one site facilitates binding to the
other sites.
h=n means complete cooperation (all sites are either filled or empty, no molecules with
only some sites filled are allowed).
h > n has never been observed, this situation is considered impossible on theoretical grounds.
7.4. Enzyme inhibition
So far we have looked upon enzymes as totally specific, binding only their substrate or,
at most, some allosteric regulator. If this were the case, you would not have to study
pharmacology, for most pharmaceuticals work by binding to enzymes. Binding can be re-
versible, then we say the enzyme is inhibited. Removal of the inhibitor (e.g. by dialysis
or gel filtration) restores enzyme activity. Alternatively, the interaction may be essentially
irreversible, then we say the enzyme is inactivated (see next section).
Depending on the mode of interaction between inhibitor, substrate and enzyme we dis-
tinguish several forms of inhibition. Note: The nomenclature was worked out by W. W.
Cleland in 1963 (Biochim. Biophys. Acta 67 104-196), but is often misrepresented in text-
books. You may therefore have learned things differently in previous courses. The nomen-
clature represented here is however used by professional enzymologists and largely agrees
with a proposed IUBMB standard.
7.4.1. Competitive inhibition
In competitive inhibition substrate and inhibitor compete for the enzyme, i.e., they can not
bind at the same time:
S
kcat
E ES E + P
competitive inhibition
I Ks
Ki
EI
From a competitive
S
inhibition mechanism it has often been concluded that substrate and
inhibitor share the same binding
kcat site on the enzyme. This however is incorrect, if substrate
binding
E changes the conformation
ES + enzyme
ofE the P in such a way that the inhibitor can no
Ks vice versa then the inhibitionuncompetitive
longer bind and mechanism inhibition
will also be competitive:
I
Kii
EIS
150
S
kcat
E ES E + P
non-competitive inhibition
I Ks I
Ki Kii
Kss
EI EIS
Uncompetitive inhibition 7.4.2
00
11 00
11 00
11
00
11 00
11 00
11
00
11 S
00
11 00
11
S
00
11
I
00
11
I 00
11
I
P
00
11
00
11
00
11 S
00
11
I
Enzyme Enzyme Enzyme Enzyme
S
S
P
S
Enzyme Enzyme Enzyme
I I I
Of course, if substrate and inhibitor share the same binding site, then the inhibition mech-
anism must be competitive, as no 2 objects can occupy the same space at the same time.
Because high [S] can exclude the inhibitor from binding to the enzyme the effect of any
given [I] can be counteracted by increasing [S]. However, as the [I] increases, higher and
higher [S] are required to achieve a given v. Thus Vmax is not changed in competitive
inhibition (see fig. 7.7). In the Lineweaver-Burk-plot all lines intersect at a common
point on the y-axis, that is, at a common 1/Vmax . If one plots the slopes of the lines in
the Lineweaver-Burk-plot as a function of [I] (not its reciprocal!) the data points are
on a straight line, Ki can be determined from its intersection with the x-axis (secondary
plot).
Example for the medical use of competitive inhibition
Ingested methanol is oxidized by alcohol dehydrogenase to methanal (formaldehyde),

which is highly toxic to the optic nerve, leading to blindness. Ethanol inhibits methanol
oxidation competitively and hence protects the optic nerve from damage. Ethanol concen-
tration has to be kept very high (close to lethal) for several days, until the methanol has
been excreted by the kidneys.
Important pharmaceuticals like methotrexate or sulphonamides also act as competitive

inhibitors, these will be discussed with the enzymes on which they act.
151
Figure 7.7.: Competitive inhibition. Top: plot of v vs [S] at several [I]. Bottom:
Lineweaver-Burk-transformation and secondary plot. Figures taken from
[Buxbaum, 2007].
no inhibition
1 [I] = 0.5 * Ki
[I] = 1 * Ki
[I] = 2 * Ki
[I] = 4 * Ki
[I] = 6 * Ki
[I] = 10 * Ki
0.75
v / Vmax
0.5
0.25
0
0 1 2 3 4 5 6 7 8 9 10 11 12
[S] / Km
Lineweaver-Burk-plot, competitive inhibition

secondary plot, competitive inhibition
10
12
8 10
slope in Lineweaver-Burk-plot
7
8
6
1 / v
5 6
4 no inhibition
[I] = 0.5 * Ki
[I] = 1 * Ki 4
3 [I] = 2 * Ki
[I] = 4 * Ki
[I] = 6 * Ki
2 [I] = 10 * Ki
2
-Ki
1
0 0
-1 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10
1 / [S] [I]/Ki
152
Uncompetitive inhibition 7.4.2
Figure 7.8.: Uncompetitive inhibition. Top: plot of v vs [S] at several [I]. Bottom:
Lineweaver-Burk-transformation and secondary plot. Figures taken from
[Buxbaum, 2007].
uncompetitive inhibition
no inhibition
1 [I] = 0.5 * Kii
[I] = 1 * Kii
[I] = 2 * Kii
[I] = 4 * Kii
[I] = 6 * Kii
[I] = 10 * Kii
0.75
v / Vmax
0.5
0.25
0
0 1 2 3 4 5 6 7 8 9 10
[S] / Km
Lineweaver-Burk-plot, uncompetitive inhibition

secondary plot, uncompetitive inhibition
12
18
y-intercept in Lineweaver-Burk-plot
10
15
8
12
1 / v
6
9
no inhibition 4
6 [I] = 0.5 * Ki
[I] = 1 * Ki
[I] = 2 * Ki
[I] = 4 * Ki
[I] = 6 * Ki 2
3 [I] = 10 * Ki -Kii
0 0
-1 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10
1 / [S] [I]/Kii
153
S
7.4.3 kcat Biochemistry and Genetics
E ES E + P
I Ks
7.4.2. Uncompetitive inhibition
Ki
In uncompetitive
EI inhibition the inhibitor reacts exclusively with the enzyme-substrate com-
plex, not with the free enzyme:
S
kcat
E ES E + P
Ks uncompetitive inhibition
I
Kii
EIS
If we increase [S], we increase [ES] and this will lead to higher [ESI]. Increasing [I] will
S
pull ES into ESI, following LekcatChatelier’s principle more E must be converted to ES to
maintain the equilibrium constant K . As a result, the inhibitor increases the apparent
E ES E s+ P
affinity of the
Ks enzyme forI the substrate and vice
non-competitive
versa. inhibition
I
Ki Kii
Since we can not combat the effect of inhibitor by increasing [S], Vmax must decrease
K
S
with increasing [I] (see fig. 7.8). Because the intersections on the y- and x- axis in the
ss
EI EIS kcat
Lineweaver-Burk-plot are the reciprocals of K and V , respectively, both increase (go
from 0) with increasing E[I].+Characteristic
m max
E ES P
further away
S K competitivefor uncompetitive inhibition are the
inhibition
I s
parallel lines in the Lineweaver-Burk-plot. Plotting the y-intercepts of the Lineweaver-
Burk-plot Ki against [I] in a secondary plot gives a straight line, intersecting the x-axis at
−Kii . S
EI kcat
E
Uncompetitive ES E + P that the enzyme acts as an oligomer.
SK inhibition is rare and is a sign mixed inhibition
I s I k
K Kii cat
Ei ES E + P
Kss
Ks k* uncompetitive inhibition
7.4.3. Noncompetitive I inhibition
EI EIS
K ii
EI + P
S
In noncompetitive inhibition both the free enzyme and the enzyme-substrate complex can
EIS
bind the inhibitor:
S
kcat
E ES E + P
non-competitive inhibition
I Ks I
Ki Kii
Kss
EI EIS
S
The four dissociation constants are related by the “law of micro-reversibility”, which
S
kcat
E ES E + P
mixed inhibition
154
I Ks I
Ki Kii
Kss k*
EI EIS EI + P
S
Figure 7.9.: Noncompetitive inhibition. Top: plot of v vs [S] at several [I]. Bottom: Lineweaver-Burk-transformation
and secondary plot for the three possible cases, Ki > Kii , Ki = Kii and Ki < Kii . Figures taken from
[Buxbaum, 2007].
non-competitive inhibition Ki = Kii
no inhibition
1 [I] = 0.5 * Ki
[I] = 1 * Ki
[I] = 2 * Ki
[I] = 4 * Ki
[I] = 6 * Ki
[I] = 10 * Ki
0.75
0.5
v / Vmax
0.25
0
0 1 2 3 4 5 6 7 8 9 10
[S] / Km
Lineweaver-Burk-plot, non-competitive inhibition (Ki > Kii) Lineweaver-Burk-plot, non-competitive inhibition (Ki = Kii) Lineweaver-Burk-plot, non-competitive inhibition (Ki < Kii)
20 20 20
18 18 18
16 16 16
14 14 14
12 12 12
10 10 10
Noncompetitive inhibition
1 / v
1 / v
1 / v
8 8 8
6 no inhibition 6 no inhibition 6 no inhibition

[I] = 0.5 * Ki [I] = 0.5 * Ki [I] = 0.5 * Ki
[I] = 1 * Ki [I] = 1 * Ki [I] = 1 * Ki
4 [I] = 2 * Ki 4 [I] = 2 * Ki 4 [I] = 2 * Ki
[I] = 4 * Ki [I] = 4 * Ki [I] = 4 * Ki
[I] = 6 * Ki [I] = 6 * Ki [I] = 6 * Ki
2 [I] = 10 * Ki 2 [I] = 10 * Ki 2 [I] = 10 * Ki
0 0 0
-2 -2 -2
-3 -2 -1 0 1 2 3 4 5 6 7 8 9 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 -3 -2 -1 0 1 2 3 4 5 6 7 8 9
1 / [S] 1 / [S] 1 / [S]
secondary plot, non-competitive inhibition (Ki > Kii) secondary plot, non-competitive inhibition (Ki < Kii)
secondary plot, non-competitive inhibition (Ki = Kii)
12
12 12
10
10 10
y-intercept slope
slope y-intercept
8
8 8
6
6 6
4 4 4
7.4.3
155
2 2 2
Ki Kii Ki, Kii Kii Ki
slope and y-intercept in Lineweaver-Burk-plot

0 0 0
-2 0 2 4 6 8 10 0 2 4 6 8 10 -2 0 2 4 6 8 10
[I] [I]/Ki [I]
can be derived from the definition of the constants:

Ki K
= s (7.9)
Kii Kss
What does this mean? Lets look at the three possible cases in turn:
Ki = Kii This means that the affinity of the enzyme for the inhibitor is independent of
whether or not it has substrate bound. In other words, substrate binding does not
change the conformation of the enzyme in such a way that the affinity for the inhibitor
would be changed. If this is the case however, then the opposite must also be true: Ks
= Kss . In the Lineweaver-Burk-plot all lines intersect in a common point on the
x-axis, because Ks and Km are related and if Ks is not changed by inhibitor binding,
Km can’t be either.
Ki > Kii In this case the affinity of E for the inhibitor is smaller than the affinity of ES
(remember that dissociation constants are reciprocals of the affinity!). In other words,
binding of substrate changes the conformation of the enzyme so that binding of the
inhibitor is facilitated. This however must also mean that the affinity of EI for the
substrate is higher than that of E, binding of I increases the affinity and lowers the
dissociation constant (and hence the apparent Km ).
Ki < Kii This is the opposite case from above, binding of substrate changes the conforma-
tion of the enzyme in such a way that binding of the inhibitor is made more difficult.
Then in turn binding of the inhibitor must change the enzyme conformation so that
binding of the substrate becomes more difficult, the dissociation constant (and hence
the apparent Km ) is increased.
Ki and Kii are determined from secondary plots, Kss is then calculated from the law of
micro-reversibility.
O
H
H3C N S S NH2
C
O
O N N A pharmaceutical working by non-competitive inhibition is ac-
etazolamide (Dianox), which binds to an essential Zn2+ ion in carbonic anhydrase. The
enzyme catalyzes the reaction CO2 + H2 O * ) HCO−3 + H , for example on the luminal mem-
+
branes of kidney tubule cells. The bicarbonate in the primary urine is broken down to car-
bon dioxide, which diffuses through the membrane into the cell and then into the blood.
If carbonic anhydrase is inhibited, more bicarbonate ions stay in the urine, Na+ and K+
follow for electro-neutrality and water osmotically. As a result urine volume is increased
(diuresis). By a similar mechanism the drug is effective in treating increased intraoccular
pressure (glaucoma) and increased intra-cranial pressure (resulting in absence seizures).
Currently it is also on clinical trials for acute mountain sickness which may befall people
at great heights where the air pressure is reduced and hence the amount of oxygen in a
156
S
kcat
E ES E + P
uncompetitive inhibition
Ks
I
Kii Enzyme inactivation 7.5
EIS
given volume of air. The hypoxemia results in faster breathing (hyperventilation), this
in turn to aS loss of carbon dioxide resulting in alkalosis. Inhibition of carbonic anhydrase
slows the decomposition of bicarbonate
kcat into carbon dioxide and hence helps to stabilize
blood EpH. ES E + P
Ks non-competitive inhibition
I I
Ki Kii
Kss inhibition
7.4.4. Mixed
EI EIS
is similar toS the noncompetitive, except that the EIS-complex has some remaining enzy-
matic activity:
S
kcat
E ES E + P
mixed inhibition
I Ks I
Ki Kii
Kss k*
EI EIS EI + P
S
The Lineweaver-Burk-plot also looks similar to the noncompetitive case (all three pos-
sibilities exist too), but the secondary diagram is curved. This allows mixed and noncom-
petitive inhibition to be easily distinguished. Mixed inhibition is rare in practice, pharma-
cologically speaking it would be useless: If we need to inhibit an enzyme to help a patient,
we don’t want it to retain activity after binding the inhibitor! Note however that mixed
inhibition is the most universal mechanism of inhibition, by simply setting some rate con-
stants to 0 we can get any of the other mechanisms from it. In physiology however you will
learn about partial (ant)agonists for receptors.
7.5. Enzyme inactivation
From the medical point of view, inhibitors have a big disadvantage: Because their interaction
with enzymes is reversible their effect diminishes as the inhibitor is excreted or metabolized.
Inactivators on the other hand completely blow the enzyme to kingdom come, making
them much more attractive pharmaceuticals. Inactivation often, but not necessarily, involves
covalent modification of the enzyme.
There are two classes of inactivators:
unspecific destroy protein structure. Acids or heavy metal salts belong into this group.
Obviously one would not normally use them pharmaceutically.
157
Figure 7.10.: Mixed inhibition. Top: plot of v vs [S] at several [I]. Bottom: Lineweaver-
Burk-transformation and secondary plot. Figures taken from [Buxbaum,
2007].
mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii
0.75
v / Vmax
f(x)
0.5
no inhibition
[I] = 0.5 * Ki
[I] = 1 * Ki
[I] = 2 * Ki
[I] = 4 * Ki
0.25 [I] = 6 * Ki
[I] = 10 * Ki
0
0 1 2 3 4 5 6 7 8 9 10 11 12
[S] / Km
Lineweaver-Burk-plot, mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii

secondary plot, mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii
10
2
1.5
7
6
1/v
slope
5 1
4
no inhibition
I = 0.5 Ki
I = 1.0 Ki
3 I = 2.0 Ki
I = 4.0 Ki 0.5
I = 6.0 Ki
2 I = 10.0 Ki
0 0
-1 0 1 2 3 4 5 6 7 8 9 -1 0 1 2 3 4 5 6 7 8 9 10
1 / [S] [I]/Ki
158
Enzyme inactivation 7.6
Figure 7.11.: Lactam-antibiotics work by inactivation of transpeptidase, an enzyme required

for bacterial cell wall synthesis.
O
H H
N S CH3
N CH3
+ HO Ser-Enzyme
O
COO-
Penicillin Transpeptidase
-
COO CH3
CH3
S N
O C H
C C O Ser-Enzyme
C N H C
H2 H
O
Inactivated enzyme
specific these compounds interact with one protein only, the protein can be protected by
the presence of its substrate.
A special case in the latter group are the suicide substrates, which bind to the substrate
site of an enzyme and are there converted into the active species by the enzymes own
k+1 k+2
GGGGGGGB
catalytic activity: E + I F GG E · IGGGGGGGAE−I. Since both the binding site and the enzy-
k−1
matic activity are required, suicide substrates are very specific for their target and often
have little side effects.
The reaction velocity of suicide inactivation is determined by slow conversion of first com-
plex, this is a first order reaction:
v = k+2 [E · I] = −d[E]/dt (7.10)
Integration yields
[E]t = [E]0 ∗ e−k+2 ∗t → ln([E]t ) = ln([E]0 ) − k+2 ∗ t (7.11)
Thus if one plots the logarithm of remaining activity as a function of time, one gets a
straight line. If the experiment is repeated with different inactivator concentrations, one
can plot the slopes of the lines as a function of [I], this results in a hyperbola, simply
because the conversion of E · I into E−I requires time. The rational is the same as with the
HMM-equation (see fig. 7.13).
159
Figure 7.12.: Aspirin inactivates Cox-1 by Ser-acetylation, it also competitively inhibits

Cox-2. In platelets no re-synthesis of Cox-1, thus coagulation is prevented for
the entire life time of the platelet!
O O O
C C C
OH OH O CH3
OH O C CH3 OH
O
salicylic acid Acetyl salicylate Methyl salicylate
(aspirin®) (oil of wintergreen)
Figure 7.13.: Inactivation of an enzyme by a suicide substrate. For details see text. Figures
taken from [Buxbaum, 2007].
time course
concentration dependence
100
[I]/Ki = 0.1
= 0.2
= 0.5 1
= 1.0
= 2.0
= 5.0
= 10
normalised rate constant k / kmax
10 0.75
[E] (% of [E]0)
0.5
0.25
0.1 0
0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 10
time (rel. units) normalised inactivator conc. [I] / Ki
160
Enzymes with multiple substrates or products 7.6
7.6. Enzymes with multiple substrates or products
So far we have looked at enzymes with one substrate and one product. If this were all,
biochemistry would be kind of boring. To write down the mechanism of reactions with
multiple substrates or products Cleland has introduced a simple notation. Lets look at
the single substrate reaction (E + S *
) ES * ) E + P) first:
) EP *
S P
( ES
EP )
E E
The reaction is drawn at a long horizontal arrow, with vertical arrows denoting the binding
of substrates or dissociation of products. Reactions inside the enzyme — without entering
or leaving compounds — are written in brackets, these are called inner complex.
If an enzyme uses two substrates it may be irrelevant which of them binds first, this is
called an random bi mechanism:
S1 S2 P2 P1
ES1 EP1
S2ES1
( P2EP1 )
E E
S2E P2E
S2 S1 P1 P2
The same of course may also be true for two products.

On the other hand, the enzyme may also require that one particular substrate binds first,
only the conformational changes associated with that binding open the second substrate
binding site:
S1 S2 P
S2ES1
ES1 ( EP
)
E E
This is called an ordered bi mechanism, here drawn with a single product.
161
The third possibility is the so called ping pong mechanism, where the first substrate binds,
transfers a functional group to the enzyme (or to a prosthetic group) and then leaves as the
first product. Only then is the second substrate bound, accepts the functional group and
leaves as the second product:
S1-X P1 S2 P2-X
ES1-X X-ES2
( X-EP ) X-E ( EP -X )
1 2
E E
Many transferases work according to this mechanism, catalase (2H2 O2 → 2H2 O + O2 ) is
another example:
H2O2 H2O H2O2 H2O
.
5+ O2
E-Fe O
( )
3+ 4+
( )
E-Fe H2O2 E-Fe O H2O2
E-Fe
5+
O H2O
.
E-Fe
4+
O
3+
E-Fe O2 H2O
E-Fe
3+
O2
3+ 3+
E-Fe E-Fe
Note the ordered release of the last products!
Enzymes with more than two substrates work in the same way, instead of bi- one would
use ter-, quad- and so on.
7.7. How do enzymes do it?
There are a only a few fundamental mechanisms by which enzymes achieve acceleration of
reactions:
Orientation Reactants are bound by the enzyme in a spatial orientation that facilitates
reaction.
Reduction of reaction order Reactants are held together by the enzyme much longer than
in random collisions. Reactions of n-th order between molecules become 1st order
reaction inside a molecule.
Acid/base catalysis Unfavorable charge development on intermediate is prevented by do-

nation of a proton from H3 O+ (specific acid catalysis), HA (general acid catalysis) or
its abstraction by OH− (specific base catalysis) or B : (general base catalysis). pKa -
values of amino acid side-chains are adjusted by the environment inside the protein
to facilitate the reaction. Example: Glu pKa in xylanase is between 4.6 and 6.7.
Redox-reactions on cofactors Redox-potential is again adjusted by protein environment,

e.g. Fe3+ + e− → Fe2+ has E 00 of −432 to 771 mV.
162
Protease reaction mechanism 7.7.1
Electrostatic catalysis an electrical charge on the intermediate can be stabilized by a

nearby opposite charge. Example: Zn-containing dehydrogenases. Electrical field strength
inside proteins can be in the order of 10 MV/cm, separation of 1 unit charge over 1 Å
lowers ∆E a by 9.6 kJ/mol.
Rack-mechanism Bonds of the substrate are strained during formation of the ES‡ inter-
mediate.
Quantum theory Electrons or protons tunnel through the energy barrier, rather than cross-
ing it.
The classical model of substrate binding, the lock-and-key model, was introduced by E.
Fischer in 1894. In this model the substrate fits into a preformed binding site within the
enzyme. However, this would actually increase the activation energy rather than reducing
it:
without catalyst lock-and-key mechanism induced fit mechanism
|
S ES |
free energy G
free energy G
free energy G
ΔGB
ΔEa ES |
EP ES EP ΔEa
ΔEa
E+S E+S
S
ΔG ΔG ΔG
ΔGB
E+P E+P
P
ES
Reaction progress Reaction progress Reaction progress
In 1959 D.E. Koshland proposes the induced fit hypothesis to solve this problem. In
this model the enzyme binds not the substrate, but the transition state. Thus part of the
energy required to convert the substrate into the transition state is compensated by the
binding energy to the enzyme, the activation energy is the difference between activation and
binding energy, rather than its sum as in the lock and key model. According to the induced
fit hypothesis the binding site in the enzyme is not preformed, but rather the enzyme adapts
itself to the transition state as this is formed. Application: Antibodies against a transition
state analogue may have catalytic properties (catalytic antibodies).
7.7.1. Protease reaction mechanism
Peptides and proteins are stable, because during their hydrolysis a high-energy intermediate
is formed, which converts back to the original compound much easier than to the next
intermediate:
163
C O
N H
H N H
R´
R
R´
X O C O
H N H
X O C O
H N H
+
R´
R´
R
+
+
X O C O
X O
H
R
Acids and bases can accelerate the conversion of the first into the second, more stable
intermediate and hence peptide bond splitting, that is why they are caustic to our skin!
Rather than working with extreme pH, proteases in our body stabilize the transition state
by distributing the charges onto nearby amino acids, as shown here for Ser-proteases:
195
R H N Ser
195
57
Ser O: + C O 193
His H N Gly
H N H
R
H N N
102
Enzyme-substate complex
O
Asp C
O
H N Ser
R H N Ser His Ser O
+ H N Gly
Ser O C O H
His H N Gly
H N H H N N Free enzyme
R O
H
N N Transition state 1 Asp C
O R
O C O
Asp C
O OH
R
R H N Ser H N Ser
His Ser O C O
His Ser O C O
H N Gly H N Gly
OH
N H
H + H
+
R H N N
H N N
O
O Asp C
Asp C O Transition state 2
O H
N H
R
R
H N Ser
His Ser O C O R
H N Gly H N Ser
His Ser O C O
H N N Acyl-enzyme intermediate H N Gly
O
O N H H
H N
Asp C
O O
Asp C
O
H2O
Ser-195 (rather than water) acts as a nucleophile and forms the intermediate. The positive
164
Adenosine Triphosphate (ATP) 7.8.1
charge is immediately transferred to His-57, there it is stabilized by salt bond formation

with Asp-102 (catalytic triad). The negative charge of the intermediate is stabilized by
hydrogen bonds to the amide nitrogens of Ser-195 and Gly-193 (oxanion hole).
In a second step the His-57 transfers a hydrogen onto the nitrogen of the peptide bond,
allowing the first product to dissociate. The resulting Acyl-enzyme intermediate is then
hydrolyzed by a similar mechanism.
7.8. Coenzymes
Some (not all) enzymes require a coenzyme for their reaction. There are 2 types of coen-
zymes:
1. Some coenzymes are bound permanently to the active site of the enzyme, either
covalently or noncovalently. These coenzymes are called prosthetic groups.
2. Some coenzymes are soluble molecules which associate with the enzyme active site
only during the reaction. They function as co-substrates. Like other substrates,
they become changed chemically during the reaction and have to be regenerated in a
different reaction.
7.8.1. Adenosine Triphosphate (ATP)
ATP is a co-substrate in reactions of energy metabolism. Chemically, it is a nucleotide:

NH2
N N
Adenine
O O O H2 N N
-
O P O P O P O C O
O O O
Polyphosphate Ribose
OH OH
ATP can serve as the energetic currency of the cell because it contains 2 energy-rich phos-
phoanhydride bonds, each with a free energy content of 30.5 kJ/mol. ATP is synthesized
from ADP + phosphate during catabolic reactions, most of this by oxidative phosphoryla-
tion in the mitochondria.
ATP hydrolysis drives:
• Biosynthetic (anabolic) reactions
165
• Active transport across membranes

• Cell motility and muscle contraction
There are 2 modes of ATP hydrolysis:
1. ATP + H2 O → ADP + Phosphate
2. ATP + H2 O → AMP + Pyrophosphate
In the second mode the pyrophosphate (PPi ) is quickly hydrolyzed by soluble pyrophos-
phatases, making the reaction irreversible at the expense of an energy rich bond.
ATP is used for:
• Nucleic acid synthesis. ATP is an immediate precursor for RNA synthesis and an
indirect precursor for DNA synthesis.
• Phosphorylation reactions. ATP can transfer its last phosphate to an acceptor mole-
cule. These reactions are catalyzed by kinases, a type of transferase. Example:
H2C OH H2C OH
HO CH + ATP HO CH O + ADP
C OH C O P O
H2 H2
O
Glycerol Glycerol-3-phosphate
• Coupling to endergonic reactions. Endergonic reactions cannot proceed in the cell un-
less the enzyme couples the endergonic reaction to ATP hydrolysis. Example: Palmitic
acid + CoA−SH → Palmitoyl-CoA + H2 O, ∆G00 = +32.8 kJ/mol.
This reaction cannot proceed in the indicated direction. The reaction that actually
takes place in the body is: Palmitic acid + CoA−SH + ATP → Palmitoyl-CoA +
AMP + Pyrophosphate ∆G00 = +2.8 kJ/mol.
This reaction proceeds in the indicated direction because its ∆G00 is close to zero and
one of the products (pyrophosphate) is rapidly removed from the equilibrium.
Important for the reaction equilibrium:
• The reaction proceeds whenever the actual ∆G (not the ∆G00 !) is negative. Therefore
the reaction can be driven in the right direction when the concentration of a substrate
is very high and/or that of a product is very low. In the above example, pyrophosphate
is very low. Also, the [ATP] / [AMP] ratio in the cell is about 100, and this lowers
the real ∆G by about 11.3 kJ/mol.
• When ATP is hydrolyzed in the reaction, the ∆G00 is reduced by 30.5 kJ/mol. This
is usually sufficient to drive the reaction.
166
Redox Coenzymes 7.8.2
In the cell, the adenine nucleotides are in equilibrium through the adenylate kinase (myok-
inase) reaction: ATP + AMP * ) 2 ADP.
The energy status of the cell can be described by the cellular [ATP] /[ADP] ratio, or by
the energy charge:
[AT P ] + 1/2[ADP ]
ν= (7.12)
[AT P ] + [ADP ] + [AM P ]
The nucleotides guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine
triphosphate (CTP) are also used as coenzymes in some energy-dependent reactions.
7.8.2. Redox Coenzymes
Some coenzymes transfer either naked electrons or electrons + protons (hydrogen atoms)
during redox reactions. Nicotinamide-adenine dinucleotide (NAD+ ) and its phosphorylated
derivative NADP+ are cosubstrates in dehydrogenase reactions. Structure:
NH2 H O
H H O
Adenine Nicotinamide
N N NH2 NH2
+
..
N N OH2 O N + -
H ,2 e N
O H2C O P O P O C O R
O O
Ribose Ribose
OH OH OH OH
The flavin nucleotides flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD)
are also hydrogen carriers, but they function as tightly bound prosthetic groups of the
flavoproteins. Structure of FAD:
167
O
H3C N
NH
H3C N N O
CH2 Flavin
Adenine NH2
HC OH Ribitol
} Riboflavin
N N HC OH H O
O O HC OH +
H+e
-
H3C N
N N C NH
O H2C O P O P O CH2
O O H3C N N O
R
Ribose
OH OH
FADH, semiquinone radical
Flavin adenine dinucleotide (FAD), oxidised
(FMN = Riboflavin + 1 Phosphate)
+ -
H+e
H O
H3C N
NH
H3C N N O
R H
FADH2, fully reduced
Straight electrons (without protons) can be transferred by many iron-containing proteins.

In the iron-sulfur proteins the iron is bound to cysteine side chains, and in the heme
proteins it is part of the heme group. The iron switches between the ferrous (Fe2+ ) and the
ferric (Fe3+ ) state during the electron transfer. Other metal ions are also found in some
enzymes, including zinc, copper and manganese. In some enzymes the metal ion participates
in electron transfers, but in others it acts as a Lewis acid (electron pair acceptor).
7.8.3. Other Coenzymes
There are many other coenzymes, each with its own special function. Examples:
Coenzyme A (CoA) is a cosubstrate. It contains a sulfhydryl group which forms thioester

bonds with organic acids. Example:
GMP
GTP PPi
O
CoA SH + H3C COO
-
CoA S C CH3
168
Enzymes in clinical diagnostics 7.9
Figure 7.14.: The optical test of Warburg is based on the different absorbance of NAD(P)
and NAD(P)H at 350 nm. This allows both substrate concentrations and en-
zyme activities to be determined. Figures taken from [Buxbaum, 2007].
height: proportional to substrate concentration

absorbance 345 nm
Spectrum of NAD and NADH (0.5 mM in water)
2.5
NAD
NADH
2 slope:
proportional to
enyzme activity
1.5
Absorbance
0.5
time
0
250 300 350 400 sample was
wavelength (nm)
added here
S-Adenosylmethionine (SAM) is a cosubstrate that contains a methyl group. This methyl

group is transferred to an acceptor during enzymatic reactions.
Many, but not all, coenzymes contain a vitamin as part of their structure: Nicotinamide
(niacin) in NAD+ and NADP+ , ribloflavin in FMN and FAD, pantothenic acid in coenzyme
A.
7.9. Enzymes in clinical diagnostics
If cells are damaged, the cell content, including the enzymes present inside the cell, are
released into the blood stream. Because each enzyme molecule can turn over many, many
substrate molecules, enzyme activities present in serum (the liquid obtained by letting
blood coagulate) can be detected with high sensitivity and specificity. Since different organs
have different functions, they also have different enzymes, and even if two organs have the
same enzymatic activity, it may be carried out by different isoenzymes. Therefore, it is
possible to test for organ damage by testing for appropriate enzymatic activities in a blood
sample, a cheap, simple and relatively non-invasive technique. This is important for example
for the diagnosis of myocardial infarct, liver and pancreas damage, muscular dystrophy and
some cancers (see chapter 10 on page 203 for details).
169
Very commonly used is the optical test of Warburg, which is based on the fact that the
nicotinamide group of NAD+ or NADP+ does not absorb at 350 nm in the oxidized state,
upon reduction however it does (see fig. 7.14). The difference between the absorbance at
the beginning and the end of the reaction is proportional to the substrate concentration,
the rate of absorbance change is proportional to the enzyme concentration.
Many enzymes however do not use NAD(P) in their reaction. For example creatine kinase
performs the following reaction in muscle cells:
Creatine kinase
Creatine + ATP FGGGGGGGGGGGGGGGGGGGGGB
GG Creatinephosphate + ADP.
The enzyme is released into the blood stream after muscle damage, especially after an acute
myocardial infarct. Thus if a patient reports to you with chest pain (angina pectoris) you
can measure the activity of creatine kinase to determine whether or not the patient has an
infarct. The reaction itself however does not lead to a change in absorbance. Therefore it
is coupled with two other reactions:
Pyruvate kinase
ADP + Phosphoenolpyruvate F GGGGGGGGGGGGGGGGGGGGGB
GG Pyruvate + ATP
and
Lactate dehydrogenase
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGB
Pyruvate + NADH + H+ F GG Lactate + NAD+ .
Thus each molecule of ADP produced in the first reaction leads to the production of exactly
one molecule of NADH + H+ in the third. The reagents are sold premixed by supply
companies, only a serum or plasma sample needs to be added.
7.10. Membrane Transport
Regulated transport across membranes is required for the exchange of nutrients, metabolic
intermediates, and waste products; to maintain ion gradients; and to transfer nutrients,
electrolytes, and water across absorptive epithelia.
7.10.1. Passive transport
Passive transport does not require an energy source and is always down the electrochemical
gradient. It includes passive diffusion (no carrier involved) and facilitated diffusion (through
a carrier).
Passive diffusion across the lipid bilayer depends on the physical properties (size, lipid
solubility) of the diffusing molecule. No energy required. Transport rate depends linearly
on substrate concentration.
Examples for passive diffusion:
170
Passive transport 7.10.1
Figure 7.15.: Modes of membrane transport. For details see text. Figure taken from
[Buxbaum, 2007].
O2
H
ADP + Pi
2K
• Most membranes are relatively permeable to water, although there are also water
channels in some cells.
• Osmosis is the passive diffusion of water from a compartment with lower solute con-
centration to one with higher solute concentration.
• Biological membranes are permeable to various extents to some very small hydrophilic
molecules: urea, methanol, ethanol.
• Blood gases (O2 , N2 , CO2 ) are permeable, but the bicarbonate ion is not.
• Membranes are permeable for small lipid-soluble substances (fatty acids, steroid hor-
mones, some drugs). If the molecule is ionizable, only the uncharged form diffuses.
Carrier-mediated transport (facilitated diffusion, uniport) requires an integral membrane

protein (“carrier” that interacts with the solute. Carrier-mediated transport shows:
• Substrate specificity
• Saturability (hyperbolic relationship between rate and concentration)
• Specific inhibition and regulation
171
The carrier forms a gated channel that undergoes a conformational change during the
transport cycle.
Examples of Facilitated Diffusion
• Glucose uptake by liver, erythrocytes, etc. is effected by an electroneutral uniport

carrier.
• The blood-brain barrier transport of glucose and amino acids.
7.10.2. Active transport
Active transport is always carrier-mediated. It can accumulate a solute against its elec-
trochemical gradient. Primary active transport hydrolyzes ATP while secondary active
transport transports a second solute down its electrochemical gradient (Co-transport): The
carrier transports at least two different solutes, either as
Symport Two solutes transported in the same direction.
Antiport Two solutes transported in opposite directions.
either as
Electroneutral transport:]No net transport of electrical charges.
Electrogenic transport: Net transport of electrical charges.
Examples of co-transport
Symport Sodium cotransport of amino acids and glucose in the intestinal mucosa and the
renal tubules, and of amino acid neurotransmitters into the nerve terminal.
Antiport (=“exchange diffusion”) Sodium-calcium exchange across plasma membrane of

many cells. Shuttles in the inner mitochondrial membrane.
primary active transport in mammals involves ATP-hydrolysis. In plants and bacteria

other energy rich compounds may be used to fuel the reaction (e.g. phosphoenol pyruvate,
pyrophosphate).
Na+ /K+ -ATPase (sodium-potassium ATPase) is in the plasma membrane of all cells
with highest activity in brain and muscle. The sodium-potassium ATPase is an
electrogenic antiporter, with 3 sodium pumped out of the cell and 2 potassium
pumped into the cell. The transport cycle requires the phosphorylation of an
aspartate side chain in the carrier by ATP. It transports Na+ out of and K+
into the cell. Membrane ATPases consume 10–30 % of the basal metabolic rate.
Cardiotonic steroids inhibit the sodium-potassium ATPase, thereby reducing
172
Ion concentrations 7.11.1
the sodium gradient across the plasma membrane. This impairs the sodium-
dependent calcium-extrusion mechanism. Increased intracellular calcium leads
to an increased force of contraction of the heart (positive inotropic effect).
Ca2+ -ATPase is present in the sarcoplasmic reticulum of muscle fibers and in the
plasma membrane of many cells. It pumps calcium out of the cytoplasm and
works by a mechanism similar to that of the Na+ /K+ - ATPase.
7.11. Homeostasis of the Intracellular Environment
Cellular processes depend on temperature, pH and in many cases, the correct concentrations
of inorganic ions. Differences between intra-and extracellular environment:
7.11.1. Ion concentrations
Ion Extracellular (mM) Cytoplasmic (mM)

Na+ 140.0 10.0
K+ 4.7 140.0
Mg2+ 1.4 30.0
Ca2+ 2.5 0.001
Cl− 113.0 4.0
HPO2−
4 2.0 11.0
HCO− 3 2.8 10.0
The calcium concentration is very low in the cytoplasm but much higher in mitochondria
and ER. Phosphate is high in the cell because of its role in energy metabolism (ATP
synthesis!). The transmembrane gradients of sodium and potassium are required for cell
excitability.
The pH is about 7.4 in blood plasma and extracellular fluid, but near-neutral in the cyto-
plasm: pH = 6.8 at 37 °C, pH = 7.0 at 25 °C. The pH difference across the plasma membrane
favors the transfer of carbon dioxide out of the cell, and it facilitates the removal of acidic
products such as lactic acid.
As far as possible, reducing conditions are maintained inside the cell. This is needed to
protect proteins, lipids, and nucleic acids from oxidative damage.
Most of the cellular metabolites are charged at pH 7. This is because the important acidic
groups (carboxy and phosphate) have pKa s below 7, and the important basic groups (amino
groups) have pKa s above 7. It means that most metabolites require carriers to cross mem-
branes. Physiological buffer systems include:
• Phosphate
173
• Bicarbonate
• Protein
Of these three systems, proteins are considered the most important because of the large
quantity of protein present in the body (about 12 kg). The amino acid histidine is most
important because its side chain pKa is not too far from the physiological cellular pH.
Also, unlike the phosphate system, the histidine side chain dissociates with a temperature
dependence similar to that of water.
The buffer systems can be overwhelmed in disease states, leading to acidosis or alkalosis.
Types:
Respiratory acidosis Retention of carbon dioxide which forms carbonic acid. In lung dis-
eases.
Respiratory alkalosis Abnormality low carbon dioxide and carbonic acid because of hyper-
ventilation.
Metabolic acidosis Overproduction of organic acids (lactic acid, ketone bodies...), or fail-
ure of the kidneys to excrete excess protons.
Metabolic alkalosis Abnormal loss of acid, for example by vomiting acidic stomach con-
tents.
7.12. Useful web resources
BRENDA enzymological database http://brenda.bc.uni-koeln.de/
IUBMB http://www.chem.qmw.ac.uk/iubmb/
UniProt Universal Protein Resource (sequences), http://www.uniprot.org
KEGG Kyoto Encyclopedia of Genes and Genomes (pathway maps), http://www.genome

.jp/kegg/pathway.html
OMIM online Mendelian inheritance in man (inherited diseases), http://www.ncbi.nlm

.nih.gov/sites/entrez?db=omim
174
1) Enzyme turnover rate: An enzyme is turning over at a substrate concentration of

[S] = 3 × Km . The reaction velocity will be
A) 0.25 × Vmax
B) 0.33 × Vmax
C) 0.50 × Vmax
D) 0.75 × Vmax
E) 1.00 × Vmax
2) Enzyme reaction velocity Under conditions of substrate saturation you double [E].
The velocity v
A) remains constant.
B) is halved.
C) is doubled.
D) is increased 10-fold.
E) is reduced 100-fold.
3) Enzyme turnover number 5 µg of a pure enzyme (MW = 50 000 Da) give Vmax =
10 µmol/min. What is the turnover number of the enzyme?
A) 10 × 103 min−1
B) 25 × 103 min−1
C) 50 × 103 min−1
D) 75 × 103 min−1
E) 100 × 103 min−1
175
4) Enzyme activity, HMM-equation Two enzymes compete for the same substrate. Most
of the substrate will be turned over by the enzyme with
A) the highest molecular weight.
B) the highest Km value.
C) the highest activity and the lowest Km value.
D) the lowest activity and highest Km value.
E) the highest specificity for the substrate.
5) Action of pharmaceutical You are employed by a drug company developing a new

antibiotic against tuberculosis. Testing the interaction of a candidate substance with an
enzyme the bacterium needs for division you obtain the diagram above. Which of the
following terms best describes the interaction between the potential drug and the target
enzyme?
antibiotic and enzyme
100
0.5
remaining enzyme activity (% of original)
0.4
k (1/s)
0.3
0.2
0.1
0
0 5 10 15 20
10 antibiotic [mM]
20 mM
10 mM
5 mM
3 mM
2 mM
1 mM
0.5 mM
0.3 mM
1
0 5 10 15 20 25
time (min)
A) Competitive inhibition.
B) Non-competitive inhibition.
C) Uncompetitive inhibition.
D) Mixed competitive inhibition.
E) Inactivation by a suicide substrate.
176
6) Metabolic and thermodynamic direction The enzyme glucose phosphate isomerase

catalyzes the reaction Fru-6P * ) Glu-6P, ∆G = −1.8 kJ/mol. For this reaction to proceed
in the physiological direction (toward Fru-6P) one has to
A) remove [Glu-6P] in a second reaction
B) remove [Fru-6P] in a second reaction
C) do nothing, as reactions proceed towards positive ∆G
D) increase the [Fru-6P]
E) increase the enzyme concentration
7) Henri-Michaelis-Menten law, Lineweaver-Burk plots The diagram shows the Lineweaver-

Burk-plot of an experiment. The interaction is
Lineweaver-Burk plot
4
[I] = 0.0 mM
3.5 [I] = 0.5 mM
[I] = 1.0 mM
[I] = 2.0 mM
[I] = 4.0 mM
3 [I] = 6.0 mM
[I] = 10 mM
2.5
1 / v (s/nmol)
1.5
0.5
0
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3
1 / [S] (1/mM)
A) the competitive inhibition of an enzyme with a Km of 1 mM.
B) the competitive inhibition of an enzyme with a Km of 2 mM.
C) the non-competitive inhibition of an enzyme with a Km of 1 mM.
D) the uncompetitive inhibition of an enzyme with a Km of 1 mM.
E) the uncompetitive inhibition of an enzyme with a Km of 2 mM.
177
8) Hill-equation, co-operative binding Oxygen binding to hemoglobin (in % of maxi-

mum) is a sigmoidal function of oxygen concentration (in kPa partial pressure). Assuming
a Hill-coefficient h = 2.9 and K0.5 = 13 kPa, what is the saturation of hemoglobin in working
muscle, where the oxygen concentration is 1 kPa?
A) 0 %
B) 7 %
C) 20 %
D) 50 %
E) 100 %
9) difference between inhibition and inactivation Some phosphoesters are used as insec-
ticides (Parathion, E605) and chemical weapons (VX, sarin, tabun). They act by covalently
modifying Ser-groups in the active center of acetylcholine esterase in the synaptic gap of
the neuro-muscular junction. This reaction most likely results in a
A competitive inhibition
B uncompetitive inhibition
C non-competitive inhibition
D mixed inhibition
E inactivation
10) Enzyme nomenclature, enzyme classes The reaction depicted is catalyzed by a

H2C OH H2C OH
HO CH + ATP HO CH O + ADP
C OH C O P O
H2 H2
O
Glycerol Glycerol-3-phosphate
A) Oxidoreductase
B) Transferase
C) Hydrolase
D) Lyase
E) Isomerase
178
Objectives 7.14
7.14. Objectives
After completion of this course unit students should be able to
• list the major classes of enzymes: Oxidoreductases, transferases, ligases, lyases, hy-
drolases and isomerases and describe the reactions performed by them.
• describe the interaction of substrates and products with an enzyme and explain how
this leads to catalysis.
• list the basic assumptions made in Michaelis-Menten kinetics.
• compare the effect of enzymes on thermodynamic and kinetic properties of biochemical
reactions.
• use the Henri-Michaelis-Menten-equation and the Hill-equation to predict the
velocity of enzymatic turnover.
• state the importance of Km and Vmax for the rate of enzymatic reactions at high and
low substrate concentrations.
• explain the role of the efficiency constant Vmax /Km
• interpret substrate-velocity and Lineweaver-Burk-plots.
• describe the effects of temperature, ionic strength and pH for the rates of enzymatic
reactions.
• describe the reaction mechanism of competitive, uncompetitive, non-competitive and
mixed inhibitions and state how these can be distinguished in a Lineweaver-Burk-
plot.
• describe the difference between inhibitor and inactivator and give examples for the
pharmacological use of both.
• describe, using examples, some of the mechanisms by which enzymes act.
• describe, using examples, the roles of co-substrates and prosthetic groups in metabolism.
• describe the structure of ATP, with emphasis on its energy rich bonds and list the
reaction types in which ATP participates.
• define the energy charge and state the relative abundances of ATP, ADP, and AMP
in healthy and metabolically stressed cells.
• relate the coenzymes NAD+ , NADP+ , FAD to the reaction types in which they par-
ticipate.
179
8. Methods in Molecular Medicine
8.1. Restriction Endonucleases
The DNA in human chromosomes is too big to be manageable in the test tube. The chro-
mosomes have to be broken down into smaller fragments of some hundred or thousand bp.
This is done with restriction endonucleases. These enzymes cleave double-stranded DNA
very selectively at palindromic sequences. The palindromes are 4–8 bp long. The longer the
recognition sequence of the enzyme, the greater is the average size of the fragments gener-
ated. Some restriction endonucleases cleave in the middle of their recognition sequence and
produce blunt-ended fragments. But most make staggered cuts, producing fragments with
short single-stranded overhangs (“cohesive ends”).
Note that the cohesive end of a restriction fragment can anneal (= base-pair) with the end
of any other fragment generated by the same restriction endonuclease. You can even link
two restriction fragments covalently. Mix the fragments, let them anneal, then add DNA
ligase.
Restriction endonucleases are bacterial enzymes that are used by the bacteria as a defense
against DNA virus. Bacteria protect sensitive sites in their own DNA by methylation.
There are lots of restriction endonucleases and a few hundred of them are commercially
available.
8.2. Probes
One of the main tools in working with DNA, is a probe that recognizes the DNA of interest
while ignoring everything else. A probe is a labeled (fluorescent or radioactive) single-
stranded nucleic acid that is complementary to the nucleic acid you are looking for. Most
important:
A cDNA probe represents the exon sequences of a gene. It will identify any restriction frag-
ment that contains expressed sequences of the gene. A cDNA is the reverse transcript
of an mRNA.
181
An oligonucleotide probe is a synthetic DNA that is complementary to a specific DNA

sequence. If used on a restriction digest of genomic DNA, it has to be at least 17 or
18 nucleotides long because otherwise the target sequence may be present in multiple
sites in the genome. Oligonucleotides of this length can be synthesized at moderately
high cost, when 32 P- or fluorescently-labeled.
The probe hybridizes with single-stranded DNA (or RNA) that is complementary to its
own base sequence. The assay conditions are said to be of high stringency when the probe
anneals only with the correct sequence but not with closely related sequences. High temper-
ature, low ionic strength and alkaline pH interfere with annealing and therefore represent
conditions of high stringency. Under proper stringency conditions, a single mismatch can
prevent the hybridization of an oligonucleotide probe with its target sequence.
8.3. Southern Blotting
A restriction digest contains thousands to millions of restriction fragments. Southern blot-

ting combines electrophoretic separation and probing to identify interesting restriction frag-
ments.
1. Treat the DNA with a restriction endonuclease.
2. Separate the restriction fragments by electrophoresis on a cross-linked gel (agarose

or polyacrylamide). These methods separate the restriction fragments by size: small
fragments move fastest. This gives you a pretty accurate estimate for the size of the
restriction fragments.
3. Dip the gel in a NaOH solution to denature the restriction fragments.
4. Transfer (“blot”) the denatured DNA to a nitrocellulose filter. Nitrocellulose binds

single-stranded DNA very tightly. Blotting produces a replica of the electrophoretic
separation on the nitrocellulose.
5. Incubate the nitrocellulose filter in a solution of the probe and wash off excess probe.
6. Visualize the bound probe by autoradiography or fluorescence scanning.
This procedure answers two questions:
1. Is a sequence complementary to the probe in your DNA extract?
2. How long is the restriction fragment detected by the probe?
182
The Polymerase Chain Reaction (PCR) 8.4
Northern blotting is a similar procedure for RNA: the RNA (usually mRNA) is separated
by gel electrophoresis, blotted to nitrocellulose, and probed. Restriction enzyme digest and
NaOH treatment is not included in Northern blot as opposed to Southern blot.
Western blotting is a method for the separation of proteins. The proteins are denatured
with the anionic detergent SDS, separated by one- or two-dimensional gel electrophoresis,
blotted to nitrocellulose, and “probed” with a (monoclonal) antibody. Eastern blotting
works similar, except that the cationic detergent CTAB is used for denaturation, hence
polarity during electrophoresis and blotting is reversed.
8.4. The Polymerase Chain Reaction (PCR)
PCR is a method for the enzymatic amplification of double-stranded DNA in vitro. This is
the second major tool in the DNA laboratory. It can be done on crude DNA extracts, but
only a selected segment is amplified. You have to know at least the flanking sequences of
the DNA you want to amplify. You need:
1. The extracted DNA.
2. Substrates for DNA synthesis: dATP, dGTP, dCTP, dTTP.
3. A heat-stable DNA polymerase (usually Taq polymerase from the thermophile bac-
terium Thermus aquaticus). This enzyme polymerizes DNA at 60 °C and survives
repeated heating to 95 °C. Like other DNA polymerases, it requires a primer to start
DNA synthesis.
4. A pair of oligonucleotide primers that hybridize with complementary sequences on

each of the DNA strands. They mark the ends of the amplified sequence.
Procedure:
1. The DNA is mixed with deoxyribonucleotides, Taq polymerase, and a very large
(>million fold) excess of the primers.
2. The DNA is denatured by heating to 95 °C.
3. The solution is cooled to 60 °C. The primers anneal and Taq polymerase synthesizes
new DNA starting from the 3’ ends of the primers. The exact temperature depends
on the primer sequences.
4. After 2 or 3 minutes, when the Taq polymerase has worked its way from one primer
to (and beyond) the level of the next, the solution is heated again to separate the
strands.
183
5. The solution is run through 20–30 cycles of heating and cooling. In theory, the amount
of DNA between the primers doubles in each cycle.
3' 5'
5' 3'
5' 3' 5'
3'
5' 3' 5' 3'
3' 5'
melt (95 °C)
5' 3'
3' 5' 3' 5'
allow primers to
3' anneal (60 °C) 5' 3'
5'
melt (95 °C)

melt (95 °C)
allow primers 3' 5' allow primers to
5' 3' 3' 5' anneal (60 °C)
to anneal (60 °C)
3' 5' 5' 3' 3' 5'
5' 3' 5' 3' 3' 5'
3' 5'
5' 3' 5'
3' 5' 3'
polymerise 3' 5'
polymerise 5' 3' 3' 5'
5' 3'
3' 5' 5' 3' 5'
3' 5' 3' 3'
5' 3' 5' 3' 5'
3' 5' 3' 5' 5' 3'
5' 3' 5' 3' 3' 5'
3' 5' 5' 3' 3' 5'
5' 3' 5' 3'
3' 5'
5' 3' polymerise
3' 5'
5'
3' 5' 3'
5' 3'
3' 5'
5' 3'
3' 5'
5' 3'
3' 5'
5' 3'
3' 5'
5' 3'
3' 5'
3' 5' 3'
5'
5' 3'
(figure from [Buxbaum, in press]) PCR generates a PCR product. This is a blunt-ended
double-stranded DNA whose ends are marked by the primers. Indeed, the primers are
incorporated in the PCR product. PCR products can be probed, but it is usually more
convenient to simply electrophorese the product and stain the gel with ethidium bromide.
There is so much more PCR product than other DNA that you can always recognize the
product as a band and you are rarely able to see the starting material in the gel.
Because of its sensitivity (only a few DNA molecules are needed), PCR is the workhorse for
all those applications where only minute amounts of DNA are available, including prenatal
diagnosis, preimplantation diagnosis, forensic applications, and the study of fossil DNA.
Limitations:
• Taq polymerase does not proofread its product, therefore the error rate is high. In
part for this reason, the method is suitable only for the amplification of short pieces
of DNA, up to 3 kbp.
• Because of its high sensitivity, contamination with extraneous DNA is a big problem.
184
Gene Mapping 8.6.1
8.5. DNA Sequencing and Deduced Functions
Historically, there were two important methods of DNA sequencing, the Maxam-Gilbert
Method and the Sanger Dideoxy Method. Today, only the Sanger method is in use. Both
methods require a single-stranded DNA, and they determine the sequence of up to several
hundred bases in one sitting.
We know our chromosomal DNA sequences, but we still need to determine which sequences
encode functional gene products, and what those gene products accomplish in the body.
Genes can be recognized in newly-sequenced DNA by telltale signs like the TATA box
(DNA = TATAAA, start codon (mRNA = AUG), stop codon (mRNA = UAA, UAG,
UGA), polyadenylation signal (mRNA = AAUAAA) and splice sites (Intron-exon junctions;
Introns begin with RNA = GU and end with AG; there are longer but degenerate consensus
sequences for these boundaries).
If you find a new gene, you can sometimes deduce its function.
• Genes, and the proteins they encode, come in families whose members are structurally
related.
• If the amino-terminus of the encoded protein is a signal sequence, the protein is most
likely a membrane protein or secreted protein.
• Hydrophobic sequences of about 20 amino acids in the encoded polypeptide suggest

membrane-spanning α-helices. - The presence of specific amino acid sequences may
suggest glycosylation or phosphorylation of a gene product.
8.6. Gene Mapping
Even with the complete genome sequence in hand, the mapping of genes is a major task in
modern genetic research. It is used to map:
• Genes for Mendelian disorders
• Genes for normal polymorphisms (blood groups, etc.).
• “Susceptibility genes” for multifactorial diseases (diabetes, Alzheimer).
• Genes affecting continuously variable traits (blood pressure, intelligence, divorce risk...)
185
8.6.1. Fluorescent in-situ Hybridization (FISH)
A strongly fluorescent cDNA probe is hybridized to a prophase or metaphase chromosome

spread. A banding technique is used, and this permits the assignment of the gene to a
specific chromosome band. This method can be used whenever a larger genomic probe (>
25 000 bp) is available. Sometimes, a cDNA probe can be used.
8.6.2. Deletion Mapping
Most disease-causing mutations are loss-of-function mutations that lead to a nonfunctional

or absent gene product. Occasionally, a very large deletion that obliterates the gene itself
and a variable amount of neighboring DNA (including, perhaps, some neighboring genes) is
the cause of an apparent single-gene disorder. If the deletion is large enough for detection
by high-resolution banding, the locus can be assigned to a chromosome band with an
accuracy of ± a few million base pairs. In other cases, a single-gene disorder is caused by a
chromosomal rearrangement when the breakpoint is within the gene.
8.6.3. Linkage Analysis
Genes that are close together on the same chromosome are genetically linked: They segregate
together during meiosis. With increased distance of the loci, however, there is an increased
chance of crossing-over. A distance on the chromosome with a recombination frequency of
1 % is called one centiMorgan. It corresponds to about 1 million base pairs of DNA.
Genes can sometimes be mapped if the locus of an unknown gene is close to that of another
gene with known position. Example: The genes for hemophilia and color blindness are about
10 centiMorgans apart on the X-chromosome; and the hemochromatosis gene is closely
linked with the genes for the HLA antigens on chromosome 6. The most important genetic
markers, however, are not genes but different anonymous DNA markers
• A classical restriction fragment length polymorphism (RFLP) is caused by a cleavage
site for a restriction endonuclease that is sometimes present and sometimes absent.
Two fragments of different length are generated that can be analyzed by Southern
blotting. These are rarely used.
• A variable number of tandem repeats (VNTR) is a short (2 to some dozen base pairs)
sequence that is tandemly repeated many times. Most VNTRs are not in the coding
regions of genes but in untranscribed spacers or introns. Good VNTRs are highly poly-
morphic, with many “alleles” in the population. Ideally, most people in the population
should be heterozygous for the VNTR. VNTRs are analyzed by Southern blotting or
PCR. These are the markers most often used. “DNA Fingerprinting” methods involve
analysis of VNTR analysis.
186
Cloning and Genomic Libraries 8.7
Mathematics: The recombination fraction τ is the probability of recombination be-

tween two loci (for example a disease gene and an RFLP). It has a numerical value between
0 and 1 and corresponds to the distance in centiMorgans.
The likelihood ratio can be calculated for different recombination fractions. It is defined as:
The probability that the observed association occurs if there is linkage (with an arbitrarily
assumed value of τ ) divided by the probability for this result if there is no linkage. A high
likelihood ratio means that the observed results are likely to be caused by linkage rather
than the random co-inheritance of two unlinked traits.
The lod score is the logarithm of the likelihood ratio. A lod score of >3 is taken as
reasonably good evidence for linkage. Formally, it corresponds to a likelihood ratio of 1000;
in reality, a better description is that a LOD score of >3 corresponds to the usual p<0.05
limit familiar from statistical analysis.
Linkage studies require large families with multiple affected family members. They can be
used even if absolutely nothing is known about the structure of the gene and its product,
but you need to know the inheritance pattern. If you don’t know on which chromosome the
gene is, you may have to make a genome-wide scan, tracking the inheritance of your gene
with a few hundred VNTRs scattered all over the 22 autosomes. Linkage studies can only
determine the approximate genomic location of a disease gene. The gene itself has to be
identified by the study of candidate genes in the relevant area and by DNA sequencing.
8.6.4. Candidate Genes
Some diseases give clues about the nature of the gene product. Many connective tissue
diseases, for example, are caused by abnormalities of extracellular matrix proteins (collagen,
elastin etc). If you have narrowed down the position of a disease gene by linkage studies,
you can obtain sequence information for genes in the region from DNA data banks. You
can then tentatively identify “suspicious” genes and see if your patients have mutations in
these candidate genes. This involves mutation scanning and/or the actual sequencing of the
candidate gene in the patients. However, not all mutations cause disease. You may stumble
on a normal polymorphism that has nothing to do with the disease!
8.7. Cloning and Genomic Libraries
DNA can be amplified by PCR. It can also be amplified by incorporation into a bacterium
where it is replicated along with the bacterial DNA. This is called cloning. A clone is a
“family” of cells that are descended asexually from a common ancestor. Foreign DNA (for
example human genomic DNA) is not incorporated into the bacterial chromosome but into
a cloning vector.
187
The cloning vector may be plasmid: a circular, double-stranded DNA that has a replica-
tion origin and that carries genes. Most bacteria naturally contain plasmids. Those plasmids
that are used as cloning vectors are R-factors that contain at least one gene for antibiotic
resistance. Also temperate bacteriophages, like λ phage, can be used as vectors. Many
cloning vectors are artificial constructs that were patched up from bits and pieces of plas-
mids and bacteriophages. The foreign DNA is ligated into the vector DNA in vitro. The
recombinant vector is then spirited into the bacterial host cell where it replicates along with
the bacterial DNA.
Cloning can be used to make a genomic library. This is a large collection of bacteria,
each containing a piece of human genomic DNA. In order to be useful, a genomic library
has to have every genomic DNA sequence represented in at least one bacterium. Simplified
procedure for constructing a genomic library with a plasmid vector:
1. Take an R-factor plasmid that contains a gene for ampicillin (type of weak penicillin)
resistance, and cleave its DNA with a restriction endonuclease. You have to use a
very selective restriction enzyme that cleaves the plasmid at only one site, outside the
resistance gene, creating a linear DNA with single-stranded cohesive ends.
2. Extract human genomic DNA and cleave it with the same restriction enzyme that
was used for the plasmid.
3. Mix the cleaved human and plasmid DNA, let them anneal with their sticky ends,
then add DNA ligase: human DNA and plasmid DNA become covalently linked into
a circle.
4. The engineered plasmid is brought into the bacterium by transformation. This is a
low-efficiency process but can be achieved in satisfactory yield in the presence of high
calcium concentration.
5. The selection of transformed clones is achieved by plating the bacteria on agar plates
in the presence of ampicillin. Only transformed bacteria can survive. Each colony is
a clone that contains a plasmid, hopefully with a piece of human DNA.
6. The colony pattern of the agar plate can be transferred to other agar plates. This is
called replica plating.
7. The colonies of a replica plate are lysed with NaOH. The denatured DNA is replica-
plated to nitrocellulose. After drying, the nitrocellulose filter is dipped into the solu-
tion of a probe which identifies a specific sequence of human DNA. This step is called
screening.
Plasmid vectors are good to propagate small pieces of DNA, up to 5000 bp or 5 kbp. Larger
chunks (≈ 15 kbp) can be cloned in λ phage: the “non-essential” phage genes are replaced
by an insert (= foreign DNA), and the engineered DNA is packaged into a phage particle in
vitro. Only DNA of the right size is packaged. The engineered phage is brought into the cell
188
cDNA Cloning and Expression Cloning 8.9
and propagated by lysogenic infection. Very large DNA pieces can be cloned in yeast cells
in the form of yeast artificial chromosomes (YACs), patched up from centromere sequences,
telomere sequences, and cloned DNA.
8.8. cDNA Cloning and Expression Cloning
Only 1–2 % of the DNA in a genomic library is coding. If you are only interested in coding
DNA, you better make a cDNA library. A cDNA is a double-stranded DNA copy of a
RNA, made by the retroviral enzyme reverse transcriptase. In genome sequencing, cDNAs
or fragments of cDNAs are referred to as expressed sequence tags (ESTs). Recipe:
1. Isolate mRNA by affinity chromatography on an oligo-dT column. The poly-dA tail
sticks to the immobilized poly-dT.
2. Add oligo-dT primers, reverse transcriptase, and deoxyribonucleotides. The oligo-dT
primer hybridizes with the poly-A tail, and the reverse transcriptase synthesizes the
cDNA.
3. Ligate the cDNA into a vector, then proceed as usual.
The tissue of origin doesn’t matter for a genomic library because all cells have the same
genome. But it is important for a cDNA library because different cells express different
genes.
Ordinarily, neither cloned genomic DNA nor cDNA is expressed in bacteria. For expression
cloning, you have to ligate:
• A human cDNA. Genomic DNA won’t do because bacteria cannot remove introns.
• A bacterial promoter. You need a strong promoter, or one that can be regulated at
will. For example, with the promoter of the lac operon, the cloned gene is expressed
only when the bacteria are kept on a glucose-free, lactose-rich diet.
• The cDNA of a bacterial ribosome-binding sequence (Shine-Delgarno sequence)
has to be ligated between the cDNA and the promoter.
• A bacterial signal sequence can be included if desired. In this case the bacteria will
secrete the protein.
With expression cloning, you can turn bacteria into lucrative factories for hormones, clotting
factors, cytokines, or any other protein. Only post-translational modifications (phospho-
rylation, glycosylation) are problematic because bacteria don’t have the right processing
enzymes. Many genetically engineered therapeutics are on the market, including human
insulin, growth hormone, interferon, clotting factor VIII, erythropoietin and tissue-type
plasminogen activator, and humanized antibodies.
189
8.9. Site-Directed Mutagenesis and Protein Engineering
The base sequence of cloned DNA can be changed intentionally. Cloned DNA is isolated with
the help of a restriction endonuclease, modified by enzymatic methods, and re-inserted into
a cloning vector. Several methods are available for the introduction of targeted mutations.
If you use expression cloning, you can produce proteins with amino acid substitutions or
other minimal changes. You can also make truncated proteins that are missing part of
the polypeptide, for example soluble variants of the spike proteins of the AIDS virus that
interfere with AIDS infection because they jam virus receptors on the cell surface. Domain
shuffling is the patching-together of parts from different genes to make a new, recombinant
gene (and protein).
Example: If you link the DNA-binding domain of the androgen receptor with the retinoic
acid-binding domain of the retinoic acid receptor, and engineer this into your tissues, you
can improve your virility by gobbling vitamin A.
8.10. Use of DNA Diagnostics
The molecular diagnosis of disease genes and normal polymorphisms is the fastest-growing
area of modern medical technology. Uses:
• Diagnosis of Mendelian disorders
• Carrier detection in relatives of patients
• Newborn screening
• Population-based screening
• Predictive testing for late-onset diseases
• Susceptibility testing for multifactorial diseases
• Prenatal and pre-implantation diagnosis
• Paternity testing
• DNA fingerprinting of criminal suspects
Technical problems for DNA diagnostics include:
• Most single-gene disorders show substantial allelic heterogeneity. Therefore, genetic
tests must screen for many mutations.
• Common diseases are usually multifactorial. Therefore, genetic testing allows only a
statistical risk estimate.
190
Southern Blotting with Allele-Specific Probes 8.10.1
8.10.1. Southern Blotting with Allele-Specific Probes
Allele-specific probes (ASOs) can be used when both the normal gene sequence and the
mutation are known: there is no allelic heterogeneity and no locus heterogeneity. Procedure
for sickle cell testing:
1. Make (or buy) two synthetic oligonucleotide probes each of the same length (>18 bp),
one complementary to the sequence of the sickle cell mutation and one complementary
to the corresponding normal sequence.
2. Treat the patient’s DNA with a restriction endonuclease that cuts left and right of
the probed sequence.
3. Divide the restriction digest in two aliquots, and electrophorese them separately in
two lanes of a gel. Then blot to nitrocellulose.
4. Apply the probe for the normal sequence to one lane, and the probe for the sickle cell
mutation to the other. The stringency has to be adjusted carefully because the two
probes differ by only one base.
You can distinguish between homozygous normal (only the normal probe binds), homozy-
gous affected (only the sickle cell probe binds) and heterozygous (both probes bind). In
this example the restriction fragments have the same length and the same electrophoretic
mobility.
Example: Adenomatous
Example: polyposis coli,polyposis
Adenomatous an autosomal
coli, andominant
autosomalcancer susceptibility
dominant cancer syn-
susceptibility
drome. Will the children insyndrome.
generationWill
III the
getchildren
cancer?in generation III get cancer?
II
III
Southern Blots
Example: Prenatal diagnosis of β-thalassemia. Has the fetus II-3

inherited the disease? Is he a carrier? Is the unaffected daughter II2 a carrier, or
homozygous normal?
191
I
II
Southern blots
III
Southern Blots
Southern Blots
Example: Prenatal diagnosis of β-thalassemia. Has the fetus II-3
inherited
Example: Prenatalthe disease?
diagnosis Is he a carrier?
of β-thalassemia. HasIsthe
thefetus
unaffected daughter
II3 inherited II2 a carrier,
the disease? Is or
homozygous
he a Example:
carrier? Is the normal?
unaffected daughter II2 a carrier, or homozygous
Prenatal diagnosis of β-thalassemia. Has the fetus II-3 normal?
inherited the disease? Is he a carrier? Is the unaffected daughter II2 a carrier, or
homozygous normal? I
I
II
II
Southern blots
Southern blots
Example: Duchenne muscular dystrophy, a severe X-linked recessive

Example: Duchenne muscular dystrophy,
Example:
muscle Duchenne
disease. muscular
Are II3 acarriers?
severea X-linked
II2 anddystrophy, recessiverecessive
severe X-linked muscle disease. Are
II2 and
muscle II3 carriers?
disease. Are II2 and II3 carriers?
I I
II II
Southern Blots
Southern Blots
77
In the case of small insertions or deletions, only one probe is needed because the restriction
77
fragments differ in length. Allele-specific or organism-specific oligonucleotides are probably
more often used in Dot blot analysis thereby saving the time-consuming electrophoresis
step.
Limitations: Most single-gene disorders show extensive allelic heterogeneity i.e., many
different mutations. Each set of allele specific oligonucleotides will only give you information
on one specific mutation. You cannot use allele-specific probes in these cases unless you
have identified the mutation in the affected family first, or are prepared to repeat the
experiment with many different set of oligonucleotides (and then sequencing would probably
be faster and cheaper). Note - A single genetic test sometimes unavailable for genetic disease
testing. See the GeneTest website for available tests and clinical testing sites for individual
conditions. However, see the section on microarrays.
192
Dot-Blotting 8.12
8.11. Use of PCR.
PCR requires less DNA than Southern blotting. It is therefore the preferred procedure for
prenatal diagnosis and pre-implantation diagnosis.
Example: Cystic fibrosis is a severe autosomal recessive disease that is most often caused
by a 3-basepair deletion. If both parents carry this mutation (disease risk: 25 %), you can
do prenatal diagnosis by Southern blotting. Alternatively, you can use PCR:
1. Amplify a short segment of the fetal DNA (<100 bp) with a primer pair that frames
each potential mutation site.
2. Separate the PCR products by gel electrophoresis. Run controls with the normal PCR
product and the PCR product with the deletion in separate lanes of the same gel.
Then stain with ethidium bromide.
If the fetal DNA yields only the normal PCR product, the fetus is homozygous normal;
if only the shortened fragment is produced, it is homozygous affected; if both bands are
present, it is heterozygous.
In the diagnosis of point mutations, the two PCR products have the same length and cannot
be distinguished on the gel. Sometimes, a restriction enzyme recognition site is changed by
the mutation; then restriction enzyme digest of the PCR product will produce fragments
of different lengths. Otherwise, allele-specific probes have to be used.
PCR can also be used in cases where we try to detect presence or absence of a given DNA
piece. First example in HIV infection: primers specific for the HIV genome will only produce
a fragment if the DNA is derived from cells infected with HIV. Second example is diseases
caused by deletion of one or more exons in a gene. Primers specific for each exon are used in
the PCR, and presence of a fragment will demonstrate presence of the exon in the starting
material. Especially in these last applications is there a major problem in securing against
accidental contamination with extraneous DNA. The product from last time you performed
the procedure can sometime float around in the laboratory in the form of microdroplets.
8.12. Dot-Blotting
Dot blotting with DNA samples Both Southern blotting and PCR with electrophoretic
separation provide information about the length of a restriction fragment or PCR product.
When this information is not needed, and you can use dot-blotting. Procedure:
1. Treat the extracted DNA with a rarely-cutting restriction endonuclease. The probed
sites have to remain intact!
2. Denature the DNA, and apply it in single dots to two nitrocellulose filters.
193
3. Probe one filter with a probe for the normal sequence, and the other one with a probe
for the mutation.
Dot blotting takes far less time than the other procedures. The DNA of many people
can be tested simultaneously on the same nitrocellulose filter. Therefore, it can be used
for screening programs. Like other allele-specific methods, the usefulness of dot-blotting is
limited by genetic heterogeneity. Also, this method tests for only one DNA sequence variant
at a time.
Dot blotting with RNA samples: The purpose of this procedure is similar to the purpose
of Northern blot, i.e., to measure the expression level of a gene in a sample from a tissue.
Procedure:
1. Purify total mRNA from the tissues of interest
2. Spot the same amount of mRNA from each sample onto a filter
3. Probe with a cDNA , a single exon, or a long oligonucleotide
4. The resulting signal will be proportional to the concentration of the specific mRNA
in the sample
Because this procedure omits the gel electrophoresis step of a Northern blot, it is not
possible from position information to see that there has been aberrant hybridization to
another mRNA, e.g., from a similar gene (you want to check β-hemoglobin; do you get
hybridization to α-hemoglobin?). Therefore, the specificity of a probe for Dot blot should
be tested in Northern blot before usage in Dot blot. Advantage of Dot-blot for mRNA is
speed, larger number of samples on a blot, and fewer steps necessary increasing the chance
that the mRNA survives to the time of hybridization (mRNA is a difficult material to work
with, RNases are rampant).
8.13. OBJECTIVES IN SUMMARY
1. Outline the procedure of DNA sequencing by the Sanger-Dideoxy and Maxam-

Gilbert methods.
2. Define what is meant by the terms palindromic sequence, cleavage specificity, blunt
and sticky ends, cloning site, high/low frequency cutters with respect to restriction
endonucleases. Name and describe the function of DNA ligase, reverse transcriptase,
polynucleotide kinase, topoisomerase, RNase and DNase.
3. Outline the procedure of Southern blotting and describe the general use of the
method.
194
OBJECTIVES IN SUMMARY 8.13
4. Outline the procedure of polymerase chain reaction (PCR) and describe the general
use of the method.
5. Define what is meant by “restriction fragment length polymorphism” and provide an
example of when RFLP analysis would be required to make a clinical decision.
6. Provide a rationale for the use of RFLP analysis and analysis of a VNTR site (Variable
Number of Tandem Repeats) in linkage studies.
7. Explain how somatic cell hybrids, in-situ hybridization methods, and linkage studies
are useful in gene mapping.
8. Define the recombination fraction and LOD scores.
9. List the steps in cloning foreign DNA into an R-factor plasmid and in λ phage.
10. Compare the procedures for generation of a genomic library, a cDNA library and
an expression library; and describe how each library can be screened for a clone of
interest.
11. Give examples of proteins produced using genetic technology, and their therapeutic
value.
12. Describe procedures for site-directed mutagenesis in cloned DNA.
13. Explain the rationale for using carrier-testing, pre-symptomatic testing, population-
based heterozygote screening, prenatal diagnosis and pre-implantation diagnosis.
14. Specify examples of when Southern blotting with allele-specific oligonucleotide
probes is useful to diagnose a known mutation.
15. List the advantages and disadvantages of using PCR-based methods for genetic diag-
nosis.
16. List the advantages and disadvantages of linkage studies for the diagnosis of genetic
diseases.
17. Calculate carrier probabilities and disease risks for Mendelian disorders using link-
age studies and closely linked RFLPs.
18. Describe the advantages and limitations of current gene therapy and antisense proto-
cols, including vector design, and clinical rationale.
19. Explain how germline gene manipulation is accomplished, and how knockout and
transgenic mice are useful in medical research.
195
Part II.
Semester one, Mini II

ROSS UNIVERSITY SCHOOL OF MEDICINE
BIOCHEMISTRY AND GENETICS I
Handout 11
9. Glycolysis: Splitting glucose in half
GLYCOLYSIS, TCA-CYCLE, OXIDATIVE PHOSPHORYATION
I. Overview
9.1. Overview
Glycolysis, TCA cycle and oxidative phosphorylation are the three stages
Glycolysis, TCA cycle and oxidative phosphorylation are the three stages in the catabolism
in the catabolism of glucose to CO2 and H2O:
of glucose to CO2 and H2 O:
Glucose
Glycolysis
2
2ATP
2 Pyruvate
2 Pyruvate 2 6 O2 12 H2O
+
NADH, H NAD+
2CO2 6 FADH2 } { FAD
2 Acetyl-CoA
2 34 ADP, 34 Pi 34 ATP, 34 H2O
TCA Cycle Respiratory chain

Oxidative phosphorylation
2 GTP 4 CO2
1. aInmolecule
In glycolysis glycolysis, a molecule
of glucose (6 carbons)ofis glucose
converted (6
to 2carbons)
molecules is
of converted
pyruvate (3 to 2
molecules
carbons).ofThis
pyruvate
pathway(3produces
carbons). This
2 ATP pathway
(from ADP +produces
inorganic 2phosphate)
ATP (fromandADP
2 +
+
NADH + H (from NAD ). Glycolysis takes place in the cytoplasm of all cells.
inorganic phosphate)
+ and+ 2 NADH (from NAD ). Glycolysis takes place in the
cytoplasm of all cells.
Pyruvate 2.
entersPyruvate
the mitochondria.
enters theThe mitochondrial
mitochondria.pyruvate
The dehydrogenase
mitochondrial converts
pyruvate
pyruvate to an acetyl residue (2 carbons) which becomes linked to coenzyme A (CoA).
dehydrogenase converts pyruvate to an acetyl residue (2 carbons) which
CO2 is released, and one NAD+ is reduced to NADH + H+ for every pyruvate. +
becomes linked to coenzyme A (CoA). CO2 is released, and one NAD is
+
In the TCA to
reduced NADH
cycle (Krebs
+ Hcycle,
for citric
everyacid cycle), acetyl-CoA reacts with oxaloacetate (4
pyruvate.
carbons)
3. toIn form
the citrate (6 carbons).
TCA cycle Thecycle,
(Krebs remaining
citricreactions of the cycle
acid cycle), regenerate
acetyl-CoA reacts
with oxaloacetate (4 carbons) to form citrate (6 carbons). The remaining
reactions of the cycle regenerate oxaloacetate from citrate. The TCA cycle forms
2 CO2, 1 GTP, 3 NADH and 1 FADH199 2.
4. The respiratory chain in the inner mitochondrial membrane uses
molecular oxygen to re-oxidize the reduced coenzymes, NADH and FADH2.
These reactions are coupled to ATP synthesis by oxidative phosphorylation.
oxaloacetate from citrate. The TCA cycle forms 2 CO2 , 1 GTP, 3 NADH + H+ and
1 FADH2 .
The respiratory chain in the inner mitochondrial membrane uses molecular oxygen to re-
oxidize the reduced coenzymes, NADH + H+ and FADH2 . These reactions are coupled
to ATP synthesis by oxidative phosphorylation.
9.2. Glycolysis
9.2.1. Reactions of glycolysis
The glycolytic reactions are summarized on page 300 of the Meisenberg & Simmons
book. Some highlights:
• Only the hexokinase, phosphofructokinase, and pyruvate kinase reactions are irre-
versible under physiological conditions.
• Hexokinase is the committed step for glucose metabolism in general, while phospho-
fructokinase is the committed step for glycolysis.
• Two ATP per molecule of glucose are consumed in the hexokinase and phosphofruc-
tokinase is the committed step for glycolysis
• Two energy-rich intermediates are formed in glycolysis: 1,3-bisphosphoglycerate and
phosphoenolpyruvate. These are used for ATP synthesis by substrate-level phospho-
rylation. Net yield is 2 ATP per molecule of glucose.
9.2.2. Regulation of glycolysis
• Regulation reactions are catalyzed by hexokinase, phosphofructokinase, and pyruvate

kinase.
• In many tissues the enzymes catalyzing the irreversible reactions are induced by in-
sulin and repressed by insulin antagonists (glucagon in the liver).
• The most important site for short-term regulation is phosphofructokinase. In the liver,
its activity is:
– inhibited by ATP and stimulated by AMP (allosterically).
– inhibited by citrate (allosterically) - inhibited by low pH.
– Stimulated by insulin and inhibited by glucagon.
200
Anaerobic glycolysis 9.3
• Other regulated steps: Hexokinase is product-inhibited by glucose 6-phosphate. Pyru-

vate kinase is allosterically inhibited by ATP.
9.2.3. Inhibition of glycolysis
Fluoride ions inhibit enolase. Sodium fluoride is used in the laboratory to inhibit glycolysis
in blood samples used for blood glucose determination.
Arsenate uncouples substrate-level phosphorylation because it competes with phosphate in

the glyceraldehyde 3-phosphate dehydrogenase reaction, forming an unstable product
with arsenate instead of phosphate in position 1 of 1,3-bisphosphoglycerate. ‘Un-
coupling’ implies that the reactions of the pathway can proceed, but without ATP
synthesis.
Inherited partial deficiencies of glycolytic enzymes are occasionally seen, most often pyru-
vate kinase. This causes hemolytic anemia.
9.2.4. Anaerobic glycolysis
Glycolysis can produce ATP in the absence of oxygen, but only if NADH + H+ is converted
back to NAD+ in the lactate dehydrogenase reaction:
NADH + H+ NAD+
- -
COO COO
C O HC OH
Lactate dehydrogenase
CH3 CH3
The LDH reaction is reversible under aerobic conditions but is irreversible under anaerobic
conditions when NADH + H+ accumulates. Lactate is a metabolic dead end. It can re-enter
the metabolic pathways only via pyruvate.
Anaerobic glycolysis, with formation of lactic acid, is the only metabolic pathway that can
produce ATP under anaerobic conditions in humans. It is important in:
• Cells lacking mitochondria (erythrocytes).
• Cells suffering from hypoxia (ischemic tissue).
• Exercising muscle
Lactic acidosis is the most common form of metabolic acidosis. All conditions in which
oxidative metabolism is impaired (pulmonary failure, circulatory collapse, cyanide poison-
ing...) cause lactic acidosis.
201
1. State the importance of glycolysis for aerobic metabolism of glucose and the tissues
and subcellular location where glycolysis takes place.
2. Know the major reactions and intermediates of glycolysis.
3. Define the term “substrate level phosphorylation”.
4. Write the overall balance of aerobic and anaerobic glycolysis including the number of
ATP and NADH + H+ formed.
5. Name the irreversible reactions of glycolysis and the way they are regulated physio-
logically and the most important situations in which lactic acid accumulates, giving
reasons for this accumulation.
202
10. Plasma Proteins
10.1. Plasma Proteins: Overview
Plasma is obtained by the centrifugation of whole blood. It is typically 51–55 % of the blood
volume in males and 57–59 % in females, the remainder being the packed red cell volume,
corresponding to the hematocrit. Plasma contains 6–8 % proteins, including fibrinogen and
clotting factors. Serum is obtained by removing fibrin and clotting factors from plasma.
10.1.1. Functions of plasma proteins
Colloid-osmotic (oncotic) pressure. Important to counteract pressure filtration of plasma

into the interstitial spaces. Necessary to prevent edema.
Transport of small metabolites Important for the transport of water-insoluble compounds
(cholesterol esters, retinol), and to prevent the renal excretion of valuable water-
soluble molecules and ions (hemin, iron, cobalamin). Also transport of many hor-
mones: steroid and thyroid hormones, vitamin D.
Defense The immunoglobulins and the components of the complement system are the most
notable examples.
Blood coagulation. Most of the clotting factors are proteases which eventually convert
soluble fibrinogen to insoluble fibrin.
Protease inhibitors regulate proteolytic processes, in blood clotting and inflammation.
Sources of plasma proteins: Most come from the liver, except immunoglobulins which are
made by plasma cells.
Half-lives are a few days to a few weeks. Pinocytosis is a non-selective route of removal and
degradation. In addition, plasma glycoproteins gradually lose the sialic acid residues from
the ends of their oligosaccharide chains while in circulation, and the resulting asialoglyco-
proteins are taken up into the liver by receptor-mediated endocytosis. With the exception
of albumin, almost all plasma proteins are glycoproteins.
Chemistry: Molecular weights range from <60 000 to over 700 000. Most have a pI in the
acidic range. Acidic pI combined with high molecular weight prevents renal excretion.
203
Major Plasma Components:

Protein Fraction Concentration (mg/dL) Function
Transthyretin Prealbumin 10–40 Binds T4, also
with RBP
Albumin Albumin 3500–5000 Colloid osmotic
pressure, binds
many ligands
α1-acid glycoprotein α1 55–140 ?
Retinol-binding protein α1 3–6 Transports retinol
α1-antiprotease α1 200–400 Protease inhibitor
Thyroxine-binding globulin α1 1–2 Binds thyroid
hormones
Transcortin α1 3–3.5 Binds glucocorti-
coids
Ceruloplasmin α2 15–60 Copper trans-
port?
Haptoglobin α2 100–200 Binds hemoglobin
α2 -macroglobin α2 150–420 Protease inhibitor
Hemopexin β 50–100 Binds heme
Fibrinogen β 200–400 Clot formation
C-reactive γ <1 Binding/removal
of antigens?
Immunoglobulins γ, also α2 & β 700–1500 Binding and re-
moval of antigens
10.2. Separation of Plasma Proteins
Electrophoresis is the most common procedure. The plasma proteins move to the anode at
mildly alkaline pH (8.6). After staining, the relative amounts of proteins in the different
fractions can be determined by densitometric scanning: 5 fractions are separated:
1. Albumin (55–68 %)
2. α1 -globulins (6–7 %)
3. α2 -globulins (8–9 %)
4. β-globulins (13–14 %)
5. γ-globulins (11–12 %)
Only the albumin fraction is reasonably homogenous. All other fractions are mixtures of
several proteins.
204
Transport Proteins 10.2.2
Immunoelectrophoresis combines electrophoretic separation with the use of antibodies to

identify individual plasma proteins. It is often used to identify the nature of an abnormal
protein peak (“paraprotein”) in patients with myeloproliferative disorders.
10.2.1. Albumin
Single polypeptide with 585 amino acid residues, 17 disulfide bonds, no carbohydrate. MW
66 000 Da, pI = 4.8. High water-solubility, low viscosity of aqueous solutions. Total amount
in the body: 250–350 g. Concentration in plasma: 3.5 g/dL. Also present in interstitial fluid:
40 % of total albumin is in plasma, 60 % in interstitium. Half-life: 17 d. Functions:
Osmotic effect: Albumin provides 75–80 % of the colloid-osmotic (oncotic) pressure of the
plasma. A decrease of the albumin concentration below 2 % causes edema.
Transport: Albumin binds fatty acids, bilirubin, thyroxine, steroid hormones, dicoumarol,
penicillin, aspirin, heme, calcium magnesium etc. There are many different binding
sites for these ligands. Binding is non-covalent and reversible.
Protein binding is important when plasma drug levels are determined in the clinical labo-
ratory: in patients with decreased serum albumin, an increased proportion of the drug is
in the free, unbound form. Chemical assays determine the total drug level, but only the
unbound form of a drug (or hormone) is biologically active.
Analbuminemia is a rare genetic disorder in which albumin is absent or greatly reduced.
With abnormalities in lipid metabolism but, surprisingly, little or no edema.
10.2.2. Transport Proteins
Transthyretin (prealbumin) migrates faster than albumin in electrophoresis. Binds thy-

roxine. Binds the RBP-retinol complex and prevents its renal excretion.
Retinol binding protein (RBP) transports retinol from the liver to other tissues.
Thyroxine binding globulin (TBG) transports thyroxine and T3. 100- fold higher affinity
for T4 than prealbumin.
Transcortin transports corticosteroids.
Sex Hormone Binding Globulin binds androgens and estrogens.
Haptoglobin binds hemoglobin. Prevents the renal excretion of hemoglobin after intravas-
cular hemolysis.
Hemopexin binds heme and hematin. Prevents their renal excretion.
Transferrin transports iron.
205
The hormone-binding proteins are physiological buffers that regulate the plasma concentra-
tion of free (unbound) hormone. Others (transferrin, hemopexin, haptoglobin) bind their
ligand and are then removed by endocytosis, followed by utilization or degradation of the
ligand and degradation or recycling of the carrier protein.
In intravascular hemolysis, oxyhemoglobin dissociates into αβ-dimers which would be

lost through the kidneys in the absence of haptoglobin. Also, heme tends to dissociate from
the apo-protein, and the free heme, after oxidation to hematin, binds to hemopexin. The
hemoglobin-haptoglobin complex is taken up and degraded by reticuloendothelial cells, and
the hematin-hemopexin complex by hepatocytes.
The levels of haptoglobin and hemopexin are decreased in patients with intravascular hemol-
ysis. This is used to differentiate hemoglobinuria from myoglobinuria in patients with a
positive test for “blood” in the urine. The urine test cannot distinguish between hemoglobin
and myoglobin, but haptoglobin is low only in hemoglobinuria.
10.2.3. Protease Inhibitors
α1 -antiprotease (α1-antitrypsin) is the major component of the α1 -globulin fraction.

Also present in external secretions (bronchial mucus). Inhibits a variety of serine proteases.
Important to limit proteolysis during inflammation. α1 -antiprotease deficiency is an autoso-
mal recessive trait that affects 1 in 7000 Caucasians (1 in 700 in Sweden). With early-onset
lung emphysema, also infantile liver cirrhosis in some patients. Homozygotes (identified by
genetic screening) have to avoid smoking. Also, a methionine residue in α1 -antiprotease
which is necessary for protease binding is oxidized by cigarette smoke.
Laboratory tests for α1 -antiprotease deficiency include plasma protein electrophoresis (de-
creased α1 -peak) and trypsin inhibitory capacity (TIC) of plasma.
α2 -macroglobulin is a major component of the α2 -globulin fraction. Wide range of anti-

proteolytic activity. No human deficiency states are known.
10.3. Plasma Proteins in Disease States
Acute phase reactants increase in response to inflammation or tissue necrosis in infec-

tions, trauma, surgery, neoplasms, autoimmune diseases etc. They include α2 -antiprotease,
haptoglobin, ceruloplasmin, fibrinogen and C-reactive protein.
Characteristic electrophoretic patterns occur in various diseases.
206
Plasma Components for Clinical Use 10.3.1
Electrophoretically observed changes from normal concentrations:

Condition Albumin α1 α2 β γ
Liver Cirrhosis ↓ ↑↑
Immediate response pattern ↓ ↑
Delayed response pattern ↓ ↑ ↑
Nephrotic syndrome ↓↓ ↑↑ ↓↓
Monoclonal gammopathy ↓ ↑↑↑
Protein malnutrition ↓↓ ↓ ↓ ↓ ↓
Iron deficiency anemia ↑
Albumin concentrations are decreased in many pathological conditions. Only dehydration
results in an increase.
Relative or even absolute increases in α2 are typical in nephrotic syndrome and protein-
losing enteropathy. In these conditions, plasma are lost in relation to their molecular size:
α2 -macroglobulin is retained because it is big (MW 725 000 Da). The α2 -fraction is also
increased when haptoglobin (an acute phase reactant) is increased in response to stress.
Individual Proteins
• Iron-deficiency anemia leads to increased transferrin concentration (increased β-
globulin fraction). However, the iron-saturation of transferrin (normal = 30 %) is
increased in hemochromatosis and decreased in iron-deficiency. People with very low
transferrin levels or congenital atransferrinemia (rare) are susceptible to bacterial
sepsis because iron is a limiting factor for bacterial growth in the blood plasma.
• Ceruloplasmin is a copper-containing protein of uncertain function. Decreased in
Wilson’s disease (hepatolenticular degeneration), an inherited defect of copper ex-
cretion into bile.
• C-reactive protein (CRP) is the most sensitive acute phase reactant. Levels rise
profoundly during infection. Its function is unknown, but it binds to some components
of bacterial cell surfaces (including pneumococcal C-protein), activates complement,
binds to T-lymphocytes, inhibits clot retraction and platelet aggregation.
• Certain proteins not normally present in plasma are useful as markers for specific
diseases. α-fetoprotein, which is normally present in fetal plasma but not in adults,
is found in most patients with hepatocellular carcinoma.
10.3.1. Plasma Components for Clinical Use
Fresh Frozen Plasma is used for hypovolemia, shock, and clotting disorders. It requires
20 min for thawing.
207
Cryoprecipitate is enriched in factor VIII and fibrinogen. Used for clotting disorders. Avail-
able as lyophilized powder.
Albumin is available in 5 % and 35 % solutions. For hypovolemia, shock, extensive burns,

cerebral edema, protein-losing conditions. Practically no risk of hepatitis transmission.
Immune serum globulin is available as 16.5 % solution for i.m. injection. For hypo-gammaglobulinemia,
also for prophylaxis of viral hepatitis. Also plasma-free blood cells (RBCs, platelets,
granulocytes) are, of course, available to the modern physician.
10.4. Clinical Enzymology
Principle: Most of the diagnostically useful serum enzymes are intracellular enzymes that
are released into the blood only when the cells get damaged. The enzymes are cleared from
the circulation with half-lives of one to several days. A diagnostically useful enzyme should
have higher or lower concentration due to a specific disease or condition. Many clinically
useful enzymes have isoenzymes forms: Isoenzymes are chemically and biologically distinct
forms of an enzyme, often tissue-specific. They catalyze the same reaction but can be
distinguished by electrophoresis or differential sensitivity to inhibitors.
Mechanism: Tissue enzymes can be released by:
Tissue necrosis: Myocardial infarction, hepatitis, acute pancreatitis.
Increased membrane permeability: Muscular dystrophy, dermato-myositis, angina pec-

toris.
Increased Tissue Source or Release from Tissue: Neoplasms, psoriasis, Paget’s disease,
healing fractures.
Impaired Enzyme Excretion: Obstructive jaundice.
Examples:
Plasma cholinesterase (“ pseudocholinesterase”) is a normal constituent of plasma. It can

degrade certain drugs, including cocaine. Uses:
• Cholinesterase is inhibited in organophosphorus poisoning (insecticides, nerve

gases).
• Activity is depressed in liver diseases (hepatitis, cirrhosis).
208
Clinical Enzymology 10.4
• Cholinesterase activity should be determined in patients to be treated with the

muscle relaxant succinylcholine. Cholinesterase normally inactivates the drug,
but is deficient in some patients.
Alanine transaminase (ALT) and aspartate transaminase (AST) are enzymes of amino
acid metabolism that are most abundant in the liver. Aspartate transaminase and to
a lesser extent alanine transaminase also occur in other tissues such as skeletal muscle
and myocardium. Both enzymes are elevated in liver diseases, AST also after acute
myocardial infarction.
Alkaline phosphatase (ALP) is increased in 2 types of diseases:
Osteoblastic bone diseases. ALP is an osteoblast enzyme that promotes bone miner-
alization by hydrolyzing pyrophosphate. Plasma levels are increased in all bone
diseases with increased osteoblastic activity: healing fractures, osteitis defor-
mans, osteomalacia, rickets, hyperparathyroidism, bone tumors. High levels in
growing children and pregnant women during 3rd trimester.
Obstructive jaundice The role of alkaline phosphatase in the liver is not known.
Besides obstructive jaundice, space-occupying hepatic lesions due to carcinoma,
tuberculosis etc. Often result in increased plasma ALP.
Isoenzymes: Normal sources of ALP in plasma are bone, liver, intestine, and placenta
(3rd trimester of pregnancy). Enzymes from these 4 sources differ in electrophoretic
mobility, heat stability, and sensitivity to inhibitors. The clinically important distinc-
tion between bone and liver isoenzymes is possible by heating to 56 °C for 15 min.
This inactivates the bone enzyme more than the liver enzyme.
Acid phosphatase (ACP) occurs in normal prostatic tissue. A different isoenzyme is present
in erythrocytes. Used as a marker for metastatic prostatic carcinoma. Not usually ele-
vated in early stages of prostatic cancer. The prostatic enzyme is inhibited by tartrate,
the RBC enzyme by copper salts.
Lactate dehydrogenase (LDH) is widely distributed, with high levels in myocardium, kid-
ney, liver, muscle. Increased plasma levels are useful in the diagnosis of myocardial
infarction, muscular dystrophy, and pulmonary infarction (increased LDH with nor-
mal AST 1–2 days after an episode of chest pain), and in monitoring the response to
cancer therapy. Isoenzymes: LDH consists of 4 subunits. 2 types of subunit occur, H
(in heart) and M (in skeletal muscle), which form 5 isoenzymes. The isoenzymes are
numbered according to their electrophoretic mobility: LDH-1 has the greatest anodic
mobility, LDH-5 is slowest. Normal plasma contains mostly LDH-4 and -5.
Isoenzyme Composition Myocardium Erythrocytes Skeletal Muscle Liver Kidney
1 H4 ++++ +++ - - +
2 H3M ++++ +++ - - +
3 H2M2 + + + + ++
4 HM3 - - ++ ++ ++
5 H4 - - ++++ ++++ ++
209
After acute myocardial infarction, LDH-1 is higher than LDH-2: flipped LDH.
Creatine kinase (CK) is present in high concentration in skeletal muscle and myocardium,
also in brain. Very little in other organs, none in the liver. It is a dimeric enzyme,
formed from M subunits (skeletal muscle) and/or B subunits (brain).
CK-1: BB, in brain
CK-2: MB, in myocardium
CK-3: MM, in skeletal muscle, also myocardium
CK-1 moves fastest during electrophoresis. CK in normal plasma is almost exclusively
CK-3. CK-3 is elevated in muscular dystrophy, after surgery, and after i.m. injections.
Elevations of CK-2 are diagnostic for myocardial infarction. Elevations of serum CK-1
are rarely observed, even after cerebrovascular accidents.
α-Amylase and lipase leak from the pancreas into the bloodstream in acute pancreatitis.
Also elevated in patients with bowel infarction or perforation.
Acute myocardial infarction leads to elevations of CK, LDH and AST. Typical time course:
5
Elevation
above
CK
normal (1) 4
(Ratio of 3 AST
Activities)
2 LDH
1 Normal Limit
1 2 3 4 5 6 7 8
Days after chest pain episode
The “flipped LDHcan often be observed during the first few days after MI only. If CK
remains elevated beyond day 2, it is usually only the CK-3 isoenzyme.
1. Describe the normal pattern of plasma proteins obtained by electrophoresis.

2. Name the binding specificities of serum albumin, transthyretrin, retinol binding pro-
tein, the hormone binding proteins and haptoglobin and hemopexin.
3. Describe the clinical consequences of α1 -antitrypsin deficiencies.
4. Name the acute phase reactants and the changes of plasma proteins in infection.
210
Objectives in summary 10.5
5. Describe the plasma protein abnormalities as seen in electrophoresis, in patients

with acute diseases, chronic diseases, protein-losing conditions and monoclonal gam-
mopathies.
6. List the therapeutic uses of fresh frozen plasma, cryoprecipitate and albumin.
7. Name diseases for which the determination of serum enzymes is important, includ-
ing plasma cholinesterase, the transaminases, alkaline and acid phosphatases, lactate
dehydrogenase, γ-glutamyltransferase and amylase.
8. State the time frame for the elevation of CK, LDH and AST after acute myocardial
infarction.
211
11. Blood Coagulation
11.1. The Biochemistry of Blood Coagulation
Thrombosis is the term used to describe clotting of blood. This process involves the
formation of a platelet plug at the site of injury, and deposition of a network of fibrin protein.
Alternatively, thrombosis which occurs internally can lead to disease states. Patients with
inherited deficiencies of clotting factors or acquired deficiencies by environmental effects
have poor clinical indications of blood coagulation. Thrombosis is also the cause of many
age-related illnesses. Clotting is activated with increasing age when coagulation factors
levels and blood vessel weakness present an increased clotting risk.
The hematologist needs a basic science understanding of blood clotting as well as a focused
approach to family medical history, lab test data, and knowledge of how to extend these
analysis by further measurement of specific proteins and DNA sequences when required.
11.1.1. Primary hemostasis (platelet-plug formation)
The platelets are highly structured anucleate cell bodies in blood at a concentration of
1.5–4 × 105 µl−1 . Primary hemostasis is triggered by a variety of signals from damaged
or activated vascular endothelial cells and/or exposure of the underlying subendothelial
matrix. Platelets first adhere to the signaling site via adhesive ligands. The main interac-
tion is between platelet membrane receptor glycoprotein Ib-IX and the giant polymer von
Willebrand factor (vWF).
Adherent platelets flatten and activate membrane fibrinogen receptors (glycoprotein IIb-
IIIa) that bind plasma fibrinogen (Fb). The aggregated platelets are activated by turning
inside-out and exposing negatively charged phospholipids (esp. phosphatidyl serine and
phosphatidyl inositol).
11.1.2. Secondary hemostasis (fibrin clot formation)
Blood shear will disintegrate platelet plugs unless a fibrin net forms. The fibrin net is
produced by a complex sequence of enzymatic activities catalyzed by activated forms of
the factors named below. These reactions constitute a proteolytic cascade, in which one
213
enzymatic reaction catalyzes the activation of the following reaction in a chain - leading to
fibrin formation.
Tissue Factor (TF) is exposed on activated endothelial cells and leukocytes where tissue
is damaged. This protein activates Factor VII. The TF/VIIa complex binds and activates
Factor X. The resulting activated Factor Xa moves to the platelet surface. [Note that little
’a’ indicates a clotting factor protein which has been activated into an enzyme of an enzyme
cofactor.]
If Factor Xa cleaves enough thrombin from the prothrombin precursor, then coagulation will
proceed. Coagulation occurs only after thrombin stimulates activation of enough Factors
VIIIa, Va and IXa. Factors VIIIa and IXa make the ’tenase’ complex which produces a
constant source of Factor Xa. This Factor Xa is located on the platelet surface and interacts
with Factor Va to form prothrombinase complexes - rapid activation of prothrombin to
thrombin occurs by this activated complex. Thrombin cleaves fibrinogen to form a fibrin
clot, and also binds to form a structural component of the protein matrix.
Clot Regulation Thrombin binding to thrombomodulin on vascular endothelial cells in-

terrupts the function of Factors Va and VIIIa, thus slowing thrombin formation. The action
of the Protein C and S on Factors Va and VIIIa brings coagulation to a halt. By a separate
mechanism, the protein antithrombin inactivates fluid-phase thrombin, and other serine
proteases, when it is bound to heparin-like proteoglycans.
Fibrinolysis Plasmin is an important degrading enzyme, specifically of fibrin clots. It is

a serine protease that is released as plasminogen into the circulation and activated by
tissue plasminogen activator (tPA), thrombin, fibrin and factor XII (Hageman factor).
It is inactivated by a2-antiplasmin, a serine protease inhibitor. Plasmin cleaves the fibrin
polymer releasing fibrin dipeptides thus removing the clot.
11.1.3. Clinical Correlates
Patient presentation A history is focused on whether the patient’s condition is consistent

with excessive bleeding. Some common presenting conditions of patients are hematuria,
facile bruising, or bleeding in connection with dental surgery, circumcision, tonsillectomy,
nose-bleeds, and rectal bleeding. Note that patient estimates of blood loss by visual esti-
mations are often overestimates. A clinical examination should include careful inspection
of the skin, oral mucosa, musculoskeletal system, nervous system, and sites of active blood
loss. Venous blood extraction should be performed by an expert, due to the possibility of
massive compartment bleeding in patients with bleeding disorders.
214
Features of Coagulation 11.1.5
11.1.4. Laboratory Tests
Platelet counts: Anticoagulated venous blood is counted using an automated cytometer

counting particles of 2–37 µm−3 . Reduced counts are associated with prolonged bleed-
ing times.
Coagulation tests: Prothrombin time (PT): The PT tests the function of the ‘extrinsic’
and
B. Laboratory Tests final common clotting pathways. Tissue Factor is added to citrated test
plasma
1. Platelet counts:at 37 °C, and the mixture is recalcified. Clot formation occurs in 12–
15 s normally. Prolonged times indicate deficiencies. The test result depends on
a. Anticoagulated venous blood is counted using an automated
cytometer counting particles of 2-37 μM3. Reduced counts are
adequatewithconcentrations
associated of coagulation factors VII, X, V, II and fibrinogen in
prolonged bleeding times.
test plasma.
2. Coagulation tests:
a. Prothrombin time (PT): The PT tests the function of the 'extrinsic' and
Activated partial
final common thromboplastin
clotting pathways. Tissue Factortimeis added(APTT):
to citrated The APTT tests the function of
the ’intrinsic’ and final common clotting pathways. Phosphatidyl serine (PS) is
test plasma at 37 C, and the mixture is recalcified. Clot formation
occurs in 12-15s normally. Prolonged times indicate deficiencies.
added intodepends
The test result test plasma
on adequateat 37 °C. Contact
concentrations
factors VII, X, V, II and fibrinogen in test plasma.
of coagulationactivation is prevented by addition of
a strong activator (eg. kaolin). Clot formation occurs in 30–40 s.
b. Activated partial thromboplastin time (APTT): The APTT tests the
function of the 'intrinsic' and final common clotting pathways.
Phosphatidyl serine (PS) is added into test plasma at 37 C.
Thrombin time (TT): Thrombin is added to a test plasma and times to the clot end-
Contact activation is prevented by addition of a strong activator (eg.
point are measured. Abnormal TT (> 15 s) are due to low fibrinogen, or inhibitors
kaolin). Clot formation occurs in 30 - 40s.
c. Thrombin time (TT): Thrombin is added to a test plasma and times to
(eg. heparin), or an abnormal fibrinogen molecule (dysfibrinogenemia).
the clot end-point are measured. Abnormal TT (> 15s) are due to
low fibrinogen, or inhibitors (eg. heparin), or an abnormal fibrinogen
molecule (dysfibrinogenemia).
VII TF
X
IX
V
VIII Fibrinogen
XI II IIa
XII
Fibrin
APTT
PT
TT
141
These tests are best used together for differential diagnosis of specific clotting defects.
11.1.5. Features of Coagulation
Hepatocellular failure has combined effects on coagulation due to:

• Reduced plasma coagulation factors
215
• Induction of a hyperfibrinolytic state, further lowering Factor concentrations in blood.

• Splenic pooling of platelets due to portal hypertension.
Heparin is known to block the function of several clotting factors. This can be a confound-
ing factor because some medical devices are flushed with heparin prior to blood collection.
Hemostatic variables depend on age-specific reference ranges, particularly in children
and infants (who have different vitamin K levels than adults) and pregnancy.
Many clotting factors (II, VII, IX, X) require liver Vitamin K for their biosynthesis. The
membrane and calcium associations of these enzymes depend on post-translational modi-
fication of γ-carboxylation of glutamic acids. The drug dicoumarol (warfarin) interferes
with blood clotting by inhibiting Vitamin K-dependent biosynthesis of the clotting factors.
Common clinical vitamin K deficiency occurs in newborns, liver disease, and malabsorp-
tion.
11.1.6. Genetics of Blood Coagulation
Hemophilia A is an X-linked bleeding disorder resulting from deficiency of Factor VIII.

Affected individuals suffer easy bruising, prolonged bleeding from wounds and muscle
hemorrhage. Weight bearing joints suffer intracapsular bleeding leading to swelling
and inflammation, and eventually amputation. About half of mutations leading to
severe hemophilia A arise from inversions in the region Xq28. Milder forms usually
arise from missense mutations. Treatment is by recombinant Factor VIII infusions
which greatly improves life expectancy.
Hemophilia B (Christmas Disease) is an X-linked disorder caused by deficiency of vitamin
K-dependent procoagulant protein Factor IX. Clinically indistinguishable from the
more common Hemophilia A. Hemophilia most often presents during early childhood.
Patients with a long APTT and a normal PT should be tested for Factor VIII and
Factor IX levels to differentiate hemophilias A and B.
von Willebrand Disease von Willebrand Factor (vWF) is a complex multimeric
glycoprotein associated with subendothelial connective tissue. The protein functions
to form a bridge between platelets in the area of vascular damage. It also has a role in
stabilizing and increasing plasma concentration of Factor VIII. Hence, vWF patients
can present with symptoms resembling those of hemophilia or platelet dysfunction
disorders. However, the mode of inheritance is autosomal recessive and bleeding is
usually observed in mucocutaneous sites rather than joints.
This common disorder may prolong the APTT. Immunoassays for vWF protein are
used to identify the presence of vWF. This assay may therefore give normal results
216
Objectives in Brief 11.2
in mutations resulting in loss-of-function, with normal or slightly reduced amounts of

vWF protein. Treatment with Factor VIII infusions corrects the symptoms of many
patients. Purified vWF is also effective, but is more difficult to obtain due to the very
large size of the protein.
Factor XIII deficiency This enzyme deficiency is due to mutation in one of the two subunits
of a transglutaminase enzyme. This enzyme cross-links fibrin polymer by formation
of covalent bonds between glutamine and lysine side-chains. This is a rare autoso-
mal recessive disorder. Presentation is in bleeding from the umbilical cord stump in
neonates, intracranial hemorrhage, or after surgical challenge in children (often dental
extractions). Maintaining levels of Factor XIII at 10–20 % of normal values is suffi-
cient to correct the bleeding disorder. Factor XIII deficiency is often grouped with the
dysfibrinogenemias (reduced functional fibrinogen), which result in similar symptoms.
11.2. Objectives in Brief
1. Diagram the blood coagulation process identifying the role of the major participating
proteins and enzymes. Platelets, glycoprotein Ib-IX, von Willebrand factor (vWF),
fibrinogen, glycoprotein IIb-IIIa, tissue factor (TF), Factor VII, Factor X, thrombin,
Factor VIII, Factor V, Factor IX.
2. Explain the mechanisms of clot regulation and fibrinolysis.
3. Name the most common clinical tests performed for coagulation function and com-
ment on the interpretation of their result.
4. Discuss the relation of clinical tests to differential diagnosis of blood clotting disorders.
5. Name the most important genetic conditions which manifest as bleeding disorders.
Hemophilia A, Hemophilia B, von Willebrand Disease, and Factor XIII deficiency.
6. Describe the relation between disease symptoms in these inherited disorders and the
normal function of the deficient enzyme.
217
12. Blood
12.1. Blood Groups
Blood groups are genetic polymorphisms of red blood cells antigens. They are important in
blood transfusions, and some can cause maternal-fetal incompatibility.
12.1.1. The ABO system
This system contains two effective antigens, A and B. People with neither A nor B have the
H-substance (= O-antigen) instead which does not induce antibody formation in humans.
The 4 possible phenotypes (A, B, AB and O) are determined by 3 alleles of a single locus,
A, B, and O. A and B are co-dominant, O is recessive. Surprisingly, everyone who does not
have the A or B antigen on his own cells has a circulating IgM antibody to the missing
antigen, even if he has never been exposed to incompatible blood before. Most likely, these
antibodies are induced by contact with microbial cell surface carbohydrates shortly after
birth. Antigen-antibody reaction results in agglutination of RBCs.
Blood group Genotype Antibodies in Blood aggluti-
(antigens on Serum nates when mixed
RBCs) with RBCs
O OO anti-A, anti-B -
A AA or AO anti-B anti-A
B BB or BO anti-A anti-B
AB AB none anti-A, anti-B
In blood transfusions, the donor’s RBCs may be agglutinated by the recipient’s antibodies.
Donor
Recipient O A B AB
O - + + +
A - - + +
B - + - +
AB - - - -
Persons with blood group O are universal donors, those with blood group AB universal
recipients. Except in emergencies, however, patients are treated only with blood of their own
219
group to avoid any agglutination of recipient cells by donor serum. Increasingly, transfusion
with resuspended packed RBCs is done to avoid this minor type of transfusion reaction.
O and A are most common in Europe, AB is least common.
The ABO blood group substances are oligosaccharides which occur as components of sphin-
golipids and glycoproteins. Their antigenic specificity is determined by the terminal sugars.
The products of the A and B alleles, which differ by 4 amino acid substitutions, are glyco-
syltransferases which transfer N-acetylgalactosamine and galactose, respectively, to the end
of the oligosaccharide. The product of the O allele, which contains a nonsense mutation, is
non-functional.
The ABO blood group substances are present not only on RBCs, but on other cells as well.
Therefore, ABO matching is essential not only for blood transfusions but also for organ
transplantations. In addition, many people secrete ABO antigens, in glycoprotein form,
into saliva and other external secretions. This requires the secretor gene Se. The secretor
phenotype is determined by the SeSe or Sese genotype, non-secretors have sese. 23 % of
Europeans are non-secretors (sese).
12.1.2. The Rhesus System
The antigenic determinants in this system are sequence variants in three membrane proteins
encoded by two closely related genes. There are multiple alleles, some of them determine
rhesus positive (Rh+) and some rhesus negative (Rh-) phenotype. Rh+ is dominant over
Rh-. The frequency of the Rh- phenotype is about 15 % in Europe, less elsewhere. Rhesus-
negative people do not normally have an antibody to the rhesus antigen although the
transfusion of Rh+ blood can induce the formation of an IgG antibody.
Rhesus incompatibility between mother and fetus can cause Hemolytic disease of the new-
born: A Rh- mother can become immunized to the Rh antigen of her Rh+ fetus. This can
occur during birth or abortion. In a next pregnancy with a Rh+ child, the anti-Rh antibody
crosses the placental barrier and coats fetal RBCs which are subsequently destroyed. This
results in anemia and a compensatory increase of erythropoiesis. Immature RBCs are re-
leased into the fetal circulation (“erythroblastosis fetalis”). Severe cases lead to intrauterine
edema (“hydrops fetalis’) and fetal death. Hyperbilirubinemia may result in kernicterus.
ABO-incompatibility prevents sensitization to the rhesus antigen. Fetal blood cells entering
the maternal circulation are destroyed by maternal anti-A or anti-B before they can induce
the formation of the Rh-antibody. ABO incompatibility can also cause hemolytic disease
of the newborn, but this is very mild because ABO antibodies are IgM while rhesus anti-
bodies are IgG. Only the rhesus antibodies can easily cross the placenta and cause severe
hemolysis.
220
Chemical Inactivation of Hemoglobin 12.2.1
Prevention: Passive immunization of the Rh- mother against the Rh antigen at the time of
birth prevents sensitization (RhoGAM).
12.1.3. Other blood group systems
There are more than 20 other blood group systems. Most of them are important only in
blood banking, especially for patients with chronic blood diseases (thalassaemia, hemophilia)
receiving repeated blood transfusions. The time-honored use of blood groups for paternity
testing is being replaced by DNA tests.
12.2. Structure of Hemoglobin
4 polypeptides (= subunits), each with its own heme:
α2 β2 Major adult hemoglobin (HbA) (98 % in adults)
α2 δ2 Minor adult hemoglobin (HbA2 ) (2 % in adults)
α2 γ2 Fetal hemoglobin (HbF) (two types, both present in the fetus)
β, γ and δ-chains (146 amino acids) have very similar primary structures. Structural ho-
mologies exist between α-chains (141 amino acids), β-chains and myoglobin, indicating
a common evolutionary origin. The tertiary structures of these 3 polypeptides are very
similar.
Hemoglobin is similar to 4 myoglobin molecules in structure. The subunits interact by

salt bonds and hydrogen bonds. Heme binds to the apo-protein as in myoglobin. Each
hemoglobin molecule binds 4 O2 -molecules.
12.2.1. Chemical Inactivation of Hemoglobin
Methemoglobin is hemoglobin in which the Fe2+ (ferrous) is oxidized to Fe3+ (ferric). It

cannot bind oxygen. Causes for excessive methemoglobin formation:
• exposure to oxidizing chemicals (most common cause)
• deficiency of methemoglobin reductase in erythrocytes, an enzyme which reduces

methemoglobin back to normal hemoglobin
• structural abnormalities of hemoglobin affecting the binding of heme to the apo-

protein.
221
Methemoglobinemia is treated by the administration of a reducing agent (methylene blue).
Carbon Monoxide (CO) binds to the heme iron of hemoglobin and myoglobin as oxygen, but
with a 200-fold higher affinity: it prevents oxygen release from the three remaining sites and
impairs oxygen delivery. CO is a product of incomplete combustion in automobile exhaust
gas, cigarette smoke, and burning buildings. A very small amount is formed endogenously.
CO-poisoning is treated with hyperbaric oxygen. Color:
Oxygenated hemoglobin: red
Deoxy-hemoglobin: blue
CO-hemoglobin: cherry-red
Importance of apo-protein-heme association:
• The apo-protein protects heme from oxidation to hemin (hemin = heme containing
Fe3+ ).
• CO-affinity is reduced by the distal histidine. Heme in solution (without apo-protein)

has a 25 000 times higher affinity for CO than for O2 .
12.2.2. Allosteric Properties
Allosteric proteins can assume alternative conformations. They usually consist of more than
one subunit. In hemoglobin, the conformation that prevails in oxyhemoglobin is called the
R-form (R = relaxed), the conformation of deoxy-hemoglobin is called the T-form (T =
tense). O2 binding breaks the salt bonds between the subunits and rearranges the inter-
subunit hydrogen bonds, thereby changing the quaternary structure from T to R.
The R-form has a 300 fold higher O2 -affinity than the T-form. Therefore there is positive
cooperativity between O2 -binding sites: binding of O2 to one heme increases the O2 -affinity
of the other 3 hemes. This positive cooperatively results in a sigmoidal O2 -binding curve:
222
Allosteric Properties 12.2.2
venous pO2 arterial pO2
working resting
100
80
60
haemoglobin
myoglobin
w/o cooperativity
40
20
0
0 3 6 9 12 15
pO2 (kPa)
Myoglobin has no allosteric properties and no positive cooperativity (hyperbolic O2 -binding

curve).
The P50 is the O2 partial pressure resulting in 50 % saturation of the oxygen carrier. The
P50 of hemoglobin is 3.5 kPa; and of myoglobin 0.07 kPa.
A ligand is any substance that binds to the protein in question: O2 is the most important
ligand for hemoglobin.
An allosteric effector is a “regulatory ligand” that influences the equilibrium between the
alternative conformations of an allosteric protein. It binds to a site distinct from the binding
site for the functional ligand (O2 in the case of hemoglobin). A positive allosteric effector
favors the ligand-binding form, and a negative allosteric effector favors the non-ligand-
binding form.
2,3-Bisphosphoglycerate (BPG) is a negative allosteric effector of hemoglobin. In RBCs,

it is present in roughly equimolar concentration with hemoglobin. It binds to the β-chains
of hemoglobin in the T-conformation but not the R-conformation, thereby stabilizing the
T-conformation and lowering the O2 -affinity. It is in large part responsible for the lower
O2 -affinity of hemoglobin as compared to myoglobin. Its concentration increases during
adaptation to oxygen-deficient conditions (high altitude).
223
Oxygen binding to HbA: Effect of 2,3-BPG

100
80
60
40
0.0 mM
0.5 mM
1.0 mM
2.0 mM
6.0 mM
20
0
0 1 2 3 4 5 6
pO2 (kPa)
Homotropic effects in allosteric proteins are interactions between identical ligands: positive
cooperativity of O2 -binding in hemoglobin.
Heterotropic effects are interactions between different ligands: O2 and BPG in hemoglobin.
The Bohr-effect is the reduction of the O2 -affinity by increased acidity and [CO2 ] (in
exercising muscle).
Bohr-effect
100
80
60
40
pH 7.75
pH 7.50
pH 7.25
pH 7.00
20
0
0 1 2 3 4 5 6
pO2 (kPa)
Acid increases the [H+] lowering Hb oxygen-affinity because oxyhemoglobin is more acidic
than deoxy-hemoglobin:
224
The Hemoglobinopathies 12.2.3
Hb + O2 *
) Hb:O2 + n H+
Increased [H+ ] shifts the equilibrium of this reaction to the left.
CO2 reduces the oxygen-affinity through its reversible, nonenzymatic, covalent binding to
the terminal amino groups to form carbamino-hemoglobin.
R-NH2 + CO2 *
) R-NH-COO− + H+
In carbamino-hemoglobin, the T-conformation is favored and the O2 -affinity decreased.
Fetal hemoglobin (α2 γ2 ) has a higher O2 -affinity than HbA because BPG is less tightly
bound. But its O2 -affinity is not as high as that of myoglobin. Myoglobin does not respond
to BPG, H+ or CO2 .
Most of the carbon dioxide is transported as HCO−
3 , formed from CO2 + H2 O by carbonic
anhydrase in RBCs. Smaller amounts are transported as dissolved CO2 and as carbamino-
hemoglobin.
12.2.3. The Hemoglobinopathies
Globin Genes
There are 2 clusters of globin genes:

α-like genes (chromosome 16) 2 identical α-chain genes, ζ-chain gene.
β-like genes (chromosome 11) β, δ,A γ,G γ genes, A γ and G γ code for two γ-chains that
differ in only one amino acid (Ala versus Gly).
Both clusters contain nonfunctional pseudo-genes as well.
Expression: ζ and are expressed only in the embryo, α and γ in the fetus (2nd and 3rd
trimester), β and some δ in adults. At birth, 75 % of hemoglobin is HbF (α2 γ2 ) and 25 %
is HbA (α2 β2 ). HbF disappears within 4–6 months after birth.
Hemoglobin Point Mutations
More than 400 single-amino acid substitutions in α- or β-chains are known. Consequences:
• More than half of these mutations are asymptomatic.
• Mutations in the heme-binding pocket are likely to cause methemoglobin.
• Some mutant hemoglobins have an abnormally low oxygen affinity (causing cyanosis)
or an abnormally high oxygen affinity (causing polycythemia).
225
• Some abnormal hemoglobins have reduced solubility, leading to sickling.
Sickle Cell Disease
Mutation: A point mutation leading to a Glu→Val Substitution in position 6 of the β-chain

(HbS).
Properties: Normal O2 - affinity, but reduced solubility of deoxy-HbS, with sickling of

RBCs in venous (but not arterial) blood.
Inheritance: A/S heterozygotes are healthy although their RBCs can sickle under oxygen-
free conditions in the test-tube. S/S homozygotes have sickle cell disease.
Signs & Symptoms: • No problems up to 4–8 months
• Moderately severe anemia (total hemoglobin 7–11 %).
• Acute “sickling crisis”: Painful crisis (most common), splenic sequestration crisis
(infants/young children only), aplastic crisis.
• Multiple infarction: “Autosplenectomy”, renal infarcts, leg ulcers, cerebrovascular

accidents.
• Increased mortality: Infections and sequestration crisis in children, cerebrovas-

cular accidents and renal failure in adults.
Diagnosis: The blood smear shows irreversibly sickled cells; HbS solutions become turbid
in an oxygen-free environment; hemoglobin electrophoresis; DNA probes.
Treatment: Avoidance of hypoxia, fluids and analgesics during acute attack, symptomatic
treatment of complications, drugs that increase HbS-solubility or induce HbF synthe-
sis.
Prevalence: High in Africa, Arabia, India, and Mediterranean. Heterozygotes are partially
protected from malaria.
Hemoglobin C: Glu → Lys substitution in position 6 of the β-chain. Mild hemolysis in

C/C homozygotes, mild form of sickle cell disease in S/C heterozygotes.
226
The Hemoglobinopathies 12.3.1
Thalassemias
The synthesis of either α-chains or β-chains is reduced or absent, leading to anemia of

varying severity. Common in the Mediterranean, Africa, South and Southeast Asia.
α-thalassaemia: Not enough α-chains
β-thalassaemia: Not enough β-chains
Thalassaemia minor: Heterozygous thalassemia. With borderline anemia.
Thalassaemia major: Homozygous thalassemia. Severe anemia.
α-Thalassemia Usually caused by large deletions of α-chain genes.
1 gene deleted: Silent carrier. Asymptomatic
2 genes deleted: α-thalassemia minor. Borderline.
3 genes deleted: Hemoglobin H disease (HbH = β4 ). Moderately severe anemia.
4 genes deleted: Hemoglobin Barth disease (Hb Barth = γ4 ), hydrops fetalis.
β-Thalassemia More than 90 different mutations are known. Heterozygotes are borderline
normal/anemic.
Cooley’s anemia (= β-thalassemia major): Either with complete lack of β-chains (β0 -
thalassemia) or a very low amount of β-chains (β+ -thalassemia major). Milder vari-
ants are called β-thalassemia intermedia. Most “homozygotes” are actually compound
heterozygotes.
Signs & Symptoms: β0 -thalassemia with severe anemia (2–5 % hemoglobin). Only HbA2
and HbF. Microcytosis, poikilocytosis, anisocytosis, target cells. Abortive erythro-
poiesis, abnormal expansion of red bone marrow, extramedullary erythropoiesis (in
liver). Untreated patients die in childhood.
Treatment: Regular blood transfusions. Chronic infusion of desferrioxamine (iron chelator)

to prevent iron overload. Bone marrow transplantation, drugs inducing γ-chain (HbF)
expression. Bone marrow transplant.
227
12.3.1. Blood Groups
1. Describe the biochemical background for ABO and Rhesus blood groups.
2. Use the ABO, rhesus and MN blood group systems for paternity testing.
3. Predict transfusion reactions due to ABO incompatibility for all possible combinations
of donor and recipient.
4. Outline the pathogenic mechanism of hemolytic disease of the newborn due to rhesus
incompatibility or to ABO incompatibility; compare and contrast these two..
5. Describe the mechanism by which ABO incompatibility can protect from hemolytic
disease due to rhesus incompatibility, and give the rationale for the use of RhoGAM.
12.3.2. Hemoglobin and Myoglobin Biochemistry
1. Describe the structure of myoglobin with respect to α-helical structure, the role of
nonpolar interactions for the tertiary structure, and the roles of non-covalent interac-
tions, proximal histidine and distal histidine for heme binding to the apo-protein.
2. State the reversible, non-covalent nature of oxygen binding to the heme iron and the
importance of the iron being in the ferrous state.
3. Define the terms hematocrit, anemia, polycythemia, and hemolysis.
4. State the typical normal value for hematocrit and blood hemoglobin concentration.
5. Describe the subunit structure of hemoglobins A, Az, and F and specify the interac-
tions between the subunits.
6. State the chemical difference between hemoglobin and methemoglobin and name
causes of methemoglobin formation.
7. Describe the competitive interaction between carbon monoxide and oxygen in carbon
monoxide poisoning and name the therapeutic approach.
8. Define the term “allosteric protein”, “positive allosteric effector”, “negative allosteric
effector”, “homotropic effect” and “heterotropic effect”.
9. Describe the oxygen binding curves for myoglobin, adult hemoglobin and fetal hemoglobin,
and relate the sigmoidal binding curves of the hemoglobins to their allosteric proper-
ties.
228
Hemoglobinopathies 12.3.3
10. Describe the binding of 2,3-bisphosphoglycerate to hemoglobin. Describe the effects

of acidity and carbon dioxide concentration on oxygen binding in the Bohr effect.
12.3.3. Hemoglobinopathies
1. Describe the molecular defect in sickle-cell disease and how this leads to its clinical
expression.
2. Define the categories of thalassemias in terms of their associated molecular lesions
and severity. Explain how the thalassemias present, clinically.
3. List the treatment options for the major hemoglobinopathies.
229
Part III.
Semester one, Mini III

13. Mendelian Inheritance
13.1. The Patterns of Mendelian Inheritance
Definitions:
Alleles: Alternative forms (variants) of a gene.
Locus: The position of the gene on the chromosome.
Homozygous: Carrying 2 identical alleles of a gene.
Genotype: The genetic constitution. Usually used with reference to a specified gene.
Phenotype: The appearance of the individual.
Wild-type allele: The “normal” variant of a gene.
Compound heterozygote: Person carrying two different mutations in the two copies of a
gene.
Dominant allele: Allele that determines the phenotype both in the homozygous and the
heterozygous state.
Recessive allele: Allele that determines the phenotype in the homozygous but not the
heterozygous state.
Co-dominant alleles: Both phenotypes are partially expressed in the heterozygous state.
Proband (propositus): The individual who brings the family to the doctor’s attention.
The phenotype can be analyzed at various levels. In many metabolic disorders the het-
erozygotes are healthy (recessive inheritance), although the activity of the affected enzyme
is intermediate (co-dominant inheritance).
In autosomal dominant disorders, affected individuals are heterozygous. The phenotype is
observed in successive generations, affecting males and females equally. The offspring of an
affected individual have a 50 % risk of being affected. Unaffected individuals do not transmit
the disorder.
In autosomal recessive disorders, affected individuals are homozygous. The phenotype tends
to occur in more than one member of a sibship, affecting males and females equally. In most
233
cases, the affected individual is an offspring of two parents that both are unaffected het-
erozygotes. On average, one quarter of a sibship is affected. Matings between an affected
individual and an unaffected unrelated partner produce only unaffected heterozygous off-
spring unless the partner is a carrier (unaffected heterozygote).
X-linked recessive disorders are expressed mostly in males. Heterozygous females are phe-
notypically normal. On average, 50 % of males in a sibship are affected. They have inherited
the trait from their mother. There is no father-to-son transmission.
X-linked dominant disorders are expressed in successive generations. Females are affected
twice as often as males. Affected males have no normal daughters and no affected sons.
This pattern is rare.
Y-linked inheritance concerns only males, with direct father-to-son transmission. The most
important Y-linked trait is maleness. There are also infertility-causing mutations in Y-
linked genes that can be transmitted to sons by intracytoplasmic sperm injection. Notice
that the non-penetrant person shows no symptoms whatsoever.
Mitochondrial diseases are transmitted from one affected mother to all children. All children
of an affected father are normal. Sometimes, a disease-causing mitochondrial mutation is
present in only part of the patient’s mitochondria. This is called heteroplasmy.
13.2. Variations of gene transmission and expression
Definitions:
Penetrance: The likelihood that a genotype is expressed. Many autosomal dominant dis-
eases have incomplete penetrance, resulting in “skipped generations”. Penetrance can
be quantified in percentages, or as a fraction of 1. A penetrance of 0.8 means that a
person with the predisposing genotype has a likelihood of 0.8 of showing the disease
phenotype
Expressivity: The extent to which a genetic trait is expressed. Different patients may be
either mildly or severely affected although they have the same genotype. Another
phenotypic difference could be early or late age of onset.
Pleiotropy: One gene results in multiple morphological, biochemical, physical, or clinical

abnormalities.
Locus heterogeneity: The same phenotype can be produced by mutations in different

genes.
234
Functional classification of mutations 13.3.1
Allelic heterogeneity: The same phenotype can be produced by different mutations in the
same gene. This is extremely common because most disease-producing mutations
are loss-of-function mutations: any mutation that disrupts the function of the gene
product will cause the same disease.
Allelic heterogeneity usage 2: different mutations in a gene causing different phenotypes.

This is seen less commonly than usage 1, but one example is the FGFR3 gene, where
different mutations can cause 3 types of dwarfism, 2 types of craniosynostosis, and 1
type of skin mis-coloration.
Sex-limited and sex-influenced traits: Autosomal traits that are expressed exclusively or
predominantly in males or females. Example: Pattern baldness, an autosomal domi-
nant trait expressed only in males.
Anticipation: The tendency of some genetic diseases to get worse in successive generations.
This is caused by an unstable “premutation” that is prone to develop into a more
serious defect. The cases known so far are expansions of triplet repeats that become
amplified progressively once they have reached a certain length.
Imprinting: The differential expression of genetic traits depending on the maternal or pa-
ternal origin of the gene. The mechanism is incompletely understood but one of the
important players is increased DNA methylation in the copy of a gene inherited from
one of the parents.
Contiguous gene disorders: Diseases that are caused by a large deletion (“microdeletion”,
for the cytologist) that removes a group of contiguous genes. They often combine the
signs of two or more single-gene disorders. Example: Wilms tumor/aniridia.
Phenocopies: Conditions that resemble a genetic disease but are caused by non-genetic
factors, for example deafness after intrauterine rubella infection.
New mutations are common in dominant and X-linked recessive diseases, but autosomal
recessive diseases are almost always inherited. The recurrence risk after the birth of a child
with a new mutation is low, but above the population incidence because of possible germline
mosaicism.
Undisclosed nonpaternity has to be expected for about 10 % of all children.
13.3. Functional classification of mutations
Medical genetics is concerned with the functional consequences of human mutations. When
considering molecular pathogenesis of human mutations, different effects can be recog-
nized.
235
13.3.1. Loss of function
A mutation associated with a reduction or a complete loss of one or more of the normal
functions of a protein is described as a loss of function mutation. Most mutations other
than missense mutation result in a loss of function effect.
Loss of function can be associated with either dominant or recessive inheritance, but most
inborn errors of metabolism are caused by loss of function mutations which are harmless
in the heterozygous state (carrier). This indicates that 50 % of normal enzyme activity is
usually enough for normal function.
Haploinsufficiency
Loss of function mutation in the heterozygous state in which half of the normal level results
in phenotypic effects are termed haploinsufficiency mutations. The phenotypic manifesta-
tions sensitive to gene dosage are a result of mutations occurring in genes that code for
either receptors or more rarely enzymes, the functions of which are rate limiting. Exam-
ples of “haploinsufficiency” are familial hypercholesterolemia and acute intermittent
porphyrias.
There are number of autosomal dominant disorders where the mutational basis of the func-
tional abnormality is the result of haploinsufficiency, in which homozygous mutations result
in more severe phenotypic effects. This is the case for familial hypercholesterolemia.
13.3.2. Gain of function
A mutation associated with an increase in one or more of the normal function of protein
is described as a gain of function mutation. These are less common than loss of function
mutations. For example in Huntington disease the mutant protein forms cellular aggre-
gates which have a neurotoxic effect. In achondroplasia and thanatophoric dysplasia
mutations in the FGFR3 gene result in a increased cell receptor activity leading to mild
and severe suppression of bone growth. Mutations exerting gain of function usually cause
conditions that show autosomal dominant inheritance.
Dominant negative effect
A mutation has a dominant negative effect if the product of the mutant allele interferes
with the product of another allele, resulting in an adverse outcome. Dominant negative
effects usually arise when the protein product is multimeric, enabling the mutant protein
to disrupt the function of the final product. Structural proteins such as the collagens show
236
Linked Markers for Genotype Prediction 13.5
dominant negative effect in for example in osteogenesis imperfecta (“brittle bone

disease”).
The aquaporin Aqp2 is required, amongst other things, for water re-absorbtion from pri-
mary urine in the kidney. It acts as a monomer, however it is transported in a vasopressin-
dependent manner between the plasma-membrane and intracellular stores as tetramer.
Thus mutations in Aqp2 show a dominant negative effect and lead to diabetes insipidus.
13.4. Linked Markers for Genotype Prediction
Allele-specific probes and PCR can be used only if the exact mutation is known. If the
gene is known but the exact mutation is not (allelic heterogeneity), allele-specific probes
cannot be used unless the whole gene is scanned, for example with a specially-made DNA
chip. Instead, the mutation can be tracked with linked markers. A marker is a naturally
occurring variation in the chromosome, normally positioned between genes or within introns.
This variation can be detected using DNA methods. RFLPs can be used, but VNTRs are
better because they are more variable. The human genome project has produced a map of
VNTRs spanning the whole genome. Every gene in our genome has a few of them nearby.
Limitations of linkage studies:
1. You have to sample DNA from several family members.
2. There is always a chance of a crossing-over, therefore you cannot make 100 % accurate
predictions. The linked marker should be as close as possible to the gene or, ideally,
within an intron of the gene.
3. Linkage studies may be uninformative, especially if you can get DNA of only a few
family members or the marker is not sufficiently polymorphic in the family.
Linkage studies can be done with Southern blotting or PCR. The length of the restriction
fragment or the PCR product reflects the repeat number of the VNTR.
Note that in linkage studies a VNTR allele that is linked to a disease gene in one family
may be linked to the wild-type allele in another. If, in a population, a VNTR variant
is more often associated with the mutant allele than expected by chance, we call this
linkage disequilibrium. It suggests that the mutation occurred recently on a chromosome
that happened to carry this particular variant. A constellation of two or more closely linked
genetic markers is called a haplotype. In the absence of crossing-over, the gene combination
of the haplotype is inherited as one unit, like a single gene.
237
occurred recently on a chromosome that happened to carry this particular
13.5 variant. A constellation of two
Biochemistry andorGenetics
more closely linked genetic markers is called a
haplotype. In the absence of crossing-over, the gene combination of the
haplotype is inherited as one unit, like a single gene.
13.5. Pedigree Analysis
IV. PEDIGREE ANALYSIS
Autosomal dominant inheritance:
Autosomal dominant inheritance:
• If a parent is affected, each child has a 50 % risk of inheriting the disease gene.
- If a parent is affected, each child has a 50% risk of inheriting the disease
gene.the children of unaffected individuals are not at risk.
• If penetrance is complete,
- If penetrance is complete, the children of unaffected individuals are not
• If a patient has no affected
at risk.parent, he has a new mutation (assuming complete pen-
etrance). His own- children havehas
If a patient a 50no
% risk, although
affected further
parent, children
he has of his
a new parents (assuming
mutation
have a low risk. complete penetrance). His own children have a 50% risk, although
further children of his parents have a low risk.
Autosomal recessive inheritance:
Autosomal
• When analyzing recessive
the pedigree, inheritance:
start with the patient and go up and down the family
tree. - When analyzing the pedigree, start with the patient and go up and
down the family tree.
• Parents and children- of a patient
Parents andarechildren
obligatory carriers.
of a patientWhen you go up carriers.
are obligatory and down When you
the family tree, the carrier
go upriskand
is cut in half
down thewith eachtree,
family generation.
the carrier risk is cut in half with
each generation.
• Most patients are produced by matings
- Most patients arebetween two by
produced carriers:
matings1/4 between
affected, 1/4
two ho-
carriers: 1/4
mozygous normal, 1/2 carriers.
affected,For
1/4a homozygous
sibling of an affected,
normal,there is 2/3 risk For
1/2 carriers. of being
a sibling of an
a carrier if an unaffected status isthere
affected, known.is 2/3 risk of being a carrier if an unaffected status is
known. in patients.
• New mutations are uncommon
- New mutations are uncommon in patients.
• If the disease is rare,- individuals outside
If the disease is the
rare,family are most
individuals likely homozygous
outside nor-most likely
the family are
mal. For a more accurate prediction ofnormal.
homozygous the disease
Forrisk, you accurate
a more have to know the carrier
prediction of the disease
frequency in the population.
risk, you have to know the carrier frequency in the population.
Probability of carriers, assuming that outsiders are homozygous normal:
Probability of carriers, assuming that outsiders are homozygous normal:
1/4 1/4
1/2 1/2 1/4

1/2
2/3
1/3
1/2
1/6
1/4
238 88
X-linked recessive inheritance 13.5.1
Probability of affected child (both parents unaffected):
Probability (father is carrier) x Probability (mother is carrier) x (1/4)
Probability of affected child (both parents unaffected): Probability (father is carrier) x
Example:
Probability (mothercarrier frequency
is carrier) in Example:
x (1/4) the population is frequency
carrier 2%. in the population is 2 %.
Disease risk: 2/3 x 1/50 x 1/4 = 1/300
1/50 2/3
Probability of affected child if one parent is affected: (1/2) x Probability

(unaffected parent is a carrier)
Probability of affected child if one parent is affected: (1/2) x Probability (unaffected parent
is a carrier)
Probability of an affected child if both parents are affected: 100%, except in
cases of locus heterogeneity where the children are unaffected double
Probability of an affected child if both parents are affected: 100 %, except in cases of locus
heterozygotes.
heterogeneity where the children are unaffected double heterozygotes.
X-linked recessive inheritance:
The disease gene is passed on with the X-chromosome: From the mother
to sons and daughters, from the father to daughters but not sons.
- Unaffected males don’t carry the gene.
13.5.1. X-linked recessive inheritance
- The patient’s mother is an obligatory carrier, unless the patient has a
new mutation.
- Going down the family tree in the female line, the carrier risk is cut in
The disease gene ishalfpassed
with on with
each the X-chromosome: From the mother to sons and daugh-
generation.
ters, from the father to daughters but
- All daughters of an affected not sons.
father and half of the daughters of a
carrier mother are carriers.
- Riskmales
• Unaffected of an don’t
affected son:
carry theOne-half
gene. of the probability that the mother is
a carrier.
- For carrier mother with offspring of unknown sex, risk is 1/2 for
• The patient’smother
motherpassing
is an obligatory carrier,
the bad allele on,unless
times the
1/2 patient
that thehas a new is
offspring mutation.
a son (daughter does not become affected, only carriers).
- New
• Going down the mutations
family are
treecommon for many
in the female line,X-linked recessive
the carrier diseases.
risk is cut in half with each
generation.
Probability of carriers:
• All daughters of an affected father and half of the daughters of a carrier mother are
carriers.
• Risk of an affected son: One-half of the probability that the mother is a carrier.
1/2
• For carrier
1/4 mother with offspring of unknown sex, risk is 1/2 for mother passing the
bad allele on, times 1/2 that the offspring is0 a son (daughter does not become affected,
only1/8
carriers).
• New mutations are common for many X-linked recessive diseases.

89
239
a son (daughter does not become affected, only carriers).
- New mutations are common for many X-linked recessive disea
Probability
Probability ofofcarriers:
carriers:
1/2
1/4
V. MODIFIED RISK: BAYES THEOREM. 0
1/8
The carrier probabilities, as determined by pedigree analysis, may have to
modified. Procedure:
1. Determine13.6.
the Modified Risk: BayestoTheorem
original probability be or not 89 to be a carrier. This is
e prior probability.The carrier probabilities, as determined by pedigree analysis, may have to be modified.
2. DetermineProcedure:
the probability of the observed situation if the proband is a
rrier or not a carrier.• Determine
This is the theoriginal
conditional probability.
probability to be or not to be aAnother
carrier. Thisway
is theto write
prior
s is probability(observation ⏐ genotype)
probability.
3. Multiply the •prior probability
Determine with
the probability theobserved
of the conditional
situation ifprobability,
the proband is aseparately
carrier or
not a carrier. This is the
each assumption (to be or not to be a carrier). This yields the
conditional probability. Another way to write this joint
is
probability(observation k genotype)
obability.
• Multiply the prior probability with the conditional probability, separately for each
4. Compare the joint for the assumption that the proband is a carrier with
assumption (to be or not to be a carrier). This yields the joint probability.
e joint probability for the assumption that he/she is not a carrier.
• Compare the joint for the assumption that the proband is a carrier with the joint
probability for the assumption that he/she is not a carrier.
ese calculations are best done in the form of a table.
These calculations are best done in the form of a table.
II
1 2 3
ample: A woman has a brother with hemophilia. She has 4 healthy sons and
affected children.240How likely is it that II-2 carries the hemophilia gene?
II-2 is a carrier II-2 is not a carrier
Sum
1/2 1/2
ob. 1/2 x 1/2 x 1/2 x 1/2 = 1/16 1
1/2 x 1/16 = 1/32 1/2 x 1= 1/2 = 16/32 17/32
Example: A woman has a brother with hemophilia. She has 4 healthy sons and no affected
children. How likely is it that II-2 carries the hemophilia gene?
prior probability 1/2 1/2 Sum
conditional probability 1/2 × 1/2 × 1/2 × 1/2 = 1/16 1
joint probability 1/2 × 1/16 = 1/32 1/2 × 1 = 1/2 = 16/32 17/32
resulting probability 16/17
Compare the joint probabilities: 1 chance that she is a carrier : 16 chances that she is not a
carrier. The carrier probability is 1 in 17 (about 6 %). [A more mathematical explanation:
the two joint probabilities do not sum to one, which they really have to do (if you have only
two possibilities, one or the other has to happen). Therefore we divide by the sum and get
to the values in resulting probability, and now the two numbers add to one.]
In this example, the prior probability is the original probability that II2 has inherited the
gene. The conditional probability is the probability that all 4 sons are healthy, assuming that
she is a carrier or is not a carrier. In other cases, the conditional probability is determined by
the outcome of a laboratory test (how likely is it that the carrier test is negative even though
the proband is actually a carrier?). Most laboratory tests have false negatives and
false positives! Or it is determined by the patient’s health in a disorder with incomplete
penetrance or age-dependent onset (how likely is it that the patient is healthy at his age even
though he carries the disease gene?). Computers are better than physicians at diagnosing
diseases because they master Bayes theorem!
1. Recognize the patterns of Mendelian inheritance from pedigree charts.

2. Predict the carrier probabilities in pedigrees with autosomal recessive and X-linked
recessive diseases.
3. Calculate disease risk in families with established Mendelian disorders.
4. Use Bayes’ theorem for the calculation of modified risk for Mendelian disorders.
5. Define the terms penetrance, expressivity, pleiotropy, locus heterogeneity, allelic het-
erogeneity, phenocopy and anticipation.
241
14. Krebs- (TCA-) cycle: Burning the
carbon skeleton
14.1. The Pyruvate Dehydrogenase Reaction
Pyruvate enters the mitochondrion on a specific carrier in the inner membrane.
14.1.1. The pyruvate dehydrogenase complex
Pyruvate dehydrogenase is a multi-enzyme complex in the mitochondrial matrix. It contains

3 components, each with its own prosthetic group:
• Pyruvate dehydrogenase (thiamine pyrophosphate).
• Dihydrolipoyl transacetylase (lipoic acid).
• Dihydrolipoyl dehydrogenase (FAD).
NAD+ and CoA are soluble cofactors.
14.1.2. Overall reaction
Pyruvate + NAD+ + CoA-SH → Acetyl-CoA + NADH + H+ + CO2

The reaction is irreversible. There is no way to convert acetyl-CoA back to pyruvate.
14.1.3. Functional impairment
• Vitamin deficiencies, especially thiamine.

• Arsenic poisoning: Arsenite binds tightly to lipoic acid.
• Partial genetic deficiencies, with lactic acidosis, encephalopathy, optical atrophy, spinocere-
bellar ataxia. The nervous system is very sensitive to impairments of pyruvate dehy-
drogenase because it depends on glucose oxidation for its energy.
243
14.2. The TCA Cycle
14.2.1. Reactions
The reaction sequence is summarized on page 308 of the Meisenberg & Simmons book.
The enzymes are soluble in the mitochondrial matrix except succinate dehydrogenase (SDH)
which is in the inner mitochondrial membrane. Generally, all cells that have mitochondria
also have a TCA cycle.
14.2.2. Products of the TCA cycle
The TCA cycle produces 2 CO2 , 3 NADH + H+ , 1 GTP and 1 FADH2 .

Effectively, the 2 carbons of the acetyl residue in acetyl-CoA are oxidized to CO2 . The
GTP, formed by substrate-level phosphorylation, is energetically equivalent to ATP. The
most important products are NADH + H+ and FADH2 which feed into the respiratory
chain.
14.2.3. Regulation of pyruvate dehydrogenase and TCA cycle
• Many enzymes in the oxidative mitochondrial pathways are subject to feedback inhi-
bition by NADH + H+ and ATP.
• Irreversible reactions are catalyzed by
– Pyruvate dehydrogenase
– Citrate synthase
– Isocitrate dehydrogenase
– α-ketoglutarate dehydrogenase
• Pyruvate dehydrogenase is
– inhibited by its products acetyl-CoA and NADH + H+ .
– inhibited by high and activated by low energy charge
– inactivated by phosphorylation.
– Phosphorylation is stimulated by high [ATP] / [ADP], [Acetyl-CoA] / [CoA],
and [NADH + H+ ] / [NAD+ ] ratios.
• Citrate synthase is inhibited by ATP and citrate.
244
Other reactions of TCA cycle intermediates 14.5
• Isocitrate dehydrogenase is stimulated by ADP and inhibited by a high [NADH +

H+ ] / [NAD+ ] ratio.
• α-Ketoglutarate dehydrogenase is inhibited by its products NADH + H+ and succinyl-

CoA, and by high energy charge (note similarities to pyruvate dehydrogenase!)
14.3. Inhibition of the TCA cycle
Fluoroacetate is converted enzymatically first to fluoroacetyl-CoA and then to fluorocitrate.

Fluorocitrate inhibits aconitase.
14.3.1. Other reactions of TCA cycle intermediates
Biosynthetic reactions
α-Ketoglutarate and oxaloacetate are both precursors and degradation products of gluta-
mate and aspartate, respectively.
Succinyl-CoA is a precursor in the biosynthesis of heme.
Oxaloacetate can be converted to glucose in the liver (gluconeogenesis).
Anaplerotic reactions
Anaplerotic reactions produce TCA cycle intermediates. They are required because oxaloac-
etate has to be present to ensure continued functioning of the cycle, even when TCA cycle
intermediates are drained off for biosynthesis.
Most amino acids are degraded to TCA cycle intermediates.
Pyruvate carboxylase produces oxaloacetate from pyruvate:
pyruvate
Pyruvate + CO2 + ATP + H2 O GGGGGGGGGGGGGA Oxaloacetate + ADP + Pi
carboxylase
The enzyme contains biotin. All ATP-dependent carboxylations require biotin. Pyruvate
carboxylase is also important in gluconeogenesis. It is activated by acetyl-CoA.
245
14.4. Shuttles Across the Inner Mitochondrial Membrane
14.5. Shuttles for electrons from cytoplasmic NADH + H+
Most of the NADH + H+ and FADH2 that fuel the respiratory chain are made in the mito-
chondrion itself: pyruvate dehydrogenase, TCA cycle, β-oxidation of fatty acids. NADH +
H+ is not transported across the inner mitochondrial membrane. Electrons from cytoplasmic
NADH + H+ can be channeled into the respiratory chain by the glycerol phosphate shuttle:
Cytoplasm Membrane Mitochondrial

Pyruvate Matirix
NADH, H+ FADH2 Q
H2C OH
O O
C O P O
H2
O
Glycolysis Dihydroxyacetone-phosphate
FAD QH2
NAD+
H2C OH
HO O
C O P O
H2
O
Glycerol-phosphate
Glucose
Glycerol phosphate is reoxidized to dihydroxyacetone phosphate by a

flavoprotein in the inner mitochondrial membrane, without ever entering the
Glycerol phosphate ismatrix.
mitochondrial re-oxidized
Thistoflavoprotein
dihydroxyacetone
transfersphosphate by a flavoprotein
the hydrogen to coenzyme in the
Q
inner mitochondrial membrane, without ever entering the mitochondrial matrix.
(ubiquinone), a component of the respiratory chain. The P/O ratio is 2 (2 ATP This flavo-
proteinformed
transfers
perthe hydrogen
FADH 2).
to coenzyme Q (ubiquinone), a component of the respiratory
chain. The P/O In ratio is 2 (2 ATP formed
the malate-aspartate per FADH
shuttle, ).
the 2hydrogen is transported into the
mitochondrial matrix as malate. Because oxaloacetate is not transported across
the membrane, the carbons of malate are shuttled back into the cytoplasm as
In the malate-aspartate
aspartate: shuttle, the hydrogen is transported into the mitochondrial matrix
as malate. Because oxaloacetate Cytoplasm
is not transported acrossMitochondrial
the membrane, the carbons of
malate are shuttled back into the cytoplasm as aspartate: Matrix
Pyruvate
NADH, H+ Oxaloacetate Oxaloacetate NADH, H+
246
Glycolysis
NAD+ Malate Malate NAD+

Glucose
α-Ketoglutarate α-Ketoglutarate
mitochondrial matrix. This flavoprotein transfers the hydrogen to coenzyme Q
(ubiquinone), a component of the respiratory chain. The P/O ratio is 2 (2 ATP
formed per FADH2).
In the malate-aspartate shuttle, the hydrogen is transported into the
mitochondrial matrix as malate. Objectives
Because oxaloacetate is not transported across
14.6
the membrane, the carbons of malate are shuttled back into the cytoplasm as
aspartate:
Cytoplasm Mitochondrial
Matrix
Pyruvate
NADH, H+ Oxaloacetate Oxaloacetate NADH, H+
Glycolysis
NAD+ Malate Malate NAD+

Glucose
α-Ketoglutarate α-Ketoglutarate
Glutamate Glutamate
Aspartate Aspartate
14.5.1. Other substrates and products 121
Most TCA cycle intermediates are transported across the inner mitochondrial membrane
by antiport carriers (‘translocases’). Also ATP, ADP, and inorganic phosphate are trans-
ported. Acetyl-CoA, oxaloacetate and NAD+ are not transported. O2 is diffusible (no carrier
required).
14.6. Objectives
1. Describe the subcellular location of pyruvate dehydrogenase, its coenzyme require-

ments, and the balance of the reaction.
2. Predict the effects of pyruvate dehydrogenase inhibition with respect to substrate
accumulation and oxygen system involvement.
3. List the sequence of intermediates in the TCA cycle and identify those reactions that
produce important products like NADH + H+ , FADH2 and GTP.
4. Give reasons why anaplerotic reactions are sometimes required to maintain the TCA
cycle.
247
15. Respiratory Chain, Oxidative
Phosphorylation and Reactive Oxygen
Species
15.1. Respiratory Chain: Burning Hydrogen
15.1.1. Overall reactions
The overall reactions in the respiratory chain are:

FADH2 + 1/2 O2 → FAD + H2 O ∆G00 = −200 kJ/mol
NADH + H+ + 1/2 O2 → NAD+ + H2 O ∆G00 = −219 kJ/mol
Actually, the electrons of the reduced coenzymes are channeled through a bucket brigade
of electron carriers which form the respiratory chain. The exergonic redox reactions in
the respiratory chain are used to create a proton gradient across the inner mitochondrial
membrane. The dissipation of this proton gradient is coupled to the synthesis of ATP by
oxidative phosphorylation.
15.1.2. The redox potential
Redox reactions are 2-substrate reactions in which electrons are transferred from one sub-
strate to the other. The tendency for a reduced substrate to donate electrons is expressed as
its standard redox potential (E 0 or E 00 ), which is measured in volts. A low E 00 means high
“reducing power”. Under standard conditions, electrons are transferred from the substrate
with lower E 00 to the one with higher E 00 .
E 00 is driving force of the reaction:
∆E 00 = Eoxidant
00 00
− Ereductant (15.1)
∆E 00 is not a property of a redox couple but of the reaction. It is related to ∆G00 :

∆G00 = −n × F × ∆E 00 (15.2)
with n = number of electrons transferred and F = Faraday constant.
249
15.1.3. Components of the electron transport chain
• Flavoproteins in the respiratory chain contain FMN.
• Iron-sulfur proteins contain non-heme iron bound to cysteine SH-groups, also H2 S.

Fe is reversibly oxidized to Fe3+ and reduced to Fe2+ .
• Cytochromes contain an iron-porphyrin as prosthetic group, either heme (Cyt. b, c,

c1) or heme a (Cyt a, a3). Cyt a3 also contains (non-heme) copper. The heme iron
transfers electrons by reversibly changing between the Fe2+ and Fe3+ forms.
• Ubiquinone (coenzyme Q) is a hydrophobic coenzyme which is mobile in the mem-

brane. Structure:
H+ H+
O O e- OH
e-
H3C O O CH3 H3C O O CH3 H3C O O CH3
H3C O CH3 H3C O CH3 H3C O CH3

O OH OH
Ubiquinon (Q) Semiquinone radical (QH*) Ubiquinol (QH2)
• Organization of the respiratory chain: Soluble components of the respiratory chain

are
NAD+ /NADH + H+ : The most important substrate.
Ubiquinone: In the lipid phase of the membrane.
Cytochrome c: A peripheral membrane protein.
Molecular oxygen (O2 ): Terminal electron acceptor.
All other components are integral membrane proteins. Direction of electron flow is shown
below (left to right):
250
Phosphorylation sites 15.2.2
Redox potential of some components:

Redox-pair E 00 (mV)
NAD /NADH + H
+ + -320
Ubiquinone/Ubiquinol +100
Cytochrome c Fe3+ /Fe2+ +220
1/2 O2 + 2 H+ / H2 O +820
15.2. Making ATP from electricity
15.2.1. Phosphorylation sites
Oxidative phosphorylation produces 3 ATP per NADH + H+ , and 2 ATP per FADH2 : the
P/O ratios are 3 and 2, respectively. The P/O ratio is the number of high-energy phosphate
bonds synthesized per 1/2 O2 consumed. Electron flow through the following sites results
in proton gradient formation, which can be used for phosphorylation:
• NADH + H+ - Q reductase
• QH2 - Cytochrome c reductase
• Cytochrome oxidase
Electron flow and phosphorylation are tightly coupled. Electrons will not flow through the
respiratory chain unless ATP can be formed from ADP and inorganic phosphate.
251
15.2.2. Mechanism of oxidative phosphorylation
1. The redox reactions in the respiratory chain are used to pump protons out of the
mitochondrial matrix. This creates a proton gradient of 1 pH unit, and it helps to
maintain a membrane potential of 200 mV, positive outside.
2. Protons move back into the mitochondrial matrix through a proton channel (Fo com-
ponent) which is coupled to an ATP-synthesizing enzyme (F1 component).
15.2.3. Energy yield from glucose
Anaerobically (Glucose → 2 Lactate): 2 ATP.
Aerobically (Glucose + 6 O2 → 6 CO2 + 6 H2 O):

Pathway Intermediate ATP
Glycolysis 2
2 NADH + H + 6
Pyruvate dehydrogenase: 2 NADH + H+ 6
TCA-cycle: 2 GTP 2
6 NADH + H+ 18
2 FADH2 4
38
The actual energy yield of oxidative metabolism is closer to 30 ATP because the substrate
shuttles in the inner mitochondrial membrane require energy (they dissipate the membrane
potential).
15.2.4. Regulation of electron flow and phosphorylation
Required are: reduced coenzymes, oxygen, inorganic phosphate, and ADP. ADP is rate-
limiting under most conditions.
15.2.5. Inhibition of oxidative phosphorylation
• Inhibitors of electron transport:
Amytal, rotenone: NADH - Q reductase
Antimycin A: QH2 - Cytochrome c reductase.
Cyanide, azide, CO, H2 S: Cytochrome oxidase.
252
Reactive Oxygen Derivatives 15.3
• Oligomycin inhibits the Fo /F1 ATPase (= mitochondrial ATP synthase). Because

electron flow and ATP synthesis are coupled, electron flow ceases as well.
• Most uncouplers of oxidative phosphorylation are weak hydrophobic acids which carry
protons across the membrane, thus dissipating the proton gradient. ATP synthesis
stops but electron flow is accelerated. Examples: 2,4-dinitrophenol, pentachlorophenol.
Valinomycin uncouples by transporting potassium ions across the inner mitochondrial
membrane, thus dissipating its membrane potential.
• A complete lack of oxygen (anoxia) may deplete cellular ATP stores within minutes.
Anoxia and hypoxia (relative lack of oxygen) are most commonly caused by ischemia
(interruption of blood supply). Cell death results after a few minutes (neurons), about
an hour (myocardium, liver, kidney), or several hours (fibroblasts, epidermis, skeletal
muscle). Typical effects:
– Decreased energy charge
– Increased glycolysis, mostly from stored glycogen
– Decreased pH because of lactic acid formation
– Impaired ion pumping. Osmotic imbalance with cellular edema (swelling) and
mitochondrial swelling.
– Increased membrane permeability, both plasma membrane and organelles.
– Release of lysosomal enzymes, autolysis.
15.3. Reactive Oxygen Derivatives
The partial reduction of O2 , by less than 4 electrons at a time, leads to highly reactive and
toxic products:
Superoxide radical O2 + e− → O−·
2
Hydroperoxide radical O−·

2 + H → HO2
+ ·
Hydrogen peroxide 2 HO·2 → H2 O2 + O2

Hydroxy radical H2 O2 *
) 2 HO· , (Fenton-reaction)
Hypochlorite H2 O2 + Cl− → H2 O + OCl−
Radical: Compound with an unpaired electron (indicated by · ), very reactive (“molecular
terrorist”)!
Superoxide radical is probably formed as a byproduct of many oxygenase reactions, but
most of it is derived from the respiratory chain.
253
Sources of H2 O2
• Nonenzymatic formation from the superoxide radical, or directly from cytochrome

oxidase.
• Flavoproteins outside the mitochondria form H2 O2 . Flavoproteins in the inner mito-

chondrial membrane (SDH, mitochondrial glycerol phosphate dehydrogenase) transfer
their electrons (hydrogen) to ubiquinone:
Substrate Product
(Reduced) (Oxidized)
Substrate Product
FAD FADH2
FAD FADH2
QH2 Q
Respiratory
QH2 Chain Q
Respiratory
1/2 O2 Chain H2O
1/2 O2 H2O2 is also formed in the respiratory

c) H2O burst of macrophages by the
• H2 O2 is also formed in the respiratory burst of macrophages by the action of NADH
action of NADH oxidase.
c) + H2O2 is also formed in the respiratory burst of macrophages by the
+Nonmitochondrial
H oxidase.
action of NADH oxidase. regenerate their prosthetic group by a
flavoproteins
reaction with molecular oxygen:
• Non-mitochondrial flavoproteins
Nonmitochondrial flavoproteins regenerate
regenerate their their prosthetic
prosthetic group
group by a by a reaction with
reaction with molecular
molecular oxygen:
Substrate oxygen: Product
Substrate Product
FAD FADH2
FAD FADH2
H2O2 O2
H2O2
Through free-radical chain reactions, reactive O2oxygen products can induce
the nonenzymatic oxidation of polyunsaturated fatty acids and, probably,
Through free-radical
mutagenesis. This may be chain reactions,
important in reactive oxygen products
aging, degenerative can induce
diseases, and
Through
the free-radical
nonenzymatic
carcinogenesis. is chain
It oxidation reactions, reactive
for acuteoxygen
of polyunsaturated
also responsible fatty products
acids
oxygen can induce the nonenzymatic
and, Protective
toxicity. probably,
mutagenesis. This may be important in aging, degenerative diseases, and
oxidation
mechanisms:of polyunsaturated
carcinogenesis. It is alsodismutase
fatty acids and, probably,
responsible isfor aacute
mutagenesis. This may be important
oxygen enzyme,
toxicity. Protective
a) Superoxide ubiquitous both in
inmechanisms:
aging, degenerative diseases, and carcinogenesis. It
mitochondria and cytoplasm. It is present in all aerobic is alsoorganisms,
responsible for acute oxygen
toxicity.a) Superoxide dismutase
but not in obligatory is aReaction:
anaerobes. ubiquitous enzyme, both in
mitochondria and cytoplasm. It is present in all aerobic organisms,
but
2 O2not H+
¯ +in 2obligatory anaerobes.
O2 + Reaction:
H2O2
+
2 O2¯ + 2ismechanisms
15.3.1.b) Protective
Catalase H
found in blood O2 +most
and H2Otissues.
2 It is most abundant in
peroxisomes. Reaction:
b) Catalase is found in blood and most tissues. It is most abundant in
peroxisomes. Reaction:
126
254
126
Objectives 15.4
Superoxide dismutase is a ubiquitous enzyme, both in mitochondria and cytoplasm. It

is present in all aerobic organisms, but not in obligatory anaerobes. Reaction:
2 O−
2 + 2 H → O2 + H2 O2
+
Catalase is found in blood and most tissues. It is most abundant in peroxisomes. Reac-
tion:
2 H 2 O2 → 2 H 2 O + O 2
Peroxidases are enzymes that destroy hydrogen peroxide by reacting it with an organic
substrate. Some peroxidases contain the rare amino acid selenocysteine in their reaction
center.
Many organic molecules protect against oxidative damage by reacting spontaneously with
free radicals to terminate free-radical chain reactions. They include vitamin E, ascorbate,
the retinoids, uric acid, and bilirubin. Proteins also protect against free radical generation
by binding to metals such as iron (ferritin) and copper and zinc (metallothioneins). The
free-ion form of these metals is maintained by these storage proteins at extremely low
concentrations to prevent free radical damage.
15.4. Objectives
1. Define the terms “oxidation” and “reduction”. State, in qualitative terms, the relation-
ship between the change in standard redox potential and the free NAD+ ratio for the
regulation of the major energy-producing catabolic pathways.
2. State the ATP yield and energetic efficiency of glucose oxidation.
3. List examples of site- energy change of redox reaction.
4. List the major types of hydrogen and electron carriers in the respiratory chain and
describe the functional organization and subcellular location of the respiratory chain.
5. Know the P/O ratios for the respiratory chain oxidation of NADH + H+ and FADH2 .
6. Describe the mechanism of oxidative phosphorylation according to the chemosmotic

hypothesis.
7. Identify the limiting factors for the rate of oxidative phosphorylation in different
physiological states.
255
8. State the roles of energy charge and NADH + H+ /specific inhibitors and uncouplers
of oxidative phosphorylation. Predict the effects of these inhibitors and uncouplers on
cellular energy charge, NADH + H+ /NAD+ ratio, thermogenesis, glycolytic activity
and lactic acid formation.
256
16. Single-Gene Disorders and Traits
Several thousand single-gene disorders have been described. Most are rare, with population
incidences on the order of 1 in 10 000 or less. They affect at least 1 % of all newborns.
Most of these diseases are autosomal dominant or autosomal recessive, but 10 % are X-
linked recessive. The molecular defect is known for most of the more important single-gene
disorders. Also some harmless traits (for example eye color) are single-gene, and there are
many normal polymorphisms with mendelian inheritance, for example blood groups.
16.1. Skeletal and Connective Tissue Diseases
Osteogenesis Imperfecta This clinical entity is marked by brittle bones, blue sclera, and
conductive hearing loss later in life. There is a wide range of severity, from perinatal lethal
to mildly affected, with or without dwarfism and deformities.
Inheritance: Usually autosomal dominant, rarely recessive.
Molecular defect: >80 different mutations in type I collagen genes are known, most of
them point mutations replacing a glycine by another amino acid.
Molecular diagnosis: Linked markers can be used for prenatal and pre-symptomatic diag-
nosis in many families. Recurrence risk for the perinatal lethal form is 3 % (germline
mosaicism!), and ultrasound monitoring of subsequent pregnancies is recommended.
Ehlers-Danlos Syndrome This is a heterogeneous group of diseases with stretchy skin

and loose joints. Easy bruisability and cigarette paper scars occur in most types. The three
most common types are inherited as autosomal dominant traits but X-linked and autosomal
recessive forms are known; overall incidence is 1 in 150 000.
Marfan Syndrome There are 3 kinds of abnormality:

• Long limbs, arachnodactyly.
• Ocular abnormalities, with short-sightedness and/or ectopia lentis.
• Aneurysm of the ascending aorta, mitral valve defects, aortic dissection.
257
Expressivity is highly variable. Phenocopies are common, and the syndrome may be con-
fused with other genetic disorders (homocysteinuria, Ehlers-Danlos syndrome).
Molecular defect: Mutations in the gene for the connective tissue protein fibrillin.
Molecular diagnosis: There is much allelic heterogeneity. Therefore linked markers are
most commonly used.
Treatment: Avoidance of vigorous exercise and aggressive treatment of hypertension.
16.2. Skeletal Dysplasias and Dysostoses
Skeletal dysplasias are disorders with abnormal body proportions, frequently dwarfism.
Dysostoses are abnormalities of individual bones or groups of bones. > 100 different dys-
plasias have been described, with different modes of inheritance. Examples:
Apert syndrome is a malformation syndrome with craniosynostosis (abnormal fusion of

cranial sutures), syndactyly, cardiac septation defects (atrial or ventricular), progres-
sive hearing loss, delayed development, mental deficiency, and increased sweat pro-
duction. Caused by new mutations in the gene for a fibroblast growth factor receptor
(FGFR2). Incidence 1 in 10 000.
Achondroplasia is the most common form of short-limbed dwarfism. Incidence is 1 in 10 000

even though 7/8 are caused by a new mutation. Height 120–135 cm with marked short-
ening of the proximal parts of the extremities (rhizomelia), depressed nasal bridge,
bulging forehead, and lumbar lordosis. Complete penetrance. Patients are fertile and
have a normal life span. Homozygotes (some of the children of two achondroplas-
tics) are seriously malformed and die shortly after birth. Most cases are caused by
a Gly → Arg substitution in the transmembrane domain of a fibroblast growth fac-
tor receptor (FGFR3) that is expressed in chondrocytes. This abnormal receptor is
constitutively active. Other FGFR3 mutations cause two other forms of dwarfism
(hypochondroplasia and thanatophoric dysplasia ), two forms of craniosynos-
tosis (Muenke and Cruzons ), and a skin coloration disorder (acanthosis nigricans).
Prenatal diagnosis of achondroplasia is possible with ultrasound or allele-specific
probes - but what do you do when achondroplastic parents want to use prenatal
diagnosis to ensure that their child is also a dwarf?
The differential diagnosis of skeletal dysplasias and dwarfism requires X-rays and
other diagnostic tests. Patients should be referred to a specialist for the diagnostic
workup.
258
Other Muscular Dystrophies 16.3.1
Hypophosphatemia (Vitamin D Resistant Rickets) The patients have bone demineral-

ization and deformities as in rickets, but do not respond to normal doses of Vitamin D.
This condition is caused by a renal transport defect for phosphate. It is X-linked, with
incomplete penetrance in heterozygous females.
16.3. Diseases Of Muscles and Peripheral Nerves
Muscular dystrophies are inherited muscle diseases with progressive muscle wasting and
weakness. Differential diagnosis includes peripheral neuropathies, spinal muscular atrophies,
inflammatory diseases (myositis, dermatomyositis), inborn errors of muscle metabolism, and
myasthenia gravis. The muscle diseases can be distinguished from the neuropathies by ele-
vated blood levels of creatine kinase. Other diagnostic tests are muscle biopsy, electromyo-
graphy, and measurements of peripheral nerve conduction velocity.
Duchenne Muscular Dystrophy (DMD) This X-linked recessive disease is the most com-
mon (1 in 4000 males) and most deadly muscular dystrophy. Patients are normal for 1 or
2 years after birth, before developing progressive muscle weakness and wasting. There is
pseudohypertrophy of the calf muscles, inability to walk by age 10–12, death at 20 a (res-
piratory or cardiac failure), mild mental impairment, and massive creatine kinase elevation
even in the pre-symptomatic stage.
Molecular defect: The gene is huge, with 79 exons spread over 2.2 × 106 bp. This is the
largest gene in the genome! It codes for dystrophin, a structural protein that connects
the sarcolemma with the cytoskeleton. It is also present in the brain. Most mutations
are large deletions.
Molecular diagnosis: Deletions in patients can be detected by Southern blotting or PCR.
If the nature of the mutation is unknown, linked markers can be used for carrier
detection. Many carrier females have mildly elevated creatine kinase, and 8–10 %
show symptoms. This is an unusually large proportion for an X-linked disease (about
5 % is a more common number).
Gene therapy: Somatic gene therapy aimed at myoblasts is in the experimental stage.
Technical problem: The dystrophin gene is so large (13 000 bp coding sequence) that
it is hard to fit into retroviral vectors.
16.3.1. Other Muscular Dystrophies
Becker muscular dystrophy: Mutations in the dystrophin gene (frameshift), but milder
than Duchenne.
259
Limb-girdle muscular dystrophy: Autosomal recessive, juvenile or adult onset and slowly
progressive, and no mental impairment. Severity varies, slower progression cf. DMD. Defect
in one of the four sarcoglycan genes.
Facio-scapulo-humeral muscular dystrophy:!fascio-scapulo-humeral Autosomal domi-

nant with variable age of onset and variable severity.
16.3.2. Myotonic Dystrophy
Autosomal dominant, incidence 1 in 10 000. Weakness and wasting of facial muscles, ster-
nomastoid and distal limb muscles, and myotonia. Also cataracts and cardiac conduction
defects and endocrine changes. Severe cases with hypotonia from birth, pouting expression,
and mild mental deficiency. Two forms:
DM1 is caused by the amplification of a trinucleotide repeat in the 3’-untranslated region

of a gene for a protein kinase (myotonin kinase).
DM2 is caused by a CCTG repeat in an intron of ZNF9 (an RNA binding protein)
The severe cases have only been described for DM1. Age of onset and severity depend on the
repeat number. The repeat, once amplified, is unstable in female meiosis, with pronounced
anticipation when inherited from the mother. PROMM is a milder phenotype (proximal
myotonic myopathy) also caused by these mutations.
16.3.3. Peripheral Neuropathies
Inherited motor + sensory peripheral neuropathies (HMSNs) are known as Charcot-

Marie-Tooth (CMT) syndrome. Inheritance is autosomal dominant, rarely autosomal
recessive or X-linked recessive. Overall incidence 1 in 2500. Most common: CMT type
IA.
Autosomal dominant, caused by duplication of a 1.5 Mb portion of chromosome 17p. The

duplicated part contains a gene for a myelin membrane protein. Peripheral nerve conduction
velocity is reduced in this and some other types of CMT disease. Mild to moderate handicap
with weakness of peroneal muscles and foot drop, some sensory loss, normal life span.
260
Mental Retardation 16.4.2
16.4. CNS Disorders
16.4.1. Hereditary ataxias
This is a heterogeneous group, with intermittent or progressive ataxia and usually associated
with other neurological problems.
Friedreich’s ataxia (incidence 1 in 40 000) is an autosomal recessive disease, chronic

progressive with onset usually between 4 and 20 years. There is degeneration of dorsal root
ganglion cells and spinocerebellar tract, with progressive ataxia, sensory neuropathy, and
areflexia. Most patients die in their 30s–50s. The disease is caused by the expansion of a
GAA trinucleotide repeat in the first intron of a gene. The trinucleotide is amplified from
a normal length of < 22 to > 200. Some patients have no trinucleotide expansion but an
inactivating point mutation in one copy of the gene.
Other ataxias:
• Deficiency of pyruvate dehydrogenase or pyruvate carboxylase
• Deficiencies of urea cycle enzymes. - Hartnup’s disease
• Abetalipoproteinemia
• Ataxia-telangiectasia, a chromosome breakage syndrome with rash immunodeficiency,
and malignancies.
16.4.2. Mental Retardation
Some mentally retarded patients have an identifiable single-gene disorder. Most common
(1 in 2000 males):
Fragile X Mental Retardation Affected males have mental retardation of variable severity.
Mild dysmorphic features: long face, large ears and jaw, and large testes. Some carrier
females are also mentally deficient, and some males with the mutation are not retarded
(“normal transmitting males”). This disease have alternatively been described as an X-linked
recessive or as a dominant disease with reduced penetrance; the proportion of manifesting
carrier females have been reported at anywhere between 20 % and 50 %; manifesting carrier
females generally have very long repeats, longer than the average expressing male.
Molecular defect: A triplet repeat CGG in the 5’-untranslated region of a gene is amplified.
Normal people have 6–54 copies of the repeat (average 29). It is amplified to 60–200 re-
peats in normal transmitting males and > 200 in retarded males. The large amplifications
261
become heavily methylated. This silences the gene. Once the sequence is amplified to > 60
copies, it becomes unstable in female meiosis, and further amplifications occur in successive
generations. Very large repeats are also unstable in mitosis. Therefore, there may be ex-
tensive mosaicism (different repeat numbers in different somatic cells). Also patients with
loss-of-function mutations (deletions, frameshifts) in this gene are retarded. The affected
gene (FMR1) is expressed in the brain, but otherwise its function is unknown.
Diagnosis: Affected males and carrier females have a fragile site in Xq27. The fragile site is
seen only when cells are cultured in folate-deficient media. Southern blotting and PCR are
now the preferred procedures in affected families. These methods provide a good estimate of
the repeat number, but PCR generally cannot produce a product from the longer repeats.
Screening by dot-blotting or microarray is technically difficult.
Huntington’s Disease
This is a neurodegenerative disease with cell death in the corpus striatum and (less severe)
the cerebral cortex. The first signs are vague personality changes, often with poor judg-
ment and/or irresponsible behavior. This is followed by a motor disorder with chorea and
athetosis, accompanied by progressive dementia. Most patients die 15–20 years after onset
of the motor disorder. 20 % of cases are diagnosed before 26 years of age, 50 % before 35,
and 80 % before 43. Penetrance is 100 % in patients living long enough.
Incidence: Variable, about 1 in 20 000 in the US, 1 in 333 000 in Japan. New mutations are
rare.
Molecular defect: Amplification of a CAG trinucleotide repeat in the coding region of a

gene (coding for glutamine). Normal people have 10–29 copies of the repeat, patients
36–120. Expansion of the repeat can occur in male meiosis. Repeat number correlates
with severity and age of onset. The normal function of the gene is unknown.
Diagnosis: PCR or (rarely) Southern blotting.
16.5. Blood Diseases
16.5.1. Clotting Disorders
There are several inherited deficiencies of clotting factors. Most important:
262
Occulocutaneous albinism 16.6.2
Hemophilia A (“classical” hemophilia). This condition is X-linked recessive. There is not

much spontaneous bleeding, but prolonged bleeding occurs after minor injuries. Repeated
bleeding into joints can lead to arthritis. With modern treatment (transfusion of factor VIII
during bleeding episodes), the life expectancy is only mildly reduced.
Molecular defect: Many different mutations in the factor VIII gene have been identified.
Diagnosis: Carrier detection by RIA of factor VIII is unreliable. Allelic heterogeneity makes
the use of allele-specific methods difficult, therefore gene tracking with linked markers is
used for carrier testing and prenatal diagnosis. Complementation tests can be used to
differentiate hemophilia A from other bleeding disorders.
16.5.2. Structural defects of RBCs
In hereditary spherocytosis and elliptocytosis, RBCs are spherical or elliptical. These RBCs
have a reduced life span in vivo because they are removed by splenic macrophages, and they
have increased fragility in vitro. Patients are either asymptomatic or have mild anemia.
These conditions are caused by inherited abnormalities of spectrin, band 3 protein, or other
components of the membrane skeleton. The spectrin content of RBCs is always reduced
because any spectrin that is not tied into the membrane skeleton is degraded.
Inheritance: Autosomal dominant in most patients.
Diagnosis: Hematology (RBC shape). DNA probes or linkage studies are useless because
of extensive heterogeneity (both locus and allelic heterogeneity).
Treatment: No treatment is required in mild cases. Splenectomy is indicated in anemic
patients.
16.6. Skin Diseases
16.6.1. Occulocutaneous albinism
Albinos have white skin, white or yellow hair, and pink iris in typical cases. They get
sunburn, are at risk for skin cancer, and may have mild visual disturbances.
Inheritance: All forms are autosomal recessive.
Molecular defect: Nearly half of all albinos cannot make tyrosinase (“tyrosinase-negative”
albinism). Most tyrosinase-positive albinos have a mutation in a gene that codes for a
membrane protein, probably a transporter for tyrosine in the melanosome membrane.
Treatment: Straw hat and sunglasses.
263
16.6.2. Epidermolysis bullosa (EB)
This is a group of skin blistering diseases with damage to the dermal-epidermal junction in
response to mild trauma. Most forms are autosomal dominant. Most common: EB simplex,
caused by defects in the genes for keratin 5 or keratin 14 in the basal layer of the epidermis.
16.7. Polycystic Kidney Disease
Adult polycystic kidney disease is a relatively common (1 in 1000) autosomal dominant

trait, with cyst development in kidneys, liver, pancreas and spleen. 50 % of patients develop
kidney failure by age 70. 10 % of all patients with end-stage renal disease have polycystic
kidney disease. Prenatal diagnosis with allele-specific probes or linked markers is possible.
Renal cysts can be detected with ultrasound in pre-symptomatic patients. This disease can
be caused by mutations in 2 different genes (locus heterogeneity).
Infantile polycystic kidney disease is a rare autosomal recessive disease, with death in early
childhood. Prenatal diagnosis is possible by ultrasound.
16.8. Cystic Fibrosis
Cystic fibrosis (mucoviscidosis) is the most common lethal single-gene disorder in the white
population (1 in 2000). Exocrine glands of skin, GI-tract, respiratory tract and male repro-
ductive tract are affected, with hyperviscosity of secretions. Specific problems:
• Neonatal bowel obstruction (meconium ileus) in 5–10 % of cases.
• Pancreatic insufficiency (cyst formation and fibrosis), with malabsorption and steat-
orrhea even in babies.
• Bronchial obstruction and recurrent lung infections ( Ps. aeruginosa and Staph. au-
reus).
• Increased CI− , Na+ and K+ in sweat. Babies taste salty.
• Male infertility, atresia of the vas deferens.
This is a severe disease, but with good medical care many patients survive into their 40s.
Pancreatic enzyme replacement, dietary management, bronchodilators, and prophylactic
antibiotics are the mainstay of treatment.
264
“Harmless” Mendelian Traits 16.10
Molecular defect: The gene product is a cAMP-regulated chloride channel in exocrine

glands (CFTR = cystic fibrosis transmembrane regulator). A 3-base-pair deletion is
most common (∆F508 , 70 % in Northern Europe), but > 100 other mutations have
been identified. Some of these mutations lead to a milder form of the disease; the
mildest symptoms described include only the male infertility.
Carrier detection: Molecular probes are available for the more common mutations, but not
the exotic ones.
Prenatal diagnosis: By PCR and/or allele-specific probes.
Gene therapy: Somatic gene therapy aimed at the respiratory epithelium (using adenoviral
vectors) has been attempted.
16.9. Blindness and Deafness
Severe congenital deafness (deaf-mutism) occurs with a frequency of 1 in 2000. 60 % are

autosomal recessive, 10 % autosomal dominant or X-linked, 30 % phenocopies. There is
extensive locus heterogeneity. Recurrence risk after an affected child is born to hearing
parents: 1 in 7. After two affected children: 1 in 4.
Congenital blindness is rare, but several retinal diseases that lead to blindness later in life are
single-gene. Most common phenotype: Retinitis pigmentosa, with night blindness, tunnel
vision, blindness with variable age of onset. Overall incidence is 1 in 5000. Inheritance is
autosomal dominant (15 %), autosomal recessive (70 %) or X-linked recessive (15 %). Some
patients with autosomal dominant retinitis pigmentosa have mutations in the gene for
rhodopsin. The most common forms of acquired blindness (macular degeneration, glaucoma,
cataract, diabetic retinopathy) are not single-gene but multifactorial.
Genetic counseling is difficult both for congenital deafness and for retinitis pigmentosa
because of the extensive locus heterogeneity and allelic heterogeneity.
16.10. “Harmless” Mendelian Traits
Some normal variations and minor “abnormal” conditions are inherited as simple Mendelian
traits.
Red-green color blindness This is a common X-linked recessive condition affecting 5–10 %
of all males. There are two forms: Protanopia (red blindness, incidence 1 %), and
deuteranopia (green blindness, incidence 5 %). Partial defects are called protanom-
aly (1 %) and deuteranomaly (1.5 %). The affected genes code for the red and green
pigments in cones. They are close together on the X chromosome. Color blindness
265
is diagnosed with color charts. Defects of the blue pigment (coded by an autosomal
gene) are rare, as is total color blindness (failure of cone development).
Male pattern baldness This trait is autosomal dominant with male-limited expression in
most families, with hair loss in young men, usually at 20–35 years of age. Expression
is male limited because testosterone is necessary for the phenotype; a castrate will
not get bald. More limited hair loss in older men is usually polygenic.
Eye color There are two or three major interacting genes. In most (but not all) families,
brown is dominant over gray/green. Green/blue is thought to depend on a second
gene, with green dominant over blue.
Cerumen (ear wax) Most Europeans and Africans have wet ear wax, but 80 % of Japanese
and variable proportions of other orientals have dry ear wax. Wet ear wax is dominant
over the dry type.
16.11. Tuberous Sclerosis
Tuberous sclerosis is an inherited neuro-cutaneous disorder with incidence about 1/5000

in some populations that involves many organ systems. Clinical picture includes epilepsy,
learning difficulties, behavioral problems, and skin lesions. One source of pathology is benign
tumors of especially skin, kidney, and brain. Penetrance is high, but some of the symptoms
are so subtle that only a dedicated search will detect the disease in a person; there is
widely variable expressivity. Genetic background shows locus heterogeneity, at least 2 and
probably 4 loci involved; the two well-characterized loci are classified as tumor suppressor
genes and the gene products are involved in intracellular regulation of e.g., insulin signaling.
Symptoms as stated are variable, but lowered IQ, epilepsy and seizures, skin patches with
angiofibromas, hypopigmented maculaes, Shagreen patches, or a distinctive brown fibrous
patch on the forehead are all commonly occurring in this disease. Renal angiomyolipoma or
cysts are common. Cardiac rhabdomyoma is a cause for prenatal death (sometimes seen as
more than one spontaneous abortion when one parent is affected), but if the child survives,
rhabdomyomas will often spontaneously regress. A benign tumor is a problem especially if
it occurs inside the skull, but sometimes the characteristic benign tumors actually develop
into malignant and metastatic states.
16.12. Phenylketonuria (PKU)
PKU is caused by increased levels of phenylalanine in the blood and the typical cases in-
volve mutations in the phenylalanine hydroxylase gene (PAH) inherited in an autosomal
266
Hemochromatosis 16.13
recessive fashion. The homozygous child is normal at birth, but fail to achieve developmen-
tal milestones and develop microcephaly and progressive impairment of cerebral function.
Hyperactivity, seizures and severe mental retardation are major symptoms later in life.
A “mousy” odor, hypopigmentation and eczema are other findings. To completely avoid
the development of symptoms, treatment must be initiated by the third week after birth;
treatment consists of a special diet low in phenylalanine and high in tyrosine, and mon-
itoring the plasma concentrations of these two amino acids. Ideally, treatment should be
continued throughout life, but at least until development is complete. If a woman has been
developing normally and then stopped the diet in adulthood, she needs to restart the diet
well before getting pregnant; otherwise the child will be born with microcephaly and many
other malformations, and after birth show severe neurodevelopmental delay and growth
retardation.
The incidence of PKU has traditionally been listed as 1/10 000 in medical genetics text-
books, which still might be a valid number for European Caucasians. In the USA, recent
estimates for classical PKU range from 1/13 500 to 1/19 000. The disease is more common in
Caucasians and Native Americans, while the incidence is lower in African Americans, His-
panics and Asian Americans (a different source mentions “Orientals” as a high risk group).
Non-PKU hyperphenylalaninemia (caused by mutations in PAH with some residual enzyme
activity) is somewhat rarer.
16.13. Hemochromatosis
The clinical features of hemochromatosis include cirrhosis of the liver, diabetes, hyper-
melanotic pigmentation of the skin, and heart failure. Primary hepatocellular carcinoma,
complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes.
Since hemochromatosis is a relatively easily treated disorder if diagnosed, this is a form of
preventable cancer.
At least 5 iron-overload disorders labeled hemochromatosis have been identified on the basis
of clinical, biochemical, and genetic characteristics.
Classic hemochromatosis (HFE) An autosomal recessive disorder, is most often caused

by mutation in the gene for ferroportin on chromosome 6p21.3. It has also been found to
be caused by mutation in the gene encoding hemojuvelin, which maps to 1q21. This form is
common in Caucasians of Northern European descent, incidences of 1/200–1/300 and 1/72
in NE Quebec.
267
Juvenile hemochromatosis , or hemochromatosis type 2 (HFE2) Also autosomal reces-

sive. One form, designated HFE2A, is caused by mutation in the HJV gene. A second form,
designated HFE2B, is caused by mutation in the gene encoding hepcidin antimicrobial pep-
tide, which maps to 19q13. This and the other minor forms of HFE are rare. One form is
autosomal dominant.
Chromium, an essential trace mineral required for normal insulin function, is transported
bound to transferrin and competes with iron for that binding. It has been found that less
chromium is retained in patients with hemochromatosis than in controls, suggesting that
the diabetes of hemochromatosis may be due in part to chromium deficiency.
Treatment of the iron overload is through phlebotomy about once a week until the iron
content has gone down, and less often after. Desferrioxamine can be given if anemia devel-
ops. Iron intake should be avoided. The different symptoms should be treated as per best
practice.
16.14. Diseases Caused by Unusual Mutations
16.14.1. Imprinting-related Syndromes
Imprinting is not completely understood, but DNA methylation at CG sequences is involved.

A highly methylated region often changes chromatin structure and transcription is turned
off. More than 20 loci are known to be imprinted on one allele in normal subjects. The
imprinting pattern also depends on which parent the chromosome is derived from. In some
cases, a locus will be imprinted if derived from the father but not the mother, and in other
cases, vice-versa.
Prader-Willi syndrome is characterized by hypotonia in infants, and by obesity, hy-

pogonadism, short stature, small hands and feet and mental retardation in children and
adults. It is usually caused by deletion in the long arm of chromosome 15 inherited from
the father.
Angelman syndrome (“happy puppet syndrome”) includes mental retardation, ataxia

and hyperactivity with jerky movements, and inappropriate laughter. It is caused by the
same deletion as in Prader-Willi syndrome, but inherited from the mother.
Both Prader-Willi and Angelman syndromes are caused by effects on imprinting.
The Prader-Willi/Angelman region is unusual in that the region contains two im-
printed regions very close together but non-overlapping. The common deletion overlapping
both imprinting regions, if inherited from the mother, leads to lack of an active gene for the
268
“Angelman gene” (ie. the E6-associated protein ubiquitin-protein ligase gene [UBE3A]). A
different gene is inactive if the deletion is inherited from the father and the undeleted copy
from the mother. A more unusual cause of either Angelman or Prader-Willi syndrome
is uniparental disomy (5–20 % of cases)! If both copies of chromosome 15 are inherited from
the father, they will have Angelman syndrome, if they are inherited from the mother it
leads to Prader-Willi syndrome. Angelman caused by uniparental disomy is milder
than Angelman caused by deletion.
Beckwith-Wiedemann syndrome (BWS) is characterized by overgrowth, resulting in

exomphalos, macroglossia, and gigantism in the neonate. Hemihypertrophy, visceromegaly,
adrenocortical cytomegaly, and dysplasia of the renal medulla are additional features.
Adrenal carcinoma, nephroblastoma (Wilm’s tumor), hepatoblastoma, and rhabdomyosar-
coma occur with increased frequency (≈ 5 %) particularly in those with hemihypertrophy.
There may be linear indentations of the ear lobe and/or peculiar posterior helical ear pits.
The phenotype tends to normalize with age. In normal subjects, only the paternal copy of
the growth factor IGF-2 on chromosome 11 is expressed while the maternal copy is silenced
through imprinting. BSW is most often caused by loss of imprinting, leading to transcription
of both copies of IGF-2.
16.14.2. Lepore Hemoglobins
Some patients with β-thalassaemia have a Lepore hemoglobin: They are lacking the normal
genes for β and δ chains but have a new gene instead that starts as δ and ends as β.
Because it has the promoter of the δ chain gene, it is expressed at a low rate, accounting
for the β-thalassaemia. Some normal individuals have an anti-Lepore mutation: There
is a complete β-chain gene, a complete δ-chain gene and an abnormal gene that starts as
β and ends as δ. These mutations are caused by unequal crossing-over during prophase of
meiosis I. This type of mutation is important during evolution. It can not only create novel
fusion genes, but also gene duplications. It is responsible for the existence of gene clusters
in our genome.
1. State the approximate incidence, the phenotypic features and the molecular lesions of
the most important Mendelian disorders (Osteogenesis Imperfecta, Ehlers-Danlos
Syndrome, Achondroplasia, Marfan Syndrome, Duchenne Muscular Dystrophy,
Myotonic Dystrophy, Charcot-Marie-Tooth Disease, Cystic Fibrosis, Polycystic
Kidney Disease, Occulocutaneous Albinism, Red-green Color Blindness, Hemophilia
269
A, Hereditary Spherocytosis and Elliptocytosis, Huntington’s Disease, Friedre-

ich’s Ataxia, Fragile X Mental Retardation, Deafness and Retinitis pigmentosa, Tuber-
ous Sclerosis, Phenylketonuria, Hemochromatosis).
270
17. Hormone Biochemistry
17.1. Types of Extracellular Messenger
The transduction of a chemical signal from cell to cell may be
Endocrine: The messenger is a hormone that is transported by the blood. In most but not
all cases, the hormone is formed in a specialized endocrine gland.
Paracrine: The messenger acts on neighboring cells in the tissue where it is formed. Exam-
ple: prostaglandins.
Autocrine: The messenger is secreted, but acts on the synthesizing cell. Many paracrine
messengers are also autocrine.
Neurotransmission is a specialized kind of paracrine signaling in which the extracellular

messenger, or neurotransmitter, is secreted by a neuron at a specialized junction called
a synapse.
17.2. Hormone Receptors
A hormone receptor is a cellular protein which binds the hormone non-covalently and me-
diates its physiological effects. It may be an integral protein of the plasma membrane, or it
may be intracellular. Receptor binding is always the first step in hormone action. Hormone
binding induces a conformational change in the receptor protein, and this conformational
change triggers the cellular response. Receptor binding is:
• High-affinity
• Reversible
• Specific for the hormone or class of hormones
• Saturable
271
The dissociation constant KD corresponds to the hormone concentration at which half of

the receptor sites are occupied. It describes the affinity between receptor and hormone.
The maximal binding Bmax corresponds to the number of receptors.
The presence or absence of a receptor determines whether or not a cell responds to a specific
hormone. The magnitude of the response depends on the number of receptor-hormone
complexes. Hormone action depends on the number of receptors (Bmax ), their affinity for
the hormone (KD ), and the coupling of the receptor with its downstream targets. All
of these may be regulated physiologically. Also many drugs act on receptors that were
originally designed for hormones or neurotransmitters.
17.3. Types of Hormone Receptor
Some extracellular messengers can freely diffuse across membranes, but most cannot. Water-
soluble messengers have to bind to a receptor in the plasma membrane. Types:
Steroid hormone receptors are zinc-finger proteins (see fig. 4.1 on page 93). They are lo-
cated either in the nucleus or the cytoplasm of unstimulated cells. Without hormones,
the receptor protein does not bind DNA. With the bound hormone, however, it reg-
ulates transcription by binding to response elements on the DNA. The receptors for
thyroid hormones, retinoic acid, and vitamin D work by the same mechanism. Since
effects are mediated through protein synthesis and the proteins have fairly long life
spans, this type of action cannot work on a minute-by-minute basis.
Neurotransmitter receptors mediating the fast actions of neurotransmitters on the mem-

brane potential are ligand-gated ion channels. These channels are closed in the absence
of the neurotransmitter, but open within a millisecond or so when the transmitter
binds. The opening of sodium or calcium channels causes depolarization (excitation).
Example: nicotinic acetylcholine receptor in the neuromuscular junction. The open-
ing of chloride or potassium channels causes hyperpolarization (inhibition). Example:
GABA-A receptor in the brain.
Receptors with enzymatic activity are inactive in the unstimulated state, but catalyze a
reaction when the extracellular messenger is bound. Example: Receptors for growth
factors and insulin phosphorylate tyrosine side chains in proteins.
G-protein linked receptors mediate most of the classical hormone effects. They usually
trigger the formation of a second messenger.
272
Receptors Coupled to G-Protein 17.5
17.4. Receptors Coupled to G-Protein
These cell surface receptors belong to the family of the 7-transmembrane receptors: they
criss-cross the membrane seven times.
G-proteins are attached to the hormone receptor on the cytoplasmic surface of the plasma
membrane. There are several families of G-proteins which are associated with different
receptors and act on different effector systems. These G-proteins have 3 subunits: α, β and
γ. The β and γ subunits are closely bound to each other, but the α-subunit can easily
dissociate from βγ. The α-subunit has a binding site for GTP and GDP. In the resting
state, the complete heterotrimeric G-protein, with GDP bound to the α-subunit, is bound
to the intracellular domain of the hormone receptor. After hormone binding:
1. The conformation of both the receptor and the attached G-protein changes.
2. This results in the dissociation of GDP from the α-subunit and its replacement by
GTP.
3. The α-subunit -GTP complex dissociates from βγ.
4. Both the α-GTP complex and the βγ leave the hormone receptor and move along the
inner surface of the plasma membrane.
5. α-GTP and βγ bind to effectors on the inner surface of the plasma membrane. Most
effectors are enzymes for the synthesis of a second messenger, but some G-proteins
act on ion channels in the plasma membrane.
6. The α-subunit has an intrinsic GTPase activity which hydrolyzes the bound GTP.
GDP remains bound to the α-subunit, but the α-GDP complex no longer binds the
effector. It binds to βγ and the receptor instead.
7. Upon re-stimulation of the receptor, GTP binds in exchange for GDP, and the cycle
is repeated.
The GTPase activity of the α-subunit is necessary to terminate hormone action. Types of
G-protein: The G-proteins are classified according to the type of α-subunit they contain:
Gs -protein (s = stimulatory) stimulate adenylate cyclase.
Gi -proteins (i = inhibitory) inhibit adenylate cyclase.
Gq -proteins stimulate a phosphatidylinositol-specific phospholipase C.
G12 -proteins regulate ion channels.
273
17.5. Cyclic AMP (cAMP)
cAMP is formed by adenylate cyclase:

O O O H2 H2
-
O P O P O P O C O Adenine O C O Adenine
O O O adenylate cyclase
+ PPi
(on plasma membrane) O P
OH OH O O OH
ATP cAMP
The active site of adenylate cyclase is on the inner (cytoplasmic) surface of the plasma
membrane and its activity is always under hormonal control.
cAMP is inactivated by phosphodiesterase:

H2 O H2
O C O Adenine O P O C O Adenine
phosphodiesterase O
+ H2O + H
+
O P
O O OH OH OH
Several phosphodiesterases exist. Some of them are also controlled by hormones. Phospho-
diesterases are inhibited by methylxanthines (caffeine, theophylline, aminophylline).
The effects of cAMP are mediated by protein kinase A (PKA):

Cat
cAMP
Reg Cat
Reg cAMP
Reg Cat
+ 4 cAMP
Reg cAMP
Cat
cAMP
inactive Protein kinase A catalytically active

subunits
This enzyme phosphorylates specific proteins on serine or threonine residues, including some
enzymes (glycogen synthase, phosphorylase kinase etc) and nuclear proteins. The most im-
portant nuclear targets are transcription factors of the CREB family (CREB = cAMP
response element binding). CREB proteins bind to the cAMP response element. The phos-
phorylation of DNA-bound CREB proteins by protein kinase A stimulates transcription.
274
Calcium and Phosphatidylinositol 17.6
cAMP mediates the effects of calcitonin, PTH, TSH, epinephrine (β-receptors only), vaso-
pressin (on kidney), glucagon, ACTH, and many other hormones. Clinical problems:
• Patients with pseudohypoparathyroidism show signs of PTH deficiency (hypocal-

cemia, tetany,) but unlike true hypoparathyroidism, other abnormal signs (short
stature, mental and neurological problems) are also present, and PTH levels are nor-
mal or elevated. Many of these patients have a defective Gs protein, with inefficient
coupling of hormone receptor to adenylate cyclase.
• The symptoms of cholera are induced by the exotoxin of the bacterium Vibrio cholerae.
Cholera toxin binds to the surface of intestinal mucosal cells, and one of its subunits
enters the cell. This subunit is an enzyme which ADP-ribosylates a specific arginine
side chain in the αs -subunit of Gs :
αs -subunit + NAD+ → αs -subunit-ribose-P-P-ribose-adenine + Nicotinamide
This covalently modified αs -subunit can still bind GTP and activate adenylate cyclase,
but it has lost its GTPase activity. This results in prolonged stimulation of adenylate
cyclase. The typical diarrhea of cholera is mediated by excessive accumulation of
cAMP in the intestinal mucosal cells. Also one of the toxins of enterotoxic E. coli, the
cause of traveler’s diarrhea (“Montezuma’s revenge”), acts by a similar mechanism.
Some hormones (endorphins, α2 -adrenergic agonists, D2 -dopamine etc.) decrease cel-

lular cAMP levels. Their effect is mediated by an inhibitory G-protein (G1 ). Pertussis
toxin (Bordetella pertussis causes whooping cough) modifies G1 covalently, thereby
locking it in the inactive GDP-form.
17.6. Calcium and Phosphatidylinositol
Calcium concentrations in the cytoplasm are normally very low (< 1 × 10−6 M). Some
hormones and neurotransmitters, such as epinephrine (α1 -receptors), acetylcholine (mus-
carinic receptors), histamine (H1 -receptors), angiotensin II, serotonin, and vasopressin in
blood vessels, cause the release of calcium from the ER, thereby increasing its cytoplasmic
concentration.
Through a G-protein, the activated hormone receptor stimulates a phosphoinositide-specific

phospholipase C, which cleaves the membrane lipid phosphatidylinositol-4,5-bisphosphate
into inositol -1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol.
IP3 diffuses to the ER membrane where it opens a calcium channel. Calcium affects cellular
processes by binding to various regulatory proteins:
Troponin C mediates contraction of striated muscle.
275
Calmodulin is structurally related to troponin C. Present in all nucleated cells. The calcium-
calmodulin complex activates many enzymes, including phosphorylase kinase, the
calcium pump in the sarcoplasmic reticulum, brain phosphodiesterase, and myosin-
light-chain kinase in smooth muscle.
Protein kinase C (PKC) is attached to the inner surface of the plasma membrane, with
substrate specificity different from protein kinase A. Activated by calcium, diacylglyc-
erol (a product of phospholipase C!) and phospholipids. There are many isoenzymes
of protein kinase C with different regulatory properties. Pharmacologically, protein
kinase C is activated by phorbol esters (from croton oil), which act as tumor promot-
ers.
Muscle contraction is always triggered by calcium. In striated muscle, it is mediated by tro-

ponin C. Smooth muscle has no troponin, but calcium binds to calmodulin and this complex
stimulates myosin light chain kinase. The phosphorylation of the myosin light chains by this
enzyme causes contraction. cAMP opposes the calcium effect in smooth muscle. In vascular
smooth muscle, α1 -adrenergic agonists, vasopressin and angiotensin contract by increas-
ing calcium, β-adrenergic agonists relax by increasing cAMP. In bronchial smooth mus-
cle, histamine (H1 receptor) and acetylcholine (muscarinic receptor) contract by increasing
Ca2+ , β-adrenergic agonists relax by increasing cAMP. Phosphodiesterase inhibitors (theo-
phylline, aminophylline) are good for asthma because they increase cAMP.
Exocytosis of water soluble products from storage vesicles in secretory cells is triggered by
calcium: transmitters from nerve endings, histamine from mast cells, insulin from β-cells,
zymogens from the exocrine pancreas.
Cell growth and/or mitosis is often stimulated by the phospholipase (C)-(IP3)-calcium

system.
Besides the phospholipase C/IP3 system, there are other ways by which an external stimulus
can elevate intracellular calcium:
• The NMDA receptor (a glutamate receptor in the brain is a ligand-gated calcium

channel.
• An activated G-protein opens a calcium channel. Example: A calcium channel in the

myocardium is opened by the activated Gs -protein in response to epinephrine.
• An extracellular messenger depolarizes the plasma membrane, and this opens voltage-
gated calcium channels. Examples: Neurotransmitter release and smooth muscle con-
traction. Voltage-gated calcium channels in smooth muscle cells can be blocked phar-
macologically by calcium channel blockers.
276
Desensitization of Receptors 17.9
17.7. Cyclic GMP (cGMP).
cGMP is formed by guanylate cyclase, an enzyme which occurs in both membrane-bound

and soluble forms. cGMP is inactivated by various phosphodiesterases. It acts by activating
protein kinases G. Second messenger functions:
Atrial natriuretic factor (ANF) is a polypeptide hormone from the heart which causes
natriuresis, vasodilation and a decrease of aldosterone synthesis. The ANF receptor
has an intracellular guanylate cyclase domain that becomes active after ligand binding.
Nitric Oxide (NO·), known as “endothelium-derived relaxing factor”, is formed in endothe-

lial cells by an NO· synthase which uses arginine as a substrate. Vasodilators such
as acetylcholine (muscarinic receptors), bradykinin, and histamine activate the NO·
synthase through calcium-calmodulin. NO· diffuses from the endothelium to the vas-
cular smooth muscle cells where it activates a soluble (cytoplasmic) from of guanylate
cyclase, causing vasodilation. Nitrovasodilator drugs such as nitroglycerin are metab-
olized to nitric oxide.
In retinal rod cells, visible light activates rhodopsin, a member of the 7-transmembrane
receptor family. Rhodopsin is coupled to the G-protein transducin. The α-subunit-
GTP-complex of transducin activates a cGMP-specific phosphodiesterase: the extra-
cellular stimulus (in this case not a hormone but light) decreases cellular cGMP levels.
The decrease in cGMP hyperpolarizes the membrane.
17.8. Desensitization of Receptors
Prolonged agonist exposure can desensitize receptors:
Receptor phosphorylation: The agonist-induced conformation of the receptor may be sen-

sitive to phosphorylation, which converts the receptor to a “useless” form. Exam-
ple: Activated (but not inactive) β-adrenergic receptors become phosphorylated by
β-adrenergic receptor kinase (BARK). The phosphorylated receptor can still bind
epinephrine, but it can no longer interact with the Gs -protein. Also protein kinase A
can phosphorylate and thereby inactive the β-adrenergic receptor.
Receptor down-regulation: Agonist-stimulated receptors are physically removed from the

cell surface by receptor-mediated endocytosis. Endocytosed receptors can be recycled
to the cell surface, but some of them are degraded by lysosomal enzymes. The down-
regulation of insulin receptors is a mechanism of insulin resistance in type II (maturity-
onset) diabetes mellitus. Unlike receptor phosphorylation, down-regulation is long-
term and can be overcome only by the synthesis of new receptor.
277
1. Recognize the structural features of the major classes of steroid hormones and state
the precursor relationships of these hormones.
2. Name the substrates and products of cytochrome P-450 dependent hydroxylation
reactions.
3. Describe the sequence of steps in thyroid hormone biosynthesis and their cellular
localization.
4. Describe the sequence of reactions, cofactors and precursor relationships in the biosyn-
thesis of catecholamines, indolamines and histamine.
5. List the mechanisms for the inactivation of biogenic amines, including the mechanisms
of the inactivating reactions and type of products formed.
6. List the typical steps in the processing of pro-hormone, using pro-insulin as an exam-
ple.
7. Name the products that are formed in the cyclooxygenase and lipoxygenase pathways
of eicosanoids metabolism.
8. Name the substrates for the synthesis of acetylcholine and GABA, the products formed
in degradation of these neurotransmitters and the enzymes catalyzing these reactions.
9. Describe the typical kinetics of hormone receptors with respect to Bmax and KD .
10. Describe the mechanism of signal transduction by hormone-regulated G-proteins and
name the second messengers that activate protein kinases A, C and G.
11. Outline the sequence of events in the phosphoinositide second messenger system.
12. Name the molecular targets of cholera toxin and pertussis toxin.
13. Describe the mechanism of action for steroid and thyroid hormones.
14. Name examples of agents that act through protein/tyrosine phosphorylation, by bind-
ing ligand-gated ion channels, or by inducing the synthesis or degradation of cGMP.
278
Part IV.
Semester two, Mini I

18. Vitamins and minerals
This introduction will be necessarily brief. For more a more complete introduction to nu-
trition see [Shiles et al., 2005].
18.1. Nutrient doses

All things are poison, and nothing is without poison. Only the dose makes that
a thing is not poison.
Philippus Theophrastus Aureolus Bombastus von Hohenheim (Paracel-
sus)
“A lot helps a lot” is no proper approach to human nutrition. Substances which are required
by the body in small amounts may be very toxic if given in too large a dose. For this reason
experts in different countries have set up tables of dietary requirements. Most well known
of these are the recommended dietary allowance (RDA) set by a group of nutrition
scientists in the Food and Nutrition Board at the National Academy of Science of the
USA, which are reviewed every 5 years. These represent the best judgement of a group
of experts on the daily amounts of nutrients that are sufficient and safe for a healthy
individual [Otten et al., 2006].
The RDA is calculated from the average requirement for a particular nutrient plus 2 stan-
dard deviations (see fig. 18.1). It therefore meets the requirements of about 98 % of the
population. An average person requires 77 % of the RDA to maintain health. Failing to
eat the RDA of a nutrient does not necessarily lead to deficiency, but the probability of a
deficiency is the larger, the more uptake deviates from recommended values.
In recent years, RDA-values were complemented by estimated average requirement (EAR)

and tolerated upper intake level (UL)-values. Together with the RDA-value these form
the Dietary reference intake (DRI). EAR is the dose required by an average member of
the group studied. UL is that dose that will not cause adverse effects in the majority of
subjects, even when taken regularly over many years.
Note that if the EAR of a nutrient can not be determined it is also mathematically impos-
sible to determine the RDA. In such cases adequate intake (AI) values are given instead of
RDA-values. The AI-value is determined from the intake of a healthy reference population,
281
Nutrients: Beneficial and toxic effects
F(x)
f(x)
Dose
EAR RDA UL LD 50
Estimated Recommended Tolerated 50%
Average Dietary Upper Lethal
Requirement Allowance Intake Dose
Level
Dietary Reference Intake (DRI)
If EAR has not been determined, RDA can not be set. In this
case use AI (Adequate intake) instead of RDA.
Food and Nutrition Board of the National Academy of Sciences of the USA
EB 2007
Figure 18.1.: Estimated Average Requirement (EAR): A nutrient intake value that is
estimated to meet the requirement of half the healthy individuals in a group.
Recommended Dietary Allowance (RDA): This value is a goal for indi-
viduals. It is the daily dietary intake level that is sufficient to meet the nutrient
requirement of 97–98 % of all healthy individuals in a group. Tolerable Up-
per Intake Level (UL): The highest level of daily nutrient intake that is
likely to pose no risks of adverse heath effects to almost all individuals in
the general population. As intake increases above the UL, the risk of adverse
effects increases. EAR, RDA and UL values together form the Dietary Ref-
erence intake (DRI). Somewhere beyond the UL lies the LD50 , the dose
that would kill half of the studied population. LD50 is not part of the DRI,
however. Note the break in the dose-axis.
282
Assessing nutrient stores and needs 18.1.2
hence it is an intake that by definition will cause neither deficiency nor toxicity in the
majority of people.
Remember: A deficiency can not be diagnosed from uptake data alone, but only from
clinical, physical and biochemical data on a specific person.
18.1.1. The US Recommended Daily Allowance
Recommendations for RDA are given separately for different genders and age groups, mak-
ing the table a bit unhandy. For everyday purposes a simplified version of the recommen-
dations exist, the US recommended daily allowance (US-RDA). It is important to
clearly distinguish RDA and US-RDA, despite their similar abbreviations. The US-RDA
is taken to be the highest RDA (based on 1968 recommendations) in any gender and age
group above 4 years (in most cases the US-RDA is equivalent to the RDA of 18 year old
males). Because US-RDA values are used for labeling of food packages and changes would
be expensive to the industry, they are not updated like the RDA values, but have remained
constant since 1968.
18.1.2. Assessing nutrient stores and needs
The bodies pools of many micronutrients, in particular minerals and fat soluble vitamins, are
difficult to determine. The nutrients are stored in tissues like bone or liver, and blood levels
are fairly constant unless either the stores are so depleted that blood levels can no longer
be maintained or the stores are so full that the excess can no longer be put in there (see fig.
18.2). RDAs for such nutrients are therefore difficult to determine. One lab technique used
is to give a relatively large dose of the suspected nutrient. The amount excreted is then
measured. If retention is high, body stores were depleted. Nutritional assessment is covered
in [Gibson, 2005].
Additionally, needs for micronutrients, in particular ultratrace elements, may depend on
physiological status, and may increase upon exposure to nutritional, metabolic, hormonal
or psychological stress. In such a situation a subclinical deficiency may become manifest.
For the ultratrace elements there is an additional problem: Very sensitive analytical tech-
niques are required to determine their concentration in both food and tissue. Additionally,
it is very difficult to exclude very small concentrations of trace elements not only from food
and water, but also from cages, toys, scientific instruments etc., which might otherwise lead
to contamination. The techniques continue to be developed, one reason why we expect to
see further chemical elements declared essential in the future.
To ensure adequate supply, trace elements and vitamins are sometimes added to processed
food. We distinguish:
283
start of withdrawl onset of symptoms
blood
stores
critical concentration
Figure 18.2.: Depletion of body stores (blue, dotted line) and blood concentrations (red, full
line) of a nutrient over time. As the uptake of the nutrient ceases the body
stores are depleted at a certain rate, but the blood concentration remains
almost constant. Only after the body stores are so empty that blood concen-
tration can no longer be maintained does it drop too. Symptoms of withdrawal
appear when the blood concentration drops below a critical threshold.
284
Vitamins 18.2
Enrichment : when nutrients are added solely to replace losses during food processing
Fortification : when extra nutrients are added to the food.
18.2. Vitamins
Modern scientific understanding of nutrition started when Lavoisier identified the rela-
tionship between oxygen consumption and heat production. Liebig introduced the chemical
analysis of food. He believed that carbohydrates and fats meet the bodies energy require-
ments, while proteins are used for growth. Dumas and Lunin found that a synthetic food
composed of water, protein, carbohydrates and fat (the then known components of milk)
does not support human growth: babied fed such a diet died. Funk proposed in 1912 that
apart from these macronutrients small amounts of various substances are required, which
he called vitamines (literally: nitrogen containing compounds necessary for life). The ‘e’
was dropped from vitamine in 1920, to reflect the fact that few of these compounds are
amines, some do not even contain any nitrogen at all.
Vitamins were originally divided in fat soluble vitamin A and water soluble vitamin B. In
1917 it became clear, that vitamin B contained a heat labile substance with activity against
Beri-Beri (Vitamin B1 ) and a heat stable compound that promoted growth (Vitamin B2 ).
1925 Jansen & Donath isolated 5 g of pure vitamin B1 from 1 t of rice bran, the struc-
ture was finally solved in 1936, followed by the first synthesis. Today 300 t/a of synthetic
Vitamin B1 are produced. In total 13 vitamins have since then been isolated, identified and
synthesized, the last one was vitamin B12 in 1973.
In this context it is important to remember that vitamins isolated from natural sources and
those synthesized in the laboratory are identical substances and have, dose by dose, the
same effects inside the body. There is therefore no need to use the (sometimes very expen-
sive) “natural” vitamins for supplementation. Many “natural” vitamins even are synthetic
products mixed into a natural base (like synthetic B vitamins mixed with yeast powder or
synthetic ascorbate mixed with acerola).
Vitamins are organic substances required by an organism in small amounts (µg/d or mg/d).
They may act either as coenzymes or as participants in chemical reactions (for example
antioxidants).
Very high doses of vitamins may have additional, pharmacological or toxic effects. Research
into such effects has only started. It is certainly unnecessary, but possibly also dangerous,
to give vitamins at very high doses (exceeding 150 % US-RDA). “Megadoses” of more than
10 times US-RDA may be toxic even in the case of water soluble vitamins which can be
excreted into urine. In case of fat soluble vitamins, which accumulate in the body, such
doses are definitely dangerous.
285
O O
P P
O O O
O O
2-Methyl-butadiene (Isoprene) isopentenyl diphosphate
(activated isoprene)
HO
HO
7-Dehydrocholesterol (Provitamin D) Tocopherol (Vitamin E)
OH
Plastoquinone (Vitamin K1) Retinol (Vitamin A1)
Figure 18.3.: Fat soluble vitamins are derived from isoprene (2-methyl-butadiene). The bi-
ological building block is isopentenyl-diphosphate (activated isoprene), which
you have encountered in cholesterol synthesis. The building blocks are in-
dicated by alternating blue and red segments. Vitamin E and K additionally
contain aromatic residues produced in the shikimate-pathway, which is present
in plants but not animals.
Provitamins are compounds that can be converted into the active vitamin (for example
carotene to vitamin A). Anti-vitamins (or vitamin antagonists) are compounds which are
chemically similar to vitamins and can bind to their binding sites, without being able to ful-
fill their function. They are used as research tool, chemotherapeutic agents (e.g. methotrex-
ate) or as antibiotics (e.g. sulphonamides).
Vitamin deficiencies are still a major health problem in developing countries, but may be
seen in the poor, homeless, drug addicts and psychiatric patients even in industri-
alized nations.
18.2.1. Fat soluble vitamins
Fat soluble vitamins belong into the class of chemicals known as isoprenoids (isoprene =
2-methyl butadien, see fig. 18.3).
286
Fat soluble vitamins 18.2.1
Fat soluble vitamins are not excreted from the body, but accumulate in the liver if taken in
excess. Thus most people have stores of these vitamins and they may be able to go without
them for weeks or even months before deficiency becomes visible. On the other hand, if
these vitamins are taken up in large excess over prolonged periods, toxic effects are seen.
Fat soluble vitamins are absorbed in the small intestine dissolved in fat droplets also con-
taining bile salts. Cave: Patients with reduced fat uptake (for example with intestinal or
pancreas disease, cystic fibrosis, abetalipoproteinemia or on a low fat diet) may not receive
enough fat-soluble vitamins!
Retinal (Vitamin A)
Function Vitamin A is required for vision (part of the “visual purple” rhodopsin in the
light sensitive cells of the retina, which is bleached to “visual yellow” by light). However,
only 0.01 % of vitamin A is found in the eyes, and 90 % is stored in the liver. The remainder
is distributed throughout the body and serves to control reproduction and development:
• Epithelial cells in mucous membranes require vitamin A. In its absence, keratinized

cells without cilia are formed, which can not produce mucous. This may lead for
example to respiratory infections.
• Vitamin A controls the development of osteoblasts and osteoclasts and thereby bone
formation.
• Sperm production in males and maintenance of pregnancy in females depends on a
sufficient supply of vitamin A.
• Wound healing requires vitamin A, the mechanism is unclear.
Vitamin A is actually a group of related compounds, called retinoids. Retinol, retinal and
retinoic acid can be converted into each other by redox reactions, carotene consists of two
vitamin A molecules linked tail to tail. Oxidation of retinal to retinoic acid is irreversible in
the human body, and retinoic acid can not protect from the visual and reproductive effects
of vitamin A deficiency. However, retinoic acid is an important morphogenetic hormone,
which regulates growth and the formation of epithelia and bones.
Until 1967 vitamin A amounts were measured by their biological effects in international
units (IU). From then on “retinol equivalents” (RE) became the official unit of measurement.
1 RE is equivalent to 1 µg of retinol, 6 µg of β-carotene or 12 µg of other retinoids. This is
equivalent to 3.3 IU of vitamin A.
Clinically retinoic acid (not carotene) may be used for topical administration in acne pa-
tients (cave: may cause birth defects if given to pregnant females). β-carotene has been
shown to interfere with proliferation and progression in certain cancers (especially lung
cancer).
287
CH3 CH3 CH3

3 2
9
4 1
7
8 10
11 13
14
15
OH
6 12
5
CH3 H3C CH3

H3C CH3 Retinol CH3 CH3
2 [H]
CH3 CH3 H3C
H3C CH3 β-carotene
CH3 CH3 CH3
O
CH3 11-cis-Retinal
CH3 Retinal CH3 H
H3C
H
[O]
H3C CH3 H3C
O
CH3 CH3 CH3 O Retinal reductase Rhodopsin Opsin
Retinol isomerase
OH
Retinoic acid Light

H3C CH3
CH3 CH3 H CH3
O
H
H3C CH3
all-trans-Retinal
Figure 18.4.: Different forms of vitamin A. The alcohol retinol is oxidized first to the alde-
hyde retinal (required for vision) and finally to retinoic acid, which is a hor-
mone involved in morphogenesis. The conversion between retinal and retinol
is fully reversible, but retinoic acid can not be converted into retinal by the
human body. 11-cis-retinal is a component of rhodopsin (visual purple). Under
the influence of light it isomerizes to all-trans-retinal (visual yellow), which
dissociates from the protein component and is transported out of the retina for
regeneration. 11-cis-retinal then returns to the cone cell stacks and recombines
with opsin to form rhodopsin again. The light induced conversion of 11-cis into
all-trans retinal also starts the signaling cascade in the photoreceptor cells.
288
Food sources Yellow and orange plants, leafy vegetables, red palm oil, egg yolk, milk fat
and liver provide high concentrations of vitamin A. Synthetic β-carotene is used as food
color for example in lemonades.
In developing countries with undersupply of vitamin A staple foods may be supplemented

(cane sugar in central America, sodium glutamate in the Philippines). The use of red palm
oil for cooking and the home growth of carotene rich vegetables should be encouraged. In
some countries children < 5 a of age receive 30 000 RE of vitamin A in oil directly onto the
tongue every 6 month.
Uptake and metabolism Retinoic acid is readily absorbed in the intestine and transported
in blood bound to albumin. Target cells bind it at a surface receptor, cellular retinoic
acid binding protein (CRABP)).
Retinol is usually taken up as retinyl palmityl ester which is split by pancreas juice. Retinol
is absorbed by the intestinal epithelial cells with the aid of bile, uptake is vitamin E depen-
dent. Inside the mucosal cell the palmityl ester is reformed and secreted with chylomicrons
into the lymphatic system, from where it enters the blood stream and the liver, where it is
stored in lipid droplets.
Retinol is released (Zn2+ dependent) from the liver bound to retinol binding protein
which binds to a receptor at the surface of the target cells, cellular retinol binding
protein (CRBP).
Carotene may be either split in the intestine by pancreas juice to retinol or taken up
whole, this uptake (but not that of retinol) is regulated by vitamin A stores in the body.
Carotene is stored in adipocytes and adrenals, not in the liver. Blood carotene concentration
reflects food intake, not body stores. Excess uptake may cause a yellow tinge of the skin,
carotenodermia. It may be converted in the body to retinol, but it may also be used as
antioxidant.
Excretion Retinoic acid can be excreted by the kidneys, and some carotene and retinol
may be excreted in bile. Most retinol is stored in the liver however (about 150 000 RE in
the average adult).
Deficiency Night blindness, xerophthalmia (dry eyes), failure to produce tears, keratoma-
lacia (excessive keratin in skin and conjunctiva), opaqueness and sloughing of the corneal
epithelium (Bitot’s spots), rupture of the cornea, followed by infection and hemorrhage of
the eye. Worldwide this leads to 250 000 cases of blindness per year, mostly in children.
Many more may succumb before these symptoms develop.
289
Figure 18.5.: Bitot’s spot, eye lid changes and cataract in a patient with vitamin A de-
ficiency. Hypovitaminosis A is still the worlds most common cause of pre-
ventable blindness, even though it can be prevented for a few cents per patient
and year. Image from webeye.ophth.uiowa.edu/.
290
Other signs of vitamin A deficiency include sensitivity to infections, developmental prob-

lems, sterility, a rough, dry skin (especially around the shoulders), folliculosis (permanent
goose bumps), acne.
Changes in the gastrointestinal epithelia may lead to diarrhea. Tooth enamel may fail to
form, sense of taste and smell may be lost.
Toxicity High doses of vitamin A (more than 20 000 RE/d) given to pregnant female may
cause birth defects in the infant, for example cleft palate.
Vitamin A poisoning has been observed after a diet high in liver (historically seen in po-
lar exploration). Symptoms include headaches, drowsiness, nausea, loss of hair, dry skin,
diarrhea, anorexia, skeletal pain, resorption of bone, cessation of menstruation in females,
beginning several months after the start of high uptake.
Infants are more sensitive, symptoms may appear after 1 month and may also include hy-
drocephalus, increased intracranial pressure and hyperirritability.
Because the uptake of carotene is regulated, plant sources of vitamin A are safe.
Genetic diseases relating to vitamin A metabolism The regeneration of all-trans- to 11-

cis-retinal can not be done in the retinal rods. Instead, the all-trans-retinal is transported
to the blood stream by a specific ABC-type membrane ATPase, ABC-R. Mutations in this
transporter lead to accumulation of all-trans retinal and its metabolites in the cone cell,
which become poisoned. In the mildest cases (miss-sense mutations) this leads to Star-
gardt-disease. More severely affected patients suffer from autosomal macular dystro-
phy, in the worst cases (frame shift or splicing mutants with total loss of ABC-R function)
retinitis pigmentosa results. In autosomal macular dystrophy the extend of damage also
depends on environmental factors, which are not fully understood yet.
Calciferol (Vitamin D)
Function Calcitriole, the active form of vitamin D, is produced in the kidney under the
influence of parathormone and stimulates the synthesis of a Ca2+ and Pi binding protein in
the gut and thereby the absorption of these nutrients from food. Calcitriol also stimulates
Ca2+ and Pi resorption in the kidney and Ca2+ release from the bones.
Amounts of vitamin D in the food are measured in international units (IU), 1 µg = 40 IU.
Because the synthesis of cholecalciferol in the skin requires UV light, nutritional needs for
vitamin D are particularly high in people whose occupation or clothing habits exclude them
from sunlight.
291
26
25
27
23
21
24
18
20 22
12
19 11 13 17
CH3 14
16
15
9
2 8
10
5
3
4 6 7
HO 7-dehydro cholesterole
UV-light h*ν
(skin)
Ergocalciferole
CH2
identical! Vitamin D2, from yeast + plants
H2C
HO
Cholecalciferole
(Calciole, Vitamin D3)
OH
Liver
OH
CH2
Calcitonine
HO 25-Hydroxy cholecalciferole
(Calcidiole)
Kidney
Ca, Pi
Parathormone
OH Calcitonine OH
OH
OH (OH)
CH2 CH2
Kidney
1,25-Dihydroxy cholecalciferole
HO (Calcitriole, active forme of vitamine D) HO 24-hydroxylated vitamin D
(inactive, excreted)
Figure 18.6.: Metabolism of vitamin D. 7-dehydro cholesterol can be produced in the hu-
man liver. Opening the ring system to form cholecalciferol (vitamin D3 ) is
possible in the human skin only if it is exposed to UV light. Cholecalciferol is
converted to 25-hydroxy cholecalciferol (calcidiol) in the liver, which is then
converted (under the control of parathormone) in the kidney to the active
form of vitamin D (Calcitriol, 1,25-dihydroxy cholecalciferol). This substance
controls the uptake and metabolism of Ca2+ and P. Ergocalciferol (vitamin
D2 ) is a compound found in yeasts, which can substitute for cholecalciferol. It
is used as a vitamin D supplement.
292
Figure 18.7.: Genu varum (bend femurs) and reduced bone density in a 2-year old child
with rickets. Image from en.wikipedia.org.
Pregnant woman also have high needs for vitamin D, because about 1/2 of the fetal Ca2+ is
deposited in the last 6 weeks of pregnancy. Needs are also high during lactation, as breast
milk contains large amounts of Ca2+ .
Food sources liver, fish, yeast, milk (cave: patients with lactose intolerance!).
Uptake and metabolism Vitamin D is absorbed in the upper part of the small intestine
with 80 % efficiency, transported in chylomicrons to the blood stream, where it is attached
to α-globulin2 (vitamin D binding protein) and transported to the liver, together with
the vitamin D produced in skin. In the liver it is converted to calcidiol, transported to
the kidney and converted to calcitriol, the active form. The later reaction is controlled by
parathormone under the influence of low serum [Ca2+ ]. High serum [Ca2+ ] leads to the
release of calcitonin from the thyroid gland, which stimulates the hydroxylation of calcidiol
and calcitriol in the 24-position, leading to inactive products (see figure 18.6) .
Patients receiving anticonvulsants or tranquillisers may have larger needs for vitamin D.
Excretion Some vitamin D may be excreted in bile and feces.
293
Deficiency If not enough vitamin D is available, Ca2+ and Pi metabolism are dysregulated.
This leads to a low muscle tonus and to weak bones and teeth, which are unable to fulfill
their function. In children this is called rickets, in adults osteomalacia. Patients suffering
from rickets are easy to recognize, the leg bones bow under the weight of the body (O-legs),
the rib cage is concave and narrow and crowds the internal organs (pigeon breast). The
fontanel fails to close (should normally happen by 1 year of age), teeth erupt late, are ill
formed and decay rapidly.
Toxicity In sensitive children doses as low as 400 IU/d may lead to hypercalcemia, nausea,
weight loss and failure to grow.
Calcidiol increases the production of the interleukin IL-2 in T-lymphocytes. That inter-
feres with the maturation of TH1 - and dendritic cells, resulting in a TH2 -answer and IgE
production against environmental antigens. Thus the risk for allergies is increased, indeed,
according to some authors the increase in the morbidity from allergies is at leat in part due
to increased vitamin D supplementation in children.
During pregnancy a hypervitaminosis D may lead to impaired maturation of osteoblasts

and defective bone formation in the embryo.
Tocopherol (Vitamin E)
Function Tocopherols and tocotrienols include a large number of related compounds, of

these α-tocopherol is most active. The activity is measured in tocopherol equivalents (TE),
1 TE = 1 mg of RRR-α-tocopherol = 1.49 IU. Synthetic tocopherol supplements contain
an inactive stereoisomer, hence their activity is, weight for weight, only half of the natural
product.
Vitamin E is an antioxidant and radical scavenger. It prevents oxidation of polyunsaturated

fatty acids in cell membranes, in particular of the red blood cells and in the lungs. In
this role it partially overlaps with the function of Se. Needs for vitamin E increase with
the uptake of poly-unsaturated fatty acids (PUFA), but fortunately the best sources of
PUFA also contain a high concentration of vitamin E. Other functions for vitamin E in cell
signalling, which have been described in the literature, are now understood as consequence
of the protection of PUFA, which in turn have signalling function, e.g. arachidonic acid.
(Trabera & Atkinson, 2007).
Vitamin E will protect vitamins C and A in food against oxidation during storage.
Clinical uses of vitamin E: intermittent claudication (cramps in calf muscle) and fibrocystic
breast disease (painful but harmless knots).
294
CH3
OH
H3C
O CH3
α-tocopherol CH3 CH3
OH
this compound has the highest vitamin E effect
H3C
O CH3
α-tocopherol CH3
CH3
R
OH
H3C RH
CH3
O O glutathione
H3C ascorbate
β-tocopherol CH3
O CH3
CH3
free radical
R + H2O
OH RH
CH3
H3C O
H3C
O CH3
O CH3
OH
γ-tocopherol CH3 quinone
CH3
Figure 18.8.: Left: Tocopherols include a large number of different compounds (which might
be isolated for example from wheat germ oil), but only tocopherol α, β and
γ have vitamin E activity. Right: Oxidation of α-tocopherol by free radicals.
You have seen a similar stepwise reaction with ubiquinone (CoQ) in oxidative
phosphorylation.
295
Food sources Plant oils, nuts, wheat germ, asparagus, avocado, mango, spinach
Uptake and metabolism Vitamin E is absorbed with fat in the upper part of the small
intestine and transported in chylomicrons and LDL. It rapidly exchanges with cell mem-
branes. Patients suffering from cystic fibrosis have low fat absorption capacity and may
require supplementation with 50–400 TE/d.
Excretion Vitamin E is stored in adipocytes, muscle and liver, so little excretion occurs.
Deficiency flaky skin, weak muscle (nutritional muscular dystrophy, muscle fiber frag-
mentation), liver degeneration, loss of membrane function, hemolysis (resulting in anemia),
increased lipofuscin formation.
Hypovitaminosis E may also be involved in cancer and sterility (tocos (gr) = birth). How-
ever, a recent study (SELECT) on protection against prostate cancer had to be aborted as
no beneficial effect could be demonstrated either alone or in combination with Se. Similarly,
no beneficial effect against breast and lung cancer has been detected in other studies.
In premature infants low stores of vitamin E combined with a limited ability to absorb fat
may lead to increased sensitivity to oxygen damage, in particular to the retina (retrolental
fibroplasia). 100 TE/d α-tocopherol protect, if given either by injection or orally in a water
miscible form.
Toxicity Hypervitaminosis E is rare. Symptoms include gastrointestinal distress, nausea,

diarrhea and failure of blood clotting (made worse if vitamin D is low). Epidemiological
studies have shown increased all-cause mortality if intake exceeds 2000 mg/d, an UL of
1000 mg of α-tocopherol was established.
Vitamin E related inherited diseases In ataxia and vitamin E deficiency (AVED)

the inability of the liver to pass vitamin E into the blood stream leads to severe neurolog-
ical defects. Patients require supplementation with an injected, water-miscible vitamin E
preparation.
Vitamin K
Function Vitamin K is a group of compounds required for blood coagulation (spelled with
‘k’ in many languages other than English). The need for this compound was discovered by
Danish Carl Peter Henrik Dam in chicken 1929, the substance was characterized by
American Edward Adelbert Doisy. Both shared the 1943 Nobel price for Medicine.
296
Glu Gla
O
2,4-Naphtoquinone CO Prot CO Prot
O phytyl OH Prot N CH Prot N CH
H H
CH2 CH2
3 C C
CH3 O O HOOC H H HOOC H COOH
O Phylloquinone (plants, vitamin K1) Warfarin
OH CO2 + O
H
CH3 CH3
O
O OH
isoprenyl- R O2 R
H2O
OH γ-glutamyl carboxylase O
n=4-13 Vit. K (hydroquinone)
CH3 O O Vit. K (epoxide)
Warfarin
O Menaquinone (bacteria, vitamin K2) Phenprocoumon (Marcoumar)
2 [H] 2 [H]
Vit. K epoxide O Vit. K epoxide
O OH reductase reductase
CH3
CH3
R
O O O
O Coumarin
Menadione (synthetic, vitamin K3) Vit. K (quinone)
Figure 18.9.: Left: Different forms of vitamin K: Phylloquinone is found in plants,

menaquinone in meats. Menadione is a synthetic form of vitamin K. Note that
in the older literature quinones are spelled (etymologically correct!) chinons.
Middle: Vitamin K antagonists used as anti-coagulants (“blood thinner”) and
rat poison. Right: Conversion of Glu to Gla by γ-glutamyl carboxylase, thus
creating a Ca2+ binding site. The enzyme requires vitamin K in its reduced
form (hydroquinone) as cosubstrate, the vitamin is oxidized to the epoxide in
the process. The epoxide is returned to the active form in a 2-step process,
both steps are catalyzed by vit. K epoxide reductase. This enzyme is inhibited
by vit. K antagonists.
297
The mode of action of this vitamin however was discovered only in 1974 by several inde-
pendent groups.
Vitamin K is a cofactor in the synthesis (in the liver) of factors II (prothrombin), VII
(proconvertin), IX (thromboplastin) and X (Stuart-factor) of the blood clotting cascade.
For activity, these factors require the conversion of a glutamic acid residue to γ-carboxy-
glutamic acid (abbreviated Gla). The change from Glu to Gla creates a Ca2+ binding site
in the protein.
Osteocalcin, a Ca2+ binding protein in bone and possibly other proteins require Gla forma-
tion as well, as does Gas6, a anti-apoptotic factor in cells.
Conotoxins (from a marine snail, Conus geographus) and some snake venoms also contain
Gla. Bacteria like E. coli use menaquinone as an intermediary for anaerobic respiration,
where electrons are transferred from lactate or NADH to other electron acceptors than
oxygen, e.g. nitrate forming nitrite.
Two naturally occurring compounds have vitamin K activity, phylloquinone (vitamin K1 )
in plants and menaquinone (vitamin K2 ) in animals and bacteria. Menadione is a synthetic
compound (vitamin K3 ), which is activated to vit. K2 in the body. Like with other synthetic
K-vitamins it’s use in animal feeds is now frowned upon, it should not be used in humans.
Food sources About half of our supply of vitamin K is produced by our intestinal flora.
Salicylates, sulphonamides and antibiotics reduce intestinal synthesis.
Vitamin K is also contained in leafy green vegetables (1 Tbs of parsley = 150 % RDA) and
in smaller concentration in fruits, tubers, seeds, eggs, dairy products and meats. Cooking
can reduce vit. K content of food, steaming is recommended instead.
Uptake and metabolism Vitamin K uptake in the small intestine is aided by bile and
pancreas juice. Obstruction of the bile duct and oil based laxatives interfere with vitamin
K uptake. Efficiency of uptake is about 10 %. Body stores of vitamin K are small and
turnover is rapid. Bacterially produced vitamin K in the end gut is absorbed passively with
relatively low efficiency, even this uptake however requires bile acids.
Excretion Vitamin K may be stored in the body for about 1–2 wk maximum in liver and
adipose tisue, metabolites are excreted into the bile and to some extend also into urine.
Deficiency Deficiency is usually the result of malabsorption and leads to hemorrhaging,

stomach pain, bone malformation (especially in children) and deposition of Ca2+ -salts in
arteries. Since such deposits are part of atherosclerosis it has been speculated that vitamin
K RDA may be too low.
298
Water soluble vitamins 18.2.2
CH3
OH
H NH2 HC
NH2 O
S O O S O
N N C C O P O P O N N C C O P O P O
+ H2 H2 + H2 H2
O O O O
H 3C N CH3 H3 C N CH3
Thiaminediphosphate Hydroxyethyl-thiamine diphosphate

(activated acetaldehyde)
(activated vitamin B1)
Figure 18.10.: Thiamin diphosphate, the metabolically active form of thiamin, serves as a
cofactor for dehydrogenases and transketolases.
Toxicity has been observed only after massive overdose of synthetic vitamin K derivatives:
Hemolytic anemia, increased blood bilirubin (jaundice) followed by bilirubin accumulation
in the grey matter of the brain. This then leads to mental retardation. Vitamin K1 and K2
are considered non-toxic, a UL has not been set. Suspicions that injected vitamin K could
lead to leukemia have not been confirmed in later studies.
Vitamin K related genetic diseases Newborns are at risk for late-onset hemorrhagic
disease of the newborn (HDN) after about day 8, for this reason in many countries a
prophylactic vitamin K dose is given. Since the condition is rare the benefit is questionable,
however, it will do no harm.
18.2.2. Water soluble vitamins
The capacity of our body to store water soluble vitamins is much smaller than for the fat
soluble. Thus overdoses of water soluble vitamins tend to be less toxic, because the excess
is excreted from the body, on the other hand, vitamin deficiencies develop much faster as
there are no body stores to draw on.
Thiamin (Vitamin B1 )
Function Thiamin diphosphate, the metabolically active form of thiamin, serves as cofac-
tor for dehydrogenases and transketolases, for example:
• oxidative decarboxylation (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase,

branched chain α-ketoacid dehydrogenase) where the remaining molecule is trans-
ferred to CoA.
• transketolase (e.g. in pentose phosphate pathway)
299
• Conversion of tryptophan to niacin
• Neurotransmitter release for high frequency impulses, the mechanism is unknown
• the metabolism of ethanol and acetaldehyde
It is thought that Beri-beri is caused by the accumulation of toxic intermediates from

inhibition of transketolase.
Clinically thiamin is used to reduce nausea in pregnancy.
Food sources meat, fish, poultry, legumes, nuts, whole grain cereals, less in milk and
vegetables.
Thiamin deficiency is usually associated with the change from unprocessed to processed
grain products, for example polished rice or white flour. Thiamin (and some other nutrients)
are contained mostly in the aleuron layer of the grain, which is removed during processing.
Thiamin is sensitive to heat, oxygen, and alkali (baking soda).
Some foods contain a thiaminase, which destroys the thiamin. In fish and seafood, this
enzyme is heat labile, but black tea contains a heat stable form.
Uptake and metabolism Thiamin is taken up in the duodenum by active, Na+ coupled
transport after the hydrolysis of any phosphate groups by intestinal phosphatases. Sulfon-
amides and some antibiotics interfere with uptake.
The human body contains about 30–70 mg of thiamin, half of that is found in muscle.
Excretion in urine.
Deficiency Hypovitaminosis B1 leads to Beri-beri, a life threatening disease. Beri-beri is

still a major public health problem in some developing countries. About 1.5 × 106 people
worldwide are said to suffer from thiamin deficiency of various degrees. For example in the
Philippines there are 75 infant deaths per 100 000 births linked to Beri-beri, it is the fourth
leading cause of death.
Beri-beri is characterized by lack of motoneuron coordination (Beri-beri literally means

“I can’t, I can’t”), weakness, wasting of muscles, un-coordinated movements, convulsions,
confusion, apathy, high heart rate (tachycardia), heart hypertrophy, edema, high serum
pyruvate, cyanosis, vomiting. Babies have a thin, almost inaudible cry. Cause of death is
usually heart failure.
300
Onset is most frequently in infants of 2–5 months, very rapid and requires treatment within
hours to prevent death. Cause is usually a thiamin deficiency in the mother, who then
produces a milk with low thiamin content, which also contains pyruvic aldehyde, a toxic
metabolite.
In adults there are two forms of Beri-beri:
- wet (edematous) with fluid accumulation starting in the feet and moving upwards.
Death occurs from heart failure as the chest becomes congested with liquid.
- dry (wasting) with gradual loss of body tissue and emaciation.
Further symptoms in both forms include numbness of the legs, irritability, disorderly think-
ing, constipation (because of low muscle tone in the GI tract), nausea, anorexia.
Alcoholics, whose “liquid nutrition” does not contain vitamin B1 , who may suffer from in-
testinal degeneration (limiting uptake) and who have a high need for this vitamin to detoxify
the ethanol, may suffer from Wernicke-Korsakoff-disease. Wernicke’s encephalopa-
thy is an acute CNS dysfunction (delirium) and can be cured with thiamin. Korsakoff’s
psychosis (anterograde amnesia with confabulation) is irreversible and requires institution-
alization.
Toxicity none known for uptakes up to 100 mg/d per os. Injected thiamin seems more
dangerous and should be avoided.
Riboflavin (Vitamin B2 )
Function Riboflavin in the form of FAD and FMN is a cofactor of oxidoreductases (see
fig. 18.11).
Food sources Milk, egg, whole grain cereals, yellow vegetables, liver, kidney.
Riboflavin is stable against heat, acid and oxygen, but destroyed by alkali and exposure to
light.
Uptake and metabolism Uptake of riboflavin in the upper part of the small intestine is
regulated by thyroid hormone. Uptake efficiency is about 70 % if riboflavin is taken with a
meal, but only 15 % if taken separately. Riboflavin is converted to FMN in the intestinal
epithelial cells and transported in blood to the liver bound to albumin. There it is converted
to FAD.
301
Riboflavin (Vitamin B2)
H O H H O
H3 C N H 2 [H] H 3C N H
N N
H3 C N N O H 3C N N O
H CH2 H CH2 H
HC OH HC OH
Riboflavin HC OH HC OH
(Vitamin B2) HC OH HC OH
CH2 CH2
OH OH
H O H O
H 3C N H H3 C N H
N N
H2 N
H 3C N N O H 3C N N O N
H CH2 H CH2 N
HC OH N
HC OH
N
HC OH HC OH
OH
HC OH HC OH O
O H 2C O O OH
O P O O P O P O
O O O
Flavin mononucleotide (FMN) Flavine adenine dinucleotide (FAD)
Figure 18.11.: Riboflavin, in the form of the coenzymes FMN and FAD is a component of
oxidoreductases.
Excretion Riboflavin is excreted in urine, after the kidneys have reabsorbed the bodies
needs. Small amounts are excreted in bile.
Deficiency psychological deviations (hypochondriasis, depression, hysteria), cheilosis (cracked

and inflamed lips), glossitis (smooth, purple-red tongue), growth retardation, reduced hand
grip strength, fatty dermatitis, anemia.
A lack of vitamin B2 during embryonal development leads to malformations (cleft palate,

cataracts, shortening of the long bones and fusion of ribs).
Cave: Since riboflavine is light sensitive it gets destroyed during phototherapy of newborns
suffering from hyperbilirubinemia. Supplementation is required in such cases.
Toxicity No riboflavin toxicity has been observed up to 20 mg/d, which is the limit of
uptake capacity.
302
H3 C
O N
O
C O C
NH2
N N N
Nicotinic acid Nicotinamid Nicotin

(not a vitamin, toxin in tobaco)
O
C
NH2 O H
N NH2
+ C
N
O O
O P O
O N
HO OH
O P O N
Isoniacid
O O NH2
N (tuberculostatic drug)
N N
HO OR
R = H: NAD+
R = Pi: NADP+
H O O
H H
H C H C
NH2 2 [H] NH2 +
+ + H
H N H H N H
R R
NAD(P)+ NAD(P)H + H+
Figure 18.12.: Nicotinamide is a component of NAD+ and NADP+ , which are used as co-
factors in dehydrogenases to catalyze the transfer of reduction equivalents.
Nicotine is not a vitamin, but a toxin from tobacco. In the American litera-
ture nicotinic acid and nicotinamide are called niacin, to prevent the wrong
impression that tobacco consumption would supply a vitamin. Isoniazid is
an antivitamin used as tuberculostatic drug.
303
Nicotinamide and nicotinic acid (Niacin, Vitamin B3 )
Function Nicotinamide is a component of NAD+ and NADP+ , which are cofactors in

dehydrogenases.
Nicotinamide can be synthesized in the human body from tryptophane, 60 mg of Trp are
equivalent to 1 mg of nicotinamide (1 niacin equivalent NE). Proteins on average contain
about 1 % Trp, thus 6 g of protein are equivalent to 1 NE. Conversion of Trp to NAD(P) re-
quires thiamin, pyridoxin, riboflavin and biotin. Cave: In ~ estrogen inhibits the conversion
of Trp to niacin, making them more susceptible to pellagra than |.
Food sources Meat, legumes, nuts, fish.

Corn contains nicotinamide in a chemically bound, unavailable form. Treatment of corn
with dilute bases releases it (nixtamalization). South American Indians traditionally treat
corn with a suspension of burned lime Ca(OH)2 to achieve this effect.
Niacin is chemically stable under conditions normally employed in food preparation.
Uptake and metabolism Vitamin B3 is absorbed in stomach and upper part of the small
intestine.
Excretion in urine as methyl derivative.
Deficiency Lack of nicotinamide, leads to pellagra (Ital. pelle = skin; agra = rough)
characterized by “the four D’s”: diarrhea, dermatitis, dementia and death. Black tongue
disease in dogs is used as an animal model for pellagra. Treatment is by oral vitamin B
complex or yeast, this may be accompanied with a tranquilizer in patients with psychiatric
symptoms. Recovery should occur within a few days.
A diet high in Leu and low in Ile may also cause pellagra, this can occur in areas where
sorghum or millet is a staple food.
Isoniazid (INH, isonicotinic acid hydrazide) is a tuberculostatic drug which acts as vitamin
antagonist. Pellagra may occur in patients treated with this drug.
Alcoholics have high needs for vitamin B3 for the detoxification of ethanol.
Corn was introduced in the 17th century into southern and eastern Europe and widely
planted because of its high yields. However, the habit of nixtamalization was not trans-
ferred from the new world. This led to a pellagra epidemics in Europe and later in the
southern US, especially in spring when little other food was available. First description of
pellagra was in 1735 by Gaspar Casal, Goldberger in the 1930’s recognized pellagra as
304
Figure 18.13.: Dermatitis on sun-exposed body parts in pellagra. Image from Ashourian
& Mousdicas, NEJM 354 (2006) 1614.
305
a vitamin deficiency. The myth of vampires is claimed to originate from these epidemics:
The light-sensitive dermatitis made patients avoid daylight. After several episodes of ery-
thema, the patient’s skin becomes parchment-like. Bleeding sores in mouth and tongue
make blood run down out of the victims mouth, the glossitis makes the tongue appear
red. Brain-damage results in dementia and a manic-depressive state, making the behavior
of the victims unpredictable and potentially dangerous. Pica leads to the patient eating
life animals (zoophagia: flies, spiders, birds). Mucus membrane lesions lead to the patients
refusal to eat solid food. The high number of deaths from an unknown, chronic wasting
disease was attributed to a nosferato (from Gr. plaque carrier) seeking revenge on his/her
neighbors and family after death.
Toxicity Nicotinic acid, but not nicotinamide, is a vasodilator. Doses in excess of 50 mg

lead to flushed skin, tachycardia, hypoglycemia and burning or tingling sensations.
Excessive supplementation with vitamin B3 leads to gastrointestinal distress, nervousness,

recurring ulcers, glucose intolerance and fulminant hepatitis.
Under strict medical supervision, 1–2 g/d may be used to treat high blood cholesterol and
schizophrenia (“orthomolecular therapy”), higher doses have no beneficial effect, but single
doses of up to 9 g do not seem to cause acute toxicity.
Pantothenic acid (Vitamin B5 )
Function Pantothenic acid is a component of Coenzyme A, which is required as acyl

carrier. The energy-rich thioester bond preserves the energy of C−C-bonds split in making
the acyl-CoA.
Pantothenic acid is clinically used to stimulate gastrointestinal mobility after surgery. Pan-
tothenol is used in cosmetics, its mechanism of action (if any) is unclear.
Food sources all living matter (pan (Gr) = everywhere). Particularly rich are organ meats,
fish, whole grain cereals and royal jelly.
Pantothenic acid is destroyed by dry heat.
Uptake and metabolism
Excretion Out of an average uptake of 13–19 mg/d 2–7 mg appear in urine and 1–2 mg in
feces.
306
OH O CH3
OH
O N
CH3
OH
Pantothenic acid
Adenine
Pantothenic acid
Mercaptoethyl amine Ribose-3'-phosphate
O O CH3 O O
HS O P O P O N
N N O N
CH3 O O N
OH
N N
O OH
Coenzyme A
O P O
O
Figure 18.14.: Pantothenic acid is a component of Coenzyme A, which is used as acyl-group

carrier in biochemical reactions (Acetyl-, succinyl-, propionyl-, fatty acid and
HMG-CoA). The thioester bond to the acyl group is energy rich.
Deficiency Lack of pantothenic acid causes unspecific symptoms resulting from reduced
cell health, including a higher rate of infection, fatigue, insomnia, vomiting.
Reports that pantothenic acid could prevent the greying of hair have not been substanti-
ated.
Toxicity Very large doses (more than 10 g) cause diarrhea.
Pyridoxine (Vitamin B6 )
Function Pyridoxalphosphate is a cofactor in transaminase, deaminase and decarboxylase

reactions. These occur in energy metabolism, amino acid metabolism, hormone synthesis
(serotonin, histamine, taurine, GABA), cross-linking of elastin, synthesis of RNA and heme,
synthesis of arachidonic acid from linoleic acid, cholesterol synthesis and turnover, synthesis
of nicotinamide.
The requirements of vitamin B6 depend on the protein intake, 16 µg are needed per g of
protein.
307
H2C OH HC O H2C NH2

HO CH2OH HO CH2OH HO CH2OH
H3 C N H 3C N H 3C N
Pyridoxin Pyridoxal Pyridoxamin
NH2
HC O O H 2C
H2 H2 O
HO C O P O HO C O P O
O O
H 3C N H3 C N
Pyridoxal phosphate Pyridoxamine phosphate
(bound to transaminase) (bound to transaminase)
+ +
HO O HO O
C C
H2 N C H O C
R R
aminoacid keto acid
Figure 18.15.: Pyridoxalphosphate is a cofactor in transaminase, deaminase and decarboxy-

lase reactions. Pyridoxin, pyridoxal and pyridoxamine can all be used by the
human body to produce pyridoxalphosphate.
Food sources Pyridoxin is widely distributed, white meats, liver, whole grain cereals, egg
yolk, bananas, avocados, green leafy vegetables and potatoes are good sources.
Pyridoxin is sensitive to alkali, light and oxygen.
Uptake and metabolism Absorption is by passive diffusion in the jejunum with 40–80 %
efficiency depending on the food source. Pyridoxin is transported in blood bound to albu-
min.
Absorption of pyridoxin is inhibited by more than 40 drugs, including oral contraceptives

(add 10 mg/d of pyridoxin to the requirements), isoniazid, penicillamine.
Excretion In urine mostly as pyridoxic acid, in smaller amounts also as pyridoxamine,

pyridoxal and pyridoxal phosphate.
Deficiency Weakness, difficulty in walking, microcytic hypochromic anemia, insomnia,

neuromotor seizures, hyperexcitability, dermatitis. In developed countries vitamin B6 defi-
ciency is mostly seen in alcoholics.
308
Low uptake leads to increased oxalate in urine, this may lead to urinary calculi.
If deficiency occurs in the first few months of life, permanent brain damage may occur.
Infants suffering from hypovitaminosis B6 give a characteristic, piercing cry.
During pregnancy a lack of pyridoxin increases the risk of toxemia, a fatal complication.
Women who took oral contraceptives before becoming pregnant may enter pregnancy with
low stores of pyridoxin. Supplementation with pyridoxin may help to control nausea.
Large doses (up to 200 mg/d) are used for the symptomatic treatment of carpal tunnel
syndrome.
Toxicity Doses of more than 2 g/d lead to loss of motor coordination, which is reversible
upon withdrawal. Regular supplementation with more than 200 mg/d may lead to depen-
dency.
Cobalamine (Vitamin B12 )
Function Vitamin B12 is required for
• Several mutases in which a hydrogen and some group on an adjacent carbon exchange
places. This may be followed by an elimination of water or ammonia.
• Methyl group transfers and recovery of folate.
• Other reactions in microorganisms and plants.
Food sources Bacteria (cyanocobalamine), dairy products (methyl- and hydroxocobal-

amin) and fish, poultry and meat (adenosylcobalamine).
Contrary to public opinion intestinal bacteria can not supply the body with cobalamine,
as they occur in the large intestine, where little if any uptake occurs. On the contrary in
veterinary medicine antibiotics like aureomycin and penicillin are given to speed weight gain
in cows in part by killing cobalamine destroying bacteria in their gastrointestinal system
(intestinal physiology and anatomy is quite different in ruminants and man).
Uptake and metabolism Vitamin B12 is in the stomach bound to “intrinsic factor”, a heat
labile glycoprotein produced in the parietal cells of the stomach. The complex of vitamin
and intrinsic factor is then absorbed in the small intestine, the reaction depends on Ca2+ .
Uptake efficiency is 1–60 % depending on supply. In blood cobalamine is transported bound
to transcobalamine (a protein) to reduce renal loss. Cobalamine is stored in the liver, most
people have stores for several years.
309
R= CN: Cyanocobalamine (Vitamin B 12)

O NH2 O
R= 5'-desoxyadenosine: Adenosylcobalamine (Coenzyme B 12) C
C NH2
R= CH3: Methylcobalamine (cofactor for C1-transfer) R CH2 C
O H3C C H2
H2
C H2 H3C
HO OH
H2N C H2 CH3
N N C N
CH3
3+
H2 N N
O CH2 O N Co N
N C C H2
5'-desoxyadenosine H2N H2 N C H2
CH3CH3 C
CH3 C NH2
CH3
C O
C H2 CH2
H2N
O CH2
C O
HN N CH3
CH2 O
H3C C O P O N
CH3
H
O OH
H2C O
OH
O S CoA O S CoA
C B12 C
odd chain
fatty acids HC CH2 Methylmalonyl- HC CH2
O C H CoA-mutase H C O
O O
Methylmalonyl-CoA Succinyl-CoA
Figure 18.16.: Cobalamine has a heme like structure with a central Co atom. Various ligands
can occupy the 6th position of the Co coordination sphere. One reaction which
requires cobalamine is the exchange of a hydrogen atom against a neighbor-
ing group, for example in methylmalonyl-CoA mutase. If methylmalonyl-CoA
is not eliminated by this reaction, it’s concentration will increase until it is
utilized instead of malonyl-CoA for the synthesis of fatty acids. The resulting
branched chain fatty acids get incorporated into phospholipids and destabi-
lize myelin sheaths.
310
Excretion with urine, pancreas juice and bile.
Deficiency Lack of vitamin B12 leads to pernicious anemia, where red blood cell pre-
cursors can not divide for lack of DNA. This leads to fewer, very large (megaloblastic)
erythrocytes. Because of failure in fatty acid biosynthesis in the brain patients also show
demyelinization.
Pernicious anemia is usually the result of failure to produce intrinsic factor, so that uptake
efficiency is reduced. The condition was originally treated by giving the patient 300 g/d
raw liver pulp (Whipple 1920 followed up by Minot & Murphy. All three shared the
Nobel Price for Physiology and Medicine in 1934). Liver contains high concentrations of
cobalamine and the high dose overcomes the uptake deficiency. Life quality for the patients
was improved as more and more concentrated extracts could be prepared, which could be
injected i.m. (Cohn 1938). Since 1973 vitamin B12 can be made synthetically, intrinsic
factor from hog stomach is now also available.
Apart from genetically determined inability to secrete intrinsic factor, a cobalamine defi-
ciency may be caused by:
- a strict vegan (as opposed to vegetarian) diet
- removal of stomach
- defective mucosal lining of the stomach, caused by genetic problems, Fe-deficiency,

alcoholism, thyroid dysfunction
- tapeworm infections
- lack of transcobalamine, the transport protein in blood
- therapy with anticonvulsants or certain antibiotics
- reduction of gastric acid production in old age, needed to liberate cobalamine from
protein complexes in food
- Inherited lack of uptake protein in the intestine (Schilling-disease)
- Autoantibodies against transport protein in blood or cell surface receptor
Toxicity No toxic reactions have been observed so far even in relatively large doses, because
amounts exceeding the binding capacity of transcobalamin are rapidly excreted by the
kidneys. Allergies have been reported though.
311
O C O OH
O C O C C
HO C
R O R OH
.O C
R O R OH
O C O C
O O O H2O
HO C HO C O C O C
HC HC HC alkali, light, heat
HC OH
HO CH HO CH HO CH HO CH
C OH C OH C OH C OH
H2 H2 H2 H2
L-Ascorbic acid Ascorbic acid radical L-Dehydroascorbic acid Diketogulonic acid
physiologically inactive
HO O HO O
C C
CH2 CH2
CH2 CH2
O C O2 CO2 C OH NH2 O
O OH
C H2N C C C C C C
HO O H2 H H2 H2 H
OH
α-ketoglutarate Succinate
Proline hydroxylase similar: Hydroxylysine
+ +
H H H H
H H HO H
H H H H
O O
H N H N
X Gly X Gly
Peptidyl-Proline Peptidyl-4-Hydroxyproline
Figure 18.17.: L-Ascorbic acid (Vitamin C) is required for the hydroxylation of proline and
lysine in connective tissue and acts as an antioxidant. Under the influence of
light and heat diketogulonic acid is formed, this compound has no vitamin
C activity.
312
L-Ascorbic acid (Vitamin C)
Function Only L-Ascorbic acid has vitamin C function, the D-form is used under the
name of erythroic acid as a meat preservative. Ascorbic acid is required for
- the introduction of hydroxy groups into lysine and proline residues in collagen, which
are required for the correct folding of this extracellular matrix protein. The reaction is
Peptidyl-Pro + α-ketoglutarate + O2 → Peptidyl-Hydroxyproline + succinate + CO2 .
The hydroxylase requires Fe2+ (ferrous) in its catalytic center. Sometimes succinate
is formed without hydroxylation of proline or lysine. In this case Fe is oxidized to the
3+ (ferric) form, and requires reduction by ascorbic acid to regenerate the enzyme
(α-ketoglutarate + ascorbate + O2 → succinate + dehydroascorbate + CO2 + H2 O).
A similar mechanism probably explains the requirement for ascorbate in the synthesis
of tyrosine, carnitine, hydroxymethyluracil, uracil and uridine.
- formation of norepinephrine from tyrosine and serotonin from tryptophan
- fatty acid desaturation (regeneration of cytochrome b5 )
- C-terminal amidation of neuropeptides with glycine at the C-terminus (for example α-

melanotropine): Peptidyl −CO−NH−CH2 COOH + O2 + 2H+ → Peptidyl−CO−NH2
+ H2 O + OCH−COOH. This reaction is catalyzed by an ascorbate- and Cu2+ -dependent
monooxygenase.
- Val and Ile synthesis (not in humans)
- conversion of cholesterol to bile salts
- reducing dietary Fe3+ to Fe2+ for uptake
- chelating minerals like Fe2+ , Zn2+ , Ca2+ for uptake
- protecting dietary vitamins like folic acid from oxidation
- detoxification in the liver
- histamine detoxification (allergies)
- sulfate metabolism: formation of ascorbic acid sulfate
Dehydroascorbic acid is regenerated using glutathione.
Pharmacological doses of vitamin C (more than 1 g/d) are sometimes recommended to

protect against cancer and colds. Both efficacy and safety of such doses have not been
established.
313
Food sources After Lind showed in 1747 that juice from citrus fruits could cure scurvy,
it became law that any ship leaving an English harbor had to carry sufficient lime juice for
the journey, hence the nickname “limey” for British service men.
Ripe fruits and vegetables contain considerable amounts of vitamin C. Of meats, only liver
is a good source.
Cows milk contains little vitamin C, and most of that is destroyed during sterilization.
Infants raised on cows milk therefore require supplementation to avoid the rapid onset of
lethal scurvy. Most infant formulas take care of this however. Breast milk contains sufficient
vitamin C for the infants needs, provided the supply for the mother is high enough.
Ascorbic acid is frequently added to processed food as antioxidant to stabilize color and
flavor, protect sensitive nutrients like PUFA in margarine and to maintain an acidic pH.
Uptake and metabolism Ascorbic acid is absorbed in the upper part of the small intestine
both by diffusion and by Na+ -dependent uptake. Uptake efficiency depends on the dose. The
maximal serum concentration of 1.2 mg/l is reached at a dose of 45 mg/d, this is equivalent
to total body stores of 1500 mg. If uptake of vitamin C stops, body stores will decrease by
about 3 % per day, scurvy will start at 300 mg total body stores.
Smoking reduces vitamin C absorption, effectively doubling the required dose.
Excretion Vitamin C is excreted into urine, reabsorption is regulated to maintain the

serum level at 1.2–1.5 mg/l.
Deficiency Lack of vitamin C leads to scurvy. Patients become listless, tired, suffer from
body aches, muscle cramps and loss of appetite. The joints swell. The skin becomes dry,
rough and feverish. Because of the weak connective tissue small hemorrhages lead to purple
spots (petechia) on the skin. The gums will also be affected, teeth will fall out and secondary
infections cause additional damage.
Toxicity At doses in excess of 500 mg/d ascorbic acid is converted to oxalic acid, this
may lead to kidney stones in the 20 % of the population genetically predisposed to this
disease. Megadoses in pregnant woman may lead to dependency in the infants. There is
also evidence for oxidative damage caused by excess uptake of ascorbic acid (production of
superoxide radicals by the Fenton-reaction).
314
Figure 18.18.: Scurvy resulting from vitamin C deficiency. As Pro and Lys in collagen can
no longer be hydroxylated, connective tissue is weakened leading to petechia.
Pictures from phil.cdc.gov.
O O
HN N H HN N COO-
H H H H
C CH2 C CH2
H2C H S H2C H S
CH2 CH2
CH2 CH2
CH2 Biotin
CH2
C - C
HN O HCO3 - HN O
H2O
CH2 CH2
CH2 CH2
Lysin residue
CH2 CH2
O CH2
H O CH2
N H H O H
C N N H H O
C N H C N
HC O C N H
R H HC O
O R H
R O R
Protein chain
Biocytin Carboxybiocytin
Figure 18.19.: Biotin is required as cofactor for carboxylases, to which the biotin is chemi-
cally bound at a lysin residue.
315
Biotin
Function Biotin dependent carboxylases are required for synthesis of fatty acids (Acetyl-
CoA carboxylase for the production of malonyl-CoA), glucose (pyruvate carboxylase for
the synthesis of oxaloacetate), nicotinic acid, purines and prostaglandins. The metabolism
of Val, Ile, Met and Thr requires propionyl-CoA carboxylase and the metabolism of Leu
β-methylcrotonyl-CoA carboxylase. Of these enzymes acetyl-CoA carboxylase is cytosolic,
the remaining enzymes occur in mitochondria.
Biotin is also required for the formation of antibodies and pancreatic amylase.
Food sources Widely distributed. Biotin from animal sources tends to be protein bound
and fat soluble, plant biotin free and water soluble.
Rich sources are liver, kidney, egg yolk, yeast, legumes (in particular sprouted), nuts,
cauliflower and whole grains.
Biotin is heat stable, but is unstable under alkaline conditions and may be oxidized.
Uptake and metabolism Biotin bound to lysine (biocytin) is released from proteins by
proteolytic digestion. Biocytin is hydrolyzed by biotinidase and taken up in the jejunum
by active, saturatable transport and by passive diffusion. Anticonvulsants interfere with
uptake.
Biotin is transferred to a lysine group in the apo-carboxylases by holocarboxylase synthase

in a ‘ping-pong’ reaction: Biotin and ATP bind to the synthase and bound biotinyl-5’-AMP
is formed under release of pyrophosphate. Then the apo-carboxylase binds to the synthase,
and biotin is transferred to it. Finally, the holoenzyme and AMP are released.
Excretion in urine, in part as metabolites.
Deficiency Maculosquamous dermatitis, alopecia, low appetite, nausea, depression, seizures,

encephalopathy, glossitis, immune suppression.
A deficiency may occur after the ingestion of a large number of raw eggs, because the
egg white contains a factor (avidin) which binds biotin with very high affinity (Kd =
1 × 10−15 M).
Toxicity none known.
316
Inherited diseases relating to biotin Neonatal multiple carboxylase deficiency (neona-

tal MCD) is caused by a holocarboxylase synthase which has a reduced affinity for biotin.
The disease becomes apparent soon after birth.
Late onset MCD becomes apparent when the infants are weaned, that is when they switch
from free biotin in mothers milk to biocytin in normal food. It is caused by biotinidase
deficiency.
In both cases supplementation with very high doses of biotin (up to 10 mg/d) can bring
relief.
Folic acid (Vitamin M, Vitamin B9 )
Function Folic acid is a C1 -carrier, this is discussed in detail in the chapter on amino acid
metabolism.
Food sources Wheat germ, liver, kidney, yeast, mushrooms, fruits and leafy vegetables
(folio (Lat.) = leaf).
Folate is destroyed by heat, light and oxidation.
Uptake and metabolism After extraneous Glu residues have been split of, folate is ab-
sorbed with 50–90 % efficiency in the upper part of the small intestine. Uptake is vitamin
B12 dependent. Folate is then transported by the portal blood to the liver and from there
to the rest of the body. Inside the cells of the body it is conjugated with a chain of up
to 12 Glu molecules to prevent it from leaving the cells. In the liver folate is stored as
methyl folate, release requires vitamin B12 . Normal liver stores are about 7–15 mg, which
is sufficient for almost half a year.
Oral contraceptives, antitumor agents, anticonvulsants and excessive ethanol consumption

interfere with folate uptake. Tropical sprue will also lead to uptake deficiency.
Excretion because folate is converted to the polyglutamate inside the cells, very little is
excreted.
317
Figure 18.20.: Megaloblastic anemia. Images curtesy of Dr. W.M. Todd,

http://www.va.gov/telepathvisn6/Hematpth.htm.
Deficiency megaloblastic anemia, weak immune system.

During pregnancy higher risk of complications like hypertension and damage to the embryo:
neural tube defects and “small for date” births. Therefore reasonable folate supplementation
during pregnancy is recommended.
Folate deficiency is considered to be the worlds most prevalent vitamin deficiency, in par-
ticular in children, pregnant females and old people.
Toxicity Folate in very high doses interferes with the uptake of Zn2+ and may promote
the growth of certain tumors.
Folate can to some extent relieve the megaloblastic anemia and glossitis seen in pernicious
anemia. However, it exacerbates the neurological problems and makes the disease more
difficult to diagnose from blood smears. For this reason many countries limit the folate
content of vitamin supplements.
18.3. Minerals
A large number of elements have been shown to be essential for human nutrition, some of
which were thought of as contaminants only a few years ago. We distinguish
318
Mass elements 18.3.1
mass elements needed in amounts of more than 100mg/d. These include Ca, Mg, Na, K, Cl
and P. They are present in the body in concentrations of more than 50 ppm.
trace elements needed in amounts greater than 1 mg/d. These include Fe, Zn, Cu and Mn.
ultratrace elements needed in amounts of less than 1 mg/d. These include As, B, F, I, Se,
Cr, Co, Mo, Si, V, Ni, Sn.
Several other elements may also be required (suspected are Ba, Sr, Cd, Br). Traces of most
other elements are found in the body too, but it is unknown whether or not they have any
function.
Trace element concentration in food stuff is determined by soil concentration. Modern

international marketing of food, where food comes from many different regions, has made
deficiencies in trace elements much rarer.
Milk and dairy products contain little trace elements, this is also true for human milk.
Infants should therefore receive supplementation with other food from 4–5 m of age onwards,
when their body stores – acquired during pregnancy – become depleted.
Both required amounts of micronutrients and their toxicity in high doses have to be consid-
ered. Most micronutrients can be obtained in sufficient amounts from food, supplementation
should be considered for Ca, Fe and I only, unless very unusual circumstances dictate oth-
erwise. This issue is made even more complicated by the fact that there are synergistic and
competitive interactions between minerals (Se − Hg, Ca − Pb, Fe − Zn, Mo − Cu, Mo − S) and
between minerals and vitamins (Ca - vitamin B12 , Se - vitamin E).
18.3.1. Mass elements
Ca2+ and P
These elements are conveniently considered together, not only because they are both re-
quired for the formation of bone and teeth, but also because their uptake is influenced by
the presence of the respective other element.
Function About 1.5–2 % of body weight is Ca2+ (about 1 kg) and about 1 % is phosphorus.
90 % of both elements are found in bones and teeth in the form of hydroxyapatite (a
crystalline material containing Ca(OH)2 , Ca3 (PO4 )2 and CaF2 in variable amounts, with
Mg2+ , Zn2+ , Na+ , CO2−
3 also present). The crystals are incorporated into a protein matrix
(collagen in bones and keratin in teeth).
Ca2+ is also found in the blood and soft tissues (≈ 10 g), where it has several functions:
319
• Blood clotting: When platelets are injured, Ca2+ influx releases thromboplastin
from their cell membranes. This in turn stimulates the conversion of prothrombin
into thrombin.
• Stimulation of the absorption of vitamin B12 and some other nutrients.
• As intracellular messenger involved for example in the regulation of muscle contraction

and insulin secretion
• Cofactor of enzymes like acetylcholine esterase and lipase. An antioxidative enzyme

in skin is Ca2+ dependent, Ca2+ thus is useful in treating burns and slowing the aging
of skin.
• Ca2+ lowers blood pressure and may to some extend counteract high blood pressure
caused by excessive Na+ . It also lowers cholesterol levels.
• Ca2+ stabilizes DNA and RNA structure.
• Ca2+ is used for the prevention and treatment of arthritis, rheumatism, menopause
problems, menstrual cramps and nephritis, although the mechanism isn’t quite clear.
Ca2+ is a natural tranquilizer and required for good sleep. When your grandma recom-
mended you take a glass of warm milk with honey before you go to sleep, she may not have
known what she was doing, but her advice was certainly sound.
Phosphorus too has many functions in the body apart from tooth and bone formation:
• Energy metabolism
• Phospholipid formation
• Phosphorylation for signaling and transport
• DNA and RNA formation
• Component of cofactors (eg thiaminphosphate) and enzymes
• Regulation of blood pH
Food sources Ca2+ and Pi are contained in milk and dairy products, grains, fish, meat
and some vegetables. Phosphate is also contained in some processed food like soft drinks
and sausages.
320
carrier-dependent
uptake in small
Calcidiole intestine
Parathormone
Parathyroid
g/l
70m citrate release
<
from osteoclasts
Blood [Ca] Calcitriole
>7
0m
g/l parafolicular cells Calcitonine
Ca-retention
(C-cells) of thyroid in kidney
24-hydroxylated
metabolites
Osteoblast
function
Figure 18.21.: The calcium concentration in blood is closely regulated.
Uptake and metabolism Ca2+ is taken up with 10–30 % efficiency (in adults, children up
to 75 %) by active transport into the epithelial cells of the small intestine, particularly the
uppermost part, were the chyme is still slightly acidic. Oxalate, phytate, fatty acids, stress
and lack of exercise (cave: bedridden patients!) interfere with Ca2+ absorption, as do factors
that increase the transport speed of food through the intestine. Caffeine, mineralocorticoids,
thyroxin and anticonvulsants also interfere. Lactose and glucose increase Ca2+ absorption,
as does a food Ca2+ /Pi ratio between 1:2 and 2:1.
Phosphate is released from food materials by phosphatases in the digestive juices. Its uptake
in the small intestine, resorption in kidney and bone metabolism is controlled by parathor-
mone and calcitonin in much the same way as that of Ca2+ . Al(OH)3 based antacids interfere
with Pi absorption by forming insoluble AlPO4 .
Maintaining Ca2+ and Pi in a soluble form The concentration of Ca2+ (1.2 mM) and Pi
(1.3 mM) in blood serum is higher than their solubility product (nominal solubility prod-
uct of [Ca5 (PO4 )3 (OH)] is 10 × 10−53 M), i.e. these ions should precipitate in the form of
hydroxyapatite. Such precipitation of course would be fatal. Several factors are responsible
for maintaining these ions in a soluble state:
factors affecting the solubility product: The presence of NaCl and other salts in the blood
increases the solubility product. Also, at the pH of blood most of the phosphate is
not PO3−
4 .
321
low molecular weight molecules forming complexes with Ca2+ : The most important one
is pyrophosphate. Bone mineralization requires the activity of pyrophosphatases,
which make the Ca2+ available and also increase the local concentration of phosphate.
proteins forming complexes with Ca2+ : Many proteins – including serum albumin – can
bind Ca2+ with moderate affinity on acidic amino acid side chains. Thus the concen-
tration of free Ca2+ is reduced.
colloid stabilization: Certain proteins like fetuin A bind to small (50–100 nm) hydroxya-
patite particles and prevent them from precipitation. The colloid is removed from the
blood stream for example by phagocytes.
Thus humans avoid “Lot’s wife’s problem” 1 (W. Neumann). However, unwanted calcifica-
tion is involved in the pathomechanism of some diseases, most notably atherosclerosis.
Excretion Ca2+ is lost from the body in urine (100–175 mg/d), feces from gastrointestinal
secretions (130 mg/d) and sweat (20 mg/d).
Deficiency Low Ca2+ and Pi lead to bone demineralization, osteoporosis and osteo-
malacia. Low blood [Ca2+ ] additionally leads to tetany from higher nerve excitability and
high blood pressure. Low blood [Pi ] leads to fatigue and loss of appetite. Soy has been
shown in studies to reduce Ca-loss in females after menopause.
Toxicity High blood [Ca2+ ] leads to the contraction of muscle fibres (Ca-rigor). There
is circumstantial evidence linking excessive Ca2+ -uptake to prostate and ovarian cancer.
A high uptake of Pi from processed foods (soft drinks, sausages etc.) has been linked to
attention deficit disorder in children.
Na+ and K+
Again it is easier to treat these two ions together. Both are important electrolytes, Na+
occurring mainly in the extracellular, K+ mostly in the intracellular fluid. This imbalance
is maintained at the expense of metabolic energy by the Na+ /K+ -ATPase (Na+ -Pump) in
the cell membrane, which pumps 3 Na+ out of and 2 K+ into the cell for each molecule of
ATP hydrolyzed. This results in a ratio Na+ /K+ of 1:10 inside and 28:1 outside the cell.
1
In Gen. 1915−26 Lot’s wife was turned into a salt pilar because she looked back onto Sodom and Gomorrah
while they were destroyed for their sins.
322
Function Na+ maintains the osmotic pressure of blood and extracellular fluid. The con-
centration gradient of Na+ across the cell membrane is used to power secondary active
transport processes for many nutrients (for example glucose and amino acids). By neutral-
izing acids Na+ is involved in the regulation of blood pH.
K+ in the same way is involved in the regulation of the intracellular pH and osmotic pressure.
Some enzymes like the 70 kDa 70 kDa heat shock cognate (Hsc70) (“uncoating ATPase”)
require K+ as cofactor.
The disequilibrium of both Na+ and K+ results in an electrical potential across the cell
membrane. This allows signal conduction in nerves as well as the excitation of muscle and
gland cells.
Food sources Fruits, vegetables and meat contain large amounts of K+ . In the form of
alginate, iodate and nitrate K+ is used in food processing. Beware however of K+ leaching
into the cooking water.
Na+ is widely available in our food in the form of table salt, usual uptake of NaCl is
about 7–18 g/d. Only a small part of this is “discretionary salt” (salt which we add our self
and can therefore control). In addition, Na+ as bicarbonate, glutamate, citrate, phosphate,
saccharinate, benzoate, sorbate, propionate and nitrite is widely used in food processing,
these compounds account for ≈ 10 % of daily Na+ intake.
Uptake and metabolism Na+ and K+ are absorbed mostly in the small intestine.
Excretion About 90–99 % of the Na+ ingested is excreted by the kidneys, depending on
the amount taken up in food. This is regulated by aldosterone. A high blood [Na+ ] will
activate thirst receptors, to ensure a sufficient water supply for excretion. 200 mg/l Na+ is
found in sweat.
Deficiency About 500–700 mg/d of Na+ would be adequate for the bodies needs, however
a diet with only this amount would be unpalatable. Additionally, some Na+ is stored in the
body bound to bone to cover a short lack of supply. However, large losses with sweat or
diarrhea must be replaced, to prevent water intoxication.
K+ too is usually supplied in sufficient quantities in the food. However, increased losses
occur in diarrhea, vomiting and after the use of diuretics.
If not enough carbohydrate is taken up with the diet, the body has to use protein instead
to supply glucose for brain and erythrocyte function. This leads to a dangerously high
loss of water (to excrete urea) and therewith also electrolytes. This can lead to circulatory
failure.
323
Toxicity About 20 % of the US population suffer from Na+ -dependent hypertension, which
is caused or at least worsened by high dietary Na+ uptake. Hint: Replace part of the
discretionary salt with herbs and spices, and add 1/4 of those only in the last 10 min of
cooking. It also helps to keep a ratio of about 1:1 between dietary Na+ and K+ .
Outright Na+ -poisoning may occur when people drink sea water, for example after a ship
wreck . Sea water contains more Na+ than can be excreted with the water, thus increasing
thirst. Victims then drink more sea water until death occurs from kidney failure.
High blood [K+ ] leads to lack of muscular coordination, tissue breakdown and acidosis.
Eventually death occurs by kidney and heart failure. In some countries that still have capital
punishment injection of KCl solution is used for execution (death by lethal injection).
Chloride
Function Chloride is an important counter-ion for Na+ and K+ . By neutralizing bases

formed in the body it participates in the regulation of pH. This is particularly true in
erythrocytes, to compensate for the constant change between the formation and removal of
bicarbonate (chloride shift). High concentrations of chloride are found in gastric secretions
and in cerebrospinal fluid. Chloride is found mostly in the extracellular fluid, to balance
the negatively charged proteins inside the cells. About 0.15 % of body weight is Cl− .
Food sources Chloride is widely distributed in food, and added in the form of NaCl.
Excretion Excretion is by the kidneys, were reabsorption occurs when supplies are low.
Deficiency In one case several children died after being fed an infant formula from which
the chloride had been left out. Like Na+ and K+ , Cl− needs to be replaced after losses by
diarrhea, vomiting, diuresis and excessive sweating.
Mg2+
Function Mg2+ is involved in more than 300 known enzyme reactions, amongst them those
that require nucleotides or phosphate. Mg2+ is a Ca2+ antagonist in nerve activity. About
30 g are found in an adult body, 60 % of this in bone (some as magnesium phosphate in the
bone structure, the rest loosely bound as Mg2+ store). Mg2+ is 7 times more concentrated
in the intracellular than the extracellular fluid.
324
Trace elements 18.3.2
Food sources Green plants contain high amounts of Mg2+ , but some is found in most
other food as well. Water may also supply some Mg2+ . However, the Mg2+ supply in the
average diet, at least in the US, is marginal.
Uptake and metabolism Mg2+ is absorbed in the small intestine with 35–40 % efficiency.
Ca2+ , ethanol, Pi , phytate and fat decrease, vitamin D and lactate increase absorption
efficiency. Absorption and excretion of Mg2+ are regulated by the thyroid and parathyroid
glands in much the same way as Ca2+ .
Excretion In the kidney.
Deficiency Mg2+ deficiency may be caused by malnutrition, vomiting, diarrhea, surgical

trauma and high Ca2+ intake. Ethanol and diuretics increase Mg2+ loss. Consequences are
irritability, nervousness, vasodilatation, muscle cramps and convulsion (Mg2+ tetany). In
extreme cases heart failure occurs. There seems to be a connection between soft tissue
calcification and low Mg2+ intake.
Mineral losses with sweat in endurance sports may lead to muscle cramps, for which lack
of Mg2+ is largely responsible.
Toxicity High Mg2+ concentrations have an anaesthetic effect, eventually leading to coma
and death by heart failure. Clinically, this can result from kidney failure, when Mg2+ ex-
cretion is repressed.
18.3.2. Trace elements
Iron
An adult human body contains about 3 g of iron, about 70 % of this as hemoglobin, 7 % in

iron containing enzymes and 4 % in myoglobin. Most of the rest is stored in the liver. As
about 1 % of all red blood cells are turned over each day, 25 mg/d of iron are metabolized.
Most of this iron is recycled, only about 1 mg/d are lost (shed intestinal cells, urine, skin,
hair).
Loss can triple in menstruating females (usual loss during menstruation is about 35 ml
blood, equivalent to 18 mg iron. Blood loss may rise to 200 ml/cycle in clinical cases).
Pregnancy and lactation also place increased demands on iron supplies.
Hookworm infections lead to a loss of about 0.2 ml blood per day and worm, this can
amount to a blood loss of 200 ml/d in heavy infections. About 500 × 106 people world-wide
325
are infected with this parasite. Infections with Giardia can also cause iron deficiency. Both
parasites may cause deficiencies in other nutrients as well.
Food sources Iron is contained in red meat, liver, eggs and many vegetables. Only a small
part of the iron contained in food is actually resorbed. Resorption is prevented by complex
forming agents particularly in plant foods, like oxalic, tannic and phytic acid.
Iron in human breast milk (but not cows milk) can be resorbed to about 50 %, iron from
heme in red meat to about 30 %; there is apparently a special transporter for heme-iron
in the gut. Iron in many plants is resorbed only with about 10 % efficiency, in rice only
1 %. Free iron can be absorbed only in the ferrous (Fe2+ ), not in the ferric (Fe3+ ) form.
Reducing agents like ascorbate (Vitamin C) therefore increase iron resorption. Iron is most
soluble (and easiest resorbed) in an acidic environment.
Usual eating habits tend to satisfy caloric needs of the body, with the need of micronutrients
being satisfied as a side effect. Women have on the one hand a higher dietary need of iron,
but at the same time a lower need for energy (lower basal metabolism, smaller body size).
These factors conspire to make the risk of iron deficiency higher in women (probable Fe-
intake 9 mg/d) than in men (probable iron intake 11 mg/d). On the other hand, men (and
postmenopausal women) are more at risk of iron overload.
Uptake Food iron is complexed with gastroferrin when it arrives in the stomach. Heredi-
tary lack of gastroferrin leads to impaired iron resorption.
Actual resorption takes place in the small intestine (duodenum and jejunum). In the gut
cells iron is complexed with ‘protein C’. It is than transferred either to transferrin for
transport to the liver or to ‘protein S’ for short term storage in the gut cell.
Iron binding to transferrin is dependent on the iron concentration in blood and therefore
iron demand of the body. Excess iron is initially stored with protein S, from where it can be
efficiently transferred to transferrin. In case of large excess iron is transferred from protein
S to ferritin, from which it will not be mobilized again: As gut epithelial cell have a live
span of only a few days this iron is lost eventually with the stool.
Transferrin (siderophilin in older literature) is a soluble glycoprotein. It is the only known
asymmetric single polypeptide with two binding sites for the same substrate. The binding
sites are assumed to be equivalent, without co-operativity between them.
The iron-transferrin complex (holotransferrin) is bound at the cell membrane of target cells
at a specific receptor. This transferrin receptor has at neutral pH a high affinity for holo-, but
a low affinity for apotransferrin. Receptors with bound transferrin are preferentially sorted
into clathrin coated pits and endocytosed via clathrin coated vesicles. The vesicles with the
receptor/holotransferrin complex fuses with the endosome, where the complex is exposed
326
Compounds forming Fe complexes

NH2
CH2 OH
CH2 HO OH
CH2
CH2
CH2 O C
HO N O
C O CH2
CH2 OH
HC O O
CH2
H OH O C OH
C O C
HO H H
NH C C
H OH
CH2 O
C O
CH2
CH2
CH2
HO OH
CH2
OH
HO N
C O an example for tannic acids
CH2
CH2
Pi ~O O ~ Pi
C O
O~ Pi
NH Pi ~ O
CH2 Pi ~ O
CH2 O ~ Pi
CH2
Myoinosit hexaphosphate
CH2
(Phytate)
CH2
HO N
C O HO O
C C
CH3 O OH
Oxalic acid
Deferoxamine
Figure 18.22.: Tannic acid, phytate and oxalate give insoluble precipitates with iron and
interfere with its uptake (and that of other bivalent metals). Deferoxamine
is used for chelation therapy.
327
cell membrane
fusion dissociation
Sorting
Binding coated pit
Vesicle
formation
coated vesicle
budding
uncoating
and fusion
Endosome (pH ~5)
Figure 18.23.: The transferrin cycle. Iron (red) loaded transferrin (green) binds to the trans-
ferrin receptor (purple) at the cell surface, which is then internalized by
clathrin (blue) coated vesicles. In the low pH environment of the endosome
the iron dissociates, but conformational changes in both apo-transferrin and
receptor ensure that these proteins stay together for recycling to the cell
membrane. At the cell surface the apo-transferrin/receptor complex is ex-
posed to neutral pH and apo-transferrin dissociates. This frees the receptor
for a new cycle. Figure taken from [Buxbaum, 2007].
328
to low pH (about 5.5). Under these conditions transferrin rapidly looses the bound iron,
but remains bound to the receptor because at this low pH the receptor has a high affinity
for unloaded transferrin. The transferrin/receptor complex returns to the cell membrane in
recycling vesicles. At the cell surface the transferrin/receptor complex is again exposed to
neutral pH, where the affinity of the receptor for apotransferrin is drastically reduced. The
complex therefore dissociates and apotransferrin is released to the blood stream.
The ferric iron is reduced inside the endosomal system to ferrous iron by an unknown
electron donor (NADH, ascorbate, glutathione?) and then passes the endosomal membrane
into the cytoplasm.
Blood contains haptoglobin and hemopexin to scavenge any hemoglobin or heme respec-
tively resulting from hemolysis (about 10 % of red blood cells lyse in the blood stream
instead of being taken up by macrophages). The resulting complexes are then taken up by
hepatocytes for iron recycling.
Only part of the transferrin molecules in blood are loaded with iron. Total Iron Binding
Capacity (TIBC, in µM) is the concentration of bound iron plus the free capacity of the
serum to bind iron (measured by adding a known amount of iron and determining the
amount not bound by transferrin). If the iron concentration is divided by this value, the
relative iron saturation is obtained. Example: TIBC = 56 µM, serum iron concentration =
43 µM. This yields a saturation of 100 % * 43 µM / 56 µM = 77 %. Normal iron saturation
is about 30 %.
Iron bound to transferrin must be in the ferric (Fe3+ ) state, while gut cells can only resorb
ferrous iron (Fe2+ ). Oxidation from the ferrous to the ferric state is achieved by Ferroxidase
I and II. Ferroxidase I is also known as ceruloplasmin, and also acts as a copper transporting
protein.
Iron supply in the cell is regulated by the rate of synthesis of transferrin receptor and apo-
ferritin. In the case of transferrin receptor, the breakdown of mRNA is regulated, in case of
apoferritin the rate of translation. Both mRNAs contain iron responsive elements (hairpins
of 30–40 nucleotides) outside their coding sequence to afford iron dependent regulation.
Storage Ferritin is a 24 subunit protein made up of 2 types of polypeptides: The H chain

has a molecular mass of 21 kDa, the L chain of 19 kDa. The two forms of apoferritin have
different tissue distribution, the heart contains mainly ferritin H, the liver and spleen mainly
ferritin L. Ferritin can bind 20 % of its weight in iron, 2500 ions per ferritin molecule. Iron
is bound as a mixed hydroxide, phosphate and oxide complex. Iron enters the complex as
Fe2+ , and is than oxidized to the 3+ state by an unknown mechanism. A small proportion
of the ferritin leaves the cells, again by unknown mechanisms, and shows up in serum.
Serum ferritin levels mirror the body’s iron supply. However, some disorders increase serum
ferritin levels, thus covering an iron deficiency.
329
Figure 18.24.: Left: Prussian blue/safranine O staining for iron deposits (here in der-
matitis). Picture taken from Rivera, Ishihara & Mihara, Arch. Der-
matol. Res. 295 (2003) 19. Middle: Blood smear from a normal and
right: from a dog suffering from iron deficiency. Note the irregularly
shaped, irregularly sized erythrocytes (microcytic) with large central pal-
lor (hypochromic). Image from Woods, Tarpley, Johnson & Latimer,
http://www.vet.uga.edu/vpp/clerk/mwoods/.
330
If a cell contains more iron than it can store bound to ferritin, it stores the remainder as
hemosiderin. Hemosiderin is a iron oxide aggregate with organic components. It occurs in
lysosomes, and increased hemosiderin content may eventually damage the lysosomes. In
histological sections hemosiderin is stained by the Prussian Blue reaction.
Deficiency If the body lacks iron, it can no longer produce hemoglobin in sufficient
amounts. It will produce fewer, smaller red blood cells which contain less hemoglobin than
usual (microcytic hypochromic anemia). This will impair oxygen transport. Patients
will feel exhausted and short of breath. They will be of pale (or even bluish) skin color.
Some circumstantial evidence links iron deficiency with immuno-suppression and increased
infections. Serum ferritin levels below 12 µg/ml or iron saturation below 16 % indicate iron
deficiency. Treatment is by iron supplementation, usually in the form of ferrous sulphate or
gluconate (in children 6 mg/(kg day), adults 50 mg three times a day), and ideally combined
with ascorbic acid. Supplementation requires regular laboratory controls.
If the patient lives in an area where parasites such as hookworms or Giardia are endemic
(or has visited such an area), the presence of those parasite should be investigated. Other
causes for blood loss (for example intestinal cancer) also need to be excluded.
Toxicity
Too much iron is toxic to the body, causing hemochromatosis. A normal human liver con-
tains about 1 g of iron, this can increase to 40–50 g in severe cases of hemochromatosis.
Serum ferritin levels can reach 6000 µg/l (normal 20–250 µg/l). Causes for hemochromatosis
are
- congenital failure to regulate apoferritin synthesis. This creates an iron sink in the
tissue, leading to lower iron saturation and increased iron uptake.
- congenital excess iron absorption. The condition has been linked to chromosome 6 and
occurs most often in patients with HLA-A3. This may specifically affect the uptake
of heme iron.
- excess iron intake. This can occur if rusty cooking utensils are routinely used, if
iron supplements are taken in excess (poisoning with those interesting looking green
iron pills accounts for 1/5 of all poisoning cases in children in the US → keep all
medicines out of reach of children!)
- alcoholism. Alcoholic drinks like red wine contain high concentrations of iron, addi-
tionally alcohol affects iron uptake regulation.
- liver cirrhosis or portacaval shunt
- pancreatitis. Pancreas juice is involved in iron uptake regulation.
331
- professional exposure to iron, for example in miners

- reduced use of supplied iron, for example in thalassemias. Frequent blood transfusions
may add to the problem (250 mg Fe in 500 ml blood).
In iron overload, the iron is deposited throughout the body as hemosiderin precipitate. This
causes damage to the cells. Particularly affected are liver, pancreas and muscle. Symptoms
of hemochromatosis include:
- Chondritis and arthritis, in particular in fingers and hand. Initial symptom in about
half the patients.
- Increased melanin production in the skin
- Hypopituitarism, leading to dwarfism and sexual infantilism
- Gonadal atrophy (particularly in |)
- Cardiomyopathy, arrhythmias and heart failure
- Splenomegaly
- Addison’s disease (adrenal gland)
- Fibrosis and islet cell destruction, leading to Diabetes mellitus. Together with the
darker skin this leads to the syndrome of bronze diabetes.
- Liver cirrhosis, liver failure, portal hypertension and hepatocellular carcinoma
Treatment is by blood letting (phlebotomy) (500 ml/week over 2 a). Chelation therapy
with deferoxamine (an iron chelator isolated from Streptomyces ssp.) and vitamin C is
usually not recommended because of side effects.
Fluorine
Function Replacement of hydroxy-groups in hydroxyapatite in teeth by F− makes them

harder and more resistant to tooth decay. In bones, this reaction makes the hydroxyapatite
more resistant to demineralization. Thus a good fluorine supply in youth may offer women
some protection against osteoporosis in menopause.
Food sources Most of the fluorine comes from drinking water, in particular in those places
where either the fluorine content in the water is naturally high or were it is supplemented
to 1 ppm. Seafood contains some fluorine, as do vegetables grown in areas with high soil
fluorine. The amount of fluorine introduced into enamel during brushing with F− containing
toothpastes is very low, however, some of the toothpaste is invariably swallowed, and this
does offer some benefit. Wine and tea are also relatively high in fluorine.
332
Uptake and metabolism F− is absorbed with 90 % efficiency, mostly in the stomach. The
blood [F− ] is mirrored in other body fluids.
Excretion The blood [F− ] is regulated by the kidneys, about half of the normal daily
uptake is excreted.
Deficiency Drinking water [F− ] of less than 1 ppm is associated with a higher incidence
of tooth decay. It may also increase the risk of osteoporosis.
Toxicity More than 2.5 ppm of fluorine in drinking water leads to fluorosis with chalky
discolourations and brownish stains on enamel. High concentrations of fluorine are cytotoxic
(inhibition of enolase → use of fluorine to stop metabolism in blood samples).
Copper
Function Copper can occur in the +I and +II oxidation state, and is used in enzymes
catalyzing redox-reactions. Cu+ salts tend to be water insoluble and are subject to oxidation
under environmental conditions. Hence we can focus here on Cu2+ .
The human body contains 70–150 mg of Cu2+ , mostly in bones and muscle. Cu2+ aids the
uptake of iron, releases stored iron in the liver and stimulates the synthesis of hemoglobin.
Ceruloplasmin (Ferroxidase 1) and Ferroxidase 2 maintain Fe in the ferric (Fe3+ ) state for
transport. Cu2+ is found in the active center of monoamine and diamine oxidases, important
for the metabolism of serotonin, norepinephrine, dopamine, melanin, tyramine and hista-
mine. Lysyl oxidase is required for the formation of crosslinks in elastin and collagen. Blood
clotting factor V also contains Cu2+ . Superoxide dismutase (synonyms are cytocuprein in
bone, erythrocuprein in red cells, hepatocuprein in liver or cerebrocuprein in CNS) protects
the body against oxidative damage. Cu2+ is also required for the synthesis of phospholipids
and in the respiratory chain (cytochrome c oxidase). It is involved in cholesterol metabolism,
thermal regulation, immune and cardiac function, although the mechanism is not clear.
Food sources Cu2+ is widely distributed in foods of both animal and plant origin (except
milk), some may also leach into the drinking water from copper pipes. Particularly good
sources are shellfish, nuts, legumes, whole grain cereals.
333
Uptake and metabolism Dietary Cu2+ is absorbed with 25–40 % efficiency in the stomach
and upper small intestine by regulated, active transport. In the intestinal cells it is bound
to metallothionein. Cu2+ is transported as complex with albumin and transcuprin with the
portal blood to the liver, where it is either excreted into bile, stored as metallothionein
complex or used for ceruloplasmin synthesis. Ceruloplasmin binds to the cells in the body,
where the Cu2+ is absorbed. Uptake is counteracted by excess iron, zinc, molybdenum and
ascorbic acid.
Excretion in bile, controlled by the adrenal gland.
Deficiency Copper deficiency may lead to low ceruloplasmin levels in blood and thereby
to normocytic, hypochromic anemia. Leukopenia and neutropenia may also be found. Pa-
tients may suffer from osteoporosis and flaring or fractures of the metaphyses and arthritis.
Melanin formation will be lower, leading to depigmentation. Arterial, myocardial and neu-
rological disease will be found, in part as consequence from increased cholesterol level. Heart
beat may be irregular. Glucose tolerance may be lowered.
Toxicity Acute Cu2+ poisoning may be seen after accidental or suicidal intake or from
ingestion of acidic foods stored in copper containers. Contact of copper salts with burned
skin has also resulted in poisoning. Symptoms are gastric pain, nausea, vomiting, diarrhea,
coma, oliguria, hepatic necrosis, vascular collapse and death.
Genetic diseases relating to Cu2+ Wilson’s disease (hepatolenticular degeneration)

is a chronic Cu2+ poisoning caused by an autosomal, recessive disease (1:20 000 births)
in the ATP7B -gene which leads to reduced biliary excretion. The copper accumulates in
liver, brain and cornea. This is treated by chelation therapy with penicillamine (usually in
combination with Zn2+ and pyridoxin supplements). Provided therapy starts before major
tissue damage has occurred, patients may enjoy normal life expectancy.
Menke’s kinky hair syndrome, a X-linked genetic disease (1 out of several 100 000
births) results in a defective Cu2+ -ATPase (ATP7A) and is usually fatal by age 3. ATP7Ap
resides in the membrane of the Golgi-apparatus and transports Cu2+ from the cytosol
into the organelle where it is used to make Cu2+ -dependent enzymes. The enzyme can
also move to the plasma membrane to transport excess Cu2+ from the cytosol into the
blood. As a consequence of the deficiency copper is not available for its normal functions
and accumulates in intestine, spleen, muscle and kidney. The symptoms therefore are a
combination of those caused by Cu2+ toxicity and Cu2+ deficiency.
Cutis laxa results from a failure to produce lysyl oxidase and therefore functional collagen
and elastin.
334
Figure 18.25.: A Kayser-Fleischer-ring is diagnostic for Wilson’s disease. It

is caused by copper deposition in Descemet’s membrane. Image
from Fred & van Dijk, “Images of Memorable Cases: Case 9,”
http://cnx.org/content/m15007/1.2/.
335
In Downs syndrome an overproduction of superoxide dismutase may be found.
Albinism results from a failure to produce tyrosinase, a Cu2+ dependent enzyme required
for melanin biosynthesis.
A cytochrome c oxidase deficiency leads to myopathy.
Zinc
Function Zinc always occurs in the +II oxidation state. The human body contains about
1.5–2.5 g of Zn2+ , mostly in cytoplasm. There are more than 200 enzymes known which have
Zn2+ in the active center, from all 6 main enzyme classes. Zn2+ is required for the incorpora-
tion of methionine into skin proteins, important for wound healing. Insulin is stored as Zn2+
complex. Zn2+ is also required for bone development, RNA and DNA polymerases, carboan-
hydrase, metallopeptidases, NAD+ /NADP+ dependent dehydrogenases (including ethanol
metabolism), vitamin A mobilization from liver. Also found in eye, prostrate and semen.
Sufficient supply with Zn2+ increases Cd and Pb tolerance. The conversion of dietary folate
into the monoglutamate requires Zn2+ . Zn2+ -finger proteins regulate gene expression.
Food sources Highest concentrations are found in seafood, eggs and meat, which may not
be available to low income groups. Cereals and legumes are also high in Zn2+ , but cereals
contain also phytate, which makes Zn2+ unavailable (reduced by leavening of the bread).
Uptake and metabolism Zn2+ is absorbed in the upper part of the small intestine with
30–50 % efficiency (up-regulated in pregnant females). Absorption is enhanced by the amino
acids His and Cys, which form stable, absorbable complexes with Zn2+ . The uptake into the
intestinal epithelium is carrier mediated and not energy dependent. In the intestinal cells
Zn2+ is bound to metallothionein, in portal blood to albumin. In systemic blood Zn2+
is bound to α-macroglobulin, transferrin and albumin. Albumin-bound Zn seems to be the
major transport form to the cells. There is no specific Zn2+ store, and Zn2+ deficiency may
develop rapidly. However, short term needs can be bridged by muscle and bone wasting.
Chelators like penicillamine, DTPA (used to prevent Fe poisoning in thalassemia), etham-

butol (a tuberculostatic) and anticonvulsants like valproate interfere with Zn2+ uptake.
Zn2+ uptake efficiency is also reduced in intestinal problems like Crohns disease, short
bowel syndrome, jejunoileal bypass, sprue, AIDS-associated diarrhea and in alcoholic cir-
rhosis.
336
Excretion mainly with pancreas juice, bile and duodenal secretions into feces, but also
in urine (increased by EDTA and similar compounds), sweat and (in |) semen or (in ~)
menstrual secretions. Hyperzincuria may be found in diabetics (both type 1 and 2).
Deficiency About a fourth of the worlds population is estimated to suffer from zinc de-
ficiency. If supply of Zn is below requirements, total Zn concentration in serum (measured
for example by atomic absorption spectroscopy AAS) drops only slowly, since most of the
Zn is bound tightly in metalloproteins like α-2-macroglobulin. However, the so called labile
Zn (free Zn2+ and Zn bound in weak complexes to thiol groups, measurable by fluorescent
Zn-chelators like zinquin) drops within a few days.
Because of the large number of biochemical processes that require Zn, symptoms of Zn
deficiency are variable. Acne, skin lesions, retarded growth, delayed sexual development,
oligospermia, delayed wound healing, loss of appetite, loss of taste and smell (hypogeusia
and hyposmia). The eye may be affected: photophobia, night blindness, corneal edema and
clouding, conjunctivitis, xerosis and keratomalacia, with permanent damage if treatment is
late. Hair may be hypopigmented and reddish, with patchy loss. Failure of chemotaxis in
monocytes and neutrophiles causes loss of immune function. Patients suffering from Zn2+
deficiency may be irritable, lethargic, depressed, sleepy, with fine tremor, ataxic gait and
slurred speech.
Conditions like diabetes, asthma, arthritis and sickle cell anemia are worsened by Zn defi-
ciency.
Children with Zn2+ deficiency grow slower than their peers, and suffer more frequently from
infection. Studies in Vietnam, Bangladesh and Indonesia have shown that Zn2+ supplemen-
tation can reduce the incidence of pneumonia by 41 % and diarrhea by 18 %. Both diseases
are major causes of infant death.
In pregnant females Zn2+ deficiency increases the risk of pregnancy related problems like
hypertension, there is also a risk of congenital malformations in the embryo, from low birth
weight to neural tube defects like spina bifida and anencephaly.
Regionally, Zn2+ deficiency is prevalent in Egypt and Iran, because the diet there contains
large amounts of unleavened bread, which contains phytate and interferes with uptake of
Zn2+ and other trace metals.
Infections with hookworms and Giardia may cause Zn2+ (and Fe) deficiency.
Penicillamine interferes with the uptake of Zn2+ , as do high concentrations of Ca, Fe, and
Cu.
Geophagy (eating of soil) may lead to Zn2+ deficiency, if clay minerals bind this metal.
337
Toxicity No short term toxic effects have been seen with uptakes as high as 200 mg/d, but
the long term effect of such megadoses is unknown.
Very high doses of Zn2+ from galvanized cooking utensils, excess supplementation or indus-
trial pollution leads to fever, vomiting, diarrhea, impaired immune function, Fe loss from
liver stores (and consequential microcytic, hypochromic anemia), Cu2+ uptake deficiency
and to lowered HDL levels, with a risk of atherogenesis.
Genetic diseases relating to Zn2+ metabolism A rare autosomally determined failure to

produce Zn2+ -binding protein in the intestine leads to acrodermatitis enteropathica .
The condition can be treated with high oral doses (30–45 mg/d) of Zn2+ , which compensate
for the reduced absorption efficiency.
Mothers may be unable to secrete Zn2+ into the milk, in these cases infants require Zn2+
supplementation until weaned. Supplementation of the mothers does not help in such cases.
18.3.3. Ultratrace elements
Iodine
Function The human body contains 15–23 mg of I, 3/4 of this in the thyroid gland. The
remainder is found in salivary, mammary and gastric glands and in the kidneys. In severe
iodine deficiency, total body I may be less than 1 mg.
I is a component of the hormones thyroxin and thyronin. These increase basal metabolism
and heat production by up to 30 %, conversion of carotene to vitamin A, protein synthe-
sis, carbohydrate absorption in the intestine, and reproduction. They decrease cholesterol
biosynthesis.
Because of the stimulation of basal metabolism thyroxin is sometimes used as weight loss
aid. This is very dangerous and should not be done!
Food sources Seafood, saltwater fish, seaweed, vegetables grown in I-rich soil. In many
countries iodinated table salt is available. In some underdeveloped countries (where salt
iodination can not be performed reliably) programs have started where children are given
an intramuscular injection of iodine containing poppy seed oil every 2–4 a.
338
Ultratrace elements 18.3.3
Thyroxine Metabolism
NH2 OH NH2 OH NH2 OH

H2C C H2C C H2C C
H I- H+ + 2e- H O H O
O I-
H+ + 2e-
HC CH HC CH HC CH
HC CH I CH I I
OH OH OH
Tyrosine 3-Monoiodotyrosine 3,5-Diiodotyrosine
3,5-DIT 3,5-DIT
Ala Ala
NH2 OH NH2 OH
H2C C H2C C
H O H O
HC CH HC CH
I- H+ + 2e-
I I I I
O O
HC CH HC CH
I CH I I
OH OH
3,5,3'-Triiodothyronine Tyroxin
(T3) (3,5,3',5'-Tetraiodothyronine,
T4)
Figure 18.26.: Generation of the hormones T3 and T4 from the amino acid tyrosine in the
thyroid gland.
339
Maintaining thyroid hormone concentration in blood:
Tyroxin releasing Thyroid stimulating

hormone (TRH) hormone (TSH) Thyroxin Physiological
Hypothalamus + Pituitary + Thyroid + activity
−
Natural iodine cycle:
evaporation precipitation
(400 kt/a)
Sea water rain soil plants humans
rain and flood extraction
Figure 18.27.: a) The level of thyroxin in blood is tightly regulated. For details see text.
b) Global iodine cycle. Iodine evaporated from the seas and rains down onto
soil, where it can be taken up by plants. However, extraction of iodine from
the soil by rain and flood water is faster than its deposition. For this reason
geologically old soil is deficient in iodine, and people living exclusively from
plants grown on such soil suffer from iodine deficiency.
340
Uptake and metabolism I is absorbed in the small intestine, and transported in the
blood stream to the thyroid (1/3) and the kidneys. Iodine uptake into the thyroid gland is
a very efficient secondary active process powered by the Na+ /K+ -ATPase. There is a 100:1
I concentration gradient between thyroidal cells and serum.
Release of Thyroxin releasing factor from the hypothalamus increases production of thyroid
stimulating hormone in the pituitary (see fig. 18.27a). This in turn stimulates thyroxin
release from the thyroid, which in turn reduces the release of thyroid stimulating hormone.
Excretion Excess I is transported to the kidneys and the salivary gland.
Deficiency There is a natural iodine cycle operating in nature (see fig. 18.27b). Some
400 000 t/a of iodine evaporate from the seas and are transported to the soil with rain.
However, this is not sufficient to replace the loss from the soil by rain and flood extractions.
Geologically old soil, or soil in flood plains, tends to be poor in iodine, and the plants
growing on those soils will have a low I content too. People living in those areas, and
obtaining their food locally may become I deficient.
Iodine deficiency causes (simple) goiter, a hyperplasia of the thyroid gland, which can
grow up to 1 kg in weight instead of the normal 15–25g, leading to breathing difficulties.
This may be because of insufficient I supply or the presence of goitrogenic substances
like SCN− , which interfere with I metabolism. Such substances are found in raw cabbage,
peaches, almonds, soybeans, cassava and peanuts. Some pharmaceuticals also are goitro-
genic like sulphonamides and para-aminobenzoic acid.
Eventually iodine deficiency will lead to mental retardation, in extreme cases to cretinism.
There are about 1 × 109 people worldwide suffering from iodine deficiency, 200 × 106 people
suffering from simple goiter (of which 4 % are caused by goitrogenic chemicals). 25 × 106
people worldwide suffer from mental defects resulting from gross iodine deficiency, of those
5 × 106 are suffering from overt cretinism. This makes I deficiency a significant health prob-
lem especially in developing countries, which is shameful as it can be prevented easily and
with small financial input by adding traces of sodium iodate to cooking salt.
Low I supply during pregnancy leads to cretinism in the infant (dwarfed, mentally retarded,
thick and dry skin, protruding abdomen). This can be treated only soon after birth, other-
wise permanent damage occurs. Stillbirths are also common.
If I is low during childhood, myxedema may develop in adults, characterized by sparse hair,
dry and yellow skin, low voice, psychomotor defects and poor cold tolerance. Newer research
indicates that this may actually be a condition caused by a combined iodine and selenium
deficiency. The enzymes that produce and inactivate T3 and T4 contain Se in their active
center.
341
Figure 18.28.: Left: Goiter is the result of iodine deficiency and mainly found in isolated
populations with little trade with the outside world. Middle: Myxedema can
be caused by hypo- and hyperthyroidism, the mechanism is unclear. Right:
The swellings are caused by incorporation of excess mucopolysaccharides,
which stain heavily with alcian blue. Pictures from the archives of Martin
Finborud (1861-1930), J. Chung-Leddon, Dermatol. Online J. 7:1 (2001)
and Hunzeker et al., Dermatol. Online J. 14:10 (2008)
Toxicity In some populations even very high dietary iodine intakes seem to be well toler-
ated (up to 80 mg/d in Japanese communities where seaweed is a major part of the diet).
In other populations uptake as low as 0.1 mg/d may cause problems.
Acute increase of I supply leads to a repression of thyroid hormone synthesis ( Wolff-
Chaikoff-effect ). If the high supply persists, iodine transport into the thyroid is reduced
to allow normal hormone production rates. In some cases this adaptation does not occur,
leading to goiter and iodine myxedema, sometimes also to inflammation of the salivary
glands (sialoadenitis) and mouth sores.
Molybdenum and tungsten
Function 9 mg of Mo are found in the human body, in liver, kidneys, the adrenals and in
red blood cells. Mo is a cofactor in the metabolism of C, N and S, it occurs for example in
xanthine oxidase, sulphite oxidase and aldehyde oxidase. It promotes the retention of F−
and therefore prevents tooth decay.
Mo is a very rare element in the earths crust, but because of its good water solubility
it is the most abundant transition metal in sea water, from which early life emerged. It
can occur in the oxidation states IV, V and VI, and thus catalyze the transfer of single
342
+ [O]
Cys
S O
O
O H S Mo OH IV VI
Mo Mo
N S
HN O
O P O R
H2N N N O C
H2 O
H
H2O 2 [H]
MoCo
Figure 18.29.: Molybdenum cofactor (MoCo) bound to an enzyme. This cofactor occurs
in oxidoreductases like xanthin, sulphite and aldehyde oxidase, nitrate and
DMSO reductase. Molybdenum can be oxidized by binding an oxygen atom,
and it can be reduced by giving up that oxygen to hydrogen. This reduction
can happen in one step or in two, as Mo can also have the oxidation number
V.
electrons and of electron pairs. Because of that property, it can transmit between single
electron and electron pair transfer reactions. These properties make Mo an ideal catalyst
for living organisms.
W is used in much the same way as Mo, but it forms more stable bonds with sulphur and
is more oxygen sensitive than Mo. Thus it is used in thermophilic anaerobic organisms,
while Mo is used preferentially in aerobic organisms living in moderate temperatures (like
man).
Food sources Legumes, meat and to a lesser extend whole grain cereals.
Uptake and metabolism Absorbed with 25–80 % efficiency in stomach and small intestine.
Transported in blood loosely bound to erythrocytes and α2 -macroglobulin. Stored in the
liver as “Mo cofactor” di(carboxaminomethyl)molybdopterine (MoCo, see fig. 18.29).
Excretion mainly in urine as molybdate, some also in bile. Mo excretion is promoted by

sulfate.
Deficiency Low appetite, slow growth, reduced fertility. High mortality of infant and
mother. Mental disturbancies, coma, death.
Toxicity Diarrhea, anemia, slow growth, failure of red cell maturation, gout. Prevents
utilization of Cu2+ .
343
Genetic diseases relating to Mo A (fortunately) rare condition is the inability to syn-

thesize MoCo (autosomal recessive inheritance). Most frequent is a defect in the MOCS1A
or MOCS1B genes, which prevents the synthesis of the pterin “precursor Z” from GTP
(MoCo deficiency type A). MoCo deficiency leads to the accumulation of toxic metabolites
in the body, as a consequence neurological abnormalities appear (increased muscle tone,
rigid posture, seizures) leading to death in early infancy. An experimental treatment of
type A MoCo deficiency by i.p. or i.v. injection of precursor Z isolated from E. coli has
recently been described. No treatment is known for type B and C MoCo deficiency, since
MoCo itself is too unstable for isolation.
Selenium
Function 15mg of Se are stored in the human body, mainly in the liver. Se as Sec is an
essential component in several enzymes:
glutathione peroxidase (cGPx) is important for protection against oxidants, double-minus

mice show increased sensitivity against poisons like Paraquat and Diquat, which gen-
erate peroxides.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) Function is not quite clear.

Could possibly be involved in the regulation of prostaglandin- and leucotrien synthe-
sis by regulating the peroxide tonus in the cell. PHGPx has an additional function in
sperm production: It co-polymerizes with SH-containing proteins to form the capsula
(thus becoming a structural protein rather than an enzyme).
Selenoprotein P in plasma, with unknown function. It contains 11 Sec groups and could
be a scavenger for peroxides.
Iodothyronine deiodinases activate Tyr to T3 and deactivate T3 and T4 in the thyroid

gland. The iodination reaction requires peroxide, thus thyreocytes express also cGPx
and PHGPx to balance the peroxide tonus. Myxedematous cretinism seems to be
caused by a combined iodine and selenium deficiency.
Thioredoxin reductases DNA-synthesis (co-substrates of ribonucleotide reductase), an-

tioxidant. Knock-out mice die in early embryonal development.
Selenophosphate synthase Synthesis of the seleno carrier protein required for the synthesis
of Sec tRNA.
Other possible functions of Se include liver function and energy metabolism. At least in
bacteria, Se may occur as 5-methylaminomethyl-2-selenouridine in some tRNAs.
In animals high Se protects against some cancers, but human studies so far were inconclu-
sive. Se may also offer some protection against chemicals like Paraquat, Hg, Cd and Ag.
344
In the vicinity of the Sec in selenoproteins a Trp- and a Gln-residue will be found, these
lead to the deprotonation of the selenol group to −Se− . This group is able to reduce for
example H2 O2 with very high speed (in excess of 10 × 107 M−1 sec−1 ) and is later recycled
by thiols. The reason for the incorporation of Sec into the active center of proteins instead
of Cys is the much lower pKa value of the −SeH group (5.2) compared to the −SH group
(8.2). The reaction proceeds as follows:
+XOOH +GSH +GSH

R−SeH GGGGGGGGA R−Se− GGGGGGGGA R−Se−OX GGGGGGGGA R−Se−S−G GGGGGGGGA R−SeH
−H + −OH − −XOH −GSSG
At physiological pH (7.4) the selenol-group is fully ionized, the thiol group is not. Conse-
quently experimental replacement of Sec with Cys in peroxidases leads to an enzyme that
is several 100-fold less active and has a pH-optimum of about 9.
Food sources Organ meats, seafood, muscle meats, cereals and dairy products (in order
of decreasing concentration). Se content is reduced by milling of cereals and cooking. Plant
Se content depend on the Se content of the soil in which they were grown.
Uptake and metabolism In plants both Sec and Se-Met are produced unregulated as a
consequence of the Se content in soil. Animals do not synthesize Se-Met, after uptake this
amino acid is either metabolized or build into proteins randomly instead of Met.
Sec however is synthesized in a regulated fashion. For this, the hydroxy-group of Ser-
tRNAsec is phosphorylated by a specific kinase, the phosphate is than exchanged against
a HSe-group from a reduced Se carrier protein. This tRNA is complementary to the UGA
stop codon, incorporation of Sec requires a special translation factor with high homology to
EF-Tu. It is not entirely clear when the Opal -codon is interpreted as “stop translation” and
when as “Sec”, apparently special stem-loop structures in the mRNA in the 3’-untranslated
region control this from a distance.
Absorption efficiency of dietary Se ranges from 50 % for inorganic Se compounds to 100 %

for Se-Met. Uptake and transport mechanism are unclear. There is essentially no free Sec
(not incorporated in proteins) in our body, as it gets rapidly destroyed by Sec β-lyase.
Excretion Se is normally excreted in urine in a variety of compounds including trimethyl

selonium. High doses of Se lead to the formation of dimethyl selenide, which is breathed
out, giving the breath a characteristic, radish like smell.
345
NH2 O NH2 O
HO C C C Pi O C C C
H2 H H2 H
O Rib Ade O Rib Ade
ATP ADP
ACU ACU
Ser-tRNAsec Selenium carrier

(complementary to the protein
UGA "opal" stop codon)
Pi
NH2 O
H Se C C C
H2 H
O Rib Ade
ACU
Se-Cys tRNA
Figure 18.30.: In mammals selenocysteine is synthesized on its tRNA. This tRNA is com-
plementary to the opal stop codon. Unlike selenocysteine selenomethionine
is build into proteins randomly by competition with methionine.
346
Objectives in summary 18.4
Deficiency Heart muscle degeneration (Cardiomyopathy, Keshan disease) characterized

by multifocal necrosis, fibrous replacement, myocytolysis. Se supplementation can prevent
the progress of the disease, but not cure preexisting damage. In the CGPx(-/-) mouse
non-pathogenic Coxsackie-virus quickly mutate to virulent strains because of the increased
hydroperoxide tonus, these cause heart conditions very much like those seen in Keshan
disease.
Se-deficiency is also a possible cause of Kashin-Beck disease, an osteoarthritis in pread-
olescence. The disease is characterized by necrotic degeneration of chondrocytes, dwarfism
and joint deformation. General symptoms include muscle pain, defective finger nails and red
blood cells. Other (co)-causes may include iodine deficiency and mycotoxins from spoiled
food.
Toxicity Intakes between 24–200 µg/d do not seem to cause problems. However, chronic
intakes in excess of that lead to dermatitis, loss of hair, nail deformation and loss (alkali
disease). Acute intoxication leads to nausea, diarrhea, irritability, fatigue, peripheral neu-
ropathy, and vomiting. This seems to be caused by the formation of dimethyl selenide. In
animal studies high Se increases the sensitivity to aflatoxin and acetaminophen. Excess Se
is also carcinogenic.
At the end of this course, students should be able to

• explain the dose-effect relationships in micronutrients, using the terms estimated av-
erage requirement (EAR), recommended dietary allowance (RDA), tolerated upper
intake level (UL), Dietary reference intake (DRI), adequate intake (AI) and US rec-
ommended daily allowance (US-RDA).
• define the terms enrichment, fortification, supplementation, vitamin, anti-vitamin,
provitamin, mass element, trace element and ultratrace element.
• explain how the solubility of vitamins in fat or water determines their toxicity and
the risk for the development of deficiencies.
• explain the function of the various vitamins, their food sources, the signs and symp-
toms of deficiencies and toxicity and the diseases associated with their metabolism.
• explain the function of the various minerals, their food sources, the signs and symp-
toms of deficiencies and toxicity and the diseases associated with their metabolism.
• appreciate the role of a balanced nutrition.
347
19. Carbohydrate Metabolism
19.1. Gluconeogenesis
Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. It occurs in

liver and kidney and is necessary to maintain the blood glucose level when dietary car-
bohydrates are in short supply and glycogen reserves are depleted. It uses the reversible
reactions of glycolysis, but irreversible pyruvate kinase, phosphofructokinase, and glucoki-
nase (hexokinase) reactions have to be bypassed.
19.1.1. The first bypass: From pyruvate to phosphoenolpyruvate (PEP)
ATP ADP CO2

-
HCO3 Pi GTP GDP
Pyruvate Oxaloacetate Phosphoenolpyruvate

These two reactions bypass the pyruvate kinase reaction of glycolysis. The input of two
high-energy phosphate bonds makes the formation of PEP from pyruvate energetically
possible.
19.1.2. The second and third bypasses
Phosphofructokinase and glucokinase are bypassed by fructose 1,6-bisphosphatase and glu-

cose 6-phosphatase:
349
Glycolysis Gluconeogenesis
ATP Glucose Pi
Glucokinase Glucose-6-phosphatase
ADP Glucose-6-phosphate
Pi
ATP Fructose-6-phosphate
Phosphofructokinase Fructose-1,6-bisphosphatase
ADP Fructose-1,6-bisphosphate
19.1.3. Substrates of gluconeogenesis

3. Substrates of gluconeogenesis
Glucose
Amino Acids
Glycerol
Aspartate Oxaloacetate TCA cycle intermediates
Lactate Pyruvate Alanine

Acetyl-CoA and fatty acids are not substrates of gluconeogenesis!
Acetyl-CoA and fatty acids are not substrates of gluconeogenesis!
4. Energy
19.1.4. balance
Energy balance
The formation of 1 molecule of glucose from 2 molecules of lactate requires:
2 ATP by pyruvate carboxylase
The formation of 1 molecule of glucose from 2 molecules of lactate requires:
2 GTP by PEP carboxykinase
2 ATP
2 ATP by by pyruvate carboxylase
phosphoglycerate kinase
2 GTP by PEP carboxykinase
2 ATP
Theby phosphoglycerate
energy kinasecomes from fatty acid oxidation.
for gluconeogenesis
6 high energy phosphates
5. Regulation of gluconeogenesis
The energy for gluconeogenesis comes from fatty acid oxidation.
1. Adaptive control: The levels of glycolytic enzymes are high in the well-fed
state (high insulin), levels of gluconeogenic enzymes are high in the fasting
state (high glucagon).
19.1.5.2. Regulation of gluconeogenesis
Allosteric effectors: ATP, citrate, and acetyl-CoA favor gluconeogenesis,
AMP and ADP favor glycolysis.
3. control:
Adaptive Phosphorylation:
The levelsShort-term hormonal
of glycolytic effects
enzymes areare mediated
high in the by enzyme
well-fed state (high
phosphorylation.
insulin), levels of gluconeogenic enzymes are high in the fasting state (high glucagon).
- Pyruvate kinase is inactivated by cAMP-induced phosphorylation.
- Insulin increases and glucagon decreases the concentration of fructose
2, 6-bisphosphate, an allosteric activator of phosphofructokinase and
inhibitor of fructose 1,6-bisphosphatase.
350
II. GLYCOGEN METABOLISM
Glycogen is stored in most cells of the body but is most plentiful in liver (up to
8%) and muscle (1%). Liver glycogen is a carbohydrate reserved for the maintenance
of blood glucose; muscle glycogen is used only by the muscle itself during exercise.
1. Structure
A polysaccharide of glucose residues linked by α-1, 4 glycosidic bonds. Highly
Synthesis 19.2.3
Allosteric effectors: ATP, citrate, and acetyl-CoA favor gluconeogenesis, AMP and ADP
favor glycolysis.
Phosphorylation: Short-term hormonal effects are mediated by enzyme phosphorylation.
Pyruvate kinase is inactivated by cAMP-induced phosphorylation.
Insulin increases and glucagon decreases the concentration of fructose-2,6-bisphosphate,

an allosteric activator of phosphofructokinase and inhibitor of fructose 1,6-bisphosphatase.
19.2. Glycogen Metabolism
Glycogen is stored in most cells of the body but is most plentiful in liver (up to 8 %)
and muscle (1 %). Liver glycogen is a carbohydrate reserved for the maintenance of blood
glucose; muscle glycogen is used only by the muscle itself during exercise.
19.2.1. Structure
A polysaccharide of glucose residues linked by α-1,4-glycosidic bonds. Highly branched (one

branch point every 12 residues) with α-1,6-glycosidic bonds at the branch points.
19.2.2.
2. Synthesis
Synthesis
Phosphoglucomutase
Glucose 6-phosphate Glucose 1-phosphate
UTP
Glucose 1-phosphate
uridyl-transferase
PPi
UDP (Glc)n
(Glc)n+1 UDP-glucose
Glycogen Synthase
At equilibrium about 5% of all glucose phosphate is glucose 1-phosphate and
At
95%equilibrium about 5 % of
is glucose 6-phosphate. Theall glucose
bond betweenphosphate is glucose
UDP and glucose 1-phosphate and 95 % is glu-
is moderately
cose 6-phosphate.
energy-rich The bond between UDP and glucose is moderately energy-rich (12–
(3-4 kcal/mol).
Glycogen synthase makes the α-1, 4 glycosidic bonds. The branches with their
17 kJ/mol).
α-1, 6-glycosidic bonds are made by a branching enzyme which transfers an
Glycogen synthase
oligosaccharide makes
from the the α-1,4-glycosidic
nonreducing end of the moleculebonds.
to a C-6The branches with their α-1,6-glycosidic
carbon.
bonds
3. are made by a branching enzyme which transfers an oligosaccharide from the non-
Degradation.
reducing end of phosphorylase
Glycogen the molecule to a C-6 glucose
removes carbon.residues by phosphorolytic
cleavage from the nonreducing end, forming glucose 1-phosphate. It cleaves only α-1,
4-glycosidic bonds. The branches are removed by a debranching enzyme which
transfers pieces of 3 glucose units from the branch points and hydrolyzes the α-1, 6-
glycosidic bonds. Because of hydrolysis of the α-1, 6 bonds 8% of the glucose in 351
glycogen is released as free glucose rather than glucose 1-phosphate.
4. Difference between liver and muscle

The liver has glucose 6-phosphatase. Therefore it makes free glucose from
glycogen via glucose 1-phosphate and glucose 6-phosphate. The liver synthesizes
glycogen after a carbohydrate-rich meal and degrades it between meals to maintain the
blood glucose level. Liver glycogen lasts for 12 – 24 h.
Muscle has no glucose 6-phosphatase. Therefore muscle glycogen can be used
only locally, for glycolysis. It lasts for 2 hours during a marathon race.
19.2.3. Degradation
Glycogen phosphorylase removes glucose residues by phosphorolytic cleavage from the non-
reducing end, forming glucose-1-phosphate. It cleaves only α-1,4-glycosidic bonds. The
branches are removed by a debranching enzyme which transfers pieces of 3 glucose units
from the branch points and hydrolyzes the α-1,6-glycosidic bonds. Because of hydrolysis
of the α-1,6 bonds 8 % of the glucose in glycogen is released as free glucose rather than
glucose-1-phosphate.
19.2.4. Difference between liver and muscle
The liver has glucose-6-phosphatase. Therefore it makes free glucose from glycogen via
glucose-1-phosphate and glucose-6-phosphate. The liver synthesizes glycogen after a carbohydrate-
rich meal and degrades it between meals to maintain the blood glucose level. Liver glycogen
lasts for 12–24 h.
Muscle has no glucose-6-phosphatase. Therefore muscle glycogen can be used only locally,
for glycolysis. It lasts for 2 h during a marathon race.
19.2.5. Regulation of glycogen metabolism
The enzymes of glycogen metabolism are controlled by:
• Hormone-induced phosphorylation.
• Allosteric effectors.
Glycogen synthase is inactivated and glycogen phosphorylase is activated by
phosphorylation:
Glycogen synthase is inactivated and glycogen phosphorylase is activated by phosphoryla-
tion:
ATP ADP - Glucose 6-phosphate
Protein Kinase
Glycogen synthase a Glycogen synthase b
(dephosph'd: active) (phosph'd: less active)
Protein Phosphatase
++
Ca Pi
+ AMP - Glucose 6-phosphate ATP ADP - Glucose
(in muscle) (in muscle) (in liver)
Protein Kinase
Glycogen phosphorylase b Glycogen phosphorylase a
(dephosph'd: less active) (phosph'd: active)
Protein Phosphatase
Pi
Glucose (liver), AMP (extrahepatic tissues), and glucose 6-phosphate are

allosteric effectors - indicated effects are shown with gray arrows above.
352 Phosphorylation is induced by calcium and cAMP. In the liver, cAMP is
elevated by glucagon and calcium by epinephrine (α-receptors). In muscle, cAMP is
elevated by epinephrine (β-receptors), and calcium is elevated during excitation-
contraction coupling. Dephosphorylation is effected by phosphatase-1 which is
stimulated by insulin and inhibited by cAMP. Insulin also antagonizes glucagon directly
by lowering the cAMP concentration.
INSULIN Glucagon (liver) Acetylcholine (muscle)

Epinephrine (muscle, liver) Epinephrine (liver)
Plasma
+ AMP - Glucose 6-phosphate ATP
(in muscle) (in muscle)
Protein Kinase
Glycogen phosphorylase b
(dephosph'd: less active)
Glycogen storage diseases 19.2.6 Protein Phosphatase
Pi
Glucose (liver), AMP (extrahepatic tissues), and glucose-6-phosphate are allosteric effectors
Glucose (liver), AMP (extrahepatic tissu
- indicated effects are shown with gray arrows above. allosteric effectors - indicated effects are shown wi
Phosphorylation is induced by calcium
Phosphorylation is induced by calcium and cAMP. In the liver, cAMP is elevated elevated by
by glucagon and calcium by epinephrin
elevated by epinephrine (β-receptors), and ca
glucagon and calcium by epinephrine (β-receptors). In muscle, cAMP is elevatedcontraction
by epinephrine
coupling. Dephosphorylation is e
(β-receptors), and calcium is elevated during excitation-contraction coupling. Dephosphory-
stimulated by insulin and inhibited by cAMP. Insu
by lowering the cAMP concentration.
lation is effected by phosphatase-1 which is stimulated by insulin and inhibited by cAMP. In-
INSULIN Glucagon (liver)
Epinephrine (muscle, liv
Plasma
Membrane + +
cAMP
- +
Phosphatase-1 Protein Kinase A
+ +
Phosphorylase Kina
- + -
Glycogen Phosphorylase + Glyc
sulin also antagonizes glucagon directly by lowering the cAMP concentration.

156
19.2.6. Glycogen storage diseases
Problem: Recessively inherited deficiency of a glycogen degrading enzyme, with accumula-

tion of glycogen. 3 types:
Hepatic: Hepatomegaly, fasting hypoglycemia
Myopathic: Muscle weakness, muscle cramps on exertion.
Generalized: Brain or myocardium are also affected, besides liver and skeletal muscle.
Von Gierke’s disease (type I): Deficiency of glucose-6-phosphatase in liver and kid-
ney. Severe hepatomegaly and fasting hypoglycemia, ketosis, hyperuricemia, lactic acidosis.
Treated with regular carbohydrate feeding.
McArdle’s disease (type V): Deficiency of glycogen phosphorylase in muscle. Muscle

pain and cramps on exertion, sometimes intermittent myoglobinuria, muscle wasting in
some older patients. Little or no increase of blood lactate after exercise. No treatment
required.
353
6. Glycogen storage diseases
Problem: Recessivelyinherited deficiency of a glycogen degrading enzyme,
19.3.1
with accumulation of glycogen.
3 types:
Hepatic: Hepatomegaly, fasting hypoglycemia
Myopathic: Muscle weakness, muscle cramps on exertion.
Pompe’s Generalized: Brain
II): or muscle.
disease (typeskeletal myocardium are
Deficiency ofalso affected, besides
a lysosomal liver and (‘acid maltase’)
α-1,4-glucosidase
in all tissues. Glycogen accumulates in lysosomes. Cardiac involvement, with death before
ageand2 a.a) Von Gierke’s disease (type I): Deficiency of glucose 6-phosphatase in liver
kidney. Severe hepatomegaly and fasting hypoglycemia, ketosis, hyperuricemia,
lactic acidosis. Treated with regular carbohydrate feeding.
b) McArdle’s disease (type V): Deficiency of glycogen phosphorylase in
muscle. Muscle pain and cramps on exertion, sometimes intermittent myoglobinuria,
muscle wasting in some older patients. Little or no increase of blood lactate after
19.3. Dietary Fructose and Galactose
exercise. No treatment required.
c) Pompe’s disease (type II): Deficiency of a lysosomal α-1, 4-glucosidase
(‘acid maltase’) in all tissues. Glycogen accumulates in lysosomes. Cardiac
The liver is with
involvement, the death
mostbefore
important
age 2. site of fructose and galactose metabolism.
III. DIETARY FRUCTOSE AND GALATOSE
19.3.1.TheFructose
liver is the most important site of fructose and galactose metabolism.
metabolism
1. Fructose metabolism
Only a small fraction of the dietary fructose is phosphorylated by hexokinase in
Only a smalltissues.
extrahepatic fractionInofliver,
thekidney
dietaryandfructose
intestine, is phosphorylated
fructose by hexokinase
is phosphorylated by in extrahepatic
tissues. In liver, kidney and intestine, fructose is phosphorylated by fructokinase:
fructokinase:
Glucose Fructose
Pi ATP
Fructokinase
ADP
Glucose 6-Phosphate Fructose 1-Phosphate
Aldolase B
Fructose 6-Phosphate Dihydroxyacetone- Phosphate Glyceraldehyde

Pi
Aldolase ATP
ADP
Fructose 1,6-bis-Phosphate Glyceraldehyde 3-Phosphate
Lactate Pyruvate
Triglyceride Acetyl-CoA
157
Fructose is glycolyzed more rapidly than glucose because phosphofructokinase is bypassed.
Also, fructose-1-phosphate can accumulate in the liver because fructokinase has a higher
activity than aldolase B. The use of fructose instead of glucose in parenteral nutrition
causes lactic acidosis, liver damage and hypertriglyceridemia.
Essential fructosuria is a benign condition, caused by an inherited deficiency of fructok-

inase. Fructose is high in blood and urine after a fructose-containing meal.
Aldolase B deficiency leads to hereditary fructose intolerance (HFI), with hypoglycemia

and nausea after eating fructose. Liver damage is possible, but affected children develop a
strong aversion to sweets, and affected adults have very good teeth.
354
Fructose is glycolyzed more rapidly than glucose because phosphofructokinase
is bypassed. Also, fructose 1-phosphate can accumulate in the liver because
fructokinase has a higher activity than aldolase B. The use of fructose instead of
glucose in parenteral nutrition causes lactic acidosis, liver damage and
hypertriglyceridemia.
Reactions
Essential fructosuria is a benign condition, caused by an inherited deficiency of 19.4.1
fructokinase. Fructose is high in blood and urine after a fructose-containing meal.
Aldolase B deficiency leads to hereditary fructose intolerance (HFI), with
hypoglycemia and nausea after eating fructose. Liver damage is possible, but affected
Fructose-1,6-bisphosphatase deficiency
children develop a strong aversion to sweets, also
and affected leads
adults have toveryfructose
good teeth.intolerance, but patients
also have hypoglycemia.
Fructose In the deficiency
1, 6-bisphosphatase 2 forms ofalsofructose
leads to intolerance phosphorylated sugars build
fructose intolerance,
but patients also have hypoglycemia. In the 2 forms of fructose intolerance
up in the liver.sugars
phosphorylated Thisbuild
depletes inorganic
up in the liver. This phosphate, causing
depletes inorganic livercausing
phosphate, damage. Fructose-1-phosphate
stimulates
liver damage.glucokinase and inhibits
Fructose 1-phosphate glycogen
stimulates phosphorylase,
glucokinase causing hypoglycemia.
and inhibits glycogen
phosphorylase, causing hypoglycemia.
2. Galactose metabolism
Pathway: Galactose metabolism

19.3.2.
Galactose
ATP
Galactokinase
ADP
Galactose 1-Phosphate
UDP-glucose
Galactose 1-Phosphate Uridyl-transferase
Glucose 1-Phosphate
UDP-galactose
UDP-galactose-4-epimerase
Glucose 6-Phosphate UDP-glucose
In the Inabsence of dietary

the absence of dietarygalactose, thereversible
galactose, the reversible epimerase
epimerase reaction
reaction supplies supplies UDP-galactose
UDP-galactose for biosynthetic reactions.
for biosynthetic
Galactosemiareactions.
is caused by an inherited deficiency of galactose 1-phosphate-
uridyl-transferase. Vomiting, jaundice and CNS dysfunction develop within weeks after
birth. Later: Liver failure, cataracts (lens opacities), mental deficiency. The
Galactosemia
accumulation of is caused1-phosphate
galactose by an inherited
causes deficiency
liver damage of galactose-1-phosphate-uridyl-transferase.
(depletion of inorganic
Vomiting, jaundice and CNS dysfunction develop within weeks after birth. Later: Liver fail-
ure, cataracts (lens opacities), mental deficiency. The accumulation of galactose-1-phosphate
158
causes liver damage (depletion of inorganic phosphate!); galactitol, formed by aldose reduc-
tase in the lens, causes cataracts. Patients have reducing sugar in the urine, but enzymatic
tests for glucose are negative. A milk-free diet permits normal development.
19.4. The Pentose Phosphate Pathway
19.4.1. Reactions
The pentose phosphate pathway oxidizes glucose with formation of NADPH + H+ , used
for reductive biosynthesis and the antioxidant defenses of the cell. It also produces ribose-
5-phosphate, a precursor for nucleotide synthesis. It has an oxidative and a nonoxidative
355
branch. Reactions of the oxidative branch:

+
H2 H H2
+
O3P O C NADP NADPH O3P O C
O O
OH OH O
OH OH Glc-6-P dehydrogenase OH
OH OH
Glc-6-P 6-P-gluconolactone
H2O
+
CO2 H
+
H -
COO
NADPH NADP
+
H2C OH HC OH
O C HO CH
HC OH HC OH
6-P-gluconate dehydrogenase
HC OH HC OH
C O PO3 C O PO3
H2 H2
Ribulose-5-P 6-P-gluconate
The nonoxidative branch links ribulose-5-phosphate to glycolysis and gluconeogenesis in a

sequence of freely reversible reactions. Transaldolase and the thiamine-dependent transke-
tolase are the most important enzymes.
19.4.2. Products and regulation
For each CO2 released, the oxidative branch forms 2 NADPH + H+ .
The reactions of the oxidative branch are irreversible, therefore they can maintain a high
cellular [NADPH + H+ ]/ [NADP+ ] ratio.
Glucose-6-phosphate dehydrogenase is inhibited by a high [NADPH + H+ ]/ [NADP+ ] ratio.

There is also enzyme induction in the well-fed state.
19.4.3. Physiological role
High pentose phosphate pathway activity is seen in tissues that make reductive biosynthesis
(liver, adipose tissue, lactating mammary gland, adrenal cortex) and in tissues exposed to
oxidative stress (cornea, RBCs).
356
Glucose-6-phosphate dehydrogenase deficiency 19.5.1
When the cell needs a lot of NADPH + H+ , the products of the nonoxidative branch are
recycled to glucose-6-phosphate, and the cycle can repeat itself. When the cell needs a lot
of ribose, ribose-5-phosphate is formed not only by the oxidative branch, but also through
the reversible reactions of the nonoxidative branch.
19.4.4. Glucose-6-phosphate dehydrogenase deficiency
A partial deficiency of glucose-6-phosphate dehydrogenase in red blood cells is common in

Africa, the middle East, and South Asia. X-linked recessive inheritance. Asymptomatic,
but acute hemolysis develops in response to primaquine (an antimalarial) and some other
drugs; also after eating broad beans (favism). About 1 × 108 males are affected worldwide,
including 11 % of black Americans.
RBCs require NADPH + H+ for protection from oxidative damage. NADPH + H+ main-
tains the tripeptide glutathione in the reduced state. Glutathione can be used to destroy
hydrogen peroxide (H2 O2 ) in the glutathione peroxidase reaction, reduced glutathione has
to be regenerated by the NADPH + H+ -dependent enzyme glutathione reductase:
H2O2
2 H2O
Glutathion
peroxidase
γGlu γGlu γGlu γGlu
Cys SH + HS Cys Cys S S Cys

Gly Gly Gly Gly
Glutathion
reductase
+
NADP NADPH
+
H
357
19.5. The ‘Minor’ Pathways
19.5.1. The Polyol Pathway
The polyol pathway provides an endogenous source of fructose. Fructose occurs, for exam-
ple, in seminal fluid in concentrations up to 200 mg/dL.
O + +
HC H H2C OH H H2C OH
NADPH +
HC OH NAD
+
NADH
HC OH NADP C O
HO CH HO CH HO CH
HC OH Aldose HC OH Sorbitol HC OH
HC OH reductase HC OH dehydrogenase HC OH
C OH C OH C OH
H2 H2 H2
D-glucose D-sorbitol D-fructose
19.5.2. Synthesis of Amino Sugars
Amino sugars are components of glycolipids, glycoproteins and proteoglycans. They can be
synthesized in the body:
The nitrogen is derived from the side-chain of glutamine.
The acetyl group on the nitrogen of many amino sugars comes from acetyl-CoA.
UDP-derivatives are the activated forms of the amino sugars for biosynthetic reactions.
19.5.3. The uronic acid pathway.
Glucuronic acid is synthesized from glucose in an NAD+ -dependent reaction. It is a con-

stituent of glycosaminoglycans, and in the liver it is used for the conjugation of bilirubin
and some drugs. The enzymes of the glucuronic acid pathway are induced by many drugs
in the liver.
19.6. Practice Questions
The liver has to maintain an adequate blood glucose level in the fasting state. Try to predict
the effects of inherited deficiencies of liver enzymes on the fasting blood glucose level:
1. Glucokinase
358
2. Glycogen phosphorylase
3. Glucose 6-phosphatase
4. Fructose 1, 6-bisphosphatase
5. Aldolase B
After eating 100 g of fructose, the blood levels of lactic acid and triglycerides are higher
than after 100 g of glucose. Why?
Assume that pyruvate dehydrogenase is deficient in all cells. Which tissue would suffer
most?
Arsenite, the most toxic form of arsenic, binds tightly to the two sulfhydryl groups in
dihydrolipoic acid and thereby inhibits lipoic acid dependent reactions. Which metabolites
are elevated in the blood after arsenite poisoning? Can arsenic be determined in dead
bodies?
Which metabolites accumulate in the blood of patients with thiamine deficiency, and how
would you use this for the diagnosis of thiamine deficiency? Which RBC enzyme can be
assayed to test for thiamine deficiency, and how would you design such a test?
How does cyanide poisoning affect cellular energy charge, [NADH + H+ ]/ [NAD+ ] ratio, the
oxidation state of respiratory chain components, TCA cycle activity, glycogen metabolism
and glycolysis, blood pH and body temperature? Would poisoning with pentachlorophenol
(a wood preservative that uncouples oxidative phosphorylation) do the same?
1. Name the organs of gluconeogenesis and the physiological conditions in which gluco-
neogenesis is important.
2. Identify the reactions of gluconeogenesis that bypass the irreversible reactions of gly-
colysis.
3. State the energy requirements of gluconeogenesis, and name important metabolic

processes that supply this energy in the gluconeogenic tissues.
4. Describe the effects of hormones and metabolites on gluconeogenesis, both in short

term and long term and predict the clinical effects of deficiencies of individual gluco-
neogenic enzymes.
5. Describe the functions of glycogen in liver, muscle and other tissues.
359
6. List the sequence of intermediates in glycogen synthesis and glycogen degradation,

with special emphasis on effects of metabolites and of phosphorylation/dephosphorylation
on the catalytic activities of glycogen synthase and glycogen phosphorylase.
7. Predict the clinical effects of deficiencies of glycogen degrading enzymes in liver and
muscle.
8. List the sequence of reactions by which fructose and galactose are channeled into the
glycolytic pathway, and the major tissues where these reactions take place.
9. Describe the clinical presentations and the treatment for patients with deficiencies for
fructose and galactose metabolizing enzymes.
10. Name the most important product of the oxidative branch of the pentose phosphate
pathway, its role in metabolism and the consequences of a partial deficiency of glucose-
6-phosphate dehydrogenase in red blood cells.
11. Know that subjects with an atypical form of transketolase and thiamine deficiency
get Wernicke-Korsakoff syndrome, and that amino sugars and glucuronic acid can be
synthesized endogenously from phosphorylated monosaccharides.
360
20. Lipid Metabolism
20.1. Structures
Most naturally occurring fatty acids are unbranched and have an even number of carbons.
The carbons are either numbered, or they are designated by Greek letters. The last carbon
in the chain is called the ω (omega) carbon:
ω β
COOH
H3C (CH2)16 COOH = H3C
α
Stearic acid
The carbons of saturated fatty acids are linked only by single bonds. Mono-unsaturated
fatty acids have one C=C double bond, and polyunsaturated fatty acids have more than
one. Positions of double bonds are specified by their distance from the carboxy end. A ∆9
double bond, for example, is between carbons 9 and 10.
Humans cannot introduce double bonds beyond position ∆9. Therefore some polyunsatu-
rated fatty acids, notably linoleic acid and linolenic acid are nutritionally essential. The
biosynthetic class of these fatty acids is defined by the position of the double bond closest
to the omega carbon.
361
Examples of saturated fatty acids
COOH
4 Butyric acid CH3-(CH2)2-COOH H3C
COOH
14 Myristic acid CH3-(CH2)12-COOH H3C
COOH
16 Palmitic acid CH3-(CH2)14-COOH H3C
18 Stearic acid CH3-(CH2)16-COOH COOH

H3C
COOH
20 Arachidic acid CH3-(CH2)18-COOH H3C
Examples of unsatturated fatty acids
Palmitoleic acid ω7 16:1,9 H3C COOH
Oleic acid ω9 18:1,9 H3C COOH
H3C
Linoleic acid w6 18:2,9,12 COOH
α-Linolenic acid ω3 18:3,9,12,15 H3C COOH
H3C
Arachidonic acid ω6 20:4,5,8,11,14 COOH
The carboxy group of the fatty acids has a pKa value close to 4.8. Only cis-double bonds are
present in mammalian metabolism. However, bacteria also produce trans-fatty acids. Since
the fatty acids in cows milk were produced by their intestinal bacteria milk fat contains
about 4 % trans-fatty acids. Trans double bonds also occur in hydrogenated fats, such as
margarine and peanut butter (at about the same concentration as in milk fat). Cis-double
bonds decrease the melting points of the fatty acids and triglycerides containing them:
O
O
H2C O
O CH O
C O P O R
H2
O
unsaturated fatty acid in 2-position of glycerol
kink at cis-double bond disturbs alignment of molecules

-> lower melting point.
Polyunsaturated fatty acids are subject to nonenzymatic auto-oxidation or peroxidation.

The process is mediated by free-radical chain reactions. It occurs outside the body, where
it causes fats to turn rancid, as well as in the body. Lipid peroxidation can be suppressed
by antioxidants such as vitamins A, C and E. Some forms of lipofuscin (“age pigment”) are
formed by nonenzymatic reactions involving partially oxidized fatty acids.
362
Adipose tissue 20.3.1
20.2. Utilization of Dietary Fat
The products of pancreatic lipase (fatty acids and 2-monoacylglycerol are absorbed. The
absorption of long-chain fatty acids (> 16 carbons) requires bile salts.
In the mucosal cells, the fatty acids are activated to their CoA-thioesters:
The carboxy group of the fatty acids has aPP pKi value close to 4.8. Only cis double
bonds are present in naturally occurring fatty acids. Trans double bonds occur in
ATP AMP
hydrogenated fats, such as margarine and peanut butter. Cis double-bonds
O decrease the
melting points
- of the fatty acids and triglycerides containing them.
R COO +
Polyunsaturated CoA acids are subject to nonenzymatic
HS fatty
Acyl-CoA synthetase
CoA
R C Sauto-oxidation or
peroxidation. The process is mediated by free-radical chain reactions. It occurs outside
the body, where it causes fats to turn rancid, as well as in the body. Lipid peroxidation can
be suppressed by antioxidants such as vitamins A, C and E. Some forms of lipofuscin
The
(“agemucosal
pigment”)cells
are synthesize triglycerides
formed by nonenzymatic from activated
reactions fatty acids
involving partially andfatty
oxidized 2-monoglyceride.
acids.
In the ER, these triglycerides are packaged into chylomicrons, large lipoproteins containing
98–99 %II. lipid and 1–2 %OF
UTILIZATION protein.
DIETARY The
FAT.chylomicrons are released into the lymph and reach
the bloodstream via the thoracic duct. Once in the blood, the lifespan of chylomicrons is
The products of pancreatic lipase (fatty acids and 2-monoacylglycerol are absorbed.
less than 10 min.
The absorption of long-chain fatty acids (>16 carbons) requires bile salts.
In the mucosal cells, the fatty acids are activated to their CoA-thioesters:
Chylomicron triglycerides areATP hydrolyzed
AMP, PPtoi fatty acids and 2-monoacylglycerol by lipopro-
O
tein lipaseR (LPL), an
COO- + HS CoA
enzyme on the lumenal surface of the capillary endothelium. It is
R C S CoA
present in most tissues but not in brain
Acyl-CoA and liver. Different tissues express different isoen-
synthetase
zyme forms of LPL. The LPL activity in adipose
The mucosal cells synthesize triglycerides from tissue,
activatedbut
fattynot muscle
acids and and
2- myocardium,
monoglyceride. In the ER, these triglycerides are packaged into chylomicrons, large
islipoproteins
increasedcontaining
by insulin. This directs dietary fat to adipose tissue in the well-fed state. Treat-
98 – 99% lipid and 1-2% protein. The chylomicrons are released
ment of lymph
into the patients
and with
reach heparin releasesviaLPL
the bloodstream into the
the thoracic circulation.
duct. Once in the blood, the
lifespan of chylomicrons is less than 10 minutes.
Chylomicron triglycerides are hydrolyzed to fatty acids and 2-monoacylglycerol by
lipoprotein lipase (LPL), an enzyme on the lumenal surface of the capillary endothelium.
It is present in most tissues but not in brian and liver. Different tissues express different
isoenzyme forms of LPL. The LPL activity in adipose tissue, but not muscle and
20.3. Adipose tissue
myocardium, is increased by insulin. This directs dietary fat to adipose tissue in the well-
fed state. Treatment of patients with heparin releases LPL into the circulation.
III. Adipose tissue

The The pathway
pathway of fat of fat synthesisin
synthesis in adipose
adiposetissue differs
tissue from from
differs that in that
the intestine:
in the intestine:
Glucose Dihydroxyacetone Phosphate
NADH
Glycerol phosphate-
2 Acyl-CoA dehydrogenase
NAD+
Phosphatidic acid Glycerol 3-phosphate
Pi Acyl-CoA
Triglyceride
164
363
20.3.1. Triglyceride
Glycerol phosphate, which has to be made from glucose, is an important limiting factor.
Therefore fat synthesis is stimulated by insulin, which is required for glucose transport into
adipose cells. Most of the acyl-CoA comes from triglycerides in chylomicrons and VLDL.
Lipolysis (fat hydrolysis) is initiated by hormone-sensitive adipose tissue lipase. This en-
zyme is activated by cAMP-dependent phosphorylation. Norepinephrine and epinephrine
raise cAMP in adipose tissue by an action on β-receptors. Thyroid hormone, glucocor-
ticoids and growth hormone promote lipolysis by inducing the synthesis of proteins in-
volved in cAMP-responsiveness. Insulin inhibits the hormone-sensitive lipase. Therefore
fat is Glycerol
degraded phosphate, which has
during fasting to be
(low made from
insulin) andglucose,
during isphysical
an important limiting
exercise and stress (high
factor. Therefore fat synthesis is stimulated by insulin, which is required for glucose
norepinephrine and epinephrine).
transport into adipose cells. Most of the acyl-CoA comes from triglycerides in chylomicrons
and VLDL.
The fatty acids
Lipolysis (fatformed during
hydrolysis) lipolysis
is initiated are released intoadipose
by hormone-sensitive the blood.
tissueThey
lipase.are transported
to other
This tissues
enzyme non-covalently
is activated bound to serum
by cAMP-dependent albumin. Plasma
phosphorylation. free fatty
Norepinephrine and acids levels are
epinephrine raise cAMP in adipose tissue by an action on β-receptors. Thyroid hormone,
lowest after a carbohydrate meal and highest in long term fasting.
glucocorticoids and growth hormone promote lipolysis by inducing the synthesis of proteins
involved in cAMP-responsiveness. Insulin inhibits the hormone-sensitive lipase. Therefore
fat is degraded during fasting (low insulin) and during physical exercise and stress (high
norepinephrine and epinephrine).
20.4. Fatty Acid Oxidation
The fatty acids formed during lipolysis are released into the blood. They are
transported to other tissues noncovalently bound to serum albumin. Plasma free fattty acids
levels are lowest after a carbohydrate meal and highest in long term fasting.
With the exception of specialized cells such as neurons and RBCs, all cells can oxidize fatty
IV. Fatty Acid Oxidation.
acids. The fatty acids are either albumin-bound “free” (unesterified) fatty acids, or they are
derivedWithfrom triglycerides
the exception in lipoproteins.
of specialized cells suchThe use of lipoprotein
as neurons and RBCs, alltriglycerides
cells can depends on
oxidize fatty acids. The fatty acids are either albumin-bound “free” (unesterified)
LPL, and free fatty acids are used in proportion to their plasma concentration. fatty acids,
or they are derived from triglycerides in lipoproteins. The use of lipoprotein triglycerides
depends on LPL, and free fatty acids are used in proportion to their plasma concentration.
Oxidation takes place in the mitochondria. Long-chain fatty acids (> 14 carbons) are ac-
Oxidation takes place in the mitochondria. Long-chain fatty acids (>14 carbons) are
tivated as
activated as CoA-thioesters
CoA-thioesters in cytoplasm
in the the cytoplasm
and thenand theninto
shuttled shuttled into the mitochondrion
the mitochondrion as as
acyl-carnitine:
acyl-carnitine.
Cytoplasm Mitochondrion
Carnitine Carnitine
Acyl-CoA Acyl-CoA
CAT-1 CAT-2
CoA-SH CoA-SH
Acyl-carnitine Acyl-carnitine
The carnitine shuttle is rate-limiting for β-oxidation. Carnitine-acyl-transferase I

(CAT-I) is inhibited by malonyl-CoA, an intermediate of fatty acid synthesis.
The carnitine shuttle is rate-limiting for β-oxidation. Carnitine-acyl-transferase I (CAT-I)
In β-oxidation, C-3 (the β-carbon) of the CoA-activated fatty acid is oxidized, and the
is inhibited
fatty by malonyl-CoA,
acid shortened an intermediate
in 2 carbon decrements. of carbons
These two fatty acid synthesis.
are released as acetyl-
CoA. One FADH2 and one NADH are produced in each cycle (see next pg):
In β-oxidation,
O
C-3 FAD
(theFADH
β-carbon)Oof the HCoA-activated
2 O
OH O fatty acid is oxidized, and the fatty
2
acid shortened
R C C C in 2 carbon decrements.
S CoA These
R C C C S CoA
H H
R two
C C carbons
H H
C S CoA are released as acetyl-CoA. One
H2 H2 2 2 2
NAD
Alcohol Dehydrogenase
NADH, H+
O Acetyl-CoA CoA-SH
OH O
R C S CoA R C C C S CoA
H H2
364
Energy yield from β-oxidation of one palmitic acid:
165
Fatty Acid Oxidation 20.4
FADH2 and one NADH + H+ are produced in each cycle:

O
Carnitine
Acyl-carnitine
CoA SH
Carnitine acyl
transferase II
Carnitine
O
S CoA
Acyl-CoA
FAD
Acyl-CoA
dehydrogenase
FADH2
O
S CoA
trans-∆2-enoyl-CoA
H2O
Enoyl-CoA
hydratase
OH O
S CoA
L-β-hydroxyacyl-CoA
+
NAD
Hydroxyacyl-CoA
dehydrogenase
NADH + H+
O O
S CoA
β-ketoacyl-CoA
CoA-SH
Thiolase
O O
+ S CoA
S CoA
Acyl-CoA (-2 C) Acetyl-CoA
Energy yield from β-oxidation of one palmitic acid:
365
Metabolite equivalent to
7 FADH 14 ATP
7 NADH
7 FADH -------------------------21 ATP> 14 ATP
8 acetyl
7 NADHCoA-------------------------
96 ATP> 21 ATP
Activation
8 acetylof fatty
CoA acid -2 ATP
------------------- > 96 ATP
Activation of fatty acid -----------------
7 FADH ------------------------- 129 ATP> -2 ATP
> 14 ATP
7 NADH ------------------------- 129 ATP
Efficiency of ATP synthesis: 40 %. > 21 ATP
8 acetyl CoA ------------------- > 96 ATP
Efficiency of ATP synthesis: 40%.
Activation
Unsaturatedof fatty
Unsaturated fattyacid
fatty acids-----------------
acids are channeled
are channeled> -2into
ATP
into β-oxidation
β-oxidation by by specialized
specialized enzyme
enzyme systems. Odd-
systems.chain fatty acids
Odd-chain fattyleave
acidsone leave one 129
propionyl-CoA ATPremaining
propionyl-CoA at the at
remaining endtheof end
β-oxidation.
of β- Propionyl-
CoA
oxidation. channeled channeled
Propionyl-CoA into the TCA cycle:
into the TCA cycle:
Efficiency of ATP COsynthesis:
2 40%.
Unsaturated
Propionyl CoA fatty acids are channeled into β-oxidation
Methylmalonyl-CoA by specialized enzyme
Succinyl-CoA
systems. Odd-chain fatty acids leave one propionyl-CoA remaining at the end of β-
Propionyl-CoA Methylmalonyl-CoA
carboxylase mutase (B12)
oxidation. Propionyl-CoA channeled into the TCA cycle:
(biotin)
CO2
Propionyl CoA
Branched-chain Methylmalonyl-CoA α-oxidation.
Succinyl-CoA
Branched-chainfatty acids
fatty require
acids a specialized
require pathway
a specialized called
pathway called α-oxidation.
Very long fatty Propionyl-CoA
acids (>20 carbons) require peroxisomal β-oxidation. Very long fatty
Methylmalonyl-CoA
acids accumulate in carboxylase
Very long fatty patients with
acids (> 20 Zellweger syndrome,
carbons) require a rare,(Brecessively
mutase
peroxisomal 12)
β-oxidation. inherited
Very long fatty acids
absence of peroxisomes.
(biotin)
accumulate in patients with Zellweger syndrome, a rare, recessively inherited absence of
V.peroxisomes.
Ketogenesis.
Branched-chain fatty acids require a specialized pathway called α-oxidation.
“Ketone bodies” include acetoacetate, β-hydroxybutyrate and acetone. Acetoacetate
and Very long fatty acids
β-hydroxybutyrate are (>20 carbons)products
water-soluble formed from β-oxidation.
require peroxisomal acetyl-CoA inVery
liverlong fatty
acids
mitochondria. They are released into the blood for transport to other tissues where they can inherited
accumulate in patients with Zellweger syndrome, a rare, recessively
absence of20.5.
peroxisomes.
be oxidized. Ketogenesis
Acetone is formed nonenzymatically from acetoacetate. It has no known
biological function and is exhaled through the lungs. Large amounts of ketone bodies are
formed
V. during long-term fasting and in diabetics. Biosynthetic pathway:
Ketogenesis.
Acetyl-CoA CoA
“Ketone
“Ketone bodies”
bodies” include
CoA
include acetoacetate,
acetoacetate, β-hydroxybutyrate
β-hydroxybutyrate and acetone.
and acetone.Acetoacetate
Acetoacetate and β-
hydroxybutyrate are water-soluble products formed
and β-hydroxybutyrate are water-soluble products formed from acetyl-CoA in liver
2 Acetyl-CoA Acetoacetyl-CoA HMG-CoA from acetyl-CoA in liver mitochondria.
TheyThey
mitochondria. are released into the
are released intoblood for transport
the blood to other
for transport tissuestissues
to other where where
they can be can
they oxidized.
Acetyl-CoA
Acetone
be oxidized. Acetoneis formed nonenzymatically
is formed nonenzymaticallyfrom acetoacetate. It has no known
from acetoacetate. It hasbiological
no knownfunction
β-Hydroxybutyrate Acetoacetate
biological and is exhaled
function and isthrough
exhaled thethrough
lungs. Large amounts
the lungs. of ketone
Large amounts bodies are formed
of ketone during
bodies are long-
termlong-term
formed during fasting and in diabetics.
fasting Biosynthetic
and in diabetics.
NAD +
pathway:
Biosynthetic
NADH, H+ pathway:
CoA Acetyl-CoA CoA
Extrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetyl-
CoA. 2 Acetyl-CoA Acetoacetyl-CoA HMG-CoA
VI. Aberrations Of Fatty Acid Metabolism. Acetyl-CoA

β-Hydroxybutyrate Acetoacetate
Essential fatty acid deficiency is seen only in patients on total parenteral nutrition
or with severe fat malabsorption. NAD+ NADH, H+
Extrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetyl-

Extrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetyl-CoA.
CoA.
166
VI. Aberrations Of Fatty Acid Metabolism.
Essential
366 fatty acid deficiency is seen only in patients on total parenteral nutrition
or with severe fat malabsorption.
166
From Carbohydrate To Fat 20.7
20.6. Aberrations Of Fatty Acid Metabolism
Essential fatty acid deficiency is seen only in patients on total parenteral nutrition or with
severe fat malabsorption.
Defects of mitochondrial β-oxidation can affect muscle or liver. If muscle is affected: muscle
weakness, cramping, myoglobinuria, and fat accumulation in the cytoplasm. If the liver
is affected: Hypoketotic hypoglycemia during fasting. Although most fatty acids are not
substrates of gluconeogenesis, fatty acid oxidation supplies the energy for gluconeogenesis.
β-oxidation is impaired in:
Carnitine deficiency: Either generalized or affecting only muscle. Responds to exogenous
carnitine, or to a diet with medium-chain instead of long-chain fatty acids.
Carnitine-acyl transferase deficiency: Similar to carnitine deficiency, usually limited to
muscle.
Medium-chain acyl-CoA dehydrogenase deficiency: Both liver and muscle are affected.
Hypoketotic hypoglycemia develops during fasting.
Refsum’s disease is a rare inherited deficiency of α-oxidation. It leads to an accumulation
of phytanic acid, a branched-chain fatty acid from green vegetables and ruminant fat.
20.7. From Carbohydrate To Fat
Excess carbohydrate and protein can be converted to fat in the liver. Acetyl-CoA (from
carbohydrate or protein) is turned into fatty acids, and the fatty acids are esterified into
triglycerides. These triglycerides are secreted in VLDL for use by other tissues.
The immediate substrate for fatty acid biosynthesis is malonyl-CoA, synthesized from
acetyl-CoA by the cytoplasmic acetyl-CoA carboxylase:
HCO3
- ADP
ATP P H2O
i
O O
-
H3C C S CoA OOC C C S CoA
Acetyl-CoA carboxylase H2
(Biotin)
In addition to malonyl-CoA, fatty acid synthesis requires NADPH + H+ for the reductive
reactions.
The cytoplasmic fatty acid synthase complex contains two important SH−groups to which
the growing fatty acid and the incoming malonyl group are bound covalently. One is in a
367
cysteine side chain, the other in phosphopantetheine, a covalently bound prosthetic group.
In each cycle, 2 carbons (from malonyl-CoA) are added to the fatty acid. Reductive reactions
occur while the growing chain os bound to phosphopantetheine. Palmitate (saturated, 16
carbons) is the principal product. Overall reaction:
Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + H+ → Palmitate + 8 CoA-SH + 14 NADP+

+ 7 CO2
ATP is required only for the synthesis of malonyl-CoA.
The acetyl-CoA for fatty acid synthesis is shuttled from the mitochondrion in the form
of citrate. In the cytoplasm, citrate is cleaved by ATP-citrate lyase to oxaloacetate and
acetyl-CoA. Oxaloacetate is shuttled back into the mitochondrion in a reaction sequence in
which malic enzyme (reaction: malate → pyruvate) produces NADPH + H+ .
Acetyl-CoA carboxylase, fatty acid synthase, ATP-citrate lyase, and glucose-6-phosphate

dehydrogenase in the liver are induced by carbohydrate feeding and insulin.
Acetyl-CoA carboxylase is allosterically stimulated by citrate and inhibited by acyl-CoA.

Citrate is high after a carbohydrate meal, and acyl-CoA is high during fasting and after a fat
meal. The enzyme is also inactivated by cAMP-dependent phosphorylation and activated
by insulin-stimulated dephosphorylation.
Other fatty acids are synthesized from palmitate: chain-elongation produces fatty acids with
up to 24 or 26 carbons. Desaturation is initiated in position ∆9 , and further double bonds
are introduced between this first double bond and the carboxy group.
The liver synthesizes triglycerides and forms VLDL both in the fed state, using newly
synthesized fatty acids, and during fasting using fatty acids from adipose tissue.
20.8. Phosphoglyceride Metabolism
Phosohoglycerides are synthesized from phosphatidic acid. Phosphatidic acid is “activated”

to CDP-diacylglycerol, and this reacts with alcohol. Used for the synthesis of phosphatidyli-
nositol.
Phosphoglycerides can also be synthesized from 1,2-diacylglycerol and a CDP-activated

alcohol. Used for phosphatidylethanolamine and phosphatidylcholine.
Phospholipases are required for the remodeling and catabolism of the phosphoglycerides.
They are grouped according to their cleavage specificities:
368
Phosohoglycerides are synthesized from phosphatidic acid. Phosphatidic acid is
“activated” to CDP-diacylglycerol, and this reacts with alcohol. Used for the synthesis of
phosphatidylinositol.
Phosphoglycerides can also be synthesized from 1, 2 –diacylglycerol and a CDP-
Sphingolipid Metabolismand phosphatidylcholine.
activated alcohol. Used for phosphatidylethanolamine 20.9
Phospholipases are required for the remodeling and catabolism of the
phosphoglycerides. They are grouped according to their cleavage specificities:
A1
B
A1 B
O O O O
O H2C O OC RH1 C O C R1 H2C O CH2R
C O
1
C R1
2
R2 C O CH
R2 CO O CH O HO CH HO CH O
C O P O R3
C O P O R3 C RO3 P O
C O P O R3
H2 H2 H2
O H2 O O
A2
A2 O
D
C D
C
Fatty
Fatty acids acids
can can be replaced
be replaced by the sequential
by the sequential action ofaction of a phospholipase
a phospholipase and aselec-
and a highly highly
selective acyl-transferase
tive acyl-transferase
CO2 3 SAM
Phosphatidylserine Phosphatidylethanolamine Phosphatidylcholine
Phosphatidylethanolamine + Serine Phosphatidylserine + Ethanolamine
20.9. Sphingolipid Metabolism
The sphingolipid are synthesized from ceramide (= sphingosine + fatty acid). Synthesis
168 sugars are used for the synthesis of the
takes place in the ER, where nucleotide-activated
glycosphingolipids.
Glycolipids are degraded by highly specific lysosomal exoglycosidases (+ sulfatases).

A recessively inherited deficiency in any of the glycolipid-degrading enzymes results in a
sphingolipidosis (= lipid storage disease), with an accumulation of glycolipid substrate in
lysosomes. Neurological deterioration and hepatosplenomegaly are frequent clinical signs:
Tay-Sachs disease: Deficiency of hexosaminidase A, with accumulation of ganglioside

GM2. Hepatosplenomegaly, neurological deterioration, blindness (“amaurotic idiocy”),
cherry-red spot on the macula of the eye, death at about 3 years. Most common in
Ashkenazi Jews.
Gaucher’s disease: Deficiency of glucocerebrosidase. Glucocerebroside accumulates. The

rare infantile form is similar to Tay-Sachs. The more common adult-onset form
(mostly in Ashkenazi Jews) presents with splenomegaly, thrombocytopenia, abdomi-
nal problems and bone erosion presenting in midlife, but no mental deficiency.
369
substrate in lysosomes. Neurological deterioration and hepatosplenomegaly are frequent
clinical signs.
Tay-Sachs disease: Deficiency of hexosaminidase A, with accumulation of
ganglioside GM2. Hepatosplenomegaly, neurological deterioration, blindness (“amaurotic
20.10 cherry-red spot on the macula
idiocy”), Biochemistry and death
of the eye, Genetics
at about 3 years. Most common in
Ashkenazi Jews.
Gaucher’s disease: Deficiency of glucocerebrosidase. Glucocerebroside
Other lipid storage
accumulates. diseases
The rare include
infantile is similar todisease,
form Krabbe’s metachromatic
Tay-Sachs. leukodystrophy,
The more common adult-
disease.
onset form (mostly in Ashkenazi Jews) presents with splenomegaly, thrombocytopenia,
Niemann-Pick
abdominal problems and bone erosion presenting in midlife, but no mental deficiency.
Other lipid storage diseases include Krabbe’s disease, metachromatic
leukodystrophy, Niemann-Pick disease.
20.10. Cholesterol And Bile Acids
X. CHOLESTEROL AND BILE ACIDS
Cholesterol is both issynthesized
Cholesterol endogenously
both synthesized and derived
endogenously and from
derivedanimal
from dietary sources.
animal dietary
Vegan diets are essentially cholesterol-free. Intestinal absorption is about 50 %.
sources. Vegetarian diets are essentially cholesterol-free. Intestinal absorption is aboutThe liver
accounts for of the endogenous synthesis. Endocrine glands which produce
50%. The liver accounts for 50% of the endogenous synthesis. Endocrine glands which
50 % steroid
produce steroid
hormones also havehormones
high ratesalso have high synthesis.
of cholesterol rates of cholesterol synthesis.
On a typical AmericanOn diet,a 50–65
typical%
American diet, 50 – 65% of body cholesterol
of body cholesterol is from endogenous synthesis. is from endogenous synthesis.
Acetyl-CoA HMG-CoA Mevalonate Isopentenyl-pyrophosphate

(C-2) (C-6) (C-6) (C-5)
Cholesterol Lanosterol Squalene Dimethylallyl-pyrophosphate

(C-27) (C-30) (C-30) (C-5)
The first reactions, up to HMG-CoA, are shared with the pathway of ketogenesis.
The
But first reactions,
ketogenesis up to
occurs HMG-CoA,
in the are shared
mitochondria, with the ispathway
while cholesterol of ketogenesis.
synthesized But
in Cytosol and
ketogenesis occurs in the mitochondria, while cholesterol is synthesized
ER. The 5-carbon intermediates isopentenyl-pyrophosphate and Dimethylallyl- in Cytosol and ER.
The 5-carbon intermediates
pyrophosphate isopentenyl-pyrophosphate
are the building blocks of the isoprenoids,and dimethylallyl-pyrophosphate
which include ubiquinone, lipid
are the building
groups involvedblocks of the isoprenoids,
in anchoring which include
membrane proteins, and many ubiquinone, lipid groups
plant products. involved
The reductive
insteps in the pathway
anchoring membrane require NADPH.
proteins, and many plant products. The reductive steps in the
pathwayHMG-CoA reductase
require NADPH + H(HMG-CoA
+. ---! mevalonate) catalyzes the committed step of
cholesterol biosynthesis. Free cholesterol reduces the amount and activity of the enzyme.
Fasting reduces
HMG-CoA and insulin
reductase increases
(HMG-CoA its activity. catalyzes the committed step of choles-
→ mevalonate)
terol biosynthesis. Free cholesterol reduces the amount and activity of the enzyme. Fasting
reduces and insulin increases its activity.
The steroid nucleus of cholesterol is not degraded

169 in the body. Cholesterol is excreted in the
bile as free cholesterol or after its conversion to bile acids. The committed step of bile acid
synthesis is the introduction of a hydroxy group by 7α-hydroxylase in the ER. This enzyme
is feedback-inhibited by bile acids. The primary bile acids cholic acid and chenodeoxycholic
acid are secreted as conjugates with glycerine or taurine.
In the gut, the primary bile acids are de-conjugated and reduced to the secondary bile
acids deoxycholic acid and lithocholic acid by intestinal bacteria. 98 % of the bile acids are
absorbed in the ileum. Then picked up by the liver and re-secreted into the bile. This is
called entero-hepatic circulation.
An average bile acid molecule is recycled 5–8 times per day and persists for about a week
in the entero-hepatic system.
370
Lipoprotein Composition 20.11
The steroid nucleus of cholesterol is not degraded in the body. Cholesterol is

excreted in theBiliary
bile ascholesterol has or
free cholesterol to after
be kept in solution
its conversion as acids.
to bile a component of mixed micelles. Most
The committed
gallstones
step of bile acid synthesisconsist of cholesterol.
is the introduction They form
of a hydroxy group when
by 7α-the cholesterolin the
hydroxylase content of the bile is
ER. This enzyme is feedback-inhibited by bile acids. The primary bile acids cholic acid
increased or the concentration of bile salts or phospholipid is reduced. Gallstones are most
and chenodeoxycholic acid are secreted as conjugates with glycerine or taurine.
common in fat, fertile females.
In the gut, the primary bile acids are de-conjugated and reduced to the secondary
bile acids deoxycholic acid and lithocholic acid by intestinal bacteria. 98% of the bile
acids are absorbed in the ileum. Then picked up by the liver and re-secreted into the bile.
This is called entero-hepatic circulation.
An average bile acid molecule is recycled 5-8 times per day and persists for about a
week in the enterohepatic system.
Biliary cholesterol has to be kept in solution as a component of mixed micelles.
Most gallstones20.11.
consistLipoprotein
of cholesterol. TheyComposition
form when the cholesterol content of the bile
is increased or the concentration of bile salts or phospholipid is reduced. Gallstones are
most common in fat, fertile females.
XI. LIPOPROTEIN COMPOSITON

Typical fasting lipid concentrations are:
TypicalLipid Normal
fasting lipid concentrations are:Range (mg/dL)
Total lipid 400–800
LipidTriglycerides Normal Range (mg/dL)
40–300
Total cholesterol
Total lipid 400 – 800
120–280
Cholesterol
Triglycerides esters 40 – 300 90–200
TotalPhospholipids
cholesterol 120 - 280 150–380
Cholesterol esters
Free fatty acids 90 – 200 8–14
Phospholipids 150 - 380
Free fatty acids 8 - 14
With
With the the exception
exception of free
of free fatty fatty
acids acids
(from (from
adipose adipose
tissue), tissue),
lipids lipids inare
are present thepresent in the blood
blood as lipoproteins. General structure of lipoproteins.
as lipoproteins. General structure of lipoproteins:
= Cholesterol
= Cholesterol-ester
Lipoprotein = Phospholipid
= Triglyceride
Hydrophobic
Hydrophobic lipids (triglycerides,
lipids (triglycerides, cholesterol
cholesterol esters)
esters) are are center
in the in the of
center
the of the lipoprotein
particle, and amphipatic lipids, mainly phospholipids, form a water/lipid interface. Also the
lipoprotein particle, and amphipathic lipids, mainly phospholipids, form a water/lipid
interface. Also the apolipoproteins
apolipoproteins are amphipathic.
are amphipathic. One side of One side ofisthe
the protein protein is (facing triglyceride
hydrophobic
hydrophobic (facing triglyceride core) and the other side is hydrophilic.
core) and the other side is hydrophilic.
Lipoproteins are classified according

170 to their density: protein-rich (proteins ρ ≈ 1.5 mg/ml)
particles are more dense than lipid-rich (lipids ρ ≈ 0.9 mg/ml) particles. They can also be
separated by electrophoresis at pH 8.6:
371
20.12
Lipoproteins are classified according to their density:
Biochemistry andprotein-rich
Genetics particles are more
dense than lipid-rich particles. They can also be separated by electrophoresis at pH 8.6:
Electrophoretic result at pH 8.6 Density range (g/cm3)

-
Origin Chylomicrons (no migration) < 0.95
β-lipoproteins = LDL 1.006-1.063

pre-β-lipoproteins = VLDL 0.95 - 1.006
α-Lipoproteins = HDL 1.063-1.21

+
Properties and composition of plasma lipoproteins:
Lipid percent by class

Lipoprotein
PropertiesClass
and Diameter of
composition Protein
plasma Triglyc. Phosphol
lipoproteins: Cholesterol
Lipoprotein Class (nm) Diameter (%) Protein(%) Triglyc.ester
(%) Phosphol free Chol.
(%) free (%) Chol. ester
Chylomicron 100-1000(nm)
1-2 86(%) 8 (%)3 (%)
2 (%) (%)
Very-low-density
Chylomicron 30-80 100–1000
6-10 551–2 18 8613 78 3 2
Intermediate density 25-30 15-20 25 21 28 9
Very-low-density
Low density 20-25 30–80
22 9 6–10 20 5540 18
8 13 7
Intermediate
High density (HDL-2)density
9-12 25–30
35-45 5 15–20 33 2517 21
5 28 9
High
Lowdensity (HDL-3)
density 5-9 50-55
20–25 3 22 28 912 3
20 40 8
HighWith
density (HDL-2)
the exception 9–12 (LDL, 35–45
of apoB-100 VLDL) and apoB-48 5 (chylomicrons),
33 the 17 5
High density
apolipoproteins (HDL-3)
readily exchange5–9 50–55
between different 3
lipoprotein particles. 28
Apolipoproteins 12 3
regulate enzymes of lipoprotein metabolism, mediate the cellular uptake of lipoprotein
particles, and transfer lipids between different lipoprotein classes and between lipoproteins
and cells.
With the exception

Apoprotein MolecularofWtapoB-100 (LDL, Lipoprotein
Plasma conc VLDL) and apoB-48 (chylomicrons), the apolipopro-
Functions
teins readily(kiloDaltons) (mg/ml) different
exchange between components
lipoprotein particles. Apolipoproteins regulate en-
A-I 29 130 HDL, chylomics. Activates LCAT
zymes of lipoprotein
A-II 17 metabolism,
40 mediate the cellularUnknown
HDL, chylomics. uptake of lipoprotein particles, and
transfer lipids
B-48 241 between different
variable lipoprotein classes andPrimarily
chylomicrons between lipoproteins and cells.
structural
B-100
Apo-protein513 Molecular 80Wt Plasma VLDL,
concLDL Lipoprotein
LDL uptake
com-by cells
Functions
C-I 7 6 All except LDL unknown
C-II 9 3 ponents
All except LDL Activates LPL
C-III 9 (kDa)
12 (mg/ml)
All except LDL Inactivates LPL
D A-I 19 10 29 HDL130 HDL, chylomics.
unknown Activates LCAT
E 34 5 VLDL, IDL, remnant uptake by
A-II 17 40
chylomics. HDL, chylomics.
liver B/E receptorsUnknown
B-48 241 variable chylomicrons Primarily struc-
171 tural
B-100 513 80 VLDL, LDL LDL uptake by
cells
C-I 7 6 All except LDL unknown
C-II 9 3 All except LDL Activates LPL
C-III 9 12 All except LDL Inactivates LPL
D 19 10 HDL unknown
E 34 5 VLDL, IDL, chy- remnant uptake
lomics. by liver B/E
receptors
372
Lipoprotein Metabolism 20.12
20.12. Lipoprotein Metabolism
There are three highways of lipoprotein-based lipid transport:
1. Dietary lipids from the intestine to other tissues. The vehicles are chylomicrons and
chylomicron remnants.
2. Lipids from endogenous synthesis in the liver to other tissues. The vehicles are VLDL
and LDL.
3. Reverse cholesterol transport to the liver. The initial vehicle is HDL. Unlike the other
lipids, cholesterol cannot be degraded in extrahepatic tissues. It has to be transported
to the liver for biliary excretion or conversion to bile acids.
Chylomicrons are converted to chylomicron remnants by lipoprotein lipase (LPL) in adipose

tissue, muscle and elsewhere. These remnants are endocytosed by the liver. Endocytosis
requires apoE, which binds to a receptor on the surface of hepatocytes in the space of
Disse.
The liver synthesizes 25–50 g of triglyceride per day (+ cholesterol and phospholipid). These
lipids are released into the circulation as very-low density lipoproteins (VLDL). VLDL is
released with apoB-100 as its major apolipoprotein. Most other apolipoproteins are acquired
from HDL in the circulation. Like chylomicrons, VLDL is initially metabolized in peripheral
tissues by LPL. In the blood, VLDL triglycerides have half-lives of 15–60 min (chylomicrons:
5–10 min).
Like chylomicron remnants, VLDL remnants possess apoE, and about half of them are
endocytosed through liver apoE receptors. Smaller remnants which appear in the blood as
intermediate-density lipoproteins (IDL) are processed to low-density lipoproteins (LDL).
This involves the action of hepatic lipase as well as the transfer of excess apolipoproteins to
HDL. LDL has a solitary apoB-100 molecule which mediates endocytosis through the LDL
receptor in liver (60 %) and extrahepatic tissue (40 %). LDL is the major source of external
cholesterol for extrahepatic tissues. Alternative receptors, known as scavenger receptors,
mediate LDL uptake in macrophages.
Most cells obtain cholesterol both from endogenous synthesis and endocytosed LDL. Free
cholesterol in the cell regulates three important proteins:
• Acyl-CoA-cholesterol-acyl transferase (ACAT) is induced resulting in increased for-

mation of cholesterol esters for storage.
• HMG-CoA reductase is repressed, resulting in reduced cholesterol synthesis.
• LDL receptors are down-regulated (decreased receptor synthesis). This raises the level
of circulating LDL.
373
High-density lipoprotein (HDL) occurs in several subtypes which represent different stages
of its metabolism. Nascent HDL, released by the liver, is a small, phospholipid-rich particle
resembling a disk of lipid bilayer associated with apolipoprotein. Once in the circulation,
nascent HDL acquires unesterified cholesterol from other lipoproteins and cells. The ac-
quisition of cholesterol from cells requires the transient binding of HDL to cell surfaces.
Binding and subsequent cholesterol transfer requires apoA-I, the major apolipoprotein of
HDL, and/or apoE.
HDL-cholesterol becomes esterified by lecithin-cholesterol acyl transferase (LCAT), which

transfers a fatty acid from lecithin to cholesterol. Most of the cholesterol esters are then
transferred from HDL to triglyceride-rich lipoproteins and remnant particles by the choles-
terol ester transfer protein (CETP), much of this in exchange for triglycerides. The remnants
bring the cholesterol esters to the liver. Some cholesterol also reaches the liver by direct
transfer from HDL or by the endocytosis of apoE-coated HDL particles.
20.13. Lipoproteins and Atherosclerosis
The fatty streak consists of cholesterol ester deposits in the intima of large arteries. Macrophages
take up LDL through their scavenger receptors. They prefer chemically modified LDL, for
example oxidatively damaged LDL. These receptors may be important for the removal of
aberrant or aged lipoproteins that are no longer good ligands for other lipoprotein receptors.
The uptake of LDL-cholesterol has to be balanced by the transfer of excess cholesterol from
macrophages to HDL. A foam cell develops only when the amount of cholesterol acquired
from LDL exceeds the amount released to HDL. When the foam cell dies, the intracellular
lipid droplets become extracellular.
An atheromatous plaque develops when intimal smooth muscle cells proliferate, probably
in response to the lipid deposits and to locally-released growth factors and cytokines. Early
lesions are asymptomatic, but the complications of advanced atherosclerosis (heart attack,
stroke) are responsible for 30 % of all deaths in the US.
The risk factors for atherosclerosis include age and gender (lesions progress with age,
and males are affected earlier than females), smoking, diabetes mellitus, and hypercholes-
terolemia.
A low HDL/LDL ratio predicts a high risk of atherosclerotic lesions, while a high HDL/LDL
ratio is protective. The HDL/LDL ratio is protective. The HDL/LDL ratio can be in-
creased by exercise, low caloric intake, a high dietary unsaturated/saturated fat ratio, low
dietary cholesterol, high dietary fiber, regular alcohol consumption, and some drugs includ-
ing cholesterol synthesis inhibitors cholestyramine, and niacin.
374
Lipoprotein Disorders 20.14
20.14. Lipoprotein Disorders
Abetalipoproteinemia, a rare recessive disease, is caused by the absence of a triglyceride

transfer protein in the ER. As a result, liver and intestine are unable to synthesize and
secrete apoB-rich lipoproteins. VLDL, LDL and chylomicrons are essentially absent.
This disease leads to severe fat malabsorption and signs of vitamin E deficiency with
abnormalities of RBCs, CNS and muscle.
Tangier disease is a rare recessive disease in which HDL levels are reduced to less than 5 %
of normal. There is a marked deficiency of apoA-I, the major apolipoprotein of HDL.
LDL is also reduced. There is only a mild tendency for early atherosclerosis, probably
because decreased LDL cholesterol reduces the need for reverse cholesterol transport
by HDL. The molecular cause for Tangier-disease is a defect in the ABCA1 gene,
which encodes for an ABC-type membrane transporter which transfers phosphatidyl
choline (PC) from the plasma membrane to apoA-I. The disease is named after an
island in the Chesapeake Bay, where it was discovered.
Familial hypercholesterolemia is a dominantly inherited condition (incidence 1 in 500)
caused by a deficiency of LDL receptors in liver and peripheral tissues. LDL accu-
mulates to twice normal levels and total cholesterol is increased to 250–500 mg/dL.
Xanthomas (visible subcutaneous lipid deposits) develop in most patients by age 20 a.
Early death by coronary disease is common.
CETP deficiency is a benign condition in which cholesterol esters (formed by LCAT) can-
not be transferred from HDL to other lipoproteins. HDL-cholesterol is elevated about
four-fold. Cholesterol reverse transport is still accomplished by endocytosis of apoE-
containing HDL particles or direct transfer of cholesterol esters to the liver. This trait
is common in Japan.
Hyperlipoproteinemias are grouped into five types. These are not diseases but phenotypes
which occur in a variety of contexts. Most are “multifactorial”, but some are single-
gene or are secondary to a chronic disease such as diabetes mellitus, alcoholism or
hypothyroidism.
Type I hyperlipoproteinemia, or hyperchylomicronemia, is a rare form in which chy-
lomicron hydrolysis is impaired. Some patients have an inherited deficiency of
lipoprotein lipase (LPL), others are lacking apoC-II, an obligatory activator or
LPL. The patients have massive hypertriglyceridemia, but not much atheroscle-
rosis. LDL is reduced. The patients have to avoid dietary fat.
Type II hyperlipoproteinemia, or hypercholesterolemia, is an elevation of LDL. Mul-
tifactorial and secondary forms are far more common than familial hypercholes-
terolemia. This pattern is a common risk factor for atherosclerosis and coronary
heart disease.
375
Type III hyperlipoproteinemia, or dysbetalipoproteinemia, is caused by homozygos-

ity for an apoE variant that is not recognized by hepatic apoE receptors. Chy-
lomicron remnants and LDL-like VLDL remnants accumulate. This pattern is
rare. The atherosclerosis risk is increased.
Type IV hyperlipoproteinemia, or hypertriglyceridemia, is an elevation of VLDL.
This common pattern is associated with diabetes mellitus, alcoholism, excess
dietary carbohydrate, progesterone-rich contraceptives, and obesity. Cholesterol
is also mildly elevated, and the atherosclerosis risk is increased.
Type V hyperlipoproteinemia is rare pattern with a combined elevation of VLDL
and chylomicrons, secondary to uncontrolled diabetes mellitus or of unknown
etiology.
Familial combined hyperlipoproteinemia may be caused by a dominantly inherited gene
which causes increased apoB-100 synthesis in the liver. Either the type II or the type
IV pattern is expressed. About 1 % of the population are thought to have this disorder.
20.15. Practice Questions
Plasma free fatty acid levels are variable. Which organ produces these fatty acids, and
under what conditions do you expect elevated levels?
Compare the fates of blood-derived free fatty acids in skeletal muscle and the liver during
fasting.
Do you know of any inborn errors of metabolism that lead to muscle weakness and muscle
cramps on exertion?
The Ackee fruit, which grows in Jamaica, contains a toxin that inhibits α-oxidation. What
are the metabolic consequences, and under what conditions can this toxin be fatal?
You start a job at a pharmaceutical company that wants to develop new anti-obesity drugs.
In the first staff meeting you are asked if any of the following drug types would be promising,
and if undesirable side effects have to be expected:
• Inhibitors of pancreatic lipase
• Inhibitors of lipoprotein lipase
• Inhibitors of hormone-sensitive lipase
• β-adrenergic receptor blockers
• Inhibitors of protein kinase A
376
Objectives In Summary 20.16
• Inhibitors of cAMP-degrading phosphodiesterases

• Inhibitors of glucose transport in adipose tissue
20.16. Objectives In Summary
1. Define the terms “saturated”, “monounsaturated” and “polyunsaturated fatty acid, and
relate the physicochemical properties of fatty acids and triglycerides to their degree
of unsaturation.
2. Describe the formation of chylomicrons in the intestinal mucosa, and know the impor-
tance of lipoprotein lipase for tissue utilization of dietary triglyceride and the changes
in its activity in different physiological states.
3. Identify the substrates for triglyceride synthesis in adipose tissue, and the hormonal
and nutritional factors that determine their availability.
4. List the most important hormonal effects on hormone sensitive adipose tissue lipase.
5. Describe the role of carnitine for the mitochondrial uptake of long-chain fatty acids.
6. Describe the sequence of reactions and the cofactors for β-oxidation, and identify the
tissues that use β-oxidation as an important energy source and the effect of disruptions
in this pathway on the functions of these tissues.
7. Identify the tissue and organelle of ketogenesis, and the conditions that favor ketoge-
nesis.
8. Identify the roles of acetyl-CoA carboxylase and the fatty acid synthase complex for
fatty acid biosynthesis, and the coenzyme and energy requirements.
9. List the influences of hormones and metabolites on the regulation of acetyl-CoA car-
boxylase, and state the tissues in which fatty acid biosynthesis takes place.
10. State the roles of cytidine triphosphate and phosphatidic acid in the biosynthesis of
phosphoglycerides.
11. Describe the functions of important phospholipids such as plasmalogens and platelet
activating factor.
12. Describe the roles of the phospholipases A1, A2, C and D.
13. Describe the general principles of glycosphingolipid synthesis and degradation and the
general features for pathogenesis of sphingolipidosis and their clinical presentation.
14. Name the most important sites of endogenous cholesterol synthesis and the most
important intermediates of the biosynthetic pathway.
377
15. Know what an “isoprenoid” is.

16. Name the structural differences between cholesterol and the bile salts and describe the
entero-hepatic circulation of bile salts and feedback inhibition of bile salt synthesis.
17. Identify conditions that predispose to the formation of gallstones.
18. Know the approximate lipid and protein content of the various lipoproteins.
19. Describe the metabolism of chylomicrons, VLDL, LDL and HDL.
20. Describe the mechanism of reverse cholesterol transport by HDL.
21. State the importance of LDL and scavenger receptors for the uptake of LDL by
macrophages and parenchymatous cells.
22. Describe the regulatory effects of intracellular cholesterol on the LDL receptors and
HMG-CoA reductase.
23. Outline the empiric relationship between lifestyle, lipoprotein levels and arterioscle-
rosis.
24. Describe the pathogenesis and clinical expressions of the major types hyperlipopro-
teinemia, including familial hypercholesterolemia.
25. Identify the mechanisms for the cholesterol and triglyceride lowering effects of cholestyra-
mine, statins and fibrates and the effects of probucol as an antioxidant.
378
Part V.
Semester two, Mini II

21. Nutritional Management of Disease
21.1. Obesity
Definition: Body weight > 20 % above ideal body weight. The body mass index (BMI) is
used for estimating obesity:
BMI = Weight (kg) / Height2 (m2 )
BMI > 27 indicates obesity. Weight gain occurs whenever energy input exceeds energy
expenditure.
Adipose tissue hyperplasia: Some obese patients including most of those with severe (“mor-
bid”) obesity, have too many fat cells. The sites of adipose tissue hyperplasia are either
abdominal (male-type) or hip and thigh (female-type). Adipose tissue hyperplasia may be
caused by hypersensitivity to growth factors during childhood and/or puberty. This type of
obesity, once established, responds poorly to dietary management. On a diet that maintains
“normal” weight, patients have metabolic patterns as in starvation.
Adipose tissue hypertrophy: Most patients with milder degrees of obesity have an increased
amount of fat per adipose cell although the number of cells is more or less normal. This
type is usually caused by over-eating.
Appetite control: Stretch receptors in the stomach trigger satiety, as does an elevated blood
glucose level. In addition, when adipose cells are “filled” they release the hormone leptin.
Leptin acts on the brain to inhibit appetite, and it increases the metabolic rate. Causes of
obesity:
Genetic, heritability: ≈ 70 % in the US
Bad eating habits: too many snacks and alcohol
Eating for psychological reasons: stress, depression...
Weight gain with each pregnancy.
Poor weaning practices: use of too much sugar (empty calories!) at an early age
Misuse of milk formula: too thick
Low metabolic rate.
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Lack of exercise.
Complications of obesity:
• Type 2 diabetes
• Gallstones
• Coronary heart disease
• Hypertension
Osteoarthritis and respiratory diseases may be exacerbated by obesity.
21.2. Weight Reduction
Weight reduction leads to:

• Lower blood pressure
• Lower blood lipids
• Lower blood sugar
• Less heart disease
Energy is required for:
basal metabolic rate (BMR): 100.5 kJ/kg body weight in men and 90.4 kJ/kg body weight
in women
Physical activity: Between 20 % of BMR (sedentary lifestyle) and 50 % of BMR (heavy
work)
Postprandial thermogenesis: 10 % of the basal metabolic rate
Example Mr. Jones weighs 90 kg, is 1.6 m tall, and has a sedentary lifestyle.
Body mass index BMI = 90 kg / (1.6 m)2 = 90 kg / 2.56 m2 = 35.1 kg/m2
Basal metabolic rate BMR = 90 kg × 100.5 kJ/kg = 9045 kJ
Physical activity: 0.2 × 9045 kJ = 1809 kJ
Thermogenesis: 0.1 × 9045 kJ = 904.5 kJ
Total energy expenditure: 11 759 kJ per day
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Types of diabetes mellitus 21.3.1
How much should Mr. Jones eat if he wants to lose 1 kg per week? The energy content of
adipose tissue is about 32.7 kJ/g (it’s about 85 % triglyceride). He has to reduce his weekly
energy by 1000 g × 32.7 kJ/g / 7 d = 4671 kJ/d below his energy expenditure: 11 759 kJ/d
- 4671 kJ/d = 7088 kJ/d daily energy intake.
How much fat, CHO and protein should he eat? Fat should be 30 % of total energy, CHO
60 %, and protein 10 %.
Fat: 0.3 × 7088 kJ/d / 38.9 kJ/g = 54.6 g, about a third each saturated, mono-unsaturated
and poly-unsaturated.
CHO: 0.6 × 7088 kJ/d / 16.7 kJ/g = 254.7 g, preferably from complex carbohydrates.
Protein: 0.1 × 7088 kJ/d / 16.7 kJ/g = 42.4 g. Protein should have a high biological value.
BMR will decrease because of loss of lean body mass: he will need fewer calories. Weight will
reach a plateau unless calories are further reduced or exercise increased. Type of exercise -
Low impact:
F 3–4 times/week
I Intense walking, swimming, running, aerobics
T 20–30 min/day at least
21.3. Diabetes Mellitus
21.3.1. Types of diabetes mellitus
Type 1 diabetes (juvenile-onset diabetes)

• Autoimmune destruction of β-cells
• Onset in childhood or adolescence
• Lifetime incidence 1 in 300
• Severe disease, insulin-dependence
Type 2 diabetes (maturity-onset diabetes)
• Poor tissue response to insulin (insulin resistance) and/or reduced glucose-stimulated
insulin release
• Adult-onset
• Lifetime incidence 4–7 %
• Less severe than type 1
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Gestational diabetes occurs in some pregnant women because placental hormones antag-
onize the insulin effects. It does not persist after birth, but many patients develop
type 2 diabetes later in life.
Heritability: Moderate in type 1, high in type 2.
Obesity: Most type 2 diabetics are obese. Most normal (non-diabetic) obese people have
increased insulin levels with decreased tissue responsiveness.
Signs and symptoms:
• Hyperglycemia and glucosuria, from increased glucose production in liver and reduced
utilization in liver, muscle and adipose tissue.
• Increased plasma free fatty acids, from fat breakdown in adipose tissue.
• Increased ketone bodies. These are formed from fatty acids in the liver.
• Diabetic coma, with severe hyperglycemia, dehydration, electrolyte imbalances and,

in type 1, acidosis (“ketoacidosis”)
• Late complications:
– Microangiopathy
– nephropathy
– retinopathy
– peripheral neuropathy
– lens opacities
Importance:
• It is the 4th leading cause of death - by disease.
• It increases coronary heart disease risk 2–4 times.
• Leading cause of kidney disease.
• Leading cause of blindness.
• Causes 1/2 of all leg amputations.
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Management of Diabetes 21.3.2
21.3.2. Management of Diabetes
Number 1 goal in management is improved metabolic control. This slows down the onset
and progression of diabetic complications. Metabolic control is monitored by:
• Blood glucose
• Glycosylated hemoglobin
• Blood lipids
• Blood pressure
• Renal function
Goals in dietary management

• Generally, to assist diabetics to make the necessary adjustments in their diets and
exercise habits to ensure metabolic control.
• Maintenance of normal blood glucose levels.
• Attaintment of optimum serum lipid levels to reduce cardiovascular disease risk.
• Provision of adequate calories to maintain or attain a healthy, realistic body weight.
Adequate calories are needed to sustain physiological needs in pregnancy & lactation
and to ensure optimal growth in children.
• To prevent or delay long-term complications of the diabetic process; vascular an-
giopathies, gangrene, kidney disease, neuropathies, and blindness.
• Education of patient and relatives.
Overall dietary recommendations for diabetics

Protein: 10–20 % of total energy intake
Fat: 10 % from saturated, 15 % from monounsat. and 10 % from polyunsat
CHO: 40–45 % of total energy intake
If there is evidence of kidney damage (proteinuria), protein should be decreased to 10 % of
energy intake., or 0.6–0.8 g/kg body weight.
Levels of fat intake in diabetics:
Type A: (Normal lipid levels) 10 % sat., 10 % mono., 10 % poly., 30 % of total energy intake
Type B (Obese diabetic) 20 % of total energy intake from fat
385
Type C: (Elevated LDL levels) Fat < 30 % of total energy intake, Step II diet N.C.E.P.,
Saturated fat < 70 % 200 mg/d
Type D: (Elevated triglycerides and VLDL) < 10 % saturated, 20 % monounsaturated, <
10 % polyunsaturated 20 % protein, 35–40 % CHO
Including promotion of physical exercise and weight loss. Use of upomega-3 fats is strongly
recommended: salmon, sardine, mackerel. Other aspects:
Carbohydrates are still controversial. Meals with a low glycemic index are preferred. The
glycemic index is the relative effect of a meal on the blood glucose level, compared
with a standard food (glucose, or bread) of the same carbohydrate content.
Sucrose shows a similar glycemic response to bread and potatoes:
• Use with caution as part of the meal plan — about 5 % of the total energy intake.
• Obese diabetics should try to abstain.
• Fructose produces a smaller rise in plasma glucose levels than sucrose, but raises
VLDL and LDL.
Fruits and milk have a lower glycemic index than most starches.
Legumes (peas and beans) have the lowest glycemic index and should be encouraged.
Sodium: Assess on individual basis.
• Diabetics with mild to moderate hypertension: 2400–3000 mg/d Na.
• If there is kidney damage, 2000 mg/d or less: ‘No salt added’.
Fiber: Certain types delay glucose absorption from small intestine, e.g. legumes. Beneficial
effects on serum lipids.
Exercise: beneficial.
• Improves the body’s ability to use glucose.
• Improves insulin responsiveness.
• Lowers cholesterol.
• Lowers blood pressure.
• Reduces body fat.
• Improves the circulation.
• Reduces stress and gives a sense of well-being.
Treatment for the obese non-insulin-dependent diabetic:
386
Coronary Heart Disease (CHD) 21.4
• Weight loss increases the number and responsiveness of insulin receptors.

• Reduce total dietary fat.
• Meal spacing at 4-5 h intervals
• Realistic weight loss program: 2100 kJ/d or less.
• Moderate regular exercise
Treatment strategy for the type 1 diabetic:
• Consistency in the quantity of food intake to match the amount of insulin.
• Consistency in the timing of meals to match the onset and duration of insulin action.
• Snacks are important, particularly at peak action of insulin (to prevent hypoglycemia)
& before and after exercise.
• Frequent self-monitoring of blood glucose is necessary if the patient is on intensive
insulin therapy: 3–4 doses per day.
• Alcohol should never be taken on an empty stomach. It impairs gluconeogenesis and
can cause hypoglycemia.
Gestational diabetes Diabetes in pregnancy is associated with fetal abnormality and still-
births. Frequent monitoring of blood glucose is important. Carbohydrates calories must be
adequate to prevent fat breakdown leading to ketosis. Reducing diets are not recommended
during pregnancy. Recommended: 126–147 kJ per kg body weight per day with frequent
snacks, particularly at bedtime.
21.4. Coronary Heart Disease (CHD)
Pathology: CHD is caused by atheromatous plaques (atherosclerotic lesions) in one or more

branches of the coronary artery. The plaque forms in the intima of the artery, typically with
an inner core of cholesterol esters surrounded by fibrosis. It narrows the lumen of the artery
and impairs blood flow. Result: exertional angina pectoris, with chest pain in response
to physical activity (increased oxygen demand of the myocardium!). Or acute myocardial
infarction, usually after the formation of a thrombus (intravascular blood clot) on the surface
of the lesion. Atherosclerosis is also responsible for other ailments, including gangrene and
some strokes.
Prevalence of CHD: Myocardial infarction accounts for 25–40 % of all deaths in affluent
countries but is rare in many third world countries. Atherosclerotic lesions take a long time
387
to develop, and therefore CHD is a disease of older people. Males are affected earlier than
females.
Development of lesions: The fatty streak is a benign, reversible precursor lesion which
consists of lipid-laden macrophages and extracellular lipid (cholesterol ester) deposits in
the intima. Fatty streaks are seen even in children. Most of them regress spontaneously,
but some develop into atheromatous plaques, possibly by triggering the proliferation of
neighboring smooth muscle cells.
Major risk factors of CHD:
• Smoking
• Hypertension
• Hypercholesterolemia
• Diabetes mellitus
Hypertension and diabetes are thought to facilitate the fibroproliferative response by “stress-
ing” the vessel wall. A high LDL/HDL ratio leads to the accumulation of cholesterol esters.
Lifestyle and diet affect the blood levels of LDL and HDL cholesterol, and thereby the risk
of atherosclerosis and CHD.
21.4.1. Dietary Guidelines for Reducing Blood Cholesterol
Eat less fat
• Limit the use of fats in food preparation. Use as little butter, margarine and oil as
possible.
• Saute vegetables in 5 ml oil + 50 ml water instead of full fat.
• Spoon off all fats after browning meats.
• Trim off the visible fats on chicken and meat before cooking.
• Keep salad dressings to minimum. Switch to low-fat dressings or “light” mayonnaise.
• Use mint sauces (garlic, lemon, juice, herbs) rather than cream sauces.
• Boil, steam, bake and poach, instead of frying.
• Make wise food choices, for example:
– Exchange whole-milk products for low-fat dairy products (low-fat cheese, e.g
Mozzarella 10 % fat, Edema, Gouda or Camembert, milk and low-fat yogurt).
– Use a low-fat spread
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Dietary Guidelines for Reducing Blood Cholesterol 21.4.1
– Use unbuttered popcorn instead of peanuts.
– Instead of fruit pies eat fresh fruit.
– Eat low-fat buns instead of cakes and pastries.
– Buy lean-cuts of meat and reduce serving sizes.
– No sausages, bacon, minced meat, salami, luncheon meat, corned beef.
SAMPLE MENU (25 % of total calories from fat):
Breakfast: 1 fresh fruit, 2 slices whole wheat toast, 2 oz Mozzarella cheese, or: wholewheat
cereal and fruit.
Lunch: Tuna salad with lettuce, cucumber, whole wheat bread, 1 cup low-fat milk, fruit.
Dinner: Grilled chicken, 1 teacup cooked brown rice boiled green beans, ground provisions.
Stir-fry ratatouille, 1 cup low-fat milk, oat bran, date cookies or raisin buns.
Use less saturated fat, i.e. 10 % of total energy intake.

Fat type Appearance Sources
Saturated Usually solid at room Butter, lard, palm oil,
temperature veg. shortening, coconut
oil, highly hydrogenated
margarine, meat, poultry,
cheese, egg yolk, dairy
products, cocoa, coconut
Monounsaturated Liquid at room tem- Canola, olive, and peanut
perature oils. Peanuts, cashews,
peanut butter and avocado
Polyunsaturated Liquid at room tem- Safflower, sunflower, corn,
perature soya bean, and cottonseed
oils. Some margarines e.g.
Becel, Fleishman, hazel-
nuts, almonds, mayonnaise
made with veg. oil
The trans-unsaturated fatty acids in hydrogenated vegetable fats (margarine, peanut but-
ter) are physically, metabolically, and nutritionally similar to saturated fatty acids. It’s not
the number of double bonds that counts, but the number of cis-double bonds!
389
Avoid (or reduce) high-cholesterol foods
Dietary cholesterol should not exceed 300 mg/d. Plants do not contain cholesterol, therefore
vegan diets are cholesterol-free.
Food source Cholesterol (mg / 100g)
Egg yolk 280 mg/yolk
Organ meats (liver, kidney) > 350
Shrimp 159
Sardines 139
Crab, mackerel 103
Lobster 92
Meats: lamb, pork, beef, veal, poultry With skin, cod, game 76–94
Clams, oysters, scallop, tuna, halibut, trout 53–65
Salmon 41
Dairy cream 129
2 % fat milk 8
Whole milk 14
Ice cream 16 % butter fat 74
Cheese 96
Butter 620
Lard 91
Mayonnaise 160
Amount of cholesterol found in lb loaf:
Commercial cakes = 110 mg
Cheese cake 9” diameter = 2053 mg
Increase the intake of dietary fiber, especially of “soluble fiber” from peas and beans. Use
also fruit and oat bran. Whole grain cereals - whole wheat bread weetabix e.g. oatmeal,
shredded wheat, alpen and vegetables are high in insoluble fibres which are particularly
helpful for the regulation of bowels.
Other measures to reduce blood cholesterol:
• Quit smoking
• Take regular exercise, at least 4 times/week, intensity will depend on tolerance levels
• Lose weight, if you are overweight
• Control blood pressure
• Cut down on alcohol
390
Prevention of Coronary Heart Disease (CHD) 21.5
21.5. Prevention of Coronary Heart Disease (CHD)
More than 500 000 Americans die from CHD annually. 1 250 000 Americans suffer from
heart attacks each year. Costs range from US$ 50–100 billion annually, and nutritional
prophylaxis and therapy is a cost-effective intervention. Risk factors for CHD:
• High LDL cholesterol
• Low HDL cholesterol
• High plasma triglycerides
• Cigarette smoking
• Hypertension
• Obesity
• Lack of exercise
• Family history
• Diabetes
• Race
• Age
• Gender
LDL cholesterol level
<129 mg/dL Normal
130–159 mg/dL Borderline
>160 mg/dL High risk
Actual versus recommended U.S. dietary intake .

Nutrient Present intake Population goals
Fat 36–38 % total energy < 30 % total energy
Saturated 13–14 % < 10 %
Monounsaturated 15 % 15 %
Polyunsaturated 10 % 10 %
Cholesterol 230–260 mg < 300 mg
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N.C.E.P National cholesterol education program .

# of risk factors medical nutrition defined LDL goals
therapy
2 or less Step I diet < 160 mg/dL
2 or more Step II diet < 130 mg/dL
Established CHD Step II plus drugs < 100 mg/dL
LDL cholesterol lev- Step II plus drugs Assess in 4 weeks, re-
els > 220 mg/dL assess in 12 weeks
Continue to monitor, to ensure that aerobic exercise is maintained at least for 30 min 3–4
times per week.
• Smoking should be discontinued
• Weight loss should be actively encouraged
• Maintain motivation and teach skills to manage the disease
Nutrient Step I diet Step II diet
Fat < 30 % of energy Same
Protein 15 % Same
CHO 55 % Same
Saturated Fat 10 % <7 %
Polyunsaturated 10 % Same
Monounsaturated 15 % Same
Cholesterol <300 mg/d <200 mg/d
Continue to monitor to prevent relapse into old habits. Reduced mortality from CHD has
been recorded on vegetarian diets, more exercise and less alcohol.
Monounsaturated fatty acids in Canola, olive and some brands of sunflower and safflower
oil tend to increase HDL cholesterol. Heart disease is less prevalent in the Mediterranean
region where olive oil is popular.
Trans-unsaturated fatty acids in margarines, shortenings and peanut butter behave like
saturated fatty acids and increase LDL while decreasing HDL.
Substitution of polyunsaturated fats for saturated fats tends to decrease LDL, but has no
effect on HDL. Excess polyunsaturates may pose a cancer risk and impair immunological
function.
Populations eating a lot of soy products have a low prevalence of CHD. Isoflavins (diadzein,
genistein) are active ingredients and tend to lower LDL cholesterol.
Vitamin C may increase HDL, lower total cholesterol and protect against LDL oxidation.
Carotenes may reduce the risk of heart attacks: eat more carrots, broccoli, spinach etc. Like
vitamins C and E, the carotenes are antioxidants.
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Hypertension 21.6
Flavonoids are polyphenolics found in fruits, vegetables and red wine. They inhibit LDL
oxidation and reduce thrombotic tendency. Supplements are not recommended.
21.6. Hypertension
Importance: Hypertension is the chronic elevation of the arterial pressure. Normal: systolic
pressure 120 mm Hg, diastolic pressure 80 mm Hg. Hypertension is common, usually of
unknown origin (‘essential hypertension’) and with a strong genetic background. It causes
microvascular changes, atherosclerosis and strokes.
Blood pressure regulation: Blood pressure is determined by cardiac output and pe-
ripheral vascular resistance. Any increase in the blood volume will cause hypertension by
increasing either cardiac output or peripheral vascular resistance or both.
• Short-term regulation depends on the autonomic nervous system which adjusts car-
diac output and peripheral vascular resistance. The sympathetic system accelerates
the heart and causes vasoconstriction in many parts of the body, and the parasympa-
thetic system slows down the heart and causes vasodilation. The autonomic nervous
system is controlled by brainstem centers. Baroreceptors (pressure receptors) in the
walls of aorta and carotid arteries provide specific input to these centers.
• Long-term regulation adjusts the blood volume, and it depends on the kidneys. Im-
portant: the plasma contains fixed concentrations of electrolytes, mostly sodium and
chloride, and these are in equilibrium with the interstitial fluid. Therefore any gain or
loss of extracellular fluid volume (blood plasma + interstitial fluid) is possible only
by adding or removing sodium and chloride (‘salt’). The normal kidney responds to
increased blood pressure by excreting more water and salt (pressure diuresis). Also,
when the blood pressure in the afferent arterioles of the kidney drops, the protease
renin is released which generates angiotensin. Angiotensin is a hormone-like sub-
stance which increases the blood pressure by constricting arterioles throughout the
body, thereby increasing the peripheral vascular resistance in the short term (within
minutes). More importantly, it reduces renal blood flow, thereby reducing water and
salt excretion directly, and it induces the release of aldosterone from the adrenal
cortex. This hormone causes salt retention.
Abnormalities in hypertension: Patients with essential hypertension have normal car-
diac output but increased peripheral vascular resistance. They have a reduced renal blood
flow because of high renal vascular resistance.
Their kidneys can excrete a normal amount of salt and water, but only if the blood pressure
is abnormally high. If their blood pressure is artificially lowered (for example by removing
blood), salt and water excretion are reduced until the blood pressure has again reached an
abnormally high level.
393
Essential hypertension has been linked to increased production of ouabain and digoxin
in the adrenal gland. Both are glycosylated steroids involved in blood pressure regulation.
Digoxin from plant sources has long been used to treat congestive heart failure in elderly
patients (increases blood pressure and cardiac output, reduces heart rate).
Dietary Management of Hypertension
60 million people in the US are hypertensive, 38 % Afro-American and 29 % White Ameri-

can.
Essential Hypertension is classified according to the diastolic pressure:
Mild 90–140 mm Hg
Moderate 105–115 mm Hg
Severe > 115 mm Hg
Consequences of hypertension
• Heart failure
• Strokes
• Heart attacks
• Renal failure
• Aortic aneurysm
Risk factors for hypertension

• Sedentary lifestyle
• High-sodium diet
• High-fat, low fiber diet
• Obesity
• Cigarette smoking
• Excessive alcohol intake
• Stress
• Heredity
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Hypertension 21.6
Manipulations which help to lower blood pressure
• Low sodium intake
• High potassium intake
• High calcium intake
• Increase in fiber
• Increased unsaturated fat
• Exercise
• Relaxation
• Reduced alcohol intake
• Weight reduction
Sources of sodium 75 % of dietary sodium in the US comes from processed foods: cakes,
biscuits, pretzels, pickled food, ketchup... Also some antacids contain sodium.
Compare the sodium content of
3 oz fresh cooked pork: 59 mg Na+
3 oz cooked ham: 114 mg Na+
Amount Food Na+ (mg)

1 teaspoon Worcester sauce 60
1 oz cheddar 176
1 oz parmesan cheese 528
1 cup chicken noodle soup 1100
1/2 cup canned tomato juice 440
1 oz processed american cheese 400
1 large olive 130
1 teaspoon soy sauce 330
1 cup milk 120
1 level teaspoon m.s.g 500
1 teaspoon baking powder 370
1 teaspoon bicarbonate of soda 1000
1 level teaspoon salt 2000
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General categories of sodium-controlled diets

250 mg/d Very severe, rarely used
<1000 mg/d Severe
2400–4500mg/d Moderate
2000–3000mg/d Most commonly used
Lower sodium intake leads to decreased blood pressure in sodium-sensitive people (10–20 %
of the population), and reduced extracellular fluid volume.
21.7. The Liver
Functional anatomy The liver receives a two-fold blood supply: the portal vein supplies
blood that has passed through the capillaries of the splanchnic organs already; and the
hepatic artery supplies fresh arterial blood. Water-soluble nutrients are absorbed from the
small intestine into the blood and carried to the liver before they reach other parts of
the body. Only lipids bypass the liver: they are transported in chylomicrons through the
thoracic duct. The functional unit of the liver is the liver lobule, a cylindrical structure about
1 mm across and several mm long which is arranged around a central vein. Blood enters the
lobule from the periphery and capillaries called sinusoids. The sinusoids have a fenestrated
endothelium which facilitates the access of nutrients and other blood constituents to the
hepatocytes.
Functions The liver maintains a constant internal environment in the face of a fluctuating
nutrient supply. It also serves many specialized functions:
Regulation of blood glucose: The liver consumes glucose after a meal, converting the ex-
cess to glycogen and fat, and it produces glucose during fasting.
Metabolism of non-nutritive food components (including drugs) to water-soluble prod-
ucts that can be excreted in bile or urine, alcohol is metabolized to acetic acid or
oxidized to CO2 and H2 O.
Storage of nutrients: Vitamin A, vitamin B12, iron.
Conversion of toxic ammonia (NH3) to harmless urea.
Production of bile Bile acids are important for fat absorption, and some waste products
are excreted in the bile.
Synthesis of plasma proteins (exception: immunoglobulins)
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Disease of the Biliary System 21.7.2
Garbage removal: Worn-out plasma proteins and antigen-antibody complexes are removed
by the liver, either by hepatocytes or by macrophages (Kupffer cells) in the walls of
the sinusoids. Kupffer cells can also eat up bacteria that may enter the blood from
the GI-tract.
21.7.1. Disease of the Biliary System
The liver produces about 600–800 ml of bile daily. An amount of 40–70 ml is stored in
the gallbladder. The bile contains bile salts, cholesterol, phospholipids, and bile pigments
(glucuronic acid conjugates of bilirubin and other breakdown products of heme). Bladder
bile is more concentrated than hepatic bile because electrolytes and water are absorbed
from the bile through the gallbladder epithelium, thus increasing the concentrations of the
other components.
Fat in the duodenum stimulates the release of cholecystokinin, a hormone which induces
contraction of gallbladder and subsequent release of bile into the common bile duct and
into the duodenum.
Most gallstones consist of cholesterol. They are associated with obesity. Obese patients with
gallstones have too much biliary cholesterol. A high-fat diet may predispose to gallstone
formation.
Cholecystitis is inflammation of the gallbladder, caused by bacterial infection or gall-

stones. Some bacteria produce enzymes which deconjugate bilirubin.
Unconjugated bilirubin is insoluble and forms pigment stones. Acute cholecystitis presents
with pain, fever, vomiting and malaise. The diet should be fat-free and high in calories
until the acute phase has subsided. A patient can become dehydrated and weak if these
immediate steps are not taken.
In chronic cholecystitis, a moderate fat-free diet should be given, pending surgery. Too much
fat will cause the gallbladder to contract too frequently and cause the stones to shift. This
can induce severe pain (colic).
Cholestasis is interruption of the bile flow. It can be caused by hepatic disease (intra-
hepatic cholestasis) or obstruction of the common bile duct (posthepatic cholestasis. With
fat intolerance, fatty stools and malabsorption of fat-soluble vitamins (vitamins A, D, E
and K). Vitamin K deficiency appears early in acute cholestasis because this vitamin is not
stored in the body, resulting in impaired blood clotting. Chronic cholestasis can lead to
liver cirrhosis.
397
21.7.2. Parenchymal Liver Disease
Fatty liver is a reversible lesion that appears whenever the liver produces more fat than it
can export as VLDL. This happens in many liver diseases. Alcoholics get fatty liver because
alcohol inhibits fatty acid oxidation, thereby diverting fatty acids into fat synthesis. People
with protein-calorie malnutrition get fatty liver because their liver receives a lot of fatty
acids from adipose tissue and some of them become esterified to fat, and because the
synthesis of VLDL apo-proteins is impaired by amino acid deficiency.
Liver cirrhosis is the outcome of many liver diseases. Most cases result from chronic al-
coholism and/or chronic active hepatitis. Cirrhosis may also result from liver toxins such
as carbon tetrachloride or drugs, and from severe acute viral infections (hepatitis, yellow
fever).
In the cirrhotic liver, much of the normal tissue has been replaced by fibrous connective
tissue. The cirrhotic liver may appear as a firm fibrous mass with orange colored nodules
projecting from its surface. Blood flow is impaired, causing portal hypertension. This re-
sults in development of a collateral circulation through retroperitoneal veins, periumbilical
veins, lower esophageal veins, and rectal veins. These veins dilate. Some patients develop
dangerous (often fatal) esophageal bleeding. Blood clotting is impaired formation of clotting
factors.
Signs and symptoms include abdominal edema (ascites), jaundice, vitamin deficiencies, en-
cephalopathy (impaired intellectual function with confusion and stupor), nausea, vomiting,
anorexia, and epigastric pain. Biochemical features include
• Raised serum bilirubin
• Low serum albumin
• Raised serum alkaline phosphatase
• Raised serum transaminases
• Prolonged prothrombin time
• Raised serum ammonia
Treatment is symptomatic. In the absence of impending coma and as long as blood ammonia
is normal, a protein-saving diet (enough energy from CHO and fat that protein-catabolism
is not required, enough protein of high biological value to cover anabolic needs) is rec-
ommended in order to correct severe undernutrition, regenerate functional liver tissue and
replenish plasma proteins. However, if signs of encephalopathy or hepatic coma appear, the
protein is adjusted to individual tolerance. Recommended sodium: 500 −-1000 mg/d Na+ .
Vitamins must be supplied according to individual need and deficiency. Moderate fat is
used. Alcohol is strictly forbidden.
398
The Kidney 21.8
Hepatic coma is seen in patients with advanced liver cirrhosis, and also in patients with
severe acute liver diseases (toxins, viral infections). It results from the inability of the
severely diseased liver to maintain a normal internal environment. Ammonia accumulation
is especially dangerous. Gastrointestinal bleeding can precipitate hepatic coma because it
supplies intestinal bacteria with extra protein which is fermented to ammonia.
Hepatic coma develops when ammonia-laden blood cannot pass through the cirrhotic liver
and is rerouted through the collateral circulation. It re-enters the systemic blood flow, still
carrying its ammonia load, and produces ammonia intoxication.
Clinical manifestations of hepatic coma include apathy, confusion, inappropriate behavior,
drowsiness, ‘absent stare’, slurred speech, and tumor in the outstretched hand. The breath
may have fecal odor.
Treatment is aimed at the prevention of bleeding and reduction of ammonia formation.
Dietary Management Protein must be withdrawn from the diet completely and energy
requirements met by CHO and fat, initially parenterally through a peripheral vein. Example:
1–2 l 10 % dextrose in 24 h. Parentrovite I and II, No Intralipid. Simultaneously a nasogastric
feed may also be given to provide energy.
21.8. The Kidney
Functions The kidneys play a key role in homeostasis (maintenance of a constant internal
environment) and in the excretion of waste products:
• Excess water and electrolytes are excreted. Thereby the kidneys controls extracellular
fluid volume (plasma + interstitial fluid) and the correct ionic composition of the
plasma.
• Protons are excreted or retained to maintain the blood pH at the normal value of 7.4.
• Nitrogenous wastes are excreted: urea from amino acids, uric acid from purines, cre-
atinine from creatine. Urea excretion is directly proportional to the dietary protein
intake.
• Many drugs are excreted by the kidneys.
• The kidney is also an endocrine organ. It produces:
Calcitriol the active form of vitamin D
Renin a protease that makes angiotensin
Erythropoietin a growth factor that stimulates RBC formation in the bone marrow
399
Functional anatomy The kidneys weigh only 300 g (0.4 % of the body weight) but receive
1.2 l blood per minute (25 % of the cardiac output). The functional unit is the nephron.
The nephron starts with a small capillary bundle called glomerulus which is embedded
in a cup-like structure called Bowman’s capsule. Water, electrolytes and small molecules
are filtered into the space between glomerulus and Bowman’s capsule, and blood cells and
plasma proteins are retained in the blood. About 150–200 l of fluid are formed this way
every day. This primary filtrate is passed through a long, convoluted tubular system where
useful substances (glucose, amino acids) and most of the water and electrolytes are retrieved
by active transport across the tubular epithelium. Also protons are transported across the
tubular epithelium, and under most Conditions the urine is neutral or mildly acidic (pH
5.0–7.0).
21.8.1. Glomerulonephritis and Nephrotic Syndrome
Acute glomerulonephritis is an acute immunological response, usually after a strepto-

coccal sore throat. There is acute inflammation of the glomeruli with congestion of renal
blood flow and reduced glomerular filtration rate. Typical features:
• Reduced urinary volume
• Proteinuria, presence of blood cells in the urine, casts of protein precipitated in the
tubular system
• Edema of face and hands in the morning and ankles in the evening
• Raised blood pressure, headache, malaise
Treatment
• Drugs for hypertension and diuresis
• Fluid intake must be carefully monitored
• 1000–1500 mg sodium/day
• Protein restriction to 40 g/d if blood urea nitrogen (BUN) is high
Sample meal plan:
Milk 0.25 l/d
Breakfast 1 egg, Toast and tea, Fruit juice
Lunch 50 g meat or fish salt free, small portion of vegetables, Rice cooked without salt
Dinner Salt-free meat or chicken (30 g), Bread and low-salt margarine
400
End-Stage Renal Disease 21.8.2
Nephrotic syndrome is characterized by proteinuria, hypoalbuminemia, and peripheral

edema. The glomerular basement membrane is damaged, and plasma proteins are lost in
the urine. Some patients lose 20 g of protein per day, and their serum albumin level drops to
1.5 g/dL. It can occur as a complication of malarial infection, following certain types of acute
glomerular nephritis, in diabetes, autoimmune diseases (systemic lupus erythematosus), and
renal vein thrombosis. Nephrotic syndrome can progress to renal failure, even if it is under
control. The loss of fluid from the vascular space into the interstitium reduces blood volume
and blood pressure. As a homeostatic response, the renin-angiotensin system is activated,
and the release of aldosterone causes the retention of sodium and water.
Dietary management plays an important role, along with diuretics and anti-inflammatory
steroids. Protein: 1 g protein/kg ideal body weight plus 1 g protein for every gram lost in
the urine. Energy: 840 J for every gram of nitrogen lost can achieve satisfactory nitrogen
balance. Sodium: 1800–2300 mg/d.
Many patients have hyperlipidemia. This should be treated by replacing animal fat with
polyunsaturated fat.
21.8.2. End-Stage Renal Disease
Renal failure can be caused by pyelonephritis, glomerulonephritis, hypertension, polycystic

kidney disease, obstruction of the urinary tract, and diabetes mellitus.
Major problems:
• Uremia, with accumulation of nitrogenous waste products and with general and es-
pecially CNS toxicity
• Water retention, with increased blood volume and hypertension
• Phosphate, magnesium, and potassium are retained. But calcium may be low because
of impaired intestinal absorption (lack of calcitriol!).
• The bones become demineralized (renal osteodystrophy) because of chronic acidosis

and low calcitriol.
• Anemia develops, because erythropoietin is missing.
• Drugs that are normally excreted by the kidneys are poorly tolerated by patients with
renal failure.
The dietary management of renal failure is based on the restriction of everything that is
normally excreted by the kidneys: potassium, phosphate, magnesium, protein (urea forma-
tion!). Specific recommendations:
401
Protein: Protein should be restricted to prevent the accumulation of nitrogenous wastes.

However, uremic patients have increased protein breakdown as a result of uremic tox-
icity, chronic acidosis, and high levels of stress hormones (glucocorticoids!), therefore
they are prone to develop a negative nitrogen balance. Current recommendation for
conservative management is 0.6 g protein/kg body weight. This may be supplemented
with small amounts of essential amino acids or their alpha-keto acids. 70 % of protein
should be of high biological value. It should be spaced out evenly over the day to allow
better utilization. Milk should be included to provide a source of calcium. Nuts and
beans are not recommended because of their high potassium and phosphate content.
Energy: 170 J/kg body weight. Carbohydrate should never be in short supply, to prevent
excessive breakdown of body protein.
Sodium: 1500–2000 mg/d. No added salt.
Potassium: 1500–3000 mg/d, about 600 mg should come from vegetables and fruits low in
potassium. Potassium-containing salt substitutes must be avoided.
Phosphorus: Restriction of protein will automatically reduce phosphorus. Use bran with
caution because of its high phosphate content.
Many uremic patients have a type IV hyperlipoproteinemia. This should be treated by

replacing animal fat with polyunsaturated fat.
21.9. Nutritional Therapy In Surgery And Injury
The trauma associated with surgery always increase the need for nutrients and the circum-
stances usually restrict food intake temporarily. Following surgery there is a catabolic phase,
and certain hormones are secreted. Vasopressin causes water retention. Aldosterone reduces
renal blood flow. Glucocorticoids induce protein breakdown in many tissues, and gluconeo-
genesis in the liver. Thyroxine increases the metabolic rate. Epinephrine and norepinephrine
induce fat breakdown in adipose tissue and glycogen breakdown in muscle and liver.
The increased metabolism requires nutrients, and there may be extra requirements for the
replacement of losses: hemorrhage, fistulae, serous exudates from burns.
During fever, basal metabolic rate increases by 10 % for every degree centigrade rise in body
temperature.
Water: Adequate fluid is necessary to prevent dehydration. Large water losses may occur
from vomiting, hemorrhage, fever, exudates, and diuresis. A total of 2000 ml water
per day is needed in uncomplicated cases, but up to 7000 ml in complicated cases,
such as drainage or sepsis.
402
Nutritional Therapy In Surgery And Injury 21.9
Protein: this is the most important nutritional concern in surgical patients. A negative
nitrogen balance of up to 20 g/d may occur. Added protein losses may occur in hem-
orrhage or exudates. Protein synthesis is required for
• Wound healing
• Maintenance of plasma protein levels, prevents circulatory shock and edema
• The synthesis of immunoglobulins and other proteins for body defenses
The protein intake should be near 1.9 g/kg/d in uncomplicated cases to 2.5 g/kg/d in
cases of severe stress.
Energy: An adequate amount of carbohydrate is essential to ensure the use of protein for
building tissue and to supply the energy required for increased metabolic demands;
170–210 J/kg/d.
Vitamins: Vitamin C is necessary for collagen synthesis during wound healing; B vitamins
are coenzymes for enzymes in energy metabolism; and vitamin K is needed for blood
clotting.
403
22. Amino Acid Metabolism
22.1. Protein metabolism
About 300 g of protein are synthesized each day in our body, this is balanced by proteolysis
of an equal amount of protein. This turnover is required in part by replacement of proteins
within cells, but also to form new cells in organs with rapid cell replacement (e.g. blood,
intestinal epithelium) and requires about 5 % of our basal metabolism. More protein syn-
thesis is required in growth, pregnancy, lactation or to build muscle mass in training
athletes. Amino acids are also required for the synthesis of metabolites like heme, hormones,
nucleotides, coenzymes, melanin and biogenic amines.
To ensure an adequate supply of amino acids it is recommended that adults eat about 0.6–
0.8 g kg−1 d−1 protein, for infants this rises to 2.2 g kg−1 d−1 . Protein intake above these
recommended values does not offer any advantages with respect to strength or overall
health, but is associated with several health risks (for a review see [Metges and Barth,
2000]). However, available data do not yet permit setting upper intake levels.
Our body can not store free amino acids, any excess has to be catabolized. This generates
17 kJ/g metabolic energy.
To assess nutrition with respect to protein we look at the balance between nitrogen
• uptake (≈ 16 % of protein intake)
Figure 22.1.: Nitrogen balance in an average US-citizen. Amino acids taken up with daily
food are largely catabolized, producing urea, glucose and ketone bodies.
urea
Body protein
NH 3
250-300 g/d 250-300 g/d Glucose
~ 100 g/d ~ 100 g/d
Dietary protein Amino acid pool Carbon chains Ketone bodies
CO2 + H2 O
Metabolites
405
• loss
– urine
– feces
– milk (lactating ~ only)
– “other losses” (≈ 5 mg kg−1 d−1 )
Then there are three possible situations:
uptake > loss positive balance (growth, pregnancy)
uptake = loss equilibrium
uptake < loss negative balance, wasting of tissue (malnutrition, stress)
22.1.1. Biological value of proteins
There are 20 common proteinogenic amino acids (plus selenocysteine and pyrrolysine, which
we do not discuss in this chapter). Of these, most can be produced in our body, however,
some can not and need to be taken up, these we call essential amino acids:
Amino acid amount (g/d)
Arginine children only
Histidine unknown
Isoleucine 1.30
Leucine 2.02
Lysine 1.50
Methionine 2.02
Phenylalanine 2.02
Threonine 0.91
Tryptophane 0.46
Valine 1.50
Arginine can be produced in our body (as we will see later), but the amount is insufficient
to support rapid growth in children. Histidine is stored in muscle cells as carnosine, a short
peptide (βAla-His) which serves as buffer substance. In case of His-deficiency breakdown of
carnosine will prevent a negative nitrogen balance for a long time, therefore the daily need
could not be established experimentally.
Because we can not store free amino acids all of them need to be supplied in the required
amounts at the time of protein synthesis. The protein in our food therefore needs to have
approximately the same amino acid composition as the proteins in our body, we say: it
should have a high biological value. There are several ways to express the biological
value of a protein, the most important of these are:
406
Biological value of proteins 22.1.1
protein score for all amino acid determine contend/required ratio, take lowest value.
NPU (net protein utilization): ratio of nitrogen retained to nitrogen supplied. Takes di-
gestibility into account.
Here are the values for some important food sources:

Source protein score NPU
Human milk 1.00 0.95
Beef steak 0.98 0.93
Egg 1.00 0.87
Cow milk 0.95 0.81
Corn 0.49 0.36
white rice 0.67 0.63
Wheat 0.47 0.30
In general, the closer a species is related to us evolutionary, the closer its protein composition
will be to our own, and the higher the biological value of its proteins.
Mixing of food to achieve high biological value
Soy protein has the following composition:

Amino acid content (g/100g) required (g/d) ratio
Arginine 7.72 - -
Histidine 2.33 - -
Isoleucine 5.31 1.30 4.08
Leucine 7.98 2.02 3.95
Lysine 6.65 1.50 4.43
Methionine 1.40 2.02 0.69
Phenylalanine 5.08 2.02 2.51
Threonine 3.90 0.91 4.29
Tryptophane 1.53 0.46 3.33
Valine 5.34 1.50 3.56
The protein score is 0.69, with Met being the limiting amino acid.
Rice on the other hand has the following composition:
407
Amino acid content (g/100g) required (g/d) ratio

Arginine 7.90 - -
Histidine 2.70 - -
Isoleucine 4.10 1.30 3.15
Leucine 8.90 2.02 4.41
Lysine 4.00 1.50 2.67
Methionine 3.20 2.02 1.58
Phenylalanine 5.50 2.02 2.72
Threonine 3.90 0.91 4.29
Tryptophane 0.31 0.46 0.67
Valine 6.50 1.50 4.33
Thus the protein score is 0.67, with Trp being the limiting amino acid.
Neither soy nor rice, both important food sources, could serve human nutritional needs
alone. A mixture of both on the other hand would serve beautifully, the Met being supplied
by rice and the Trp by soy. Thus it is not necessary that each individual foodstuff has
the ideal mixture of amino acids, but the overall mixture in a meal should have. Many
indigenous people with limited access to animal protein use this principle, for example
soy/rice in Asia and beans/corn in the Americas. Note however that since amino acids
can not be stored the supply of essential amino acids can not be distributed over several
meals.
22.2. Nitrogen metabolism
22.2.1. Nitrogen transfer
If more amino acids are supplied with food than required for protein synthesis, or if the
amino acids in food can not be used completely because of poor biological value, the re-
maining amino acids must be catabolized as they can not be stored in our body. Catabolism
occurs in two steps: first the amino group is removed and converted to urea. Second the
carbon skeleton is degraded. The latter step will be discussed in the next section.
To remove the amino group from an amino acid our body has three possibilities (see fig.
22.2):
transamination In this reversible reaction the amino group is transferred onto a keto-acid,
usually α-ketoglutarate. The reaction products are the ketoacid corresponding to the
amino acid and glutamate.
Lys, Thr and Pro can not undergo transamination. Their metabolism will be discussed
in the next section.
408
Nitrogen transfer 22.2.1
Figure 22.2.: Metabolism of amino acids by transaminases, oxidases and glutamate dehy-
drogenase. For details see text.
not: K,T, P α-ketoglutarate ketoacid glutamate
- -
COO COO
- - +
COO O C COO H2N CH
H3N
+
CH + CH2 O C + CH2
R CH2 R CH2
- Transaminase -
COO COO
O
+
CH H3N
H2 CH2 H2
HO C O Pi HO C O Pi
H3C N ping-pong-mechanism
H3C N
Pyridoxal phosphate (Vit B6) Pyridoxamine phosphate
H2O NH4+
NAD(P)+ NAD(P)H + H+
- -
COO COO
+
H2N CH O C
CH2 Glutamate dehydrogenase CH2
CH2 CH2
- -
COO COO
Remember:
NADPH is used for anabolic pathways (NADPH/NADP + > 1)
NAD+ is used for catabolic pathways (NADH/NAD + < 1)
O2 H2O2
H2O NH4+ Glu α-ketoglutarate
- - -
COO COO COO
+ +
HC N H3 O C H3N CH
R d-amino acid oxidase transaminase
R R
no longer chiral !
409
Transamination is the archetypical example for a ping-pong reaction mechanism (see

section 7.6 on page 161). Transaminases contain pyridoxalphosphate, the active form
of vitamin B6 . The amino acid is bound, gets the keto-oxygen in exchange for its
amino group and leaves. The enzyme now has pyridoxamine phosphate in its active
center, after binding of α-ketoglutarate it will transfer the amino group onto this
compound, in exchange for the keto-group. Release of Glu closes the reaction cycle.
oxidation by amino acid oxidases is used to convert d-amino acids (for example from the
murein sacculus of bacteria) into the corresponding l-isomer. The amino acid is ox-
idized to the corresponding α-ketoacid using molecular oxygen. Side products are
ammonium and hydrogen peroxide. Hydrogen peroxide is a reactive oxygen species
(ROS), which needs to be detoxified (see section 15.3 on page 253). The reaction is
localized in the peroxisome. The ketoacid is no longer chiral, it can either undergo
degradation, or it can be converted into the corresponding l-amino acid by transami-
nation.
glutamate dehydrogenase cleaves the amino group from Glu by reducing nicotinamide.
The reaction is reversible and can be used both for the degradation and production
of Glu. However, cleavage of Glu into α-ketoglutarate and ammonia is performed by
reducing NAD+ to NADH + H+ while the production of Glu from α-ketoglutarate
and ammonia is performed by oxidizing NADPH + H+ to NADP+ . The reason is that
in the cell NAD+ > NADH + H+ (which favors reduction of the nucleotide) while
NADP+ < NADPH + H+ , which favors its oxidation. Remember that this allows the
independent regulation of catabolic and anabolic pathways!
22.2.2. Urea-cycle
As we have seen in the previous subsection, amino acids are catabolized by removing their
amino group, producing ammonium ions in the process. Ammonium ions however are quite
toxic, they have to be removed from the body. Since ammonium has limited water solubility,
only aquatic organisms can afford to excrete it directly via the gills. Land-living organisms
have to convert it into a suitable form to remove it via the kidneys. Mammals use the highly
water-soluble urea. Reptiles and birds use uric acid instead, this compound is almost water
insoluble. Their “urine” is therefore a paste, saving weight and water.
Production of urea occurs in the liver, it is a very energy intensive process (see fig. 22.3).
For that reason some of the enzymes of this pathway are located in the mitochondria. Urea
production occurs in several enzymatic steps:
carbamoylphosphate synthetase I transfers the ammonium onto carbon dioxide, forming

carbamoylphosphate. This reaction requires 2 molecules of ATP and occurs in the mi-
tochondria. The ammonium comes mostly from glutamate dehydrogenase reactions in
410
Urea-cycle 22.2.2
Figure 22.3.: The urea cycle. For details see text.

this nitrogen mostly from blood
Urea cycle
NH4+ + CO2 + 2 ATP + H2O Deficiency in general:
hyperammonemia, lethargy
coma, agitation, Reye-syndrome
like encephalopathy
Carbamoylphosphate synthetase I
2q35, 1:800,000, early
and late onset form Liver mitochondria
O
2 ADP + Pi + 3 H+ + C Pi
H2N O
Pi + H+ NH2
CO
+
N H3 NH
CH2 Ornithine transcarbamoylase CH2
CH2 Xp21.1, recessive, female may be CH2
affected (X-inactivation, Barr-body)
CH2 1:100,000 CH2
+ +
HC H3N HC H3N
- -
COO COO
Ornithine Citrulline
NH2 progressive spastic
O C quadriplegia, mental ATP + Aspartate
NH2 retardation 6q23
Argininosuccinate
Urea this nitrogen mostly from
Arginase synthetase transamination in the liver
Induction with Shope Citrullinemia
H2O papilloma virus? chromosome 9q34 AMP + PPi + 2 H+
1:100,000
AMP + ATP 2 ADP
+ + -
N H2 Liver cytosol N H2 COO
C NH2 Argininosuccinate aciduria: C N CH
7cen-q11.2, H
NH treatment: Arg + Ornitine NH CH
-
CH2 Argininosuccinate lyase
CH2 COO
CH2 CH2
CH2 CH2
+
HC H3N Fumarate HC H3N
+
- -
COO COO
Arginine Argininosuccinate
Summary: NH4+ + CO2 + 4 ATP + 2 H2O + Aspartate = 4 ADP + 4 Pi + 5 H+ + Furmarate + Urea
411
extrahepatic tissues, especially muscle. In the form of ammonia it is freely membrane

permeable (small gas molecule!).
ornithine transcarbamoylase catalyzes the transfer of the carbamoyl-group onto ornithine,
forming citrulline in the process. Ornithine is transported into, and citrulline out of
the mitochondria by specialized transporters.
argininosuccinate synthetase transfers aspartate onto the citrulline. Note that the red
carbon in fig. 22.3 now carries two nitrogens, just as it will in urea. The reaction
again requires energy in the form of ATP. However, the ATP is split into diphos-
phate (pyrophosphate, PPi ) and AMP. Since the PPi is immediately hydrolyzed by
pyrophosphatases, its concentration in the cell is very low. This reduces the speed of
the backward reaction (remember the principle of Le Chatelier!). We will see other
examples of this expensive trick as we progress through metabolism. Mitochondrial
ATP-synthase can not handle AMP as substrate, it has to be converted first to ADP
by adenylate kinase: ATP + AMP * ) 2ADP. Thus the reaction actually consumes a
total of 2 molecules of ATP. The nitrogen of the aspartate used comes mostly from
transamination reactions in the liver.
argininosuccinate lyase cleaves fumarate from the argininosuccinate, forming arginine (re-
member from above that humans can make their own arginine?).
arginase cleaves the guanidino-group from arginine forming urea and ornithine. The or-
nithine can be re-used for the synthesis of another urea (hence urea-cycle).
Note the difference between synthase and synthetase: the latter requires energy in the form
of ATP. The total reaction of urea production is:
NH+4 + 4 ATP + 2 H2 O + Aspartate → 4 ADP + 4 Pi + 5 H + Fumarate + H2 N CO NH2 .
+ − −
Control of urea cycle

• increased amino acid breakdown
• increased liver [Glu]
• production of N-acetyl glutamate
• which allosterically activates carbamoyl phosphate synthetase
Urea cycle failure
results in hyperammonemia and encephalopathy. Especially in complete deficiency the prog-

nosis for these patients is poor. There are two possible reasons for urea cycle failure:
inherited Feeding difficulties, vomiting, ataxia, lethargy, Reye-syndrome like encephalopathies,
coma, death. Induced aversion against protein-rich foods in mild cases.
412
Urea-cycle 22.2.2
Figure 22.4.: Nitrogen sources for the urea pathway. Most of the nitrogen on the aspartate
comes from transamination reaction in the liver, while the ammonium comes
from extrahepatic sources via blood. However, if either is not available in
sufficient amounts to get rid of the other, conversion is possible.
not K, T, P
α-ketoacid amino acid
transaminase
glutamate
NH4+ + α-ketoglutarate glutamate α-ketoglutarate
dehydrogenase
aspartate transaminase
oxaloacetate aspartate
urea
furmarate
liver cirrhosis Alcoholism, viral hepatitis. Ataxia, slurred speech, tremor (asterixis), mental
derangements (Wernicke-Korsakoff-syndrome).
In either case mainstays of treatment are an alcohol free and protein reduced diet in which
essential amino acids are replaced by their α-ketoacids. Thus the nitrogen is recycled rather
than excreted. Two substances can aid in treatment:
• Benzoate + Gly → Hippurate
• Phenylacetate + Gln → Phenylacetyl-Gln
Hippurate and Phenylacetyl-Gln are excreted in urine.
For all enzymes involved in the urea cycle defect mutants have been reported in human
patients:
413
Enzyme location frequency remark

Carbamoylphosphate synthetase I 2q35 1:800 000 early and late onset forms
Ornithine transcarbamoylase Xp21.1 1:100 000 X-linked, but with X-
inactivation in ♀ possible
Argininosuccinate synthetase 9q34 1:100 000 citrullinemia
argininosuccinate lyase 7cen-q11.2 1:70 000 argininosuccinate aciduria,
treatment with Arg + Or-
nithine
Arginase 6q23 1:1 000 000 progressive spastic quadri-
plegia
22.3. Catabolism of the carbon backbone
After removal of the amino group the charbon backbone of the amino acids need to be
converted into compounds that enter the pathways of glucose and/or fatty acid metabolism.
Amino acids are divided into 3 groups depending on where their backbone enters general
catabolic pathways:
glucogenic amino acids feed into glycolysis and Krebs-cycle:
Pyruvate Ala, Cys, Gly, Ser
Oxaloacetate Asp, Asn
α-ketoglutarate Arg, Glu, Gln, His, Pro
Succinyl-CoA Met, Val
ketogenic amino acids produce acetyl-CoA (Leu, Lys)
glucogenic and ketogenic produce precursors of gluconeogenesis as well as ketone bodies

(Ile, Phe, Tyr, Trp, Thr). Usually the ratio of these precursors is fixed, only Thr can
be used to make either glucose or ketones.
Amino acids are an important source of glucose and ketone bodies during starvation and
physical exertion. In these cases body protein, in particular muscle protein, is broken down.
The glucose is used to maintain a constant blood glucose concentration, which is required
for the correct function of brain, erythrocytes and kidney cortex. The ketone bodies are
used as fuel for muscles, especially the heart. For further discussion of this homeostatic
mechanism, see chapter 31 on page 569.
414
Ala and Ser enter glycolysis 22.3.1
Medical application: The low carbohydrate, high protein (Atkins-) diet This diet
was introduced to help overweight and obese patients with weight reduction. Given the
increasing morbidity from metabolic syndrome in developed societies that is an important
goal. Let us however consider the biochemical consequences of such a diet.
Our body requires glucose as essential nutrient. Brain, erythrocytes and kidney cortex
depend on a constant supply of glucose for their correct function, a relatively small drop
in blood glucose concentration results in life-threatening hypoglycemic coma. In case of
a person on a low carbohydrate diet the body has to maintain blood glucose level by
gluconeogenesis from proteins. This is actually one of the mechanisms by which the Atkins-
diet is supposed to work: Gluconeogenesis is metabolically expensive, the energy spend on
doing this is no longer available to make body fat.
However, proteins contain both glucogenic and ketogenic amino acids, and since amino
acids can not be stored both of them have to be metabolized at the same time. This
leads to ketoacidosis, which in turn has a diuretic effect. The drop in blood pH together
with dehydration and loss of minerals can result in a dangerous physiological situation, in
extreme (and very rare) cases leading to coma and death.
It is the diuretic effect of the Atkins-diet that results in the initial rapid weight loss (1-2 kg
over a few days) of people on this diet, which is no doubt one explanation for the popularity
of this diet. The difference between loss of water and loss of fat is not necessarily clear to
all dieters.
Every diet results not only in a loss of fat, but also of lean body mass in form of muscle
protein, which can only be partly prevented by increased physical activity. In a Atkins-type
diet this effect is more pronounced than in a balanced, calory-reduced diet. In addition, the
lack of glucose puts the body into an “energy saving mode”, which makes weight loss more
difficult (see chapter 31 for the mechanism).
It is probably all these factors that conspire to make the drop-out rates in Atkins-type
diets higher than in conventional ones (see Westmann et al., 2002 for a prospective study
jointly done by members of the Atkins-institute and critics of this diet).
22.3.1. Ala and Ser enter glycolysis
Alanine The metabolism of Ala is particularly simple, transamination converts it into

pyruvate. Since transamination is reversible, Ala can also be produced from pyruvate. The
enzyme responsible, alanine:glutamate transaminase, also known as glutamate:pyruvate
transaminase (GPT), is located in liver cytosol. In case of necrotic liver damage the enzyme
is released into the blood stream, where it can be detected by appropriate laboratory tests
with high sensitivity at low costs (see section 10.4 on page 208 for details). Detection is by
415
Figure 22.5.: Alanine and serine are produced from and converted into metabolites of gly-
colysis. For details see text.
Ser-biosynthesis
O O O O
NAD+ NADH + H+ Glu αKG O O
3-Phosphoglycerate C C C
HC OH O O C O +
H3N CH O
C O P O C O P O
H2 C O P O
O H2 H2
O O H2O
3P-hydroxypyruvate 3P-Serine
ADP Pi
O O
2-Phosphoglycerate O O ATP C
C O +
H3N CH
HC O P O 3-Hydroxypyruvate
D-Glycerate C OH
HO CH2 O H2
O O Serine
O O NADH + H+ NAD= C
C αKG
O C
HC OH
C OH
C OH H2
O O H2
Glu
Phosphoenolpyruvate C O Gluconeogenesis
C O P O
CH2 O
Serine H 2O
dehydratase
αKG Glu
O O O O
O O
C C C
O O + +
H3N
+
CH C Pyruvate H3N C H2N C
CH3 alanine glutamate O C CH2 CH3

Alanine transaminase CH3 (Thr) H 2O
this enzyme is of
diagnostic significance Trp NH4+
(GPT) Gly Cys
416
Glu, Gln, Asp and Asn enter the Krebs-cycle 22.3.2
Figure 22.6.: Transamination of Glu and Asp produces α-ketoglutarate and oxaloacetate
respectively, which are intermediates of the Krebs-cycle. Gln and Asn are
produced from Glu and Asp by amidation of the terminal carboxylic acid
group in an energy-requiring reaction. Also shown is where other amino acids
enter the Krebs-cycle.
Phe Tyr
Trp
Lys
O Leu
Ile
this enzyme is H 3C C
S CoA
of clinical
Glutamine
significance (AST)
O O
Citrate C
αKG Gl +
H3N CH
Aspartate O O +
C H 3N
cis-Aconitate
O C CH2
O O
C CH2 C
H 3N
+
CH
aspartate C O NH2
CH2
glutamate O O H2O ADP
Oxaloacetate iso-Citrate
C
transaminase Pi Glutamine
O O Glutaminase synthetase
NH4+
NH4+ NH4+
NH4+ ATP
ATP
Asparagine O O
Asparaginase C
synthetase L-Malate O O +
AMP C H3N CH
PPi H2O
O C CH2
CH2 CH2
O O
C CH2 C
+
H 3N CH Phe C aa ka O O
Furmarate -
CH2 O O
C
2-Oxoglutarate Glutamate
O NH2 Tyr
Glutamate:
Asparagine Arg Pro
oxoglutarate His
Succinate Succinyl-CoA aminotransferase
this enzyme is
Ile of clinical
Met
Thr Val significance (GOT)
coupling GPT and glutamate dehydrogenase in an optical test (see section 7.9 on page 169
for how this works).
One of the most frequent causes for liver damage is alcohol consumption, a single glass of
alcoholic beverage kills enough liver cells that the enzymes released can be detected for
2–3 days. This is of forensic importance: Driving under the influence is usually punished by
withdrawal of the drivers licence, which is returned only after the culprit has demonstrated
that (s)he can live over extended periods without alcohol.
Serine Serine is produced from the glycolytic intermediate 3-phosphoglycerate by reduc-

tion, transamination and dephosphorylation (see fig. 22.5). The latter step is irreversible,
to catabolize Ser into 3-PG a different pathway is used, which uses ATP. Serine may also
be converted to pyruvate via serine dehydratase.
417
22.3.2. Glu, Gln, Asp and Asn enter the Krebs-cycle
Transamination of Glu and Asp produces α-ketoglutarate and oxaloacetate respectively,

which are intermediates of the Krebs-cycle. Like GPT the enzymes involved, glutamate:oxoglutarate
and aspartate:glutamate aminotransferase respectively, are used as diagnostic marker for
liver damage (GOT and AGT, also abbreviated AST).
Gln and Asn are produced from Glu and Asp by amidation of the terminal carboxylic acid
group. This reaction requires energy in the form of ATP. Glutamine synthetase simply
takes ATP, ammonium and Glu to produce Gln, ADP and Pi . Asparagine synthetase
however produces AMP and PPi instead, since the PPi is immediately hydrolyzed by py-
rophosphatase the reaction is driven to the products in a similar way as discussed above
with argininosuccinate synthetase.
Both synthetase reactions are irreversible, hydrolysis of Gln and Asn to Glu and Asp is
catalyzed by separate enzymes, glutaminase and asparaginase, respectively.
22.3.3. Gly, Thr and Ser
The metabolism of Gly, Thr and Ser are connected, as seen in figure 22.7. From the medical
point of view, the following issues are of particular importance:
• The reactions from Thr to Gly or succinyl-CoA are irreversible, making Thr an es-
sential amino acid.
• Deficiency of glycine cleavage enzyme leads to non-ketotic hyperglycinemia,

a rare (1:250 000) autosomal recessive disorder of amino acid metabolism. It results
in severe, often fatal, neuronal deficiencies.
• Excessive conversion of glycine to oxalate can lead to kidney stones, since Ca-oxalate
has a low solubility in water. In such cases restriction of Gly in the diet is the best
treatment.
• Trimethylamine causes the smell of “ripe” fish. It’s N-oxide is used as osmolyte by
fishes, after their death bacterial degradation leads back to the amine.
• The pathways involve two carriers of C1-bodies, SAM and folate. These will be dis-
cussed in the next subsections.
418
Gly, Thr and Ser 22.3.3
Figure 22.7.: Threonine can be converted to succinyl-CoA, pyruvate or glycine, these reac-
tions are irreversible making Thr an essential amino acid. Gly can be converted
to oxalate, trimethylamine, Ser, or cleaved to CO2 and water. Several of these
reactions require C1-carriers, either SAM (see section on Met) or THF.
- -
COO COO
- +
COO NADH H3N CH
Oxalate 2 H+
C OH
NAD+ H2
kidney stones H2O
with Ca2+ Peroxisomes
-
COO
Tetrahydrofolate (Vit. M)
CHO FADH2
NH4+
B6 Ser hydroxymethyl
Glyoxylate transferase
FAD Methylene THF + H2O
H2O
Bacteria CO2 2 SAH COO

- THF Methylene-THF
2 SAM
+ B6
+
HN (CH3)3 H3N CH2 CO2 + NH4+
glycine cleavage enzyme
Trimethylammonium-ion Glycine
- recessive deficiency (1:250,000)
OH non-ketotic hyperglycinaemia
fatal or severe mental deficiency
H2O Acetyl-CoA
N(CH3)3
Trimethylamine CoA-SH
-
COO
[O] + -
H3N CH COO
CH3 C O C O
O N CH3 CH3 CH3

NADH α-amino-β-ketobutyrate Pyruvate
CH3
Trimethylamine-N-oxide
NAD+
- - CoA-SH CO2
COO NH4+ COO O
NAD+ NADH
+
H3N CH B6 O C C S CoA
B1
HC OH threonine CH2 CH2
dehydratase α-ketobutyrate
CH3 CH3 CH3
dehydrogenase
α-ketobutyrate Propionyl-CoA
Threonine
Succinyl-CoA
419
Figure 22.8.: Folate (Vit. M) is required for the synthesis of nucleic acids. Several important
drugs interfere with folate metabolism.
H2N N N H
O
HN H2N S NH2
N CH2 O O
O N C COOH
H Sulfanilamide (Antibiotic)
Pteridine derivative N CH
H
para-Amino-benzoic acid CH2
CH2
Folic acid, Viatamin M COOH
Glutamic acid
H H
NADPH+H+ NADP+ H NADPH+H+ NADP+
H
H2N N N H H2N N N H H2N N N H
2 1 8 7
3
H
HN 5
6
HN HN
4 Dihydrofolate N CH2 Dihydrofolate N CH2
N CH2 reductase reductase
O 10 N R O HN O H HN
H 7,8-dihydrofolate R R
Folate 5,6,7,8-tetrahydrofolate
H
H
H2N N N H
H
HN
N CH2
O H N
HC R Gly metabolism
N5-formimino-THF NH
(from His-catabolism)
Formimino-THF
cyclodeaminase
NH3
H H H
H H H
H2N N N H NADPH + H+ NADP+ H2N N N H NADH + H+ NAD+ H2N N N H
H H H
HN + HN HN
N CH2 N CH2 N CH2
O C N O C N O CH3 HN
H R H2 R R
N5,N10-methylene-THF N5-methyl-tetrahydrofolate
N5,N10-methenyl-THF
(Formaldehyde, dUMP -> dTMP) (Methanol, Homocysteine -> Methionine)
ADP + Pi
N5-formyl-THF
OMe
H2O
cycloligase H2 N N OMe
Methenyl-THF
cyclohydrolase ATP
N
OMe
NH2 Trimethoprim
H H (DHF reductase inhibitor,
H H bacteriostatic)
H2N N N H H2N N N H
H H H2N N N H
HN HN
N CH2 N10-formyl-THF N CH2
isomerase N
O H N O HC HN N CH2 O
HC R O R
NH2 N C COOH
O
H3C N CH
N5-formyl-THF
H
N10-formyl-THF CH2
Methotrexate
(formic acid, purine synthesis, (Dihydrofolate reductase inhibitor CH2
formyl-Met-tRNA synthesis) cancer, arthritis) COOH
420
Gly, Thr and Ser 22.3.4
Folate as C1-carrier
Humans can not synthesize folate, it is an essential nutrient (vitamin M). Dihydrofolate re-
ductase converts dietary folate into its physiologically active form, 5,6,7,8-tetrahydrofolate.
THF is used as one carrier of C1-bodies in metabolism. Such C1-bodies originate from
amino acid metabolism and are required for the synthesis of nucleotides and, in prokary-
otes, formyl-Met. You will recall that in prokaryotes protein starts with an N-terminal
formyl-Met, rather than Met as in eukaryotes.
The conversion between the various oxidation states of the C1 (methyl-, methylene-, methenyl-
, formyl- and formimido-) looks daunting, but all you really have to remember is that these
reactions are reversible, so that any form produced can be converted into any form re-
quired.
Because nucleotide synthesis and hence cell division can not occur without THF, several
pharmaceuticals have been developed that interfere with folate metabolism and can be used
against cancer and/or bacterial infections:
Sulphonamides were the first successful broad spectrum antibiotics. The antibiotic effect
of Prontosil rubrum® (Sulfamidochrysoidin) was discovered by G.J.P. Domagk
in a screen for substances active against wound infections, which had killed many of
his contemporaries in the trenches of WWI. The first patient he treated with it was
his own daughter, suffering from a gangrenous sports injury. Her successful treatment
from a disease which otherwise would have cost her at least the arm, if not her life,
started an entire new era of medicine. For his discovery Domagk was awarded the
Nobel price for Physiology and Medicine in 1939. He was forbidden from accepting
the price by A. Hitler and even imprisoned. Only in 1947 was he able to accept the
price (the price money however was returned to the Nobel foundation, since it was
not claimed within one year).
It was later discovered that Prontosil is a pro-drug, which is converted to sulfanil-

amide in our body. Since humans can not make folate, sulfanilamide has no effect on
human folate metabolism. In bacteria however it stops the synthesis of folate because
of its similarity to p-aminobenzoic acid, one of the precursors of folate (see fig. 22.8
for the structures).
Chemically modified sulphonamides are also used to inhibit carboanhydrase as di-

uretics and to lower the pressure inside the eyes.
Trimethoprim inhibits dihydrofolate reductase, the enzyme which converts folate into THF.
The drug is often given in combination with sulphonamides to prevent drug resistance.
Methotrexate (MTX) is another inhibitor of dihydrofolate reductase. It is used to treat

malignancies and (in much lower doses) autoimmune-diseases.
421
22.3.4. The sulphur-containing amino acids: Met and Cys
Met is required not only for protein synthesis, but also to make S-adenosyl methionine
(SAM), the second important carrier of C1-bodies in metabolism (see fig. 22.9). Removal of
the methyl-group produces S-adenosyl homocysteine. Removal of the nucleoside from
SAH leads to homocysteine, from which methionine can be regenerated using a methyl-
group transferred from methyl-THF. If not enough folate is available in the body, homocys-
teine accumulates leading to vascular complications. Excess folate can mask the pernicious
anemia that results from vitamin B12 deficiency, but not its neurological consequences. Since
in the absence of anemia hypovitaminosis B12 is much harder to diagnose most developed
countries have imposed upper limits on the folate content of vitamin supplements.
If more homocysteine is produced than needs to be converted back to Met, it can be

condensed with Ser to form cystathionine. This reaction is catalyzed by cystathionine
synthase, which is defect in 1:250 000 live births. This autosomal recessive defect leads to
homocysteinuria, a disease that leads to mental retardation, dislocation of the eye lenses,
bone elongation and osteoporosis and thrombosis. In many cases the enzyme has a lowered
affinity for vitamin B6 , the disease can then be treated by very high doses of this vitamin.
Cystathionine is broken down to Cys and α-ketobutyrate, the latter being oxidatively de-
carboxylated to propionyl-CoA.
Since Cys can be produced from Met via cystathionine it is not an essential amino acid.
Excess Cys is broken down to pyruvate, Cys (and Met) are therefore glucogenic.
22.3.5. Branched-chain amino acids: Val, Ile, Leu
All three branched chain amino acids are initially catabolized in a similar way. They are
transaminated in muscle by branched chain amino acid transaminase, the resulting α-
ketoacids are transported into liver mitochondria, where they are oxidatively decarboxylated
by branched chain α-ketoacid dehydrogenase in a reaction similar to the one you
studied for pyruvate dehydrogenase.
A defect in branched chain α-ketoacid dehydrogenase leads to maple syrup urine disease,
so called because the urine has the characteristic smell of maple syrup, caused by sotolone,
a break down product of Leu which by itself is quite harmless. Note that consumption
of curry or fenugreek can also lead to sotolone in urine, please avoid prescribing costly
laboratory investigations in such cases.
Maple syrup urine disease has autosomal recessive inheritance and a prevalence of 1:200 000.
Prognosis is very poor, the disease leads to severe mental deficiency, optic atrophy, ataxia,
ADHS, axial hypotonia, exertional fatigue, metabolic acidosis, hypoglycemia, elevated liver
422
Branched-chain amino acids: Val, Ile, Leu 22.3.5
Figure 22.9.: A derivative of Met, S-adenosyl methionine (SAM), is the second carrier of
C1-bodies in metabolism.
NH2
CH3
S N
ATP PPi + Pi N
CH3
CH2 +
H2
S C O N N
CH2
+ CH2
HC H3N
- CH2
COO +
HC H3N
Methionine - OH OH
COO
Folate deficiency increases S-adenosyl methionine (SAM)
THF [homocysteine] -> vascular disease
B12
Methyl-THF
[-CH3]
SH
Ado H2O S Ado
CH2
CH2
CH2
+ CH2
HC H3N
+
- HC H3N
COO Homocysteineuria 1:200 000 -
Homocysteine recessive, COO
homocysteine accumulation S-adenosyl homocysteine (SAH)
results in mental retardation,
Serine lens dislocation, bone elongation,
osteoporosis and thrombosis
B6 Cystathione synthase
Treatment: Met restriction,
H2O mega-doses of B6
NAD+ NADH + H+
CoA-SH CO2 CH3
S
CH3 B1 CH2
H2C CH2
+ CH2 O C S CoA
HC H3N CH2 H2O
- + O C Propionyl-CoA
COO HC H3N +NH -
- B6
4 COO
COO
α-ketobutyrate
Cystathionine
γ-Cystathioninase
- - 2-
COO [O2] COO + SO3
NH4 -
+ + COO
H3N CH H3N CH
O C
CH2 CH2
- CH3
SH SO2
Cysteine Cysteinesulfinate Pyruvate
423
Figure 22.10.: Metabolism of branched-chain amino acids.

CoA-SH CO2
- -
COO αKG Glu COO NAD+ NADH
+ B6 O C B1
H 3N CH
O C S CoA
HC CH3 muscle
HC CH3
HC CH3
CH2 CH2 liver mitochondria
CH2
Valine Isobutyryl-CoA
α-Ketoisovalerate FAD
FADH2
- NADH + H+ NAD+ CoA-SH H2O

COO H2O O C S CoA
HC CH3 C CH3
Oxidation of OH hydration
HC thioester hydrolysis CH2
O NAD+
Methylmalonate Methylacrylyl-CoA
CoA-SH
semialdehyde NADH
B1 CO2 NAD+
NADH
CO2 CoA-SH - Met
O COO
B1 CO
C S CoA
CH2 CH2
CH3 CH3 Thr
Propionyl-CoA
NADH + H+ Remember: this pathway blocked

in methylmalonic aciduria
NAD+ O
C S CoA
CH2
-
H2O CH2
COO -
+ COO
H 3N CH O C S CoA Succinyl-CoA
HC CH3 C CH3
CH2 CH
CH3 CH3
Isoleucine Tiglyl-CoA
Branched chain a-ketoacid dehydrogenase
defect in maple syrup urine disease
Prevalence 1:200,000, autosomal recessive
Severe mental deficiency, optic atrophy, ataxia, AHDS, +
axial hypotonia, exertional fatigue, H 2O 2 H
metabolic acidosis, hypoglycemia, elevated liver enzymes ADP
elevated branched chain aa in serum, abdominal pain, ATP
death in early childhood
COO
-
CO2 Pi O C S CoA
+ O C S CoA H 2O
H 3N CH CH
CH HMG-CoA
CH2 C CH3
Biotin
C CH3
HC CH3 CH2
CH3 -
CH3 COO
Leucine methylcrotonyl-CoA Methylglutaryl-CoA
424
Phe and Tyr 22.3.6
enzymes and branched chain amino acid concentrations in serum and abdominal pain.
Death occurs usually in early childhood.
Val and Ile are finally broken down to propionyl-CoA, Leu to HMG-CoA. Note that
the conversion of propionyl-CoA to succinyl-CoA is defect in methylmalonic aciduria.
These patients therefore have not only problems with fatty acid metabolism, but also with
metabolism of Val, Ile, Met and Thr.
22.3.6. Phe and Tyr
The metabolism of Phe and Tyr contains a number of important inherited diseases, which
are frequent topics in board exams.
Phenylketonuria and albinism
Phe is converted to Tyr by phenylalanine hydroxylase, a mixed oxygenase. One of the

atoms of an oxygen molecule is inserted into the para-position of Phe, the other oxidizes
tetrahydrobiopterine (BioH4 ) to dihydrobiopterine (BioH2 ). The latter needs to be
converted back to BioH4 by dihydrobiopterine reductase. An inherited recessive defect
in either phenylalanine hydroxylase (1:200 000 in most populations, but up to 1:7000 in
people of European descent) or dihydrobiopterine reductase (much rarer) results in PKU,
a serious medical condition that leads to seizures, spasticity and irreversible brain damage.
Treatment is by Phe restriction until after adolescence, when the brain is fully formed and
becomes less sensitive to the breakdown products of excess Phe. Cave: Aspartam contains
Phe and Tyr becomes essential. To avoid brain damage the law in most developed countries
requires mandatory testing for PKU in newborns (for newborn screening, see section 28.1.3
on page 513).
Defects in dihydrobiopterine reductase also prevent the production of l -DOPA and 5-OH-
Trp. This affects the serotonine and catecholamine metabolism (see chapter ?? on page ??
for a discussion of hormone synthesis).
In occulocutaneous albinism the synthesis of melanin (dark pigment of skin, hair and
iris) and pheomelanine (pigment of red hair) is defect. This results in white hair and skin
and in a red iris (because the retina shines through the unpigmented iris). Affected persons
do not have the protection from UV-radiation which the presence of melanin provides. As
a result they suffer from sunburns easily and may develop skin cancer. The only known
treatment is to totally avoid exposure to the sun.
425
Figure 22.11.: Metabolism of Phe and Tyr.

NAD+
NADH + H+
Rare form of PKU: Inability
to synthesise L-dopa and
5-OH tryptophane. These
intermediates must be
Dihydrobiopterine supplied in the diet.
reductase
Note: same enzyme used in
Tyr- and Trp-hydroxylase:
H H Serotonine and catecholamine
H2N N N HN N N biosynthesis also affected
HN H H HN H H
N C C CH3 N C C CH3
H
O OH OH O OH OH
Tetrahydrobiopterine (BioH4) Dihydrobiopterine (BioH2) Melanin
Dopamin
Phenylalanine
hydroxylase Occulocutaneous
albinism
Dopa
COO Phenylketoneuria (PKU): autosomal

- -
recessive, 1:6 000 (UK) to 1 : 200 000 COO Pheomelanine
+
H3N CH (Japan). Mental retardation, seizures, H3N
+
CH
O2 + spaticity. Mandatory diaper test in most
CH2 developed countries. Treatment by Phe CH2 + H2O
restriction until after adolescence.
Cave: Aspartame! Tyr becomes essential.
Hawkinsinuria: very rare. Tyrosinaemia type II:

Acidemia and excretion of < 1 : 250 000, affects
hawkinsin. Tyr-restricted diet OH brain, eyes and skin.
during first year of live.
Autosomal dominant. αKG
Tyrosine
Alkaptoneuria: excretion of Glu aminotransferase
homogentisate in urine Tyrosinaenmia type III:
(-> black). Ochronosis very rare, mild mental
(pigment accumulation in -
retardation, ataxia COO
tissue), arthritis.
-
COO
- COO Nitisinone, NTBC O C
CH2 CH2 CH2

CO2 O2
H+ O2
O HO C
-
OOC
p-hydroxyphenyl
Homogentisate
H O 1,2-dioxigenase OH pyruvate oxidase
H Homogentisate OH
Maleoylacetoacetate Hydroxyphenyl pyruvate
Tyrosinaemia type I: cabbage-like
- smell from FAA, liver + kidney
COO failure, liver cancer. 1 : 100 000,
but 1 : 2 000 in some areas of
Maleoyl- CH2 Quebec
acetoacetate
isomerase
O HO
2
H+
- COO
-
H COO
CH2
-
OOC O Fumarylacetoacetate
CH +
CO
hydrolase CH
H - CH3
COO
Furmarylacetoacetate Fumarate Acetoacetate
426
Trp and Lys 22.3.7
Tyrosinemia and Alkaptonuria
The breakdown of Tyr is associated with several autosomal recessive deficiencies, the most
serious of those is tyrosinemia type I. The disease results from a deficiency of fumarylace-
toacetate hydrolase which results in a cabbage-like body smell from FAA, liver + kidney
failure, liver cancer. The frequency is about 1:100 000 in most populations, but because of
a founder effect 1:2000 in some areas of Quebec (Canadian students beware!).
Tyrosinemia II and -III are much less serious than type I. Type II results from a deficiency
in tyrosine aminotransferase. The disease has a frequency of 1:200 000 and results in damage
to brain, eyes and skin.
Type III results from a defect in p-hydroxyphenyl pyruvate oxidase and is very rare.
It causes mild mental deficiencies and ataxia. A very rare partial defect in PHPO leads to
hawkinsinuria: The enzyme produces a reactive intermediate, 1,2-epoxyphenyl acetic acid
instead of homogentisate. The intermediate then reacts spontaneously (epoxy-group!) with
glutathione to produce hawkinsine. Because the production of intermediate is a gain-of-
function mutation, hawkinsinuria has dominant inheritance, which is rare for metabolic
diseases. Fortunately, the disease is relatively benign, leading to acidemia only in the first
year of life (treat with Phe + Tyr-restricted diet). Afterwards, patients become quite normal
except for the excretion of hawkinsin, which gives the urine a chlorine-like smell.
Alkaptonuria is almost benign, except for an increased risk for arthritis later in life.
However, the deficiency of homogentisate oxidase leads to the excretion of homogentisate
in the urine, which forms a dark pigment when exposed to air. The pitch-black diapers tent
to alarm the parents, who will come storming into your surgery. Simply tell them that
you know what it is and that it is not dangerous. A different pigment will over the years
accumulate in the tissue of the patient, this is called ochronosis. There is no treatment
for this “disease”.
22.3.7. Trp and Lys
Catabolism of Trp starts by opening the 5-membered ring with a dioxygenase, via a couple
of steps this part of the molecule is converted to Ala. Then the 6-membered ring is opened
and converted via α-ketoadipate to acetoacetate. There are a couple of things to remember
about the catabolism of Trp:
• Xantenurate and kynurenate originate from this pathway, they cause the yellow color
of urine.
• Trp can be converted via quinolinate into nicotinamide (vitamin B3 ). The process
is not very efficient, about 60 mg of Trp need to be catabolized to produce 1 mg of
427
Figure 22.12.: Metabolism of Trp and Lys.

+ +
H2 N H3 O2
H2 N H3
- O -
C C COO C C COO
H H
H
Tryptophan C
N N O
H dioxygenase H H2O
Tryptophan N-formylkynurenine
HCOO-
Xantenurate Formamidase
(yellow colour
of urine) +
H2O
+ O2 H2 N H3
H2 N H3 NADP+ NADPH O
C C COO
- OH
O -
H2O C C COO H
H
Alanine Kynurenine-3-
B1
monoxygenase NH2 -
NH2 N COO
Kynurenine
Kynureninase OH Kynurenate
(yellow colour of urine)
- O2 H2O
COO O - -
COO COO
C
H
NH2 3-HA-3,4-dioxygenase - spontaneous -
OOC NH2 N COO
OH
2-Amino-3-carboxy muconate- Quinolinate
3-Hydroxyanthanilate 6-semialdehyde (precursor of NAD)
CO2
NAD+
NADH
H2O
NAD(P)H
NAD(P)+
NH3
-
COO
O S CoA NADH NAD+ O C
C
CO2 CoA-SH CH2
CH2 B1
Acetoacetate CH2
CH2
CH2
CH2 -
- COO
COO α-Ketoadipate
Glutaryl-CoA
Hyperlysineaemia/-uria: mental Glu
and physical retardation aminoadipate B6
aminotransferase
-
α-KG
COO -
+ α-KG COO - -
H3N CH COO COO
+ H2O NADH
NADPH NADP+ H3N CH + NAD(P)H +
CH2 - NAD+ Glutamate H3N CH NAD(P)+ H3N CH
CH2 COO
CH2 CH2 CH2
CH2 CH2
CH2 Saccharopine Saccharopine CH2 aminoadipate- CH2
dehydrogenase CH2 CH2 dehydrogenase semialdehyde
CH2 CH2 dehydrogenase
CH2
(lysine forming) H2C CH (glutamate forming) -
+
N H3 - HC COO
N COO O
H
Lysine α-Aminoadipate-6- α-Aminoadipate
Saccharopine semialdehyde
428
Pro, Arg, His and ornithine 22.3.8
nicotinamide. Since proteins on average contain about 1 % Trp about 6 g of protein

are required per mg of nicotinamide.
Lys is converted in liver mitochondria first to α-ketoadipate, then the pathway to acetoac-
etate is the same as for Trp. Since all reactions from Lys to α-ketoadipate are reversible, it
is possible to make small amounts of Lys from Trp in our body. However, this is insufficient
to meet our Lys-needs, making Lys an essential amino acid.
There is a rare deficiency associated with the first two steps of Lys-degradation, hyper-
lysinemia/uria, which results in impaired sexual development, muscular and ligamentous
asthenia (weakness), normocytic, normochromic anemia, convulsions, episodic vomiting,
rigidity and in extreme cases coma. The disease locus is 7q31.3, in humans both saccha-
ropine dehydrogenase activities are located in the same, bifunctional protein [Sacksteder
et al., 2000]. Apparently, excess Lys is a competitive inhibitor of arginase, resulting in hy-
perammonemia, which is toxic. Treatment is by low protein diet if the disease is severe
enough to warrant that.
In brain peroxisomes a second pathway for Lys degradation exists, however, no medical
conditions have been linked to it and we will not discuss it any further.
22.3.8. Pro, Arg, His and ornithine
Pro, Arg and His are all catabolized to Glu, which can then enter the Krebs-cycle as
discussed above.
The pathway from Pro is reversible, thus we can produce Pro from Glu and Pro is not
essential. Note however that the conversion between ∆5 -dehydroproline and Pro is catalyzed
by different enzymes, the catabolic enzyme is a flavoprotein, the anabolic uses NADPH +
H+ .
Excess Arg is converted via the urea cycle to ornithine, which is converted to Glu. Since
the reactions involved are reversible ornithine can be made from Glu should the flow through
the urea cycle need to be increased.
The breakdown of His to Glu is not reversible, making His an essential amino acid. However,
there is so much carnosine (βAla-His) in our muscles that His-deficiency in the diet can be
masked for a long time by carnosine breakdown. Therefore, no dietary requirement for His
could be established. There are two inherited deficiencies associated with His-catabolism,
both are completely benign. However, they may interfere with laboratory procedures, so
you have to know about them:
Histidinemia is caused by a deficiency in histidinase, the first enzyme in the pathway. The
excess His in the blood and urine does not cause health problems, but does react with
FeCl3 in the same way as Phe, giving a false positive diaper test in the screen for
429
Figure 22.13.: Pathway for the degradation of Pro, His, Arg and ornithine.
- -
COO COO
+ +
-
H3N CH H3N CH
COO
H (CH2)3 CH2
+ β-Ala-His
H2N NH (Carnosine)
N
+ C
Proline H2N NH2 N
arginine histidine
histidinase
FAD H2O
NADP+ defect 1:10,000
+
urea -> histidinaemia N H4
cycle benign, but false
positive phenylketoneuria -
FADH2 NADPH + 2 H+
urea test COO
Diagnostic: Lack of
-
H+ COO Urocanate in sweat. CH
+
- H3N CH CH
COO
H (CH2)3
N
N NH2
ornithine N
Δ5-dehydroproline H2O Urocanate
H2O
spontaneous
αKG
-
COO
Glu
+ H2O
H3N CH
(CH2)2 H+
CH
-
O COO
glutamate-γ-semialdehyde
CH2
NAD+ + ADP + Pi CH2
-
N COO
NADH + H+ + ATP
NH
N-Formiminoglutamate
COO
- glutamate formimino transferase (FIGLU)
+
H3N CH Enzyme Deficiency: habitual
excretion of FIGLU
(CH2)2 in urine: benign
- THF
COO
Formimino-THF
glutamate
Test for folate deficiency:
FIGLU in urine after
oral His load
430
Carnitine 22.4.2
Figure 22.14.: Synthesis of carnitine

- -
COO COO
+ + H
H3N CH [O]
H3N CH O C
Glycine
CH2 HO CH CH2
CH2 CH2 CH2
CH2 CH2 CH2
+
CH2 CH2 N
+ + H3C CH
N N CH3 3
H3C CH H3C CH
CH3 3 CH3 3
NAD+
ε-N-Trimethyllysine
NADH
- -
COO COO
[O]
CH2 CH2
HO CH CH2
CH2 CH2
+ +
N N
H3C CH H3C CH
CH3 3 CH3 3
Carnitine
PKU. Patients showing a positive screening result therefore need to be investigated

by more selective, but also much more complicated and expensive, methods.
Habitual FIGLU excretion occurs when the enzyme catalyzing the last step of the pathway
is defect. Usually this totally benign condition is not even noticed. However, the
appearance of FIGLU in the urine after a challenge with a large dose of His is used to
diagnose folate deficiency. Again, the enzyme deficiency would cause a false positive
result if the absence of FIGLU in urine were not confirmed before His is given.
22.4. Compounds derived from amino acids
22.4.1. Carnitine
Carnitine is required for fatty acid metabolism. It can be synthesized from Lys, and is
therefore not an essential ingredient of food. Supplementation with carnitine is not required
even for physically active persons.
431
Figure 22.15.: Creatine as energy storage molecule in muscle.

from Arg
NH2 HN PO3
2- H
ATP ADP Pi N C O
+ +
H2N C H2N C HN C
- - CH2
from Gly N C COO N C COO spontaneous N
creatine
H H
from SAM CH3 2 kinase CH3 2 CH3
Creatine Creatine phosphate Creatinine
this substance is used

this enzyme is found
to measure renal
in serum after damage
(glomerular) activity
to muscle or heart
22.4.2. Creatine and creatinine
Sudden muscular activity in flight-or-fight response requires energy in form of ATP. How-
ever, only small amounts of ATP can be stored inside a cell. Muscle cells therefore store
energy in the form of creatine phosphate, a super-energy rich compound (∆G00 =
43 kJ/mol) which is readily converted back to ATP and creatine. Creatine itself is syn-
thesized in our body from Arg, Gly and SAM.
A small percentage of the creatine phosphate stored constantly breaks down into crea-
tinine, this is given into the blood stream and excreted by the kidney. There is no re-
absorbtion of creatinine in the kidney, all creatinine filtered by the glomerulus into the
primary urine therefore ends in the urine. The ratio of creatinine concentrations in urine
and plasma multiplied by the urine production rate therefore gives the volume of blood
filtered by the kidney per unit time (glomerular filtration rate (GFR)), which is used
clinically to assess kidney function.
The enzyme responsible which catalyzes the conversion of creatine to creatine phosphate
is creatine kinase, a cytosolic enzyme which is released into the blood stream when the
cell is damaged. It is easily measured by the coupled spectrophotometric test of Warburg
(see section 7.9 on page 169) and can be used to help in the diagnosis of myocardial infarct
and myopathy (see section 10.4 on page 208).
22.4.3. Polyamines
Polyamines are synthesized in our body either by decarboxylation of Lys (cadaverine) or

from ornithine (putrescine, spermidine and spermine). The exact function of polyamines
has not been established. It is assumed that the regularly spaced positive charges from the
amino groups helps packing DNA. There also seems to be a role in the regulation of ion
channels, in particular NMDA and AMPA receptors and the inwardly-rectifying potas-
sium channels.
432
Polyamines 22.4.3
Figure 22.16.: Biosynthesis of polyamines

NH2
N +
N H3N
CH3
+
H2 CH2
S C O N N
H2C
CH2 CH2
+
CH2 H3N CH
+ -
HC H3N COO
- OH OH
COO
Ornitine
S-adenosyl methionine (SAM)
Ornitine decarboxylase
SAM decarboxylase
CO2 CO2
H3C
NH2 H3N
+
C O
+
N CH2 H2N
N
CH3 H2C CH2
H2
S
+
N CH2 Putrescine H2O2
C O N H2C
CH2 H2C CH
+
CH2 H3N O
+
C H3N
H2 OH OH O2
polyamine H2O
oxidase
+
H3N H3C S Ado
CH2
H2C +
H3N H3C
CH2 CH2 C O
+ +
H2N H2C H2N
CH2 Spermidine N1-acetyl spermidine
CH2 CH2
H2C +
H2N H2C
diamine-N-acetyl
CH2 CH2 CH2
transferase +
H2C H2C H2N
+
H2N CH2 CH2
H2C H3C S Ado H2C H2C
CH2 Spermine
H3N
+
Acetyl- CH2
H2C CoA CoA-SH H2C
+ +
H3N H3N
diamine-N-acetyl polyamine
transferase oxidase
Acetyl- +
O H3N
CoA H2 H2 H2 H2
+
H2 CH2
C +
+ C C + C C N C N
CoA-SH H3C N C N C C H2 C C H3 H2O2 H2C
H2 H2 H2 H2 H2 H2 H2 CH
O2 O
N1-acetyl spermine
H2O
433
Figure 22.17.: Mechanism of the drug Eflornithine.

Difluoromethylornitine CHF2
O
+ -
CH N H3 C C C C COO
H2 H2 H2 H2 +
Pi O C OH H3N
H
+ +
N H3 C C C C COO
-
H2 H2 H2 +
N CH3 H3N
H2O
PLP
CHF2
H2O + -
N H3 C C C C COO
H2 H2 H2 +
H N
+ H - CH
N H3 C C C C COO H2
H2 H2 H2 + P O C OH
H N
H2 CH
P O C OH N CH3
N CH3
CO2
F
CHF
+
CO2 N H3 C C C C
H2 H2 H2
H N
H H2 CH2
+
N H3 C C C C
H2 H2 H2 + P O C OH
H N
H2 CH2
N CH3
P O C OH Enzyme-Cys-SH
N CH3
HF
H2O Enzyme-S
CH
+
N H3 C C C C
H2 H2 H2 +
O H N
CH H2 CH2
H2
P O C OH + +
N H3 C C C C H3N
H2 H2 H2 H2
+
P O C OH
N CH3 N CH3
434
Inherited amino acid transporter deficiencies 22.5.2
Inhibition of polyamine synthesis by inactivation of ornithine decarboxylase with difluo-

romethylornithine (Eflornithine® ) is used to treat cancer, seizures and sleeping sickness,
an infection caused by Trypanosoma brucei. Especially in the latter role Eflornithine® has
become known as the “resurrection drug”, since it can be used to treat even the second,
meningoencephalitic, phase of the disease.
Because Eflornithine® blocks only the synthesis, but not the uptake, of polyamines, its ef-
fectiveness in treating cancer was found to be limited. Compounds that stimulate polyamine
oxidase, and hence the destruction of polyamines (synthesized by the cell or taken up), are
currently in clinical trials.
22.5. Physiology of amino acids
22.5.1. Metabolism of amino acids
Most amino acid catabolism occurs in the liver. However, branched chain amino acids are
transaminated in muscle, the corresponding α-ketoacids are then transported to the liver
for further metabolism.
Ala is produced in the anaerobic muscle and transported to the liver via the blood stream.
Thus rather than transporting two potentially dangerous metabolites – lactate and ammonia
– only the relatively harmless Ala needs to be transported. In the liver the nitrogen is
disposed of by the urea cycle, while the pyruvate is used for gluconeogenesis. This is called
the Ala-cycle.
Glu + Gln form the major energy source for the intestinal mucosa. Gln is required for the
metabolic compensation of acidosis by the kidney: Glutaminase and glutamate dehydro-
genase produce ammonia, which binds a proton to form ammonium that ends up in urine. In
addition, gluconeogenesis from α-ketoglutarate binds another 4 protons: 2 αKG + 4 H2 O + 4 H+ → Glc + 4 CO2 + 8 [H].
This reaction however occurs also in starvation and exertion, as the kidney is as important
for gluconeogenesis as the liver. The Gln-Glc cycle between muscle and kidney corresponds
to the Ala-cycle between muscle and liver.
22.5.2. Inherited amino acid transporter deficiencies
Cystinuria is caused by a defect of the transporter for the dibasic amino acids Lys, Arg,
cystine (Cys-S-S-Cys) and ornithine in the intestine and in the kidney. Intestinal uptake of
these amino acids in the form of di- and tripeptides is usually sufficient to meet the dietary
needs of the patient (especially in rich countries with high dietary protein content) but
failure of cystine-reuptake in the kidney leads to cystine-concentrations in the urine which
exceed its solubility. This results in the formation of kidney and bladder stones. Patients
435
should maintain a large urine volume and a slightly alkaline urine pH (≥ 8.5) to prevent
the crystallization of cystine.
The transporter has two subunits, a large (SLC3A1 on 2p16.3, type A cystinuria) and
a small (SLC7A9 or rBAT on 19p13.1, type B cystinuria). Mutations in both subunits
are called type AB. Depending on the mutation inheritance can be recessive (type I) or
incomplete recessive (type II and III, heterozygotes show moderate or low increases of
dibasic amino acids in urine, respectively). It is estimated that about 10 % of stone formers
are heterozygous. The overall frequency of cystinuria in the population is 1:7000, making
it one of the most prevalent genetic disorders.
Hartnup disease is caused by a defect in the transporter for large neutral amino acids
(SLC6A19 on 5p15). This results in a relative Trp-deficiency, making the patient more
susceptible to pellagra. There may also be a (usually mild) encephalopathy due to insuf-
ficient myelin-formation. In industrialized nations the signs of the disease are rarely seen
because of the super-adequate diet. The frequency is reported to be between 1:14 000 and
1:24 000.
Oasthouse disease is caused by a defect in Met-uptake in the intestine. As a consequence,

Met is metabolized by intestinal bacteria to α-hydroxybutyrate (do not mix up with the
ketone body β-hydroxybutyrate!). This compound is absorbed in the intestine, but not
metabolized in the body. Rather, it is excreted by the kidneys, giving the urine a charac-
teristic smell of drying hops (hence the name of the disease). Patients have striking white
hair, suffer from mental retardation, convulsions, tachypnea and diarrhea. The disease is
inherited in an autosomal recessive pattern, and is exceedingly rare.
22.6. Exercises
1. Protein consumption The RDA for protein intake is 56 g/d, average uptake in US
34 g/d plant and 75 g/d animal protein. Metabolism of the excess amino acids places ad-
ditional strain on liver and kidney, worsens metabolic syndrome and may cause gout. A
diet high in (animal) protein results in an overdose of saturated fat but lacks fibre. Feeding
livestock requires 40 % of the worlds grain and 95 % of its soy production, 41 × 106 t of plant
protein are required to produce the 7 × 106 t/a of animal protein consumed in the US. 2/3
of worlds arable land is used to produce feed stuff and 87 % of its water (100 000 l per kg of
beef). Livestock in US produces 1.4 × 106 t/a of manure, 130 times the human production.
Production of methane, ammonia and nitrous oxide by cattle is a significant contributor to
acid rain and global warming. Fuel consumption per kg protein is 10 times higher for beef
than plants. Thus the consumption of excess animal proteins in rich countries contributes
436
Objectives in Summary: Amino acid metabolism 22.7
to health and environmental problems and to the hunger in developing countries. With
consumption of animal proteins increasing in India and China (which between them have
about 1/3 of the worlds population) these problems are likely to increase in the future.
Question: If all animal protein consumed by the 300 × 106 citizens of the US were replaced
by plant protein, and protein uptake restricted to recommended doses, approximately how
many additional people could be fed?
A 100 million
B 200 million
C 500 million
D 1 billion
E 2 billion
2. Biological value of proteins Calculate the protein score for a mixture of 2 parts rice
and 1 part soy beans, using the composition data provided above. Which is the limiting
amino acid?
A Ile
B Lys
C Met
D Phe
E Val
22.7. Objectives in Summary: Amino acid metabolism
At the end of this lecture students should be able to

• distinguish between essential and non-essential amino acids
• describe how the amino acid pool of our body is filled and used and define the terms
“nitrogen balance” and “biological value”.
• advice patients on nutrition with respect to proteins.
• describe how the amino acid nitrogen is used and eliminated in metabolism.
• describe how the carbon chains of amino acids enter major metabolic pathways.
• define the role of alcohol in this context.
437
• describe the role of enzymes of amino acid metabolism in laboratory medicine.

• describe the role of folate in metabolism and give example for pharmaceuticals that
interfere with it’s function.
• describe the consequences of hypovitaminosis M and B12 and the interaction between
these nutrients.
• describe non-nutritional functions of amino acids.
• describe the role of polyamines in health and disease and the pharmaceutical use of
difluoromethylornithine and polyamine oxidase activators.
• describe the patho-mechanism, signs and symptoms, treatment options and probable
outcome of
– urea cycle failures
– non-ketotic hyperglycemia
– histidinase deficiency
– homocysteinuria
– maple-syrup urine disease
– methylmalonic aciduria
– phenylketonuria
– tyrosinemia type I, II and III
– hawkinsinuria
– alcaptonuria
– hyperlysinemia
– cystinuria
– cystinosis
– oasthouse disease
– Hartnup’s disease
438
23. Biochemistry of Digestion
23.1. Digestion
Most dietary nutrients are macromolecules that have to be hydrolyzed to their building
blocks in the digestive tract. The products of digestion are absorbed:
Nutrient Enzyme Product
Proteins Proteases Amino acids, di- and tripeptides
Starch Glycosidases Glucose
Disaccharides Glycosidases Monosaccharides
Triglycerides Lipases Fatty acids, 2-monoacylglycerol
Nucleic acids Nucleases Nucleosides, bases
≈ 30 g of digestive enzymes are secreted/day.
23.1.1. Digestive secretions in man
Salivary glands
pH of saliva is 6.0–7.0. It contains 3 important enzymes:
α-Amylase: Endoglycosidase, cleaves α-1,4 glycosidic bonds in starch and glycogen. Forms
maltose, maltotriose, and α-limit dextrins. Quantitatively less important than pan-
creatic isoenzyme, but keeps teeth clean.
Lysozyme: Endoglycosidase, cleaves β-1,4 glycosidic bonds in the bacterial cell wall polysac-
charide peptidoglycan. Most Gram-positive bacteria are killed by this enzyme, Gram-
negative bacteria are protected by outer membrane. Lysozyme is also present in other
body secretions, egg white, lysosomes of phagocytic cells, etc. Wound-licking is an
instinctive way to prevent wound infection.
lingual lipase digestion of small part of dietary fats
These enzymes are inactive at the acidic pH of the stomach.
439
Figure 23.1.: Starch digestion. α-amylase can digest starch to within 6–8 glucose molecules
of a branch-point. The resulting limit dextrin is digested by isoamylase.
non-reducing end
O
H
non-reducing end HO H2
H H C
H OH
O H
H2C OH O
H
O HO
HO H H H
H O
H OH
O H Amylase
α(1->4) H2C OH HO H2
H
O H C
HO H H OH
H H
H OH H O
O HO
H2C OH H
H O
O H
Amylase HO H H
HO H2
H
H OH H C
O OH
H2C OH H H
H
O O
HO H H HO Isoamylase
H H
H OH
O
O
H2C α(1->6)
H
O
HO H H
OH H
H
O
H2C OH
H
O
HO H H
H OH H OH
O
CH2
Amylase H
O
HO H H
H
H OH
O H2C OH
H
O
HO H H
H OH
O reducing end
440
Digestive secretions in man 23.1.1
Figure 23.2.: Structure of murein and its digestion by lysozyme.

cleavage site
of lysozyme
GlcNAc NAM GlcNAc NAM

OH β1->4 OH
H2C H2C β1->4
O O O OH β1->4
O O H2C OH β1->4
O O H2C
HO O O
N O OH HO
HC N
C O OH
H3C O CH3 C
l-Ala
C
O H3C O C CH
H
3
C
iso-d-Glu l-Ala O
d-Ala Gly5 l-Lys iso-d-Glu
l-Lys d-Ala d-Ala Gly5 l-Lys
iso-d-Glu l-Lys d-Ala
l-Ala iso-d-Glu
O C l-Ala
O C
441
Stomach
The pH of stomach is 1.5–3.0. Functions of gastric acid:

• Antibacterial action
• Denaturation of proteins, which facilitates the action of proteolytic enzymes.
Pepsin, the major enzyme of the stomach, is an endopeptidase with a pH optimum at
2.0–2.5. Unlike exopeptidases (aminopeptidases and carboxypeptidases) which cleave only
terminal peptide bonds, endopeptidases also cleave internal bonds, pepsin after aromatic
+ large hydrophobic aa. Pepsin produces a mix of oligopeptides (“peptones”). Only small
amounts of free amino acids are formed.
Total gastrectomy is compatible with life: Mild digestive problems, increased risk of intesti-
nal infections, but vitamin B12 supplements are required.
Pancreas
The pancreas supplies most of the soluble enzymes in the intestine. These enzymes function
at the near-neutral pH of the small intestine. Major secretions:
Bicarbonate neutralizes stomach acid, buffers to neutral pH
Endopeptidases: The pancreatic endopeptidases are serine proteases (they have a serine
in their active site), and they have different cleavage specificities:
Trypsin cleaves on the carboxy side of Lys and Arg.
Chymotrypsin cleaves on the carboxy side of hydrophobic amino acids.
Elastase cleaves on the carboxy side of small amino acids.
Carboxypeptidases: The pancreas secretes 2 zinc-containing carboxypeptidases:
Carboxypeptidases A cleaves hydrophobic amino acids from the C-terminus.
Carboxypeptidase B cleaves basic amino acids (Lys, Arg) from the C-terminus.
α-Amylase: Similar to salivary α-amylase. Same cleavage specificity but different molec-
ular structure: The two α-amylases are isoenzymes. The pancreatic enzyme is more
important for digestion than the salivary enzyme.
Lipase: The pancreatic lipase is the major enzyme of fat digestion. Reaction: Triglyceride
→ 2-Monoacylglycerol + 2 fatty acids Also required:
Co-lipase, a pancreatic protein which anchors the lipase to the surface of fat droplets.
442
Digestive secretions in man 23.1.1
Figure 23.3.: Cholate (PDB-entry 2qo4). All the OH-groups are on one face of the molecule,
making it able to interact with water. The other face is hydrophobic (hydro-
carbons) and interacts with lipid. Note that hydrogen atoms are not visible
in X-ray crystallography.
Bile salts (from liver)

Phospholipases and nucleases take care of dietary phospholipids and nucleic acids.
Pancreatic failure cause steatorrhea + generalized lipid malabsorption. Risk of deficiencies
of fat-soluble vitamins!
Liver and bile bladder
Bile salts (20–50 g/d) are required for lipid digestion in the small intestine. Like detergents
they form mixed micelles which ferry the lipids to the mucosal surface. The break-up of
lipid/detergent micelles is aided by peristaltic motion. Absence leads to steatorrhea, e.g. if
bile stones block bile duct.
The intestinal brush border
The lumenal surface of the intestinal mucosal cells (“brush border”) has membrane-bound
digestive enzymes:
Aminopeptidases complete protein digestion. Considerable amounts of di-and tripeptides
are absorbed intact and hydrolyzed by cytoplasmic enzymes in the mucosal cells.
Endo- and carboxypeptidases act on oligopeptides
Sucrase cleaves sucrose to glucose + fructose.
Lactase cleaves lactose to glucose + galactose.
Glucoamylase removes glucose from the non-reducing end of starch, starch-derived oligosac-
charides and maltose.
Isomaltase cleaves α-1,6 bonds in isomaltose and α-limit dextrins.
443
Figure 23.4.: Oligosaccharides come in two types: In the trehalose type the C1 of one sugar
is linked to the C1 of the other. Those oligosaccharides are non-reducing and
show no mutarotation. In oligosaccharides of the maltose type the anomeric
the (C1 ) carbon of one sugar is linked to a non-anomeric carbon of the other.
This still has a reducing end.
Maltose-type Trehalose-type
acetal acetal
(non-reducing) (non-reducing)
OH OH HO
H2C OH H2C CH2
O H2C O O
HO O HO OH
HO O OH HO O OH
OH HO OH HO
OH
Glc (α1->4) Glc hemiacetal Glc (α1->α1) Glc
Maltose (reducing, Trehalose
mutarotation)
OH
H2C
O OH OH
HO H2C
HO O O
OH HO CH OH
CH2 HO O OH 2
O OH
HO OH
HO HO CH2 O
OH
Glc (α1->6) Glc Glc (α1->β2) Fru

Isomaltose Sucrose
444
Lactose intolerance 23.1.4
Trehalase cleaves trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside).
23.1.2. Undigestible materials
Many plant products, including cellulose, hemicelluloses, lignin etc. are not digested. A
small proportion of these (≈5 %) is fermented by colon bacteria forming acids (lactic, acetic,
propionic acid) and gas (H2 , CH4 , CO2 ).
Carbohydrates which are not digested but rapidly fermented by colon bacteria cause flat-
ulence. Inulin (poly-fructose is the energy store of asteraceae and umbelliferae). Raffinose
(gal(α1 → 6)glc(α1 → 2)fru), stachyose (gal(α1 → 6)gal(α1 → 6)glc(α1 → 2)fru), ver-
bascose (gal(α1 → 6)gal(α1 → 6)gal(α1 → 6)glc(α1 → 2)fru) occur in legumes and bras-
sicaceae. Beano® = α-galactosidase from Aspergillus niger breaks these sugars down and
prevents the sometimes unpleasant and socially embarrassing consequences of eating these
otherwise healthy foods.
23.1.3. Lactose intolerance
In mammals lactase activity is highest in infants and declines in later life. If they ingest
milk later the undigested lactose is fermented by endgut bacteria, resulting in flatulence,
abdominal pain, and diarrhea (this is the reason why one should not give milk to cats).
In some human populations with shepherding tradition lactase activity remains high through-
out life (persistent lactase): Europeans, Arab bedouins, Tuareg, Tutsi and Fulani.In these
populations the regulatory sequences responsible for switching off lactase production in
adolescence are mutated. However, in other populations (e.g. Asians) [lactase] drops to 5–
10 % of the original level (non-persistent lactase). After ingestion of large amounts of milk
or milk products, people with non-persistent lactase show signs of lactose intolerance. They
can use only those milk products where lactose has been destroyed by fermentation (e.g.
kefir). We distinguish
Congenital hypolactasia lactase not present at birth and throughout life (inherited defi-
ciency)
primary hypolactasia decrease of lactase activity after weaning, in humans at age 5 a or

so.
secondary hypolactasia after damage to intestinal mucosa
445
Figure 23.5.: Zymogen activation of pancreatic enzymes. Trypsinogen after release into the
small intestine is activated by enteropeptidase. The resulting trypsin can then
activate all other zymogens, including trypsinogen.
Figure 23.6.: 3D-structure of chymotrypsinogen and chymotrypsin.
446
Intermediary metabolism 23.2
23.1.4. Zymogens
Digestive proteases and phospholipases are secreted as inactive precursors called zymogens.
This mechanism protects the synthesizing cells from self-digestion. Glycosidases and lipases
are not secreted as zymogens.
Pepsin: Derived from pepsinogen. A 44-amino acid peptide is cleaved from the amino end
of pepsinogen. This cleavage is catalyzed by pepsin or occurs by auto-activation at
low pH. The cleaved peptide acts as a pepsin inhibitor at neutral pH.
Pancreatic zymogens: Trypsinogen is activated to trypsin by the duodenal enzyme en-

teropeptidase. Trypsin then activates all pancreatic zymogens including trypsinogen.
As an additional safety device, the pancreas contains a trypsin inhibitor. This in-
hibitor is a small polypeptide which binds trypsin with very high affinity. In acute
pancreatitis, the pancreatic zymogens are activated within the pancreas and the pan-
creas digests itself and the surrounding adipose tissue, amylase and lipase are found
in serum.
23.2. Intermediary metabolism
Types of metabolic processes
Anabolic reactions are biosynthetic reactions in which a complex product is made. They
require metabolic energy.
Catabolic reactions are degradative reactions. They produce useful energy as ATP. ATP
is required for:
• Biosynthetic reactions
• Active transport across membranes
• Cell motility and muscle contraction
Energy value of major dietary nutrients.
Carbohydrates = 16.7 kJ/g (4.0 kcal/g)
Fat = 38.9 kJ/g (9.3 kcal/g)
Protein = 16.7 kJ/g (4.0 kcal/g)
447
On a typical American diet, about 40 % of the energy comes from carbohydrates, 40 % from
fat and 20 % from protein. In many indigenous people carbohydrate supply up to 80 % of
the daily energy intake. Recommended are about 60 % from carbohydrates, 10 % each from
saturated, mono-unsaturated and poly-unsaturated fatty acids, 10 % from proteins.
Energy requirement per day

Basal metabolic rate (BMR): 84–105 kJ/kg (20–25 kcal/kg)
Post-prandial thermogenesis: 419–1675 kJ/d (100–400kcal/day)
Muscular activity: variable
Internal organs have high metabolic rates. The brain alone accounts for 25–30 % of the
BMR. Muscle tissue (40–50 % of body weight) consumes 30 % of the BMR. Since ♂ have
on average more muscle than ♀ (who have more adipose tissue), they tend to have a higher
BMR.
Metabolic regulation Metabolic processes are organized in pathways in which each reac-
tion is catalyzed by a separate enzyme. Metabolic pathways are regulated:
Biosynthetic pathways are regulated by feedback inhibition.
Catabolic pathways are feedback-inhibited by ATP.
All metabolic pathways can be regulated by hormones.
Usually, the enzyme catalyzing the first irreversible reaction of the pathway (its committed
step) is regulated. This implies that the enzyme catalyzing the committed step is present
at lower activity than the other enzymes of the pathway. Mechanisms:
• Enzyme synthesis may be regulated.
• Allosteric regulation of key enzymes.
• Phosphorylation of key enzymes.
Compartmentalization of metabolism:
Cytoplasm storage of fat and glycogen, glycolysis, pentosephosphate-pathway
Mitochondria Krebs-cycle, oxidative phosphorylation, β-oxidation. Part of gluconeogen-
esis, heme and aa metabolism
ER and Golgi protein, cholesterol and lipid synthesis, biotransformation. Glc6-phosphatase
Lysosomes degradation of endocytosed and cellular macromolecules
448
Carbohydrate metabolism 23.4
Peroxisomes Oxidases that produce H2 O2 (FAD-dependent): very long-chain FA degrada-

tion, bile acid, cholesterol and plasmalogen synthesis. In plants: Glyoxylate shunt.
23.3. Carbohydrate metabolism
Fate of glucose: Most dietary carbohydrate is absorbed as glucose. Most of the fructose
and galactose is converted to glucose or glycogen in the liver. Metabolic fates in the cell:
• Initially, glucose becomes phosphorylated to glucose-6-phosphate. This product can

no longer leave the cell.
• The major catabolic pathway for glucose is glycolysis. It takes place in the cytoplasm.
Converts glucose to pyruvate and produces a small amount of ATP.
• Excess glucose is used for glycogen synthesis.
• There are several minor pathways of carbohydrate metabolism that make specialized
products.
Acetyl-CoA mitochondrial oxidation Under aerobic conditions, pyruvate goes into the
mitochondria where it is converted to acetyl-CoA. This metabolite is formed not only from
carbohydrates, but also from fatty acids and amino acids. Acetyl-CoA feeds into the TCA
cycle. The TCA cycle is therefore the final common pathway for the oxidation of all major
nutrients.
The catabolic pathways produce reduced coenzymes (NADH + H+ and FADH2 ). These are
re-oxidized by the respiratory chain in the inner mitochondrial membrane. This strongly
exergonic process drives ATP synthesis by oxidative phosphorylation.
Anaerobic glycolysis The conversion of glucose to lactic acid is our only possibility to
produce energy under anaerobic conditions, but lactic acid tends to accumulate and lower
the tissue pH. Importance:
• Sudden high energy demand, for example in contracting muscle.
• Absence of mitochondria, for example in erythrocytes.
• Absence of oxygen, for example during ischemia.
449
Figure 23.7.: Digestion of dietary fat (triglycerides) in the small intestine

1
O
H2C
2O
HC O
H2C O
3 O
1- palmitoyl -2-oleoyl -3- linoleoyl-glycerol example for a fat (triacylglycerol)
H2O
pancreatic lipase
HO
fatty acid
O
O
H2C
O
HC O
H2C O
OH 1,2-diacylglycerol
H2O
pancreatic lipase
HO
O fatty acid
OH
H2C
HC O
H2C O 2-monoacylglycerol
OH
Blood glucose Brain and erythrocytes require glucose at all times, therefore a constant
blood glucose level of 70–100 mg/dL has to be maintained. The liver produces glucose by
glycogen breakdown during short-term fasting and by gluconeogenesis (mostly from amino
acids) during long-term fasting. Regulation is mostly hormonal.
23.4. Fat metabolism
Triglyceride transport Fatty acids, mono- and diacylglycerol are absorbed by enterocytes
by passive diffusion across microvillar membrane, together with fat-soluble vitamins. Inside
the enterocytes FA are bound to intestinal FA binding protein to protect the cell mem-
brane from its detergent-like effects. After re-synthesis of triacylglycerols they are packed
in lipoproteins (chylomicrons) and released into lymphatics.
450
Fat metabolism 23.4
Figure 23.8.: Pancreatic lipase (cyan→purple) and colipase (yellow→green) PDB-code 1lpa.
The enzyme becomes active only when contact with the lipid-water-interface
removes the lid (deep blue helix) from the substrate site. The enzyme is a
Ser-dependent hydrolase (similar to Ser-dependent proteases!)
Table 23.1.: Types of lipoproteins.

Chylomicrons VLDL IDL LDL HDL
density (g/ml) < 0.95 < 1.006 1.006–1.019 1.019–1.063 1.063–1.210
diameter (nm) 75–1200 30–80 25–35 18–25 5–12
mass (MDa) 400 10–80 5–10 23 0.175–0.360
protein (%) 1.5–2.5 5–10 15–20 20–25 40–55
phospholipid (%) 7–9 15–20 22 15–20 20–35
triacylglycerol (%) 84–89 50–65 22 7–10 3–5
free cholesterol (%) 1–3 5–10 8 7–10 3–4
cholesteryl ester (%) 3–5 10–15 30 35–40 12
apolipoproteins A-I,A-II, B-48, B-100, C-I, B-100, C-I, B-100 A-1, A-II, C-I,
C-1, C-II, C-III, E C-II, C-III, E C-II, C-III, E C-II, C-III, D, E
Lipoproteins are non-covalent aggregates (“micelles”) of lipid and protein. The lipids have
to be transported in this form because they are not sufficiently water soluble. Lipopro-
teins have a hydrophobic core (triacylglycerol, cholesteryl ester) and an amphiphilic coat
(apolipoprotein, phospholipid, cholesterol). Their density is determined by content of fat
(0.91 g/ml), cholesterol (1.067 g/ml), phospholipid (≈1.13 g/ml) and protein (≈1.5 g/ml).
The coat is 2.0 nm thick in all cases: smaller diameter → higher density. Various lipoproteins
have different function:
Chylomicrons transport food lipids and cholesterol from intestine through the body
VLDL, IDL, LDL transport cholesterol and lipid from the liver to other tissues (“bad
cholesterol”)
HDL transport cholesterol and lipids from other tissue to liver, scavenger of cholesterol
(“good cholesterol”), liver converts cholesterol to bile acids
451
Fate of liver synthesized cholesterol:
• endogenous cholesterol and lipids packed in VLDL
• lipid broken down by lipoprotein lipase like dietary lipids
• VLDL remnants (IDL) converted to LDL
1. • Drug
LDL metabolism
supply tissues with cholesterol: receptor mediated endocytosis
The body has to dispose of xenobiotics: non-nutritive and potentially
• lysosome
harmful releases such
substances fatty acids and amino acids
as food-bourne into cytoplasm
and air-borne pollutants, drugs, and
pyrolysis products in barbecue and cigarette smoke.
• cholesterol + cholesteryl esters go to ER or droplets Water-soluble substances
can be excreted, but lipid-soluble substances have to be converted into water-
soluble products before they can be excreted in urine or bile. Drug metabolism is
most active in the conversion
Carbohydrate-to-fat liver but also occurs
After in other
a meal, excesstissues, such as the and
dietary carbohydrate lungs.
protein
Hepatic drug metabolism proceeds in 2 stages.
can be converted to fatty acids and triglyceride via acetyl-CoA, most are transported from
Stage 1 metabolism oxidizes the drug through the microsomal
the liver to other tissues as VLDL (very-low density lipoprotein). But fatty acids cannot
cytochrome P-450 enzymes (mixed-function oxidase)
be converted to glucose! Cyt-P-450
R-H + 02 + NADPH + H+ R-OH + H20 + NADP+
23.5. There are more than

Metabolism 100 different
of amino molecular
acids forms of cytochrome P-450.
and protein
They have broad and overlapping substrate specificities and can theefore oxidize
a wide variety of drugs, environmental pollutants, and even endogenous steroid
Amino acids and
hormones. system Dietary
Thisprotein protein
is induced by intake
many isdrugs,
typically
for about 100 g,
example and 30–40 g are
barbiturates.
the minimum requirement. The levels of free amino acids are low in the body,
This results in tolerance formation, often cross-tolerance when one drug induces but there are
10–15 kg of for
enzymes protein. Daily proteinofsynthesis
the metabolism another (and
drug.degradation) is about 300 g. This consumes
5 % of the
Stage 2 metabolism consists of of
basal metabolic rate. About half the amino reactions
conjugation acids can be synthesized
with glucuronicin the
body,
acid,the others needsulfate,
glutamine, to be taken up withhydrophilic
or other food (essential amino acids).
groups. The water-soluble
conjugation products are no longer biologically active and can be excreted in
urine or bile.
Catabolism of amino acids Amino acids are catabolized mostly in the liver. The nitrogen
is converted to the excretory product urea (daily production: 30–35 g from 100 g protein).
The nitrogen-free products formed from the amino acids are either oxidized to CO2 + H2 O
or used for biosynthetic reactions, including gluconeogenesis.
2. Alcohol metabolism
23.6. Alcohol Metabolism
Alcohol is derived from microbial fermentation:
Alcohol is derived from microbial fermentation:
ADP, Pi ATP
+
NAD NADH CO2 NADH NAD+
1/2 Glucose Pyruvate Acetaldehyde Ethanol
Some ethanol is formed by intestinal bacteria and metabolized as a first-

pass effect in the liver.
452 Alcohol is absorbed from the intestine, also the stomach. It is diffusible
and therfore readily distributed throughout the body. 2% are excreted by lungs
and kidneys, the rest is metabolized in the liver and to a lesser extent the
stomach. The zinc-containing cytoplasmic enzyme alcohol dehydrogenase
(ADH) is normally rate-limiting, as is the availability of NAD+. Alcohol oxidation is
a zero-order reaction down to serum levels of about 40 mg/dL. 10 g are
Alcohol Metabolism 23.6
Some ethanol is formed by intestinal bacteria and metabolized as a first-pass effect in the
liver. Alcohol is absorbed from the intestine, also the stomach. It is diffusible and therefore
readily distributed throughout the body. 2 % are excreted by lungs and kidneys, the rest is
metabolized in the liver and to a lesser extent the stomach. The zinc-containing cytoplasmic
enzyme alcohol dehydrogenase (ADH) is normally rate-limiting, as is the availability of
NAD+ .
• Km for alcohol dehydrogenase is 1 mM (0.046 h → 0 order kinetics 0.15 h/h in most

people
• Km for aldehyde dehydrogenase 10 µM: no accumulation of ethanal

metabolized each hour, and the blood level declines by 15 mg/100 mL/h.
Pathway:
Pathway:
NAD+ NADH NAD+ NADH
Ethanol Acetaldehyde Acetic acid

GTP, CoA-SH
GMP, PPi
Acetyl-CoA
The cardiac value of alcohol is 7 kcal/g. Most of the acetic acid generated
The energy
in the liver iscontent of alcohol
transported to otheristissues Most of the acetic acid generated in the liver is
for oxidation.
29 kJ/g.
transported to other tissues for oxidation. to alcohol, with facial flushing and
“Oriental flush” is a hypersensitivity
tachycardia after only one or two drinks. It is caused by a dominantly inherited
deficiency of the mitochondrial aldehyde dehydrogenase, often in combination
“Oriental flush” is a hypersensitivity to alcohol, with facial flushing and tachycardia after
with a “superactive” ADH. This trait occurs in 40% of Chinese and Japanese.
only oneDisulfiram
or two drinks. It is caused
(“Antabuse’) by a inherited
is an inhibitor dominant
of aldehyde negative used
dehydrogenase, (tetramer!)
to deficiency
oftreat
the alcoholics.
mitochondrial aldehyde dehydrogenase (Glu487Lys), often in combination with a “su-
peractive”Alcohol
ADH. metabolism
This traitresults
occursininan
40increased
% of Chinese[NADH] / [NAD+] ratio,
and Japanese. high (“Antabuse’)
Disulfiram
energy charge, and high mitochondrial acetyl-CoA. Pyruvate
is an inhibitor of aldehyde dehydrogenase, used to treat alcoholics. dehydrogenase
and TCA cycle are inhibited because of increased [NADH] / [NAD+] and [ATP] /
[ADP]. Alcohol
H2 oxidation is not feedback-inhibited.
H2 Therefore alcohol is oxidized
in preference to S S
other nutrients.
C
H3C Gluconeogenesis inhibitedC because
CH3 pyruvate and oxaloacetate are
N C S S is C N+
depleted: the high [NADH] / [NAD ]Cratio leads to a high [lactate] / [pyruvate]
H3CandCa high [malate] / [oxaloacetate]CH
ratio 3
ratio (equilibrium of LDH and malate
H H2
dehydrogenase!).
2 Hypoglycemia and lactic acidosis can develop in alcohol
intoxication.Disulfiram (Antabuse)
Drug interactions:
AlcoholAlcohol
metabolism
inducesresults in an increased
the synthesis [NADH + enzymes
of drug-metabolizing H+ ] / [NAD
in the ]liver,
+ ratio, high energy
charge,
but alsoand high their
inhibits mitochondrial acetyl-CoA.
activity acutely. Pyruvate
Barbiturates, dehydrogenase
for example, work inand the TCA cycle are
inhibited because
intocxicated but notofthe
increased [NADHImportant
sober alcoholic. + H+ ] /in[NAD + ] and [ATP] / [ADP]. Alcohol oxi-
anesthesia!
dation is not feedback-inhibited, the body wants to get rid of this toxin as soon as possible.
VI. BLOOD CELLS
Therefore alcohol is oxidized in preference to other nutrients.
Mature RBCs have no mitochondria. Glucose is metabolized by
anaerobic glycolysis (ATP formation) and the pentose-phosphate pathway
(NADPH) formation. Only 20g are consumed per day.
453
Synthesis of 2, 3-bisphosphoglycerate (BPG):
254
Gluconeogenesis is inhibited because pyruvate and oxaloacetate are depleted: the high
[NADH + H+ ] / [NAD+ ] ratio leads to a high [lactate] / [pyruvate] ratio and a high [malate]
/ [oxaloacetate] ratio (equilibrium of LDH and malate dehydrogenase!). Hypoglycemia and
lactic acidosis can develop in alcohol intoxication.
Other health effect of alcohol

• in form of distilled spirits provides energy, but no minerals, vitamins, essential AA...
• increases the fluidity of the PM → neurotoxicity
• converted to triglycerides → lipidemia, fatty liver
• Replacement of hepatocytes by connective tissue: cirrhosis
• Mitochondrial damage, leaky inner membrane
• Aldehydes modify and cross-link proteins
23.7. Intermediary metabolism
Catabolic and anabolic processes in our body are closely intertwined, which is only possible
by tight regulation. Regulation of pathways occurs by
substrate concentration according the the HMM-law. The binding/dissociation equilib-
rium is diffusion controlled, that is, almost instantaneous.
allosteric regulation (also very fast). Homo- and heterotropic effects.
enzyme modification works on an intermediate timescale (minutes). Phosphorylation/dephosphorylation
is the best-studied example, but remember that there are many other reactions as well.
enzyme synthesis/degradation is slow, on a timescale of hours to days. It is also expensive
in metabolic energy.
To make a good regulatory site, an enzyme has to meet several criteria:
• the product of the enzyme should be precursor of a single pathway (no downstream
branch-points).
• the enzyme should be the first irreversible step of the pathway. Regulating reversible
reactions is not necessary (they are not metabolically expensive). If the regulated
enzyme were behind the first irreversible step of a pathway, intermediates would
accumulate and potentially become toxic.
• the activity of the enzyme must be low compared to all other enzymes of the pathway
(it must form the bottleneck).
454
Digestion: Objectives in summary 23.9
Figure 23.9.: Regulation of metabolic pathways. Top: Feedback inhibition of an anabolic

pathway. The substrate A is converted into the product Z via a number of
intermediates. Once the concentration of Z is high enough, it shuts down
its own production. Note that the regulation could not occur at the earlier
reaction step, since that would also prevent the production of other products,
to which B is precursor. Middle: Feedback inhibition of a catabolic pathway.
Product of this pathway is ATP, which serves as feedback regulator. At the
same time, ADP may stimulate the reaction (example: phosphofructokinase).
Thus the rate of food breakdown depends on the energy requirements of a
cell. Bottom: Feedforward stimulation. The presence of a substrate activates
the pathway for its catabolism (example: Lac-operon).
A B C z Feedback inhibition (anabolic)
other products
A B C z Feedback inhibition (catabolic)
ADP ATP
A B C z Feedforward stimulation
23.8. Questions
1. How much fat can you lose per day on a 0 J diet (tap water and vitamin pills)?
2. Why is it so difficult to lose only fat and not also muscle on a strict weight reduction
diet?
3. Glucose utilization by the brain is not insulin-dependent. What does this mean for glucose
use by the brain in the fasting state?
23.9. Digestion: Objectives in summary
1. List the enzymes for the digestion of major nutrients including, starch, disaccharides,
proteins, triglycerides, phospholipids and nucleic acids, and their site of action in the ali-
455
mentary tract.
2. Name those digestive enzymes that are subject to zymogen processing, and state the
mechanisms of zymogen activation.
3. Describe the role of mixed bile salt micelles for lipid absorption in the small intestine.
4. Predict the effects of total gastrectomy, pancreatic failure and biliary obstruction on the
digestion of major nutrients.
5. Know why some people get flatulence after drinking too much milk.
6. Define the terms “anabolic”, and “catabolic”, and state the roles of cytoplasm, lysosomes,
endoplasmic reticulum and mitochondria in intermediary metabolism.
7. Describe the principles of feedback inhibition and feedforward stimulation in anabolic
and catabolic pathways.
8. Predict in general terms the consequences of genetic deficiencies, enzyme-inhibiting toxins
and vitamin/coenzyme deficiencies on different types of metabolic pathways.
9. Elaborate the characteristic features of regulatory enzymes of metabolic pathways.
456
24. Heme, Purines and Pyrimidines
24.1. Heme Biosynthesis
Heme is made in all cells according to their needs. About 250 mg/d total, 75–80 % of this
in the bone marrow, 15 % in the liver.
The uncolored porphyrinogens have −CH2− groups connecting the pyrrole rings, and the
colored (and fluorescent) porphyrins have =CH− groups. The porphyrinogens can be oxi-
dized non-enzymatically to the corresponding porphyrins by exposure to light and air.
δ-aminolevulinate synthase is the rate-limiting enzyme. In most cells, transcription and

transport of the enzyme (S1-isoform) into mitochondria are inhibited by heme and hematin
(heme with Fe in the ferric form), which also act as allosteric inhibitors. In addition, ALA
is allosterically inhibited by glucose.
Erythroblasts of the bone marrow express the S2-isoform (encoded on the X-chromosome),
which is up-regulated by EPO. The enzymatic activity is allosterically stimulated by iron.
24.1.1. Disorders of Heme Biosynthesis
Porphyrias are diseases in which abnormal quantities of porphyrin or its precursors are
excreted. They are caused by a partial deficiency of one of the biosynthetic enzymes other
than ALA synthase, either in the liver or bone marrow: heme formation is reduced, and
ALA-synthase is dis-inhibited. The accumulation of toxic intermediates causes attacks of
abdominal pain, neurologic signs, and/or cutaneous photosensitivity. Treatment is possi-
ble by hematin infusion. Also dietary carbohydrate is effective because it represses ALA-
synthase.
Intermittent acute porphyria is a hepatic porphyria, autosomal dominant with defi-

ciency of uroporphyrinogen I synthase. Porphobilinogen and δ-aminolevulinate appear in
the urine. Symptoms do not appear before puberty. Drugs and steroid hormones which
induce the synthesis of heme-containing hepatic enzymes (cytochromes P-450 enzymes)
trigger attacks of abdominal pain, vomiting, neuropsychiatric, and cardiovascular abnor-
malities. Only a minority of individuals with the genetic trait ever have attacks.
457
Tyrosinemia I lead-poisoning
(Succinylacetone) (replacement of catalytic Zn)
COOH
COOHCH2 congenital erythropoetic porphyria
COOH
O COOH δ-ALA dehydratase COOH
CH H2O 4 NH3 CH2 CH2 Uroporphyrinogen-III COOHCH2
H2 COOHCH2
CH2 2 H2O HO synthase
P C O H2 CH2 CH2
CH2 CH2 CH2 C
C H2O
H2 N Hydroxymethylbilane H2
N
+
CH3 C O H A
HOOC C C C COOH H2C C
CH2 H2 H2 H2 N
H H2C N NH HN H
+
PBGS-porphyria H PBG-desaminase HOOC C C COOH
PLP (Vit. B6) NH3 NH3 HOOC C C C COOH H2 H2
(rare) H2 H H2 H2 D NH HN B
H3N
+
C COO
- δ-ALA N
11q24, splice variants in HOOC C C C C COOH
H2 Porphobilinogen erythroblast (42 kDa) and C CH2 H2 H2 H H2 H2
H2O H2 N
Glycine (PBG) all cells (44 kDa) C C CH2
H2O CH2 CH2 Uroporphyrinogen-III H 2
acute intermittend porphyria (UROGEN)
H CH2 COOH
+ (madness of King George III) CH2 CH2
N C COOH
HC H2 COOH CH2 COOH
H2 Cytosol
P C O COOH
Figure 24.1.: Heme biosynthesis.
N
+
CH3 Uroporphyrinogen
COOH
decarboxylase
CH2 H
CH2 4 CO2
δ-ALA synthase
C 3p21: all cells (S1-isoform) Coproporphyrinogen porphyria cutanea tarda
O S CoA Xp11-21: erythroblasts (S2 isoform) oxidase COOH
Succinyl-CoA Mitochondrial matrix CH2
CH2
CH3 CH 2 H2O2
CoA SH CH3 CH2
H2 2 CO2 2 O2
COOH H2C C H2
COOH N H2C C
CH2 H N
H3C CH3 H
CH2 CH2 H3C CH3
NH HN
CH2 NH HN
H C O HOOC C C C CH2
+ H2 H2 H H hereditary coproporhyria HOOC C C C C COOH
N C COOH H C O N H2 H2 H H2 H2
H2 HC H H2O CO2 +
N CH2 C CH2 N
P C O H2 HC H2 C CH2
Coproporphyrinogen-III H2
P C O
+
+ Fasting Protoporphyrinogen IX CH2 CH3 (COPROGEN, hydrophobic)
N CH (PROTOGEN, even CH2 CH3
3 + more hydrophobic!) CH2
H N CH3 CH2
COOH
H COOH
+
+ Glucose
LIVER 3 O2
transcription Protoporphyrinogen
EPO porphyria variegata
2+ transport into mito oxidase Porphyrias:
Fe allosteric inhibition also spontaneous - dominant inheritance: 50% activity of enzyme
3 H2O2
CH -> reduced [heme] -> activation of δ-ALA synthase
Erythroblast 2
CH2 -> normally sufficient heme synthesis
CH3 CH
transcription CH3 CH
translation H but:
HC C +
2+ H -increased demand -> precursors leave cell
N Fe HC C
Sideroblastic anemia: 2H - δALA and PBG: neurologic symptoms
H3C CH3 N
- iron-loaded mitos form ring around nucleus 2+ H (neuro-visceral + neuro-psychiatric):
N Fe N HC CH
- defect in δ-ALA synthase 2 3 3
GABA antagonist!
HOOC C C C CH2 N N
- lowered affinity for pyridoxin - Uro- + Koproporphyrinogens: skin lesions by
H H H Ferrochelatase HOOC C C C CH2
2 2
N H2 H2 H H photosensitization, fragility, itch
C CH protoporphyria N
H C CH
24.1.1
lead-poisoning H
Heme CH2 CH3
Protoporphyrin IX CH2 CH3
CH
458
2 (PROTO, conjugated system,
CH2
COOH colored, red fluorescence)
COOH
Heme Degradation 24.2.1
Porphyria cutanea tarda is the most common porphyria. Uroporphyrinogen decarboxy-

lase is deficient, but symptoms occur only in patients with iron overload and liver damage.
Cutaneous photosensitivity is the most prominent symptom. Photosensitivity occurs in all
those porphyrias in which porphyrins (rather than porphyrin precursors, as in intermittent
acute porphyria) accumulate.
δ-Aminolevulinate dehydratase and ferrochelatase are inhibited in lead poisoning: δ-aminolevulinate

is increased in the urine and protoporphyrin IX in RBCs.
Laboratory tests δ-Aminolevulinate and porphobilinogen can be measured in the urine

with colorimetric tests. Coproporphyrinogen and uroporphyrinogen are spontaneously ox-
idized to the corresponding porphyrins if the urine is left standing in air. The can be
extracted from the urine, then identified spectrophotometrically or -fluorimetrically.
24.2. Heme Degradation
About 85 % of the heme in the body is present in hemoglobin. 6 g of hemoglobin are degraded
per day, most of this in the spleen. The heme becomes first biliverdin, then bilirubin. Heme
oxygenase (heme → biliverdin) is only CO-forming enzyme in the body.
Bilirubin is transported to the liver in non-covalent binding to albumin. Liver uptake re-
quires a facilitated diffusion type carrier of high capacity, the organic anion transport protein
(OATP).
The liver conjugates bilirubin to water-soluble bilirubin diglucuronide which is actively se-
creted into the bile canaliculi by the canalicular multiple organic anion transporter (cMOAT),
also known as multidrug resistance related protein 2 (Mrp2), an ABC-type transport AT-
Pase. This step is rate-limiting for hepatic bilirubin metabolism. Both the UDP-glucuronyl
transferases and bilirubin secretion into bile are stimulated by various drugs, including phe-
nobarbital. If there are problems with excretion of bilirubin diglucuronide into bile, Mrp3
will export the conjugate into the blood stream.
Bacteria in the terminal ileum and the colon remove the glucuronate residues, convert
bilirubin to colorless urobilinogens, and the urobilinogens to colored urobilins (stercobilin
etc.) which are responsible for the brown color of the stools. Some urobilinogen is absorbed
and undergoes entero-hepatic circulation. Trace amounts are also excreted in the urine.
The normal serum bilirubin concentration is < 1 mg/dL. Most of this is unconjugated.
459
CH2 Heme oxygenase (CYP450)
CH3 CH CH3 V
O
H O Globin H3C CH3
HC A C HC HN B
N N O 3+ P
3 [O] CO Fe
H3C CH3 HC
3
CH 3 C
2+
2+ CH3 N V
D N Fe N B N Fe N
H
P V V N
COOH C C C CH2
H H2 2
H
N
A D
N C CH N
C CH H O H
H C Note: about 1% of blood H3C P
hemoglobin complexed
with CO P CH3 Biliverdine (blue-green)
CH2 CH3
Verdoglobin (green)
CH2
COOH Hemoglobin (red)
Spleen macrophages
O O
2 O O UDP +
NADPH + H
OH
Figure 24.2.: Heme degradation.
2 UDP OH Biliverdin reductase

O OH +
NADP
O low water solubility -> transported by albumin

if albumin capacity exceeded:
O UDP-GT1-A1 O - jaundice (deposition in skin + sklera, from Fr. jaune = yellow)
OH CH3 - kernicterus (deposition in brain)
HO (perivenous liver cells)
O
CH3 HN
V O Morbus Meulengracht (icterus intermittens juvenilis, CH3
O CH3
CH V
H2C HO CH3
~30% remaining activity, 8% of population) 3
CH3HN P H P N O
Crigler-Najjar-Syndrom (< 10% remaining act.)
O N H H2C V H
λ = 460 nm
N H
H N N N N N
newborn jaundice H O
H3C V N C H CH3
Bilirubin diglucuronide H CH3
C N O H3C H2
(direct bilirubin) H H V
H2C CH2 2 P P
Bilirubin (orange-red)
(indirect bilirubin) Lumirubin
O
O
HO no hydrogen bridges between propionate and
O NH-groups of ring A and B -> more water soluble
OH 8 [H] -> excretion by the kidney
O
bacterial glucuronidases bacterial enzymes
OH O Glucuronic acid
O O
CH3 CH3
HN HN
CH3
CH3 E
E 2 [H] CH3
CH3 H2
H2 P CH2
P CH2 E C H
Polymers E C N N
(dark color of stool) N N H
H N C
N C bacterial enzymes H
H O H3C H2
O H3C H
P Urobilinogen
P
Stercobilin (golden)
O
CH3
re-uptake into blood,
HN 2 [H] transport to kidney
CH3
CH3 E
H2 P CH2 Urobilinogen
E C
N N
24.2.1
N H
C
H H
O HC3
P
460
Urobilin (orange-yellow)
excreted in urine -> yellow color
Hyperbilirubinemia and Jaundice 24.2.1
Figure 24.3.: Transport and metabolism of bilirubin in the hepatocyte.
ATP
Mrp3
ATP ER ABCC3
Bile
ADP + P
Mrp2 Blood
cMOAT ABCC2
ADP + P
OATP
Ligandin
Albumin
24.2.1. Hyperbilirubinemia and Jaundice
Conjugated or unconjugated bilirubin or both are elevated in various diseases. Both kinds
of bilirubin deposit in sclera and skin, causing jaundice (=icterus). However:
• Only unconjugated bilirubin can enter the brain and cause kernicterus. This occurs in
newborns when the binding capacity of serum albumin (25 mg/dL at normal albumin
concentration) is exceeded. Kernicterus can be fatal, and survivors may be left with
a motor disorder and mental deficiency.
• Only conjugate bilirubin can be excreted by the kidney. In choluric jaundice, the urine
is dark yellow-brown (i.e., urine dark and stool pale).
Serum bilirubin is measured by the van den Bergh method. A colored product is formed.
Direct (reacting) bilirubin is bilirubin-diglucuronide which can be determined directly in
aqueous solution. Indirect (reaching) bilirubin is unconjugated bilirubin. It reacts poorly
in water because of its tight binding to albumin. It is measured after the addition of an
organic solvent like methanol or ethanol.
461
Types of hyperbilirubinemia
Mixed hyperbilirubinemia 1. Hemolytic diseases can cause mild hyperbilirubinemia (usu-

ally < 4 mg/dL).
2. Physiological jaundice of the newborn is the most common form of jaundice. It

is caused by immaturity of the liver and is usually self-limited. If treatment is
required, you can use:
• Phototherapy, which destroys excess bilirubin in the skin.
• Phenobarbital, to induce the metabolizing enzymes in the liver.
• Exchange transfusion if serum bilirubin approaches 20–25 mg/dL.
3. Conjugated disease of the newborn is most often caused by rhesus incompatibil-

ity. It may require exchange transfusion immediately after birth or even before
birth.
Conjugated hyperbilirubinemia Elevated conjugated with almost normal unconjugated

bilirubin indicates biliary obstruction (cholestasis), either intrahepatic (bile canaliculi
blocked) or posthepatic (hepatic or common bile duct blocked). Obstructive jaun-
dice can be caused by gallstones, malignancies, or severe acute liver diseases. Chronic
biliary obstruction can cause liver damage (biliary cirrhosis)!
Unconjugated hyperbilirubinemia is seen in many liver diseases including infections (he-

patitis, yellow fever) and toxins (carbon tetrachloride, acetaminophen, α-amanitin in
mushroom poisoning).
Urobilinogen can be measured in urine, blood, and stools. In cholestatic jaundice, urinary
and fecal urobilinogen disappear. The stools of these patients are clay-colored while the
urine is brown. Urinary urobilinogen is high in hemolytic jaundice and in nonspecific liver
damage.
Inherited defects of bilirubin metabolism
Crigler-Najjar syndrome type I is a severe congenital form of unconjugated hyper-

bilirubinemia (20 mg/dL), usually fatal in infants. It is caused by a complete deficiency
of UDP-glucuronyl transferase.
Crigler-Najjar syndrome type II is much milder, with a lot of bilirubin monoglucuronide

in the bile. Caused by a partial deficiency of the bilirubin-conjugated enzyme.
462
Purine Biosynthesis 24.3.1
Dubin-Johnson syndrome is a defect in the secretion of conjugated bilirubin and other

amphophilic substances conjugated to glutathione or glucuronic acid (defect in the
ABCC2 transporter (canalicular multiple organic anion transporter (cMOAT)), with
conjugated hyperbilirubinemia. Course of the disease is relatively mild, treatment is
not usually required.
24.3. Purines
24.3.1. Purine Biosynthesis
Most of our purines come from endogenous synthesis:

IMP, AMP, GMP PRPP
Purine biosynthesis ADP, ATP, GDP, GTP
GART
O O O O O NH2 ATP NH2
ATP AMP Gln Glu ADP + Pi
from O P O CH2 OH O P O CH2 O P O P O O P O CH2
pentose O O O
phosphate O O O O O O NH
pathway Formyl-THF
Rib P
OH OH OH OH PPi OH OH Gly
THF
Rib-5-P Phosphoribosyl pyrophosphate Gln-PRPP Phosphoribosyl amine
(PRPP) amino transferase
O
H Glu H
-
CO2 N Gln N
COO O Asp C N H2O
O N C O C O
COO
-
C H H
N N
H H2N N H2N N HN NH O NH
H2N N Rib P Rib P Rib P Rib P
ADP+Pi ATP
Rib P AIRC
Adenylosuccinate
lyase
H2
-
Fumarate - C COO
COO HC
O O O
Formyl-THF THF NH NH2
C N C H2O C GTP GDP+Pi
H2N N HN N C C
H2N N N
HN HN
H2N N O N N N N
H N N N N
Rib P Rib P Rib P
IMPS Rib P Rib P
Inosine monophosphate Asp
Furmarate AMP
(IMP)
Note: GTP is used for the synthesis of AMP Adenylosuccinate Adenylosuccinatete
ATP is used for the synthesis of GMP synthetase lyase
H2O
IMP dehydrogenase NAD
+
GMP synthetase
AMP GTP
O ATP O
AMP+PPi
C C N
HN N HN +
NADH + H
H2N N N O N N
H AMP desaminase
Rib P Glu Gln Rib P GMP reductase
GMP
GMP Xanthosine monophosphate +
NH4
(XMP)
GDP ATP
XMP GTP
+
NH4
The amidotransferase catalyzes the committed step in purine synthesis.
Regulation is by feedback inhibition:
463
Ribose 5-P PRPP Phosphoribosylamine IMP
Inhibited Inhibited
Inhibited by by AMP by GMP
purine nucleotides
AMP GMP
VI. PURINE DEGRADATION AND SALVAGE REACTIONS.
Purines are degraded to uric acid:
Adenine nucleotides Guanine nucleotides

24.3.2. Purine Degradation
Pi Pi
Inosine Guanosine
Ribose 1- P Ribose 1-P

Purines are degraded to uric acid (see fig.
Hypoxanthine 24.4). Uric acid is the only important product of
Guanine
purine degradation in humans.
Xanthine
Oxidase
Xanthine NH3
Xanthine O2
Oxidase
24.4. Pyrimidine Metabolism
O
H
N
HN Uric Acid
O
O N N
H
Pyrimidines are synthesized form H carbamoyl phosphate and aspartate. Carbamoyl phos-
phate is made by a cytoplasmic enzyme distinct from the mitochondrial carbamoyl phos-
phate synthetase of the urea cycle. Ribose 5-phosphate (from PRPP) is added after the
Uric acid is the only important product of purine degradation in humans.
synthesis of the pyrimidine ring (see fig. 24.5).
Salvage reactions recycle the bases by reacting them with PRPP:
The cytosolic carbamoylAdenine-phosphoribosyl

phosphate synthetase is transferase
feedback-inhibited by CTP.
Adenine + PRPP AMP + PPi
Hypoxanthine-guanine
Hypoxanthine + PRPP Phosphoribosyl transferase IMP + PPi
Familial
Guanineorotic aciduria is caused
+ PRPP by a deficiency of one of the enzymes
(HGPRT) GMP +that
PPi convert orotic
acid to the uridine nucleotides. With growth retardation, megaloblastic anemia, and a
crystallineThese reactions
sediment reduce
of orotic aciduric acid
in the formation,
urine. and they
The patients areminimize the auxotrophs
pyrimidine need who
for de novo synthesis.
respond to large doses of orally-administered uridine. A secondary form of orotic aciduria
(but without dependence on
VII. PYRIMIDINE exogenous pyrimidines) also occurs in those urea cycle enzyme
METABOLISM.
deficiencies in which carbamoyl phosphate accumulates. Carbamoyl phosphate leaking out
of the mitochondria bypasses the rate-limiting step of pyrimidine biosynthesis.
233
464
465
24.5
OH Pi Rib P OH OH
N N N N N N
H2N N N Purine nucleoside H2N N N Pi HO N N
phosphorylase H2O
Rib Rib
Guanine + Rib P Xanthosine
Guanosine NH4
Guanine
PNP deficiency (#613179): T-cell deficient, Purine nucleoside
B-cells normal, neurologic problems (pyramidal desaminase
phosphorylase
signs, spaticity)
Figure 24.4.: Purine degradation
dGTP accumulation -> low ribonucleotide reductase OH

-> low [dNTP] O
N N
Pyrimidine Metabolism
HN N
HO N N
Purine degradation HO N N O
Xanthine
H2O2 O2+ H2O urate elevated uric acid -> gout
Xanthine
O2+ H2O oxidase H2O2 H
+ pKa = 5.75
NH NH4
+
OH Pi Rib P OH Mo, 4 FeS, FAD OH O
2 H2O
H
N N N N N N N N HN N
N N Adenosine Purine nucleoside
N N N N HO N N OH HO N N O
desaminase phosphorylase
Rib Rib lactim-form
Adenosine Inosine Hypoxanthine Uric acid lactam-form
ROS
ADA deficiency (#102700): severe combined immunodeficiency (SCID) in animals (except primates,
NK-, T- and B-cells cannot develop -> "bubble baby" reptils, birds) further enzymatic
[dATP] increased 100 times O2+ H2O H2O2 spontaneous
OH OH degradation to
-> ribonucleotide reductase downregulated - allantoin (mammals)
-> low [dNTP] -> no cell division CO2 -allantoate (bony fish)
additional problems with S-adenosylhomocysteine hydrolase N N
- urea (amphibians, selachians)
-> no DNA methylation -> apoptosis N N - ammonia (marine invertebrates)
Treatment: PEGylated bovine ADA (i.m.), bone marrow transplant N N HO N O
N
NH2 N
Allopurinol Oxypurinol
O N N O
suicide inactivation of xanthine oxidase by Allopurinol
Allantoin
CAD
UTP Gln + CO2
2 ATP orotate aciduria in some urea cycle defects: mitochondrial
Carbamoylphophate
carbamoylphosphate spills into cytosol and is converted to
synthetase II
PRPP orotate -> relieve of nitrogen stress
(cytosolic)
MAP
2 ADP + Pi
Growth kinases
+ Glu
factors O O H2O O
H2N O Pi H
+ H
H3N H C C O N H C O N H C
C O C C O C C O
O CH2
+ O
H2N Dihydroorotase HN CH
Asp-transcarb- CH2 2 NAD
+
C O P O C C
amoylase O
O O O O NADH + H
+
Carbamoyl N-carbamoyl- Dihydroorotate
Asp
phosphate aspartate Dihydroorotate
dehydrogenase
O
H
(inner membrane of mitos)

CTP ATP O N C
C C O
Pyrimidine biosynthesis HN
C
CH
Figure 24.5.
O
Orotate
Pi Orotate phospho- PRPP
ADP ribosyl transferase
ATP
NH2 Glu O 2 ADP 2 ATP O CO2 O PPi
Gln
C C C C
N CH HN CH HN CH HN CH
C CH Cytidylate C CH UMP kinase C CH orotidylate C C O
O N synthetase O N NDP kinase O N decarboxylase O N C
P P P Rib P P P Rib P Rib P Rib O
Orotidylate UMP synthase
CTP UTP UMP
+
NADPH + H defective in hereditary orotate aciduria
ribonucleotide (interferes with growth and development,
lack of folate: dUTP build into DNA instead of dTTP reductase
-> strand breaks from DNA repair enzymes -> cancer hypochromic anemia)
H2O + Treatment: oral uridine
Folate antagonists: Methotrexate, sulfonamides NAD
O DHF Methylene-THF O
C CH3 C
HN HN CH
C CH Thymidylate C CH
O N O N
synthase
P dRib P dRib
24.5
466
dTMP dUMP
Deoxyribonucleotides 24.6
24.5. Salvage pathways
Salvage reactions recycle the bases by reacting them with PRPP:

O
O O O HN
O P O CH2 O P O P O
O O N
O O O H
Uracil
PPi
OH OH Uracil
phosphoribosyl
PRPP
transferase (UPRT)
O
O
NH2 O O CH3
HN
HN
N N N N N N O N
O N
N N or Rib P
H2N N N N N Rib
H H H
Adenine UMP Thymidine
Hypoxanthine Guanine
Adenin
phosphoribosyl ATP
transferase Thymidine
(APRT) PPi kinase
PPi ADP
Hypoxanthin/guanine
phosphoribosyl Salvage pathways O
transferase CH3
(HGPRT) HN
vide infra
O N
O O
NH2 Rib P
N N N N TNP
N N Thymidine kinase activates Aciclovir
H2N N N to the active form, which terminates
N N
N N the chain in viral DNA synthesis
Rib P Rib P
Rib P
AMP IMP GMP
These reactions reduce uric acid formation, and they minimize the need for the energy-
expensive de novo synthesis.
24.6. Deoxyribonucleotides
Deoxyribonucleotides are required only for DNA synthesis and repair. They are present at
concentrations well below those of the corresponding ribonucleotides. Reaction:
467
Base Base
H2 H2
P P O C O P P O C O
OH OH OH H
nucleotide diphosphate (NDP) deoxynucleotide diphosphate (dNDP)
H2O activity regulation:
substrate specificity:
dATP
ATP/dATP: reduction of UDP, CDP
ATP
dTTP: reduction of GDP
dGTP: reduction of ADP
SH S
ribonucleotide ribonucleotide
reductase reductase
SH S
either: or:
S SH S SH
Glutaredoxin Glutaredoxin Thioredoxin Thioredoxin
S SH S SH
Glutaredoxin reductase
2 GSH GSSG FADH2 Thioredoxin reductase FAD
Glutathione reductase
+ + +
NADP NADPH + H + NADPH + H
NADP
24.7. Anti-neoplastic and anti-bacterial drugs acting on

nucleotide metabolism
The dihydrofolate (DHF) formed in the thymidylate synthase reaction has to be reduced
back to tetrahydrofolate:
dihydrofolate
DHF + NADPH + H+ GGGGGGGGGGGGGGGGGGA THF + NADP+
reductase
Some anti-cancer drugs inhibit nucleotide synthesis:
Fluorouracil is converted to fluorodeoxy-UMP, an irreversible inhibitor of thymidylate syn-

thase. It is also incorporated in RNA in place of uracil.
Methotrexate (amethopterin) is a folate antagonist and inhibits dihydrofolate reductase.
Azaserine is a glutamine antagonist which inhibits the use of glutamine for purine synthesis,
the IMP → GMP and the UTP → CTP reaction.
468
Hyperuricemia and Gout 24.8
Bacteria, unlike humans, can synthesize their own folic acid, and rely on this capability
to support their rapid division. Sulfonamides inhibit this reaction competitively with p-
aminobenzoic acid. Today they are usually given together with a dihydrofolate reductase
inhibitor like trimethoprim, so that any folate produced or obtained from the host can not
be converted into the physiologically active form.
Virus also require nucleotides for their reproduction by the cell, nucleotide analogues may
be used to interfere.
24.8. Hyperuricemia and Gout
300–600 mg uric acid are produced per day, and 80 % of this is excreted in the urine. The
normal plasma concentration is 2–7 mg/dL. It is about 1 mg/dL lower in females than in
males and increases with age.
Clinical problems result from the low water solubility of uric acid. Uric acid is a weak acid
with pKa of 5.8. The acid form (at low pH) is less soluble than the salt form (at neutral or
alkaline pH), but also the sodium salt is poorly soluble when [Na+ ] is high.
Solubility
• in urine: 15 mg/dL at pH 5, 150 mg/dL at pH 7
• in serum: 7 mg/dL
Sustained hyperuricemia (serum urate > 7 mg/dL) causes sodium urate deposits in soft
tissues (tophi), and in joints where they cause the inflammatory response of gouty arthritis.
About 0.5 % of adult male and 0.1 % of females are affected.
Secondary gout is caused by an underlying disease. Most important are diseases with exten-
sive tissue destruction, including chronic hemolytic anemias, myeloproliferative disorders,
psoriasis, and malignancies. Radiation or chemotherapy of malignant tumors can cause
rampant hyperuricemia.
Primary (idiopathic) gout is not caused by another disease. There is either uric acid over-
production or impaired renal excretion. Patients with more than 600 mg uric acid in the
24-hour urine (15–25 % of patients) are overproducers. Some overproducers have increased
activity of PRPP synthetase or decreased activity of HGPRT.
A complete deficiency of HGPRT causes Lesch-Nyhan syndrome, an X-linked recessive
disease with mental retardation, spasticity, self-mutilation and rampant hyperuricemia.
Treatment of gout:
Colchicine and non-steroidal anti-inflammatory drugs are used to treat the acute attack.
469
O O
F F
HN HN Gln-Analoga
N
+ + + +
O N O N FdUMP NH3 NH3 N NH3
H H2 O H H2
Fluorouracil Rib P H2N C CH - N C CH -
HC C CH -
also inhibits mRNA processing COO COO COO
C C C C C O
O H2 Cl H2 O
Glutamine Acivicin Azaserine
dTMP
dUMP
N
Purin biosynthesis N O
thymidylate O2N CH3

synthase Trimethoprim SH S N NH2
Sulfonamides
N
N N N N N
Figure 24.6.
Rib
N N N N
N5,N10-methylene THF 7,8-DHF Folate synthesis
(bacteria only) 6-Mercaptopurine Azathioprin Ribavirin
Folate cycle inhibits Gln-PRPP aminotransferase
and adenylosuccinate metabolism
Precursor of 6-Mercaptopurine inhibits IMP-DH
after phosphorylation
Pyrimidine biosynthesis
OH H2 Cyt
+
NADPH + H HO C C
DHF-reductase C
Methotrexate O N
OH OH
C1-body Aminopterin Rib
+
from aa breakdown THF NADP
Deazauridine Cyclopentyl cytosine
gets phosphorylated and gets phosphorylated
then inhibits and inhibits
H2 Cyt OMP decarboxylase CTP synthetase
H2 Cyt
2 ATP 2 ADP P P P O C O
HO C O
HO
HO
Ribonucleotide reductase O
OH
OH OH
Cytosine arabinoside araCTP H2N N
H
inhibits DNA polymerase
Hydroxyurea
competitively with dCTP
(radical scavenger)
24.8
470
Probenecid is a uricosuric drug which inhibits the renal tubular reabsorption of uric acid.
Allopurinol is an inhibitor of xanthine oxidase. Less uric acid is formed, and the patient
excretes a mix of hypoxanthine, xanthine, and uric acid.
A high-purine diet (meat) can worsen gout, as the nucleotides contained are turned into uric
acid directly by the enterocytes. Alcohol causes acidemia, which lowers uric acid solubility,
worsening gout. This is the explanation for the gout attacks sensitive patients suffer in the
morning after a night of overindulgence. Cave: diabetic ketoacidosis!
Uric acid stones account for 5–10 % of all kidney stones. Treatment: increased fluid intake,
alkalinization of the urine, allopurinol.
• Summarize the principles of heme synthesis and degradation. Explain the patho-
mechanism of acute and chronic porphyrias, sideroblastic anemia and hyperbiliru-
binemia. Sketch how these conditions can be diagnosed and treated.
• Summarize the principles of purine synthesis and degradation. Explain the patho-
mechanism of PNP-deficiency, SCID and hyperuricemias. Sketch how these conditions
can be diagnosed and treated.
• Summarize the principles of pyrimidine synthesis and degradation. Explain the patho-
mechanism of orotate aciduria and folate deficiency. Sketch how these conditions can
be diagnosed and treated.
• Describe how nucleosides and bases are salvaged and state the significance of these
pathways.
• Explain the mechanism of anti-neoplastic and anti-infectious drugs that act on nu-
cleotide metabolism.
471
25. Nutrition During the Life Cycle
25.1. Nutrition in Pregnancy and Lactation
Good eating habits are essential in pregnancy

• To ensure that the mother maintains optimal nutritional status, which will be reflected
in the way she feels and behaves and her ability to cope with normal duties.
• To permit normal growth and development of the fetus in utero.
• To ensure the delivery of a baby of normal birth weight, >2500 g. Low birth weight
is associated with ill health and infections in the neonatal period.
Stages of pregnancy:
1. The periconceptional phase, up to 4 weeks after conception. Good nutrition during
this time period is important for normal implantation, normal cell divisions in the
embryo, and development of the placenta.
2. The period of organogenesis, 4-12 weeks after conception. This period requires a
balanced nutrition to permit normal fetal development. It is also the period of greatest
sensitivity to teratogens.
3. The period of rapid growth, 12 weeks - delivery. This period requires a substantial
increase in bulk nutrients (protein, calcium, iron) to support fetal growth.
Commonly encountered problems:
• Poor eating habits up to the time of conception resulting in poor nutrient stores: iron,
vitamin A, folic acid
• Nutritionally unbalanced slimming diets at or before conception.
• The mother may have had repeated pregnancies.
• The use of drugs and alcohol, especially during the phase of organogenesis, exposes
the fetus to teratogenic risks (fetal alcohol syndrome, cocaine-baby). Excessive
alcohol intake also affects the absorption, metabolism and excretion of many nutrients,
particularly zinc, magnesium, copper and iron.
There are important physiological changes during pregnancy:
473
• In the mother:
– Metabolic rate increases.
– Enlargement and growth of uterine walls
– Development of breast tissue in preparation for breast-feeding
– Development of the placenta
– Laying down of fat stores for lactation
– Increase in maternal blood volume by 20–50 %. This requires extra synthesis of

hemoglobin and albumin.
– Pregnancy hormones are produced by the corpus luteum (maternal origin) and
the placenta (fetal origin).
– Blood loss must be anticipated during delivery.
• In the baby:
– The fetus requires nutrients for rapid growth.
– The baby has to build up iron stores for 3 months.
The physiological processes demand increases in protein, zinc, calcium, iron, iodine, fo-
late, and vitamins C, A, and B6. Folic acid is extremely important because deficiency in
early pregnancy increases the risk of neural tube defects (anencephaly and spina bifida).
Pregnancy demands no special food, but a balanced and adequate supply of all essential
nutrients.
US Recommended Daily Allowances (RDAs) .

Nutrient Not pregnant Pregnant
Energy (kJ) 9200
1st trimester 10500
2nd + 3rd trimester 11700
Protein (g) 50 60
Calcium (mg) 800 1200
Iron (mg) 15 30
Folate (µg) 180 400
Iodine use iodized salt
Vitamin C (mg) 60 95
Vitamin A (µg) 800 1300
Vitamin D (µg) 8 10
474
Nutrition in Pregnancy and Lactation 25.1
All B vitamins should be increased. For vitamin A, both excess and deficiency have to be
avoided because excess vitamin A is teratogenic.
3. Weight gain during pregnancy:
Women who enter pregnancy 10 % or more below or 20 % or more above standard weight
for height have a greater risk of poor pregnancy outcome. During the first trimester the
mother should expect to gain 900–1800 g. During the last six months a weight gain of
500 g per week is acceptable. As a rule of thumb, weight gain should be between 11 and
12.5 kg for the entire pregnancy. Underweight women and teenagers may need to gain more.
Reducing diets are not recommended during pregnancy. Women who do not gain weight
often have underweight babies. However, large increases in caloric intake are not required
because placental hormones facilitate fat breakdown in the mother’s adipose tissue.
Tissue/Fluid Weight gain (kg)

Infant at birth 3.5
Placenta 0.5
Increase in blood volume 1.8
Uterus and muscles 1.1
Increase in breast tissue 1.4
Amniotic fluid 0.9
Mother’s fat stores 1.8–3.6
Calcium: Dairy products are the most convenient source of calcium.
Zinc: Nuts, shellfish and meat supply ample amounts but a diet too high in fiber will impair
absorption.
Iron: There are substantial amounts of iron in whole wheat bread, oats and other whole
wheat cereals. A good supply of vitamin C is required for efficient iron absorption.
Because heme iron is more readily absorbed than other forms of iron, animal foods
should be included in the diet.
Minor complaints during pregnancy Relaxation of the bladder and intestinal walls is
caused by pregnancy hormones. This can lead to urinary tract infections and constipation.
At least 8 glasses of fluid should be taken to counteract urine stasis, whole wheat cereals,
vegetables and fruit should be taken to prevent constipation.
Most mothers experience lack of appetite, early morning sickness, nausea, and aversion
to certain types of food (‘pregnancy sickness’). This is typical between weeks 4 to 12, at
the time of organogenesis. Pregnancy sickness is a normal defense mechanism that causes
the mother to avoid potentially toxic foods at a time when the fetus is most sensitive to
teratogens.
475
Smoking, alcohol, and caffeine-containing beverages should be discouraged. All pregnant

women are advised to take iron supplements daily, especially as anemia is a problem in
some prenatal clinics in the US (10 %). Fruit juice should be taken with iron supplements
to achieve maximum absorption.
25.2. Breastfeeding
Human milk is adapted to the precise needs of the young. With the exception of water and
lactose, human milk and cow’s milk are dissimilar in almost all respects.
Colostrum, the thin yellowish liquid that precedes mature milk, is often present before the
end of pregnancy but is secreted mainly during the first 5 post-partum days. It contains
less fat and lactose than mature milk and more sodium, chloride, and zinc.
25.2.1. Composition of Human Milk
The protein content is about 11 g/L, one third of that in cow’s milk. Casein is particularly
low, resulting in soft curd and easy digestibility. α, β-lactoglobulin in cow’s milk is a common
food allergen in infancy. Milk allergy causes eczema, diarrhea and asthma. Human milk does
not contain β-lactoglobulin but harmless α-lactoglobulin. Human milk is higher in taurine
and cystine and lower in tyrosine and phenylalanine than cow’s milk. This composition
is important for the pre-term infant whose liver is inefficient in converting methionine to
cysteine and in metabolizing phenylalanine and tyrosine. Human milk fat contains 14 %
polyunsaturated fatty acids (linoleic acid). The cholesterol content is some 10 to 20 times
higher than in cow’s milk. Fat-soluble vitamins are present, but vitamin K is low. All other
vitamins are present in adequate amounts. Human milk is rich in lactose (7 %).
The sodium concentration of human milk is relatively low. This is important because of
the limited capacity of the newborn kidney to deal with a heavy load of solute.
The calcium is absorbed better than the calcium in cow’s milk. Iron, copper, manganese,
zinc, magnesium and iodine are present only in small amounts. The iron content of human
milk is 0.2–0.3 mg/L. Close to 50 % of this is absorbed. The low iron content is advantageous
because iron is an essential nutrient for pathogenic bacteria. Iron is made unavailable to
bacteria by tight binding to lactoferrin (in milk) and transferrin (in the blood). Excess iron
would disrupt this protective effect. Full-term babies don’t need much iron because they are
born with a 3-months supply of iron. Only premature babies require iron supplements. The
absorption of zinc from human milk is more effective than that from cow’s milk because of
its association with a different zinc-binding protein.
Human milk contains antibodies of the IgA type. Colostrum is particularly rich in IgA.
Enteric antigen-stimulated plasma cells in the mother migrate to the breast tissue where
476
Dietary recommendations for lactating mothers 25.2.2
they secrete antibodies or are themselves secreted into the milk where antibodies are pro-
duced. Breast milk is known to prevent gastroenteritis in infants. Human milk (but not
cow’s milk) also has a high content of lysozyme.
Leucocytes are present in human colostrum. 90 % are macrophages, and 10 % are IgA-
producing lymphocytes. Sterilizing breast milk will completely destroy the cellular as well
as many of the protein components of breast milk and eliminate their antibacterial effects.
Post-partum amenorrhea is favored by breastfeeding, poor nutritional state, and phys-

ical illness. Mothers in good nutritional state, however, resume cycling 2–6 months after
birth in spite of continued breast-feeding. In non-contracepting human populations (as in
other animal species), the birth interval is determined by the duration of post-partum
amenorrhea.
25.2.2. Dietary recommendations for lactating mothers
Item Non-lactating Lactating

Energy (kJ) 9200 11300
Protein (g) 50 65
Iron (mg) 15 15
Fluids Extra fluids
The amount and composition of milk is affected by the mother’s nutrition: in malnourished
mothers, lactose remains constant but the pattern of fatty acids charges. The fatty acid
composition of the milk reflects that of the mother’s diet. Concentrations of water soluble
vitamins reflect the mother’s dietary intake. Most drugs ingested by a lactating mother
will appear in her milk. Moderate alcohol consumption by lactating women is considered
acceptable.
Breast-feeding depends on the functioning of reflexes in both the baby and the mother. The
‘rooting’, ‘suckling’ and ‘swallowing’ reflexes are present in newborns. Very small premature
babies may have weak reflexes. The mother has 2 important reflexes:
The prolactin reflex involves afferent nerve impulses from the nipple and areola to the
hypothalamus and the stimulation of prolactin release by lactation.
The milk ejection reflex involves afferent impulses to the hypothalamus. The hypothala-
mus induces the release of oxytocin, a hormone which contracts the myoepithelial
cells in the mammary gland. Emotional tension and stress inhibit this reflex, and the
main cause of lactation failure is thought to be inhibition of this reflex. The suckling
of the infant and consequent release of oxytocin decrease maternal uterine bleeding
and hasten the return of the uterus to its normal size.
477
The baby should be fed ‘on demand’ rather than by the clock. Because of its small stomach
capacity and rapid growth, the baby requires frequent feeding during the first few weeks.
Breast milk is the only food required by the baby during the first four months.
25.3. Feeding the Weaning Age Group
The weaning period is a transitional period, between the time the baby is fed entirely with
milk and the time she is feeding entirely on the adult diet. This is the period between 4
months and 2.5 a of age.
• Eating habits introduced during childhood are likely to continue until adulthood,
therefore good eating habits should be introduced early.
• From the age of about 4 months, the baby is able to swallow semisolid foods. There
is a need for increased nutrients at this time, particularly energy and iron.
• Weaning should be gradual. Abrupt removal of breast milk is totally unacceptable.
Force-feeding and bullying is to be strongly discouraged as the baby will develop a
hatred for food, associating mealtime with punishment.
• New foods should be introduced one at a time to reduce the risk of allergic reactions.
• The consistency of the meals is very important. Sieved vegetables, soft fruits, or root
crops with some gravy should be offered, followed by mixtures of two types of food
(sieved beans and root crop) and later still mixtures of three or four types of food
(e.g., chicken, bean, potato, vegetables). Sieved foods should be replaced by mashed
foods by eight months and chopped foods by one year.
• Because of the small capacity of baby’s stomach, and her high nutrient needs, feedings
must be small and frequent, and nutrient-dense food must be given.
• Exploring and experimenting are normal and desirable behavior patterns at that
age. A child’s impulses, if constantly denied, can turn to shame and self-doubt. A
child wanting to feed herself, making a mess at mealtime, or refusing to eat, behaves
normally for her age. Strategies must be used to deal with these behaviors, but no
attempts should be made to stifle them.
• There are advantages and disadvantages of commercial foods and family pot. Provided
that the family pot is nutritionally balanced and free from pathogens, it has the same
benefits as the commercial varieties.
The following is a guide to the ratio of the different foods to be used during preparation.
Animal Food 1 Tablespoon
Vegetables 1 Tablespoon
478
Feeding the School Child 25.5
Starch 4 Tablespoons
Legumes 2 Tablespoons
Mothers may choose between feeding meals from the ‘family-pot’ or using one of the com-
mercially prepared varieties. Special care must be taken to ensure that baby’s share is
removed before adding seasoning and salt and that bones and pits are removed. Popcorn
and nuts are forbidden and sweet foods are not to be encouraged. The practice of allowing
babies to continue bottle-feeding in bed leads to dental caries and buck-teeth.
25.4. Feeding the School Child
The aims are:
• To ensure that dietary intakes include a balance of macro- and micronutrients which
will enable the child to achieve optimal physical and mental development.
• To establish good dietary habits that are carried over into adulthood, for the preven-
tion of diseases later in life. These diseases include coronary heart disease, hyperten-
sion, osteoporosis, diabetes, and cancer.
The recommended distribution of calories is the same as for adults: Fat 30 %, protein 10–
15 %, carbohydrate 55–60 %.
Growth is affected by the diet, but anthropometric measurements must be interpreted with
caution. Children have growth spurts that take place at different ages in different children,
most obviously at puberty. On average, the highest growth rate for boys is during the
fifteenth year, but it is much earlier for girls.
Cognitive development is affected by nutrition. Better nutrition has been credited with
most or all of the Flynn effect: the secular increase to IQ that has been observed over
the past century. The Flynn effect is evident from preschool age to adulthood. Commonly
encountered problems in school children:
• Iron deficiency anemia is caused by poor diet in times of rapid growth.
• Obesity is caused by poor eating habits and lack of exercise.
• Dental caries are caused by the frequent consumption of sucrose.
Streptococcus mutans, the most important caries bacterium, polymerizes sucrose into a
dextran that glues the bacteria to the tooth enamel. The sugars in fruits and vegetables
and the lactose in milk and dairy products are less cariogenic than sucrose.
479
25.5. Feeding Adolescents
On average, the growth spurt for females begins at age 10–11 a and reaches a peak at
12 a. In males, it typically begins at age 12–13 a and peaks at 14 a. In females fat becomes
a larger percentage of total body weight and in males the lean body mass, muscle and
bone, becomes much greater. There are also hormonal changes that produce physically
and, hopefully, mentally mature adults within 2–3 years. There is tremendous variation in
teenagers’ rates and patterns of growth.
Energy Needs A rapidly growing 15 a old boy may need about 17 700 kJ/d. Girls of the
same age have stopped growing and a girl of 15 a may need only 8400 kJ/d, if she is not to
become obese.
Iron An ample intake of iron-rich foods should be encouraged in teenagers. Iron is im-
portant for a rapidly growing body, especially a female body that needs extra iron for
menstruation. The iron loss during each menstrual period is between 10 and 25 mg. Iron
needs pose a problem with teenagers because
• Traditional snack foods are low in iron.
• Some teenagers may be vegetarians.
• Some may adopt weird diets or may be bordering on anorexia nervosa.
Calcium The requirement for calcium reaches a peak during these years. Some teenagers
reject milk as “child’s drink’ - choosing sodas instead.
Nutritious types of snack should be encouraged. Emphasis should be placed on vitamin
C in citrus fruits and vegetables, deemphasizing soft drinks which only provide empty
calories. French fries and greasy burgers are high in salt and fat. Dairy products should be
encouraged. Nuts and beans should be encouraged for their vitamin B and iron contribution
to the diet.
25.6. Nutrition in the Elderly
Features of the aging process that are important for nutrition of the elderly:
• Poverty
• Ill-fitting dentures
• Lack of appetite due to reduction in taste buds
480
Nutrition in the Elderly 25.6
• Decreased metabolic rate due to total reduction of body cells

• Constipation, indigestion, chronic diseases
Most important: The elderly need love.
Many elderly take drugs on a regular basis. Common drug interactions:
Anticonvulsants affect vitamin D metabolism and folate absorption.
Biguanides (oral anti-diabetics) reduce B12 and folate status.
Tetracycline reduces leucocyte levels of ascorbic acid and increases its urinary excretion.
Aspirin treatment over time depletes the tissue stores of ascorbic acid and induces chronic
G.I. bleeding leading to iron-deficiency anemia.
Thiazide diuretics
Iron deficiency anemia in the elderly can be caused by:
• Reduced gastric acid secretion, which impairs iron absorption.
• Heavy reliance on tea and toast. The tannins in tea prevent iron absorption.
• Chronic blood loss: ulcers, hemorrhoids.
• Prolonged use of antacids which interfere with iron absorption.
The elderly require less energy due to their decreased metabolic rate. However too low an
energy intake may lead to a decreased intake of other nutrients.
Inadequate protein intake can lead to low hemoglobin levels (anemia), with apathy and
fatigue. Encourage low-calorie sources of high-quality protein: low-fat dairy products, fish,
liver, lean minced meat. Protein should supply 12 % of the total energy intake.
Fat should be limited to keep calories down and to lessen the risk of atherosclerosis, cancer,
and arthritis. Fat should represent 20 % of total caloric intake, mainly poly- and mono-
unsaturated which will lead to a reduction in blood cholesterol.
Fiber-rich foods help prevent constipation, and they keep the blood cholesterol down.
Whole-grain cereals, root crops and fruits contribute to the energy content of the diet
while also providing significant quantities of vitamins.
Mineral deficiencies e.g. depletion of K+ , has been associated with muscle weakness and
mental confusion. Depletion of zinc can lead to decreased taste acuity, anorexia and delayed
wound healing.
An adequate intake of calcium is necessary to prevent osteoporosis. Milk and milk products
and fish with soft bones are good sources. Milk can be incorporated into the diet in porridge,
yogurt and ice cream.
481
Fluids are important. Some elderly people have a fading sense of thirst. Some fear fluids
to avoid incontinence and may become dehydrated in the process. Dehydration can lead to
confusion, headaches, and irritability.
Physical activity is very important for the well-being of the elderly. It prevents muscular
atrophy and the development of osteoporosis, and it also increases the blood flow to brain
and limbs.
Fruits and vegetables should be emphasized as sources of vitamins and minerals. Overcook-
ing leads to destruction of vitamins B and C, and vitamin E is usually destroyed in some
canned processed foods, therefore advice should be given on how to buy, cook and store
vegetables, with emphasis on food spoilage, which may lead to diarrhea.
Sunshine is important to provide vitamin D for the bones.
1. Articulate the dietary guidelines and recommendations for Americans.

2. Define the nature and types of dietary protein and its contributions to the nutritional
content of the diet.
3. Name the coenzyme forms of water-soluble vitamins niacin, riboflavin, thiamine, B6,
pantothenic acid and folic acid.
4. Describe the deficiency syndromes for niacin, thiamine, ascorbic acid, folic acid, vita-
min B12 and vitamins A, D and K.
5. Name those vitamins that function as antioxidants.
6. Outline the metabolism and inter-organ transport of vitamins A and D.
7. Explain the reasons for bone demineralization in rickets.
8. Describe the approximate distribution of iron in the body and the functions of iron
containing proteins ferritin, hemosiderin and transferring.
9. List typical situations in which iron deficiency anemia is encountered.
10. Describe the origin, clinical presentation and treatment of iron overload.
11. State the importance of hemoglobin concentration, total iron binding capacity, iron
saturation of transferring and serum ferritin as laboratory test for the evaluation of
the iron status.
12. Identify dietary sources of heme iron and non-heme iron, and explain factors which
inhibit and enhance iron absorption.
482
13. Explain the term “protein sparing action of carbohydrates”.

14. Define the metabolic rate and the factors which influence the metabolic rate.
15. Discuss energy requirements in relation to physical activity.
16. Articulate the basic principles, advantages and disadvantages of a vegetarian and
vegan diet
17. Describe the chemical composition, caloric value and food sources of fats.
18. Define osteomalacia, osteoporosis and outline their causes and prevention.
19. Take a diet history and analyze its content.
20. List the nutritional needs in pregnancy, lactation, childhood, adolescence, and the
elderly.
21. Identify measures to enhance calcium, iodine, protein, vitamins A and C, folate and
iron contents of diets during pregnancy.
22. Explain the importance of breastfeeding during infancy, and the precautions necessary
for successful bottle feeding.
23. Discuss the treatment of the diarrheal child.
24. Define obesity and describe its harmful effects and its prevention.
25. Explain the causes, prevention and treatment of kwashiorkor, marasmus and failure
to thrive in infancy.
26. Describe the types, consequences and dietary treatments of hypercholesterolemia.
27. Prepare diets to meet the caloric and other nutritional needs of patients suffering
from hypertension, diabetes type I and II, coronary heart disease, obesity, anorexia
nervosa and kwashiorkor.
28. Discuss the role of desirable lifestyles in control of chronic diseases and define the
importance of nutritional education to combat chronic diseases.
29. List the recommended guidelines for fat intake as directed by the American Health
Association.
30. Describe the role of diet in the management of renal failure, nephrotic syndrome,
hepatic coma, liver cirrhosis, hepatitis, cholecystitis, peptic ulcer, burns, post surgery,
trauma and malabsorption syndromes.
31. List the principles of enteral and parenteral nutrition. Define the major types of food
sensitivity: Celiac disease, egg, peanut and soya allergy, and lactose intolerance.
483
26. Cell Cycle Control and Cancer
26.1. Cell Cycle Control
Dividing cells proceed through the cell cycle, while resting cells are in G0. G0 is a diploid
state like G1, but it is physiologically different: Many genes that are required for cell cycle
progression are expressed in G1 but not G0. Conversely, many genes connected with the
terminal differentiated phenotype of a cell is expressed in G0 but not in G1.
Apoptosis is “programmed” cell death. It occurs during normal embryonic development, in

regenerating epithelia, in response to hormones, and in response to severe damage, especially
DNA damage. The induction of specific proteases is a key event in apoptosis.
Two mechanisms are important for cell cycle control:
• Proteins that are required for specific stages of the cell cycle are produced only at
that time. Cyclin proteins orchestrate these changes. Many enzymes of DNA synthe-
sis (DNA polymerase α, the polymerase δ subunit PCNA, thymidylate kinase) are
transcriptionally induced shortly before S phase and degraded shortly after.
• Other proteins are present throughout the cell cycle but are regulated by phosphory-
lation. Some structural proteins become phosphorylated at the start of mitosis: chro-
mosomal scaffold proteins assemble in response to phosphorylation, and the lamins of
the nuclear lamina disassemble. Also many transcription factors and other regulatory
proteins become phosphorylated and dephosphorylated at different points in the cell
cycle.
There are two main checkpoints at which the cell makes an all-or -none decision:
• The G1 checkpoint is in late G1. The cell commits itself to DNA replication.
• The G2 checkpoint is in late G2. The cell commits itself to mitosis.
Note that the processes of DNA replication and mitosis cannot be interrupted once initi-
ated. (Interrupting the cell cycle in metaphase with the drugs colchicine or colcemid will
eventually lead the cell to die.)
485
26.2. Normal Cells in Culture
Most cells can be grown outside the body in suitable nutrient media. Properties of cultured
cells:
Mitogen dependence Mitogens are growth factors that are necessary for cell division. Pu-
rified growth factors (EGF, PDGF) can be used, or serum. Serum (but not fresh
plasma) contains PDGF, released from activated platelets during blood clotting. Also
nonphysiological mitogens (phytohemagglutinin for leucocytes) can be used.
Anchorage dependence All cells, except leukocytes, require a solid support for growth in
culture.
Contact inhibition Cells are normally inhibited by neighboring cells. Therefore cultured
cells grow only until a continuous monolayer has formed.
Mortality Cultured cells divide many times in culture (50–100 times in the case of fibrob-
lasts), then go into senescence and die.
These limitations on cell growth apply to normal cells, not cancer cells.
26.3. Cyclins and the Retinoblastoma Protein
The cell cycle is regulated by the concerted activities of nuclear protein kinases and protein
phosphatases. Cyclins are the regulatory subunits of nuclear protein kinases. Important
cyclins:
Cyclin D: Present throughout the cell cycle. It is induced by mitogens. Activation of this cy-
clin provides an important link to extracellular hormones. MAP kinase and PI3K/Akt
signal transduction pathways lead to cyclin D1 activation, and cell cycle progression.
Cyclin E: A “G1-cyclin”. It brings the cell through the G1 checkpoint.
Cyclin A: Most abundant during S phase and early G2.
Cyclin B: The classical “G2-cyclin”. It brings the cell through the G2 checkpoint and is
required for the phosphorylation of chromosomal scaffold proteins and lamins. It is
degraded suddenly during mitosis.
Control of the G1 checkpoint:
• Entry into S phase requires the transcription of genes for DNA polymerases and
other proteins of DNA replication. Transcription of these genes requires the dimeric
transcription factor E2F/DP.
486
p53 and the Damage Response 26.5
• E2F/DP is bound to the promoters of the regulated genes at all times, but during
early and mid-G1 its transcriptional activator domain is masked by the tightly bound
retinoblastoma protein (pRb).
• pRb is a phosphoprotein. Only the hypophosphorylated form suppresses E2F/DP.
• At the G1 checkpoint, pRb becomes phosphorylated by complexes of cyclin D and

cyclin E with cyclin-dependent protein kinases (Cdks). As a result, pRb falls off
the transcription factor and the E2F/DP regulated genes are expressed. pRb is the
“guardian of the G1 checkpoint”.
26.4. p53 and the Damage Response
Cells have two possibilities to respond to DNA damage:
1. Passage through the G1 checkpoint is blocked, and repair enzymes are induced. The
cell must not replicate its DNA until all the damage is repaired!
2. The cell undergoes apoptosis. This prevents survival of aberrant, irreversibly damaged
cells.
The choice between these two alternatives depends on the cell type and the kind and
severity of the damage. Not only DNA damage but also other kinds of stress (heat stress,
or inhibition of transcription or translation) can trigger these responses. Sequence of events
leading to apoptosis:
• DNA damage, oxidative stress, hypoxia and telomere erosion induce the synthesis
and inhibits the degradation of the p53 protein. ATM kinase also phosphorylates p53
causing it to accumulate in the nucleus.
• p53 is a transcription factor which binds, in an oligomeric form, to the regulatory

sites of some genes. It stimulates the expression of genes for DNA repair and cell
cycle arrest, and/or for apoptosis.
• One of the p53-induced genes codes for the Cdk-inhibitor p21 (= waf-1). p21 causes
cell cycle arrest.
The mediators of apoptosis are not well known.
487
26.5. Growth Control by External Stimuli
In order to establish and maintain the normal histology of the body, every cell has to
respond to a multitude of external stimuli. External stimuli can affect:
• The growth rate of the cell.
• The rate of cell division. Growth rate and mitotic rate are usually regulated in parallel.
• Cell motility. Most cells can creep, and they do so most obviously during embryonic
development and wound healing.
• Cell shape. Both motility and shape changes require regulation of the cytoskeleton.
External stimuli include:
Cell-cell contact Primary cells in culture stop dividing when they contact other cells. There
are many different cell adhesion proteins. They are integral membrane proteins. Most
of them make homotypic interactions with the same adhesion proteins on neighboring
cells. They interact with the cytoskeleton through peripheral membrane proteins. Cell-
cell contact triggers cytoskeletal remodeling and signaling cascades into the nucleus
that mediate contact inhibition.
Cell-matrix contact Primary cells in culture have to contact a polystyrene or glass surface
in order to grow. In vivo, cells make contact with the extracellular matrix. Cell-matrix
interactions are mediated by receptors of the integrin type. Along with peripheral
membrane proteins, attached cytoskeletal proteins, and signaling proteins (protein
kinases, G-proteins), they form the focal adhesions. Cell-matrix contact affects cell
shape and motility and is required for normal growth (anchorage dependence!).
Growth factors Cells respond to soluble growth factors that stimulate or (less commonly)
inhibit cell growth and proliferation. Most growth factors act locally in the tissue
where they are formed, but some circulate as hormones. Also “ordinary” hormones
and their second messengers can affect cell growth. The Phospholipase C-IP3-calcium
system is mitogenic for many cells. cAMP inhibits the proliferation of white blood
cells and fibroblasts but stimulates many endocrine cells.
488
Mitogenic Signaling 26.6
Growth factor Tissue of origin Target tissue Effect

Nerve Growth Sympathetically Sympathetic ganglia + Growth
Factor (NGF) innervated tissues
+ Differentiation
- Mitosis
Insulin-like Liver Many cells + Growth
Growth Factor-1
(IGF-1)
+ Mitosis
Platelet-derived Platelets Many cells + Mitosis
Growth Factor
(PDGF)
Epidermal Many cells Many cells + Mitosis
Growth Fac-
tor (EGF)
Fibroblast Many cells Many cells + Mitosis
Growth Fac-
tor (FGF)
Erythropoietin Kidney Bone marrow + Differentiation
Transforming Many cells Many cells - Mitosis
Growth Factor
(TGF)
Note: IGF-1 is also produced locally in many different tissues.
26.6. Mitogenic Signaling
Mitogens trigger phosphorylation cascades that regulate the phosphorylation states of nu-
clear transcription factors and other regulators of gene expression.
Most growth factor receptors have an intracellular protein tyrosine kinase domain. The acti-
vated receptors aggregate and phosphorylate each other. The autophosphorylated receptors
bind cytoplasmic signaling proteins, thereby recruiting them to the plasma membrane, ac-
tivating them allosterically, and/or tyrosine-phosphorylating them. This triggers several
signaling pathways, including:
Activation of phospholipase C (PLC) PLC has several isoenzymes.
PLC-β is stimulated by hormone-activated G-proteins.
PLC-γ becomes tyrosine-phosphorylated and thereby activated by growth factor re-
ceptors. The diacylglycerol formed by PLC activates protein kinase C (PKC).
Activated protein kinase C stimulates NF-κB activity.
489
Activation of the MAP kinase pathway. 1. Through adapter proteins, the autophos-
phorylated growth factor receptor activates the Ras protein, a protein similar
to G-protein GTP binding proteins. Unlike the hormone-regulated G-proteins,
Ras has only one subunit, but it also cycles between an inactive GDP-bound
form and an active GTP-bound form.
2. Ras-GTP activates the Ser/Thr protein kinase Raf. [Ras-GTP also activates
PI3-Kinase which activates a protein kinase called Akt (Also known as Protein
Kinase B). Akt activation inactivates inhibition of Cyclin D1 (Cell growth is
stimulated).]
3. Raf phosphorylates and activates protein kinases of the MEK (= MAP ki-
nase/Erk kinase) type.
4. The MEKs activate proteins of the MAP kinase family by phosphorylation on

closely-spaced Thr and Tyr side-chains.
5. The MAP kinases enter the nucleus and phosphorylate nuclear proteins on Ser
and Thr side-chains. They phosphorylate cytoplasmic target proteins as well,
and they stimulate ribosomal protein synthesis non-selectively.
The MAP kinase pathway is also stimulated by insulin in many cells.
Receptors for cytokines, including interferons, interleukins, erythropoietin, prolactin, growth

hormone and leptin, recruit a Tyr-specific protein kinase (Janus kinase) which activates a
transcription factor (signal transducer and activator of transcription (STAT)).
26.7. Principles of Malignant Transformation
Neoplasia (“new growth”) is the abnormal proliferation of a cell population that is derived
from a normal somatic cell. Benign neoplastic conditions are self-limiting, but malignant
ones (“cancer”) are deadly. Cancers are classified according to their cell of origin: sarco-
mas are derived from connective tissue cells and carcinomas from epithelial cells. Cancers
originate from a normal somatic cell by malignant transformation, usually through somatic
mutations. Therefore all mutagens are also carcinogens. The cells in a tumor are de-
rived from a single aberrant somatic cell: cancers are monoclonal in origin. The malignant
phenotype is heritable at the cellular level. Properties of cancer cells:
Mitogen-independent growth
Lack of contact inhibition
De-differentiation Cancer cells are poorly differentiated, much like embryonic cells.
490
Cellular Oncogenes 26.8
Disordered growth Cancer growth is chaotic, without any respect for anatomical bound-
aries.
Genomic instability Many cancer cells have chromosomal aberrations and/or a mutator
phenotype.
Immortality given nutritional requirements.
High mitotic rate The number of mitoses in cytological specimens is important for the
prognosis of cancer.
Metastasis Cells break loose from the tumor, are carried away by lymph or blood, and
establish secondary growths.
Cancer cells can be cultured easily. They need no mitogens, there is no anchorage depen-
dence or contact inhibition, and the cultures can be propagated indefinitely.
With rare exceptions, more than one mutation is required to make a cell malignant. Tumors
can vary in their degree of malignancy even if they are derived from the same cell type. This
is because each tumor has its own personal combination of cancer-inducing mutations.
Malignant tumors can arise from benign lesions, and low-grade malignancies can sponta-
neously transform into more malignant varieties. This is called tumor progression. It is
caused by the appearance of new mutations in an already abnormal cell population. The
more malignant mutants will always outgrow their less malignant neighbors. It’s evolution
in the fast track!
Oncogenes are genes in tumor cells whose expression promotes the malignant state. Onco-
genes are derived by activating somatic mutations from normal cellular proto-oncogenes.
Mutational activation leads either to structural alterations of the gene product, or its over-
production. Rarely, an oncogene is introduced by a virus.
Tumor suppressor genes are normal cellular genes that inhibit cell proliferation or invasive-
ness. Their inactivation can make the cell malignant.
Oncogenic activation is effective even if only one copy of a cellular proto-oncogene becomes
mutationally activated, but mutations of tumor suppressor genes cause malignancy only if
both copies of the gene are knocked out.
26.8. Cellular Oncogenes
Oncogenes are derived genes from mutation of proto-oncogenes. Most oncogene products
are involved in mitogenic signaling pathways. Thus the mutations are activating, and result
in hyperstimulation of mitogenic signaling pathways.
491
• Growth factors are rare as oncogene products, but the retroviral sis (simian sarcoma)
oncogene is a truncated version of PDGF.
• Growth factor receptors are frequent oncogene products. Most of these oncogenes code
for structurally abnormal growth factor receptors that are always switched on, even in
the absence of the ligand. Examples: erb B, neu. Also overexpressed but structurally
normal growth factor receptors are common in many cancers.
• Soluble tyrosine protein kinases belong mostly to the src family. The normal src fam-
ily kinases transmit mitogenic signals from growth factor receptors in focal adhesions.
The oncogenically activated forms are overactive and/or insensitive to negative con-
trols.
• Soluble serine/theonine protein kinases include Raf-related protein kinases. The onco-
genically activated forms are structurally abnormal variants that are constitutively
active.
• Small G-proteins of the Ras family are mutationally activated in 20 % of all cancers.
The oncogenic forms have point mutations which impair the intrinsic GTPase activity:
once activated, the Ras protein is locked in the “on” conformation.
• Nuclear transcription factors are encoded by many oncogenes, including myc, jun and
fos. The myc oncogene (normally activated by mitogens as an early-response gene) is
amplified in many tumors.
26.9. Nuclear Proteins in Cancer
Abnormal regulation of the G1 checkpoint is common in cancers. Typical defects are:
• Overexpression of cyclin D1, the major D-cyclin. Also cyclin A is occasionally over-
expressed.
• Cdk4, the major catalytic partner of the D-cyclins, is overexpressed in some cancers.
• Cdk-inhibitors are inactivated in many cancers, especially INK4a (inhibitor of kinase

4), an inhibitor of Cdk4 that mediates growth-inhibiting stimuli.
• The retinoblastoma protein, pRb, is inactivated in many cancers.
Also components of the DNA damage response system are abnormal in many cancers:
• p53 is mutationally inactivated in at least half of all spontaneous tumors. This is the
most common type of abnormality in spontaneous cancers.
492
Inherited Cancer Susceptibility 26.11
• Mdm2 is a protein that inhibits p53 in normal cells. Many of those tumors that have
intact p53 overexpress Mdm2. Anti-neoplastic drugs are in trials which disrupt the
Mdm2-p53 interaction in cancer cells.
Mutational inactivation of the p53-induced Cdk-inhibitor p21 is extremely rare in cancer.
Therefore the apoptosis-inducing activity of p53 appears to be more important than its
anti-mitogenic effect for tumor suppression. Many oncogenic mutations, for example myc
amplifications, lead to apoptosis in cells with an intact p53 system. Cancers lacking p53 or
overexpressing Mdm2 have high mutation rates and are sensitive to mutagens, but they are
often resistant to anti-tumor drugs because they cannot go into apoptosis.
26.10. Virally-Induced Cancers
Virally induced cancer is rare. For specific viral carcinogenesis, the virus has to integrate its
DNA into the host cell genome. The integrated viral DNA can stimulate the expression of
cellular proto-oncogenes by promoter insertion or enhancer insertion, but some virus have
their own oncogenes.
Oncogenic retrovirus integrate their own cDNA into the host cell DNA during their
normal life cycle. Oncogenic retrovirus have a viral oncogene in addition to the viral gag, pol,
and env genes. The oncogene has been hijacked from a host cell during retroviral evolution.
Rous sarcoma virus, which causes sarcomas in chickens, contains the src oncogene. It is
fully infective, but all other known oncogenic retrovirus are defective and can replicate only
if the cell is also infected with another, intact retrovirus. Retroviral cancers are extremely
rare in humans.
Oncogenic DNA virus integrate their DNA into the host cell genome only by accident.
Some DNA virus contain oncogenes that stimulate the proliferation of the host cell during
normal infection and can make the cell cancerous if the viral DNA becomes integrated into
the host cell DNA. Some strains of human papilloma virus (wart virus) contain oncogenes
whose products inactivate p53 and pRb, the products of the major cellular tumor suppressor
genes. These viral oncogenes are unrelated to normal cellular genes. Permanently integrated
papillomavirus DNA is present in most cervical cancers, many other anogenital cancers and
in Kaposi-sarcoma.
26.11. Inherited Cancer Susceptibility
A minority of cancers, including 5 % of colon cancers and 5 % of breast cancers, occur in

patients with inherited cancer susceptibility (these numbers come from a genetics text book;
recent results indicate that at least for breast cancer, the true number is higher). Benign
493
tumors can also be caused by inherited mutations. Most cancer susceptibility syndromes
are caused by an inactivating mutation in a tumor suppressor gene. Susceptible individuals
are heterozygous for the gene defect, therefore the cancer susceptibility is inherited as
an autosomal dominant trait, normally with reduced penetrance. Somatic cells with the
heterozygous mutation grow normally, but a cell that loses the second, intact copy of the
gene is likely to become neoplastic. From the cell’s point of view, the mutation that makes
it malignant is recessive.
Inherited cancer show a number of characteristics that a clinician should keep an eye on:
these patients show up with a cancer earlier than the average patient; these patients have
more than one focus more often than other patients; there is additional support for suspicion
of an inherited cancer if another family member has an otherwise rare cancer. The increased
risk of specific types of rare cancers are characteristic for each familial cancer gene, e.g.,
BRCA1 predisposes for breast and ovarian cancer and more rarely for cancer in colon and
prostate, while BRCA2 predisposes for breast cancer, also with some risk in male carriers
while ovarian cancer is less common than in BRCA1, and furthermore BRCA2 carriers have
a low but increased risk for cancer in prostate, pancreas, throat, esophagus, colon, as well
as gall bladder and ducts.
Retinoblastoma is a rare tumor of immature retinal cells in infants. In 60 % of the cases,

only one tumor is present and there is no family history. In 40 %, multiple tumors are present
and/or there is a family history. These patients are born with an inactivating mutation of
the retinoblastoma (Rb) gene in all their cells, either as a result of a new mutation (no family
history), or inheritance from an affected parent. When a somatic mutation inactivates the
second, intact copy of the Rb gene, the cell becomes malignant. This happens in 90 % of
those with the inherited mutation (90 % penetrance), and most of the time it happens
in more than one cell. Some of the survivors get sarcomas later in life. Homozygous Rb
mutations are also seen in sporadic retinoblastoma, and in many bladder cancers, sarcomas,
and other malignancies.
Patients with Li-Fraumeni syndrome have a high risk of breast cancer, sarcomas, leukemias,
brain tumors, and adrenocortical cancer. It is caused by mutations in the p53 gene. Most
patients have point mutations affecting the DNA-binding region of the p53 protein. Cells
become cancerous when the second copy of the p53 gene is lost and additional oncogenic
mutations take place.
Breast cancer is caused by inherited cancer susceptibility in 2–5 % of all patients. Inherited
mutations in the BRCA1 and BRCA2 tumor suppressor genes cause both breast and ovarian
cancer, with a lifetime risk of 80 % (lower in some studies). The tumors of these patients
have homozygous inactivation of the tumor suppressor gene. BRCA1 and BRCA2 are also
inactivated in a few spontaneous breast cancers but less often than e.g., the APC gene is
involved in spontaneous colon cancer. Inherited cancer susceptibility has to be suspected in
cases of early-onset breast cancer with a positive family history.
494
Patients with adenomatous polyposis coli (APC) gradually develop thousands of polyps
in the colon mucosa, and one or several of them become malignant sooner or later. The
affected gene codes for a membrane protein that may be important for contact inhibition.
When the second copy of the gene becomes inactivated, a polyp forms. Additional mutations
can create a malignant tumor. Most spontaneous colon cancers also have homozygous APC
inactivation, however, few polyps are expected in cases of spontaneous APC. Prophylactic
removal of the colon is recommended for established cases of familial APC.
Hereditary non-polyposis colorectal carcinoma (HNPCC) is caused by inherited

mutations in a gene for post-replication mismatch repair. When the second copy of the
gene is inactivated by somatic mutation, the cell develops a mutator phenotype. Somatic
mutations accumulate rapidly, until the cell either dies or becomes malignant.
Most patients develop colon cancer, but the risk of other cancers is also increased. Several
(non-allelic) mismatch repair genes are affected in different patients. Mismatch repair defects
are also seen in some spontaneous cancers.
Patients with neurofibromatosis have multiple neurofibromas (nerve sheath tumors), cuta-
neous café-au-lait spots, and iris haematomas (Lisch nodules). The tumors are benign, but
some patients develop neurofibrosarcomas. The most common form of the disease (NF-1,
von Recklinghausen’s disease) is caused by inactivating mutations in the gene coding
for a Ras-GTPase-activating protein, but the pathogenic mechanism is unclear. Homozy-
gous inactivation of NF-1 is seen in malignant tumors of NF-patients, but not necessarily
in the benign neurofibromas.
Genetic screening for inherited cancer susceptibility is becoming possible, especially for
the relatively common breast cancer mutations. A test is already available for a BRCA1
mutation that occurs in 1 % of all Ashkenazi Jews and is responsible for 10 % of breast
cancers in this population.
1. Define the term checkpoint with respect to the cell cycle. Name two cell-cycle check-
points.
2. Describe the roles of cyclins, retinoblastoma protein (pRb), and mitogens in control
of the cell cycle.
3. Give examples of transcription factors, structural nuclear proteins and enzymes whose
abundance or phosphorylation state fluctuate during different stages of the cell cycle.
4. Explain the cellular process of apoptosis and its relation to the activity of the p53
protein. Give examples of when apoptosis is likely to occur.
495
5. List major signal transducing events in the MAP kinase, phospholipase C and PI3K/Akt
pathways.
6. Give examples of oncogenes encoding growth factor receptors, GTP-binding proteins,
non-receptor tyrosine kinases and transcription factors.
7. Describe the two- hit model for the inactivation of tumor repressor genes, and the
mechanism by which the mutations in proto-oncogenes cause excessive cell prolifera-
tion.
8. Compare retroviral oncogenes with cellular oncogenes and with the oncogenes of the
papillomavirus. Describe how virus can activate cellular proto-oncogenes by promoter
insertion and enhancer insertion.
9. Name two molecular lesions that are likely to cause genomic instability and a mutator
phenotype in cancer cells.
10. Define the terms hyperplasia, hypoplasia, and metastatic growth. Name two molecular
lesions which are likely to cause progression of a cancer cell toward the metastatic
state.
11. Describe the multiple-hit theory of carcinogenesis with respect to specific genetic
lesions associated with development of cancer of the colon.
12. Name at least three genes which are inactivated in common spontaneous cancers in
humans.
496
27. Immunoglobulins and Immunogenetics
27.1. Antibody Structure
Immunoglobulins (“antibodies”) account for 20 % of the plasma proteins. Most are γ-

globulins, but some migrate in the β and α2 fractions. Immunoglobulins are also present
in interstitial fluid, exocrine secretions (tears, saliva, bronchi, GI-tract), and on the surface
of B-lymphocytes. They are glycoproteins containing 2–12 % carbohydrate. They all can
bind specific antigens.
27.1.1. Structure of Immunoglobulin G (IgG)
Immunoglobulin G (IgG) consists of 2 light (L) chains (MW 23 000 Da) and 2 heavy (H)
chains (MW 50 000 Da).
The two H chains are associated with one another in the C-terminal half of the polypeptides.
Near their middle they are connected by inter-chain disulfide bonds.
Each L chain associates with the amino-terminal half of H chain, both non-covalently and
by disulfide bonding.
The polypeptides are folded into globular domains, each stabilized by an intra-chain disul-
fide bond. Each L chain has 2 domains, each H chain 4. The domains at the amino-ends of
each chain are variable in different IgG-molecules. The other domains are constant. Each
variable domain contains 3 or 4 hypervariable regions.
The variable domains are responsible for antigen-binding, the constant domains of the H
chains for the “effector functions”, such as stimulation of phagocytosis, complement activa-
tion, and placental transfer.
Between the first and second constant domains of the H chains there is a small non-globular
portion called the hinge region. It is more flexible than the rest of the molecule and accessible
to proteases. It contains the disulfide bonds between the H chains.
Papain cleaves the H chain in the hinge region, on the amino side of the disulfide bridges, to
create two Fab -fragments (ab = antigen-binding) and one Fc fragment (c = crystallizable).
497
Figure 27.1.: Structure of immunoglobulin G, the most common type of antibody. Figure
% &'!%(
"
!
!
# $$"
"
"
Figure 27.2.: The immunoglobulin fold (PDB-code ifc2) is a sandwich of two anti-parallel
β-pleated sheets.
498
Heterogeneity of Immunoglobulins 27.1.3
The Fab fragment binds the antigen, and the Fc fragment is responsible for the effector
functions.
Immunoglobulin molecules have two antigen binding sites, each formed by the variable
domains of one L chain and one H chain. Antigen binding is non-covalent and reversible.
The antigen is a foreign molecule of high molecular weight that induces the formation of
a matching antibody. The specific portion of the antigen molecule that induces antibody
formation and binds the antibody is called the antigenic determinant, or epitope. Antibodies
of all classes bind antigens, although their effector functions are different.
27.1.2. Heterogeneity of Immunoglobulins
There are 5 classes of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. Each class has its
own type of heavy chain: γ-chains for IgG, α for IgA, µ for IgM, δ for IgD and for IgE.
IgG, IgA and IgM have subclasses with slightly different heavy chains: γ1, γ2, γ3, and γ4
for IgG1, IgG2, IgG3, and IgG4, α1 and α2 for IgA1 and IgA2, and µ1 and µ2 for IgM1
and IgM2. There is only one class each for IgD and IgE.
There are 2 types of L chains, λ (lambda) and κ (kappa). 2/3 of immunoglobulin molecules
in all classes have two κ-chains, 1/3 have two λ-chains.
Polymeric forms occur in the IgA and IgM classes: IgA occurs as a monomer (MW 160 kDa)
or dimer. Serum IgM is a pentamer (MW 900 kDa). The polymeric forms contain a single
J chain (J = joining), which is disulfide-bonded to the H chains.
Secreted IgA contains a secretory component, a 70 kDa glycoprotein.
IgG 75 % of all immunoglobulins. Placental transfer. Maternal IgG protects the newborn
from infections. Weak to moderate ability to fix complement (except IgG4). Has
memory: anamnestic response.
IgA The major immunoglobulin in external secretions. Secretory IgAis a dimer consisting of
two H2 L2 units, a J-chain, and the secretory component. Fights infections on mucosal
surfaces, and scavenges antigens before they induce an IgE (allergic) response.
IgM Present as a pentamer in plasma. Strong complement binding. IgM levels rise more
rapidly than IgG in the course of an infection. Some antigens induce only an IgM-
response but no IgG-response (example: ABO blood group antigens). No anamnestic
response.
IgD Present in trace amount only. Unknown function.
IgE Binds to the surface of mast cells, basophils, and alveolar macrophages. Binding of
antigen to cell-bound IgE induces the release of histamine and heparin during allergic
responses. IgE is elevated in many allergic patients.
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Figure 27.3.: Structure of MHC-1 (PDB-code 1qse).
27.1.3. Other Ig Domain Proteins
T-Cell Receptors (TCR)
T-cell receptors occur in the plasma-membrane of T-cells and are similar to one arm
of an antibody. They bind to antigen-loaded MHC-molecules on antigen-presenting cells
(all cells for MHC-I and phagocytic cells for MHC-II). Like antibodies, they are composed
of two chains. The binding site is at the tip of the molecule, and is formed of by several
loops of the protein chains.
Other Membrane Immune Receptors: CD4 and CD8 membrane-bound receptors also con-
tain repeats of the immunoglobulin fold. These receptors act as co-receptors and stabilize
the interaction between TCR and antigen-loaded MHC. They are unique markers for T-cells
(CD4+ in Th - and CD8+ in Tk -cells).
Classes of cellular adhesion molecules (CAMs) also contain multiple repeats of the im-
munoglobulin fold which function to form cellular contacts by dimerization. Cadherins
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The Major Histocompatibility Locus (MHC) 27.2.1
D. Clinical Methods
Figure 27.4.: How to perform an ELISA. The test can be used to detect either antigens or
antibodies.
1. Enzyme-linked Immunosorbent Assay
a. Indirect ELISA measure the amount of specific antibody

b. Sandwich ELISA
also contain immunoglobulin-like measures the amount of antigen in a sample
repeats.
Fibronectin, an extracellular matrix protein, also contains repeats of the immunoglobulin
II. IMMUNOGENETICS
fold.
A. The Major Histocompatibility Locus (MHC)
Clinical Methods
The human major histocompatibility complex (MHC) or human leukocyte antigen
(HLA) region is of greater than 4 Mb of DNA (~0.1 % of the genome). Location:
Short arm ofImmunosorbent
Enzyme-linked chromosome 6 Assay .
at 6p21.3
There are greater than 200 genes located in the MHC and ~ 40 % of these genes
have immune related functions. Traditionally, these are discussed as class I,
class Immunogenetics
27.2. II and class III genes with the sequence centromere- II - III - I - telomere.
27.2.1. The Major Histocompatibility Locus (MHC)
The human major histocompatibility complex (MHC) region is of greater than 4 Mb of

DNA (≈ 0.1 % of the genome). Location: Short arm of chromosome 6 at 6p21.3
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260
Figure 27.5.: Principle of immuno-assays. In an indirect test, labeled antigen competes

with sample antigens for a limited amount of antibodies. The more (unla-
beled) sample antigen is present in the sample, the less signal is measured.
A sandwich assay uses two different antibodies, the capture antibody is im-
mobilized on the substrate and binds the antigen. The amount of antigen is
then measured using a labeled detection antibody, that binds to a different
epitope on the antigen. In a direct test, labeled antibody is used to detect
immobilized antigen. In an indirect test, the detection antibody itself is not
labeled, but the amount of bound antigen is measured by labeled secondary
reagent (antibody against the Fc -part of Ig, protein A, protein G or similar).
Figure from [Buxbaum, 2007].
FRPSHWLWLYH VDQGZLFK
GLUHNW LQGLUHNW
502
Figure 27.6.: Organization of genes in the MHC-region of chromosome 6.
There are greater than 200 genes located in the MHC and ≈ 40 % of these genes have
immune related functions. Traditionally, these are discussed as class I, class II and class III
genes with the sequence centromere- II - III - I - telomere.
Class I region Highly polymorphic functional classes : HLA-A, HLA-B, HLA-C. Class I
molecules are expressed at the cell surface of most tissues. Functions:
• antigen presentation to cytotoxic T lymphocytes (CTLs)
• signal of “electrically silent neurons” to CTL surveillance system
• No expression at materno-fetal interface facilitates survival of fetal tissues
• HLA-C involved in target recognition by natural killer cells
• HLA-E, -F, -G have more selective tissue distributions
• HLA-G is expressed on the maternal trophoblasts effecting fetal tolerance
• HFE gene implicated in hereditary hemochromatosis
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Figure 27.7.: Structure of MHC-1.
• Other non-immune genes include those of zinc-finger proteins, ubiquitin-like proteins,

olfactory receptor family, and butyrophilin family genes
Class II region Class II molecules are encoded in a region of about 800 kb of DNA with
approximately 20 genes. [Structure below has membrane segment removed.]
• HLA-DP, HLA-DQ, and HLA-DR are expressed on antigen presenting cells. They
present peptides to T helper cells.
• Many pseudo-genes of paired HLA-DP and HLA-DQ are also present
• HLA-DM proteins participate in transfer of peptides onto class II molecules within

intracellular vesicles.
• Some ABC transporter superfamily genes are present: These genes encode proteins
which transport a variety of substances across membranes: oligopeptides, proteins and
ions. TAP1 and TAP2 are genes for translocases found in the ER membrane. TAP
function is required for Class I antigen presentation, suggesting intracellular linking
of their functions.
• LMP2 and LMP7 genes encode proteins of the proteosome complex.
504
Figure 27.8.: Structure of MHC-II.
• LMP and TAP genes, as well as some class I molecules are induced by gamma inter-
feron (IFNγ), an immune-stimulator.
Class III region 1.1 Mb segment of DNA between class I and class II regions contains
approximately 70 genes. One internal DNA segment contains a duplicated gene cluster with
complex and overlapping gene arrangements. Many class III genes encode immune-related
proteins:
• PBX2 is a homeodomain protein which participates in hematopoiesis
• RAGE is ’receptor for advanced glycation end-products of proteins’. This protein

mediates monocyte migration and activation in response to advanced glycation end-
products. Binding of AGE’s to RAGE in the endothelium induces NF-κB and in-
creased vascular permeability. RAGE could be a link in diabetes to vascular compli-
cations because of increased AGE formation in diabetics.
• G15 (hLPAATα) encodes an enzyme associated with prostaglandin metabolism, and

important inflammatory mediator. Prostaglandins play important roles in production
of pain and fever, the regulation of blood pressure, induction of blood clotting, control
of several reproductive functions and regulation of the sleep-wake cycle.
• Complement components C2 and C4 participate in the classical complement path-

way, stimulating yeast, bacterial and viral recognition with mannan-binding proteins.
The C4 gene loci are highly polymorphic. Partial deficiency of C4 is associated with
increased susceptibility to systemic lupus erythematosus (SLE), scleroderma and
primary biliary cirrhosis.
505
inflammatory and immunomodulatory activities as well as being involved in tumor
cachexia.
B. Immunoglobulin Gene Structure

Exons of immunoglobulin genes are arranged in a manner that makes
possible the generation of many unique immunoglobulin genes, and transcript
splicing patterns. Shown below is a diagram with the unique segments arranged
in tandem copies. Figure 27.9.
Structure of the IGH Ig heavy chain gene at 14q32

V region DJC region
(900 kb) (350 kb)
Telomere Centromere
~100 VH segments 26 DH seg’s 11 CH seg’s

9 JH seg’s
Antigen binding diversity Antibody Class (IgM, IgD etc.)

is encoded here. Selection is encoded here. Unique
of unique VH segments CH segments encode class
occurs in B cell maturation. switching gene segments.
263
• The G9 gene encodes a sialidase important for lysosomal function. Hyposialydation

of cell surface proteins on T cells appears to be required for normal T-cell function,
and higher sialydation may result in some types of autoimmune disease.
• Heat shock proteins Hsp70-1 and -2 genes encode proteins induced by high tempera-
ture.
• The TNF ligand superfamily of cytokines are also expressed: TNF, LTα and LTβ.
TNF is produced by a variety of cells and exhibits numerous inflammatory and im-
munomodulatory activities as well as being involved in tumor cachexia.
27.2.2. Immunoglobulin Gene Structure
Exons of immunoglobulin genes are arranged in a manner that makes possible the genera-
tion of many unique immunoglobulin genes, and transcript splicing patterns. Shown below
is a diagram with the unique segments arranged in tandem copies.
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Immunoglobulin Gene Structure 27.3.1
Figure 27.10.: Somatic recombination leads to a large number of different antibodies.
Generation of Antibody Diversity
Biosynthesis of Immunoglobulins Immunoglobulins are secreted by plasma cells which

are the descendants of B-lymphocytes (B-cells). 15–30 % of lymphocytes are B-cells. T-
lymphocytes do not produce antibodies but have T-cell receptors on their surface instead,
with idiotypes similar to immunoglobulins. Therefore both B-cells and T-cells can recognize
antigens.
B-cells do not secrete immunoglobulins, but they possess surface immunoglobulins in their
plasma membrane: Initially only IgM, later IgD, then IgG, IgA or IgE. These successively
expressed antibodies have the same variable domains, but the constant domains of the H-
chain are different. This process is called class switching. Important: each B-cell makes an
antibody of only one antigen-binding specificity. We have millions of different antibodies
because we have millions of B-cells, each making its own antibody.
The differentiation of the B-cell into a plasma cell requires the binding of an antigen to
the surface immunoglobulin and stimulation from helper T-cells. Only those B-cells that
encounter their antigen become plasma cells. This is called clonal selection.
507
27.3. MHC (HLA) and Clinical Risk of Disease
27.3.1. MHC Polymorphisms and Disease Risk
The genes of the MHC are highly polymorphic possibly due to selective pressure of regional
pathogens. More than 200 allelic variants are known for some highly polymorphic genes,
and some of these alleles are associated with the incidence of disease.
Diabetes Mellitus Europeans with Type I have a higher frequency of (96 vs 45 % expected)
of the DR3 or DR4 alleles present at the Class II DRB1 locus on 6p21.3 (in one copy) and
a higher frequency had both alleles (38 % vs. 3 % expected) Also the loci DQA1 and DQB1
may be predisposing/protecting. The mechanism for predisposition and protection are not
understood.
Rheumatoid arthritis (RA) High risk is associated with specific alleles within the DR4
locus: Two alleles, DRB1∗0401 (previously called Dw4) and DRB1∗0404 (previously called
Dw14), primarily account for the DR4 association with disease in Caucasians. These alleles
all have in common the “shared epitope”, suggesting a specific protein sequence is associated
with susceptibility to RA.
Ankylosing spondylitis (AS) Thirty percent of the susceptibility to ankylosing spondylitis

appears to be encoded by HLA-B27 genes
Common variable immunodeficiency (CVID) and IgA deficiency (IgAD) susceptibility

and resistance are most closely associated with the genes within the HLA DR-DQ regions.
27.3.2. Familial Immune Disorders
Immunoglobulin subclass deficiencies Most of these are benign, due to overlapping func-
tions within immunoglobulin subclasses. Immunoglobulin A deficiency (IgAD) is the most
common, with an incidence 1 in 500. IgAD is multifactorial with HLA associated risk.
Chronic variable immunodeficiency (CVID) affects approximately 1 in 10 000 to 100 000
individuals. The patients display a marked reduction in serum levels of both IgG and IgA.
In half of the patients, IgM is also reduced. Patients are at much greater risk for recurrent
bacterial infections.
508
Bruton agammaglobulinemia X-linked inheritance. Incidence is 1/100 000. Character-

ized by failure to produce mature B lymphocyte cells and associated with a failure of Ig
heavy chain rearrangement. The defect in this disorder resides in Bruton tyrosine kinase
(BTK, also known as BPK or ATK), a key regulator in B-cell development Decreased IgM,
IgG and IgA in serum. Less than 1 % of B cell number.
DiGeorge Syndrome (Congenital Thymic Dysplasia) Most cases result from a deletion
of chromosome 22q11.2. The spectrum of immune dysfunction is wide. Defects are due to
developmental abnormalities of the thymus, cardiovascular and endocrine organs.
Adenosine Deaminase Deficiency Autosomal recessive form of severe combined immun-

odeficiency disease (SCID-ADA). Toxic metabolites of adenosine accumulate with the im-
mune cells. These metabolites inhibit normal lymphocyte proliferation. Patients have re-
duced function of both B and T lymphocytes, non-functioning circulating immature T
lymphocytes, and hypogammaglobulinemia.
Plasma Cell Dyscrasias These are neoplastic diseases which are also called paraproteine-
mias or monoclonal gammopathies. Patients show a sharp peak in the γ-globulin region
(sometimes β or α2) on serum electrophoresis. This peak (“paraprotein”) is a homogeneous
immunoglobulin, produced by a single plasma cell clone. The remainder of the γ-globulin
fraction is depressed. Types:
Multiple myeloma: Malignant proliferation of a plasma cell clone in the bone marrow.
With anemia, bone lesions, bone pain, pathological fractures, recurrent infections. The
paraprotein is most commonly IgG (50 %) or IgA (25 %). Some patients overproduce
only L-chains which are excreted in the urine: Bence-Jones protein.
Waldenstroms macroglobulinemia: Overproduction of IgM, malignant, with blood hy-
perviscosity.
Benign monoclonal gammopathy Present in 5 % of persons above age 50 and 8 % of those
over 70. May occasionally progress to multiple myeloma.
1. Describe the general structure of immunoglobulins (Igs) and state the nature and
consequences of antigen-antibody binding.
2. Name the most important functions of the five major classes of Igs.
3. Describe the Major Histocompatibility Complex (MHC).
509
4. Explain the importance of the MHC in human disease risk.

5. Describe Immunoglobulin gene structure.
6. Explain the terms class switching and clonal selection in relation to IgG gene structure.
7. Discuss the mechanism of cellular generation of antibody diversity.
8. Name the principal types of monoclonal gammopathy, and define the term “Bence-
Jones protein”.
510
28. Inherited diseases of metabolism
28.1. Introduction
28.1.1. Significance of inherited diseases of metabolism
inherited disease of metabolism (IDoM) are individually rare, with 1 case per several thou-
sand to several hundred thousand births. However, since there are 350 known diseases of
this type, together they contribute significantly to infant morbidity and mortality. About
1 in 2500 births has some form of inherited metabolic disease, and you are not unlikely to
encounter some cases during your career. The point is, however, that you do not know yet
which ones it will be.
IDoM usually are inherited in an autosomal recessive fashion. This means that they
may be enriched in inbred populations (Mennonites in Pennsylvania, Ashkenazi Jews) and
that founder-effects may be observed (e.g. tyrosinemia in Quebec). Exceptions from the
autosomal recessive inheritance (X-linked, autosomal dominant or dominant negative) will
be noted. Many important diseases (diabetes, auto-immunity) have a genetic component,
but also require environmental factors to become manifest.
Clinically IDoM are very variable, onset of symptoms may be within 24 h after birth, or it
may occur in mature people. Symptoms may develop suddenly, or slowly and insidiously.
Even patients with the same mutation, for example from the same family, may show very
different clinical pictures. Some IDoMs may be phenocopies of other diseases, that is they
present clinically like some other (non-inherited) disease. The first such pair described in the
literature were urea cycle defects, that may present like Reye-syndrome. As a consequence,
IDoM tend to be under-diagnosed.
28.1.2. Mechanism of IDoM
Inherited diseases of metabolism usually occur because of a defect in the gene for an enzyme.
This may result in:
inappropriate transcription to few/many copies of mRNA
511
splicing defect incorrect mRNA produced (e.g. spinal muscle atrophy). About 15 % of
single nucleotide mutations interfere with splicing.
translation e.g. nonsense-mutations: inappropriate Stop-signal. May be the result of a splic-
ing defect, if an intron is maintained that contains a stop-signal.
folding protein folds to slowly and is destroyed by ERAD/proteasome (e.g. Cystic fibrosis).
Those enzyme molecules that manage to fold may function perfectly fine.
amyloid formation improperly folded protein accumulates and interferes with cellular func-
tion (e.g. α1 -antitrypsin in liver).
defective protein sequence or structure does not allow function. In oligomers may lead to
dominant negative inheritance (e.g. aquaporin in diabetes insipidus).
regulation of activity site for post-translational modification destroyed or protein becomes
independent of modification (e.g. oncogenes).
regulation of survival time too low/high concentration of enzyme.
When an enzyme is defective, the following problems may occur:
• lack of product
• accumulation of substrate
• conversion of substrate to toxic substances by other enzymes
• gain of function – protein does something it shouldn’t (→ autosomal dominant!)
Sex-linked diseases
X-linked inheritance The X-chromosome is unusual, in that ~ have two, | only one copy.
To compensate for the increased gene dose, ~ inactivate one of the two X-chromosomes
as Barr-body (Lyons-hypothesis. Inactivation occurs early in embryonic development in
such a way, that some of the cells inactivate the X-chromosome from the father, others
that from the mother. This pattern is than fixed during development, so that ~ are a
somatic mosaic. Most heterozygous ~ with a recessive mutation on the X-chromosome
have the correct copy active in about 50 % of their cells, and appear normal, although they
are carriers (can transmit the defect gene to their offspring). Some however are mildly
affected (manifestation), as too few cells in their body make the correct gene product
(variable penetrance). | carrying such a mutation are hemizygote and fully affected, as
they have no correct copy to compensate. The most well known example for this mode of
inheritance is hemophilia A. If 1 : q is the frequency of the defect gene in the population,
then 1 : q |will show the disease, but only 1 : q 2 of ~ with two defect copies will be
affected.
512
Newborn screening 28.1.3
The number of Barr-bodies in a cell is the number of X-chromosomes minus one. X-

inactivation is incomplete, some genes are expressed from all copies of the X-chromosome.
Hence patients with extra X-chromosomes (47,XXX or 47,XXY) show defects.
A few X-linked diseases are dominant, e.g., Rett-syndrome (OMIM #312750), the second
most common cause of inherited mental retardation in ~ after trisomy 21. In those cases
affected | die in utero (unless 47,XXY or the like), only ~ with one defect copy express the
disease. As affected ~ hardly ever reproduce, most cases are the result of new mutations
(usually in the paternal germline).
Sex-limited diseases can only occur in one sex (e.g. testicular or ovarian defects).
Sex influenced diseases are more common and/or more sever in one sex than the other.
For example, male-pattern baldness results from a hypersensitivity of hair follicles to the
androgen dihydrotestosterone. The disease is dominant in |, recessive in ~ due to different
hormone levels.
28.1.3. Newborn screening
In many cases, damage by IDoM can be limited by an appropriate diet, which supplements
compounds that the patient can not synthesize, or limits nutrients that they can not me-
tabolize. Such dietary management in many cases may however only prevent the worsening
of damage; such damage that has already occurred, especially to the nervous system, can
not be cured. Given the suffering involved for patients and their families, and the high costs
incurred by society, screening of newborns for certain IDoM is required by law in many
developed countries. The catalogue of diseases, for which you are obliged to screen, varies
between jurisdictions, but most commonly includes the following:
• phenylketonuria
• galactosemia
• hypothyroidism
• mucopolysaccharidoses
There are some screening tests, that are inexpensive and so simple to perform that they may
also be done by community nurses or midwifes, it involves nothing more than pouring a few
drops of reagent solution onto a used diaper and observing for color changes. Their lack of
specificity is an advantage in this case, as they react to any of several severe conditions:
FeCl3 phenylketonuria, tyrosinemia, maple syrup urine disease, alcaptonuria and ketonuria.
Interference by salicylates and phenothiazines.
513
Reducing substances glucose, galactose, fructose, lactose, sialic acid. Interference by cephalothin
and ampicillin
Nitroprusside sodium ketones, cystine, homocysteine
Azure A mucopolysaccharides
Any positive result in these tests needs to be followed up with more specific investigations,
which however require a specialized laboratory.
Blood samples from a heel-prick may be used for screening, either fresh or dried on absorbent
paper. Common tests include:
• PKU, MSUD, homocystinuria, tyrosinemia
• MCAD! deficiency, biotinase deficiency, galactosemia
• adrenal hyperplasia, hypothyroidism
• sickle cell disease
The most modern technology for screening uses liquid chromatography coupled to multi-
dimensional mass spectrometry (LC/MS) to determine the identity and concentration of
virtually any compound in a sample. Any unexplained deviations from normal need follow-
up. Although the initial instrument costs are very high, this method allows high-throughput
screening of samples with minimal staff involvement.
Given all the fancy technology available to physicians nowadays one should not forget that
careful observation and a thorough physical exam of a patient can alert an astute physician
to the possibility of an IDoM. The following signs should raise suspicion:
• lethargy, convulsions, hypotonia
• hepatomegaly, also kardio- or splenomegaly
• acidemia, increased anion gap
• hyperammonemia
• hypoglycemia
• unexplained vomiting
• elevated liver enzymes
• unusual color, smell or structure of hair, eyes, skin, stool or urine
514
Newborn screening 28.1.3
Figure 28.1.: Liquid chromatography with multi-dimensional mass spectrometry is a mod-

ern technology used to determine metabolites in urine or deproteinized serum.
Molecules in the sample are separated from each other by liquid chromatogra-
phy, the effluent from the column is injected into an ionizer, which turns the
dissolved sample molecules into single ions in vacuum. Following a high elec-
tric potential the molecules fly through the first analyzer, which determines
their molecular mass. Ions of a given mass then enter a collision cell, where
they bump into He atoms. The force of the collisions makes the ions break into
fragments. Different bonds have different stabilities, hence the breaking points
are specific for each compound. The molecular weights of the fragments are
then determined in a second analyzer, giving a “fingerprint” spectrum. Thus
from LC/MS/MS you get for each compound in the sample the chromato-
graphic position and concentration, molecular weight and fragment spectrum.
This is sufficient to reliably identify and quantify (almost) all compounds in
a sample. Figure from [Buxbaum, in press].
Sample
injector
Chromatographic column (RPC, IEC)
Mobile phases
Chromatographic
sample identification
Peak identification
quantitation Data
Internet base
Photometer He gas (low pressure)
Ionizer Analyzer Collision cell 2nd Analyser
dissolved sample -> determines molecular mass fragments sample ions determines molecular mass
ion stream in vacuum of sample ions of given molecular mass of fragments (fingerprint)
515
Signs may worsen in stress (infection, exertion). Some IDoM are associated with character-
istic smells of urine, sweat or breath:
Disease Pathway Description
diabetic ketoacidosis Glc acetone, fruit
hawkinsinuria Tyr chlorine-like
isovaleric acidemia Leu sweaty feet
maple syrup urine disease Leu, Ile, Val caramel
oast house syndrome Met drying hops
methylmalonic aciduria odd-chain fa, aa ammonia
phenylketonuria Phe mouse urine
propionic acidemia Ile, Val, Thr, Met ammonia
trimethylaminuria choline, carnitine fish
tyrosinemia I Tyr cabbage
urea cycle defect amino acids ammonia
28.2. Cytosolic enzymes
Carbohydrate, amino acid and nucleotide metabolism occur mostly in the cytosol. Most of
the following diseases you will have encountered already in their respective pathways.
28.2.1. G6PDH deficiency – Favism
G6PDH is the first committed step in pentose phosphate pathway, which supplies ribose
(for nucleic acid synthesis) and NADH + H+ . The later protects the cell against oxidative
damage, it is required for the regeneration of oxidized glutathione. Especially in erythro-
cytes, which are exposed to a particularly high oxidative stress, the pentose phosphate
pathway is the only major source of NADH + H+ (only anaerobic glycolysis!).
Reduction of G6PDH activity in heterozygotes leads to an advantage in malaria-infested

regions (Mediterranean, Africa, Asia). The higher redox-potential and shorter life time of
the erythrocyte limits growth of the blood-stage of the malaria parasite. Favism is inherited
in an X-linked recessive manner, and therefore protects mainly ~.
Affected |(about 100 Mio worldwide) appear usually normal, but will suffer an acute
hemolytic crisis after ingestion of substances that lead to the formation of reactive oxygen
species. Classically, this includes broad (fava) beans (Vicia faba L.), which contain divicine
as offending ingredient. Several classes of pharmaceuticals also have members that can pre-
cipitate such a crisis, which you may remember by the acronym A4 : analgetics, antipyretics,
antimalarials and antibiotics.
516
Galactosemia 28.2.3
Figure 28.2.: Galactosemia is caused by the inability to convert ingested galactose into
glucose. The excess galactose is then converted to galactitol and galactonate
instead.
O
O O
HO
OH OH OH
HO HO aldolase HO
GalDH
OH OH OH
(via lactone) HO reductase
HO HO
OH OH OH
Galactonate Galactose Galctitol
Gal-1-P uridyl transferase (type I, 1 : 50,000)

Gal-kinase (type II, 1 :100,000)
UDP-Gal-4 epimerase (type III, rare except Japan)
OH
HO
OH
HO
OH
Glucose
During a crisis, damaged hemoglobin will precipitate forming Heinz-bodies, which are
visible in a stained blood smear. That results in membrane damage, damaged erythrocytes
are then removed by the spleen. Heinz-body anemia also occurs in other diseases with
hemoglobin damage (α-thalassemia, poisoning...).
28.2.2. Galactosemia
If ingested galactose can not be converted to glucose (due to failure of any one of the
three enzymes involved), it will be converted to galactitol and galactonate instead by other
enzymes. Those increase the osmotic pressure in the eye lens, leading to swelling, which
in turn interferes with membrane integrity. The influx of Na+ from the interstitial fluid
leads to further swelling, and finally apoptosis. The result is cataract formation. Other cell
types are also affected, leading to difficulty feeding, diarrhea, lethargy, hypotonia, jaundice,
hepatomegaly, mental retardation, verbal dyspraxia (difficulty), motor abnormalities and
ovarian failure in ~.
Management is mainly by Gal-free diet, which can reverse early cataracts, but not the
other problems. In addition, disease progression is only slowed (albeit significantly), since
our body produces Gal on its own.
517
28.2.3. Errors of fructose metabolism
There are three IDoM in fructose metabolism, of which hereditary fructose intolerance is
the most dangerous:
hereditary fructose intolerance (HFI) is caused by fructoaldolase (aldolase B) deficiency

in liver, kidney, and small intestine. It is asymptomatic until Fru, saccharose or sorbitol
is ingested, usually after weaning. The disease results in poor growth, liver and kidney
damage and, potentially, sudden death. In most cases the disease is self-limiting, because
affected kids learn to avoid foods that make them feel sick. Treatment: Fru, Saccharose and
sorbitol-free diet, then normal development.
Fructosuria is caused by hepatic fructokinase deficiency. Since the fructose is not taken
up efficiently into hepatocytes, it is excreted in urine. No pathology, hence no treatment
is required. Cave: since Fru is a reducing sugar (spontaneous conversion to Glc, especially
under alkaline conditions) fructosuria may be mistaken for diabetes mellitus in urinalysis.
Fru-1,6-bisphosphatase deficiency prevents gluconeogenesis and results in exertional or

fasting hypoglycemia + acidemia (lactate, pyruvate, ketone bodies). Management involves
a Fru-free diet and frequent meals. Under those conditions, development is normal.
28.2.4. Lactase persistence/restriction
Lactose in mammal sucklings is split to Gal + Glc by lactase on the brush-border membrane
of the small intestine. Expression of lactase is reduced after weaning to 5–10 % of suckling
levels, in humans that usually happens at age 3–5 a. In certain populations however, which
have a tradition of shepherding, mutations in the cis-acting (=DNA) elements that regulate
lactase mRNA transcription result in expression of this enzyme throughout life. The ability
to utilize milk from animals as additional food has arisen several times independently during
human history (convergent evolution).
Although lactose restriction is normal (and indeed observed in ≈ 90 % of humans), rather

than a disease, consumption of milk or milk products by such individuals has drastic and
unpleasant consequences: The undigested lactose is fermented by colonic bacteria, resulting
in flatulence, abdominal pain and diarrhea. Treatment in such cases is supportive, symptoms
vanish on their own after a couple of hours.
Other mammals too lose their ability to digest lactose after weaning, therefore please do
not feed milk to adult cats!
518
Glycogen storage diseases 28.3
28.2.5. Amino acids
Errors of amino acid metabolism have been discussed in chapter 22 and will not be repeated
here.
28.3. Glycogen storage diseases
All glycogen storage diseases are autosomal recessive, except VIII, which is X-linked. They
affect one of several isoforms of enzymes and are therefore tissue specific. Mostly affected
are liver (hepatomegaly, fasting hypoglycemia) and muscle (hypotonia). Numerical nomen-
clature of glycogenoses in the literature is confused, we follow OMIM. Most, but not all,
glycogenoses affect cytosolic enzymes.
0a defect in liver glycogen synthase GYS1. Hypoglycemia, high blood ketones, increased
free fatty acids and low levels of alanine and lactate in fasting, hyperglycemia after
meals.
0b defect in muscle glycogen synthase GYS2. Cardiomyopathy and exercise intolerance

with absence of muscle glycogen.
Ia (von Gierke) Glc-6-Pase defect in the lumen of the ER. Severe hypoglycemia after 2–
4h of fasting since no blood Glc from liver glycogen. Hyperlipidemia and xanthoma,
liver + kidney damage, gout, liver adenomas → carcinomas. Treatment: uncooked
corn-starch.
Ib defect Glc-6-P transport from cytosol into ER. Slow growth, small, hyperlipidemia
and xanthomas, hepatomegaly, liver adenoma, neutropenia. Treatment: GM-CSF, un-
cooked corn-starch
II (Pompe) acid maltase (α-1,4-glucosidase) in lysosomes of all organs. Infantile form se-
vere cardiomegaly and death by 3 a from cardiomegalia glycogenica by inactive en-
zyme. Adult form by reduced enzyme conc: slowly progressive muscle hypotonia
(wheel chair, respiratory support, sphincter), vascular damage. Treat with high pro-
tein / low carb diet, infusion of recombinant glucosidase precursor.
IIIa (Cori-Forbe) Glycogen debranching enzyme defect in muscle + liver. Hepatomegaly,

hypoglycemia, growth retardation, progressive skeletal myopathy, cardiomyopathy,
fasting hypoglycemia.
IIIb like IIIa, but liver only.
IV (Anderson) defect in branching enzyme leads to long, unbranched, insoluble glycogen

that precipitates in liver. Hepatomegaly and liver cirrhosis, death by age 5 a.
519
V (McArdle) defect in muscle phosphorylase. Exercise intolerance and muscle cramps, no

Cori-cycle, rhabdomyolysis may lead to renal failure. Often appears only in early
adulthood. Treatment: Glc before exercise, or avoid strenuous exercise.
VI (Hers) defect in liver phosphorylase. Mild to moderate hypoglycemia, mild ketosis,

growth retardation, and prominent hepatomegaly. Good prognosis.
VII (Tarui) defect in muscle (M) phosphofructokinase. Exercise intolerance with cramps
and possibly rhabdomyolysis and myoglobinuria, hemolysis as the same isoform is
also expressed in erythrocytes. Gout as Fru-6-P goes through hexose monophosphate
pathway: PRPP ↑, AMP is also increased. Compensatory increase of 2,3-DPG: O2 -
affinity ↑. Glucose intolerance. Prognosis depends on remaining enzyme activity (or
replacement by L-form in erys).
VIII defect hepatic phosphorylase kinase (X-linked). Hepatomegaly, growth retardation,

elevated liver enzymes, hypercholesterolemia, hypertriglyceridemia, and fasting hyper-
ketosis. Symptoms resolve in adolescence. Treatment with dextrothyroxine (D-T4) if
required.
XI (Fanconi-Bickel) defect glucose transporter GLUT2. Decreased Glc (+Gal) uptake/release

by liver, Glc sensing in pancreas, re-uptake in renal tubules. Growth failure, rickets,
osteoporosis, dwarfism, hepatomegaly, moon-shaped face, and fat deposits on shoul-
ders and abdomen, fractures and pancreatitis. Glucose, aa, phosphate, protein and
uric acid in urine increased, metabolic acidosis. Treatment: antiketogenic diet; water,
electrolyte and vitamin D supplementation.
28.4. Lysosomes
Lysosomes are required for the breakdown of molecules (intracellular and extracellular) that
are no longer required. Failure of any lysosomal enzyme leads to accumulation of its sub-
strate inside the lysosome, this interferes with cellular function. 50 such lysosomal storage
diseases have been described. The main problems occur with breakdown of mucopolysac-
charides (from the extracellular matrix) and sphingolipids (from the cell membrane). I-cell
disease affects all lysosomal enzymes.
Several attempts of treatment of lysosomal storage diseases have been published:
Bone marrow replacement can slow the progression of the diseases, since the amount of
circulating substrates is reduced. This limits uptake into the defective own lysosomes.
However, there is significant morbidity and mortality associated with this expensive
procedure.
520
I-cell disease 28.4.1
Figure 28.3.: I-cell disease is caused by the inability to add a phosphate group to a mannose
group in the sugar-tree of lysosomal proteins. The Man-6-P acts as a signal
for transport from the trans-Golgi to the lysosome.
Asn
Asn Asn
NH
NH NH
GlcNAc Fuc
GlcNAc GlcNAc
GlcNAc export plasma membrane
GlcNAc transport GlcNAc transport
Man or exocytosis
Man + trimming Man + addition of sugars
Man Man Man
Man Man Man Man
Man Man
Man Man Man Man Man
GlcNAc GlcNAc GlcNAc
Man
Gal Gal Gal
Glycoprotein Glycoprotein
(high-Mannose type) (core oligosaccharide)
ER cis-Golgi Glycoprotein
(complex type)
trans-Golgi
GlcNAc-Phospho- UDP GlcNAc

transferase
I-cell disease UMP
Asn GlcNAc Asn

NH NH
GlcNAc GlcNAc binding to Man-6-P receptor
enrichment in clathrin coated vesicles
GlcNAc + transport to trans-Golgi GlcNAc transport to lysosome
Man Man
GlcNAc P Man Man P Man Man
Man Man Man Man
Enzyme replacement therapy recombinant enzyme is infused into the patient, binds to
cellular Man-6-P receptors and undergoes endocytosis. That way, the enzyme ends
up in the lysosome, where it can do its work. Problem: Blood/brain barrier limits
effect against CNS damage (may be overcome by high dose: current phase I/II trial).
Somatic gene therapy The defective gene is replaced by a working copy in the affected
cells. Because of fatalities encountered, all human experiments on gene therapy are
currently on halt.
28.4.1. I-cell disease
I-cell disease is caused by the failure to phosphorylate Man in the sugar tree of lysoso-
mal enzymes in the cis-Golgi. The Man-6-P group normally acts as a sorting signal for
transport from the trans-Golgi to the lysosome. Thus all lysosomal enzymes end up in
the default pathway (export out of the cell), rather than in the lysosome, which is rendered
521
non-functional. This results in the formation of inclusion bodies (Name!). Exported lysoso-
mal enzymes do not cause damage to the body, since the pH in the interstitium (7.4) is far
away from that in the lysosome (5), so they are inactive.
I-cell disease is characterized by abnormal bone + joint development → dwarfism, he-
patosplenomegaly, heart valve defects, failure to grow and develop motor skills, corneal
clouding and death by age 7 a (congestive heart failure or respiratory tract infection).
28.4.2. Mucopolysaccharidoses and sphingolipidoses
522
523
28.4.2
Figure 28.4.: Breakdown of dermatan sulphate in lysosomes
Breakdown of dermatan sulphate

MPS II: Hunter MPS I: Hurler (complete) MPS IV: Maroteaux MPS VII: Sly
(X-linked) Scheie (partial)
Mucopolysaccharidoses and sphingolipidoses
O
H O O
GalNAc O
H H
O H
NAc
H O NAc O NAc
HO O NAc
H H
C HO HO H
H H HO
H2 C C O
H H H H C
HO β1-3 H 2 H2 H2 H H H
O
H H HO O HO O
H
HO O O NAc
H H H H
O H H
OH HO
GlcUA H O OH O OH C
O OH H H
H H H2
HOOC H
H
H HOOC HOOC HO
H H HOOC O
OH H H H H
H H H
H2O 2- OH OH O
SO4 H H 2O IduA H H2O 2- OH H2O H
O SO4 H GalNAc O OH
β1-4 O O
H O H NAc
S H O
H H
O NAc HOOC H
H H HO
GalNAc H O NAc O NAc H2O H
O NAc OH GlcUA C H
HO H H H H2
C HO H OH
H H H HO HO OH
2 C H C HO
H2 H H2 H H C H
O α1-3 H2 H H
H O O O OH
S H O HO OH
H
S H S H H
O O
S O OH
H
IduA-S H
H H
COOH
H H
COOH
OH
HO
OH
H HO
Iduronat sulphatase H α-L-iduronidase GalNac-4-sulphatase β-hexosamidase β-Glucuronidase
Figure 28.5.: Breakdown of heparan in lysosomes

S S S
Heparan GlcNS GlcUA GlcNAc GlcUA GlcNAc
H2C O S
-
O
H2C O S COO OH
-
O O O O
H2C OH COO OH OH
O O α1-4 NH
O O
OH OH C O
NH β1-4 OH
O α1-4
OH C O CH3
OH
NH β1-4 CH3
S
H2O Heparan sulphamidase
Sanfilippo A
2-
H2C O S
SO4 O
-
H2C O S COO OH
-
O O O O
H2C OH COO OH OH
O O NH
O O
OH OH C O
NH OH
O CH3
OH C O
OH
NH2
CH3
Acetyl-CoA N-acetyl transferase

Sanfilippo C
H2C O S
CoA O
-
H2C O S COO OH
-
O O O O
H2C OH COO OH OH
O O NH
O O
OH OH C O
NH OH
O CH3
OH C O
OH
NH
CH3
C O
CH3
H2O N-acetylglucosamidase
Sanfilippo B
GlcNAc
H2C O S
-
O
H2C O S COO OH
-
O O O O
COO OH OH
O O NH
O
OH NH C O
OH
HO CH3
C O
OH
CH3
H2O β-glucuronidase
Sly (MPS VII)
GlcUA
H2C O S
2-
H2C O S H2O SO4 -
O
O H2C OH COO OH
- O
H2C O S COO O O O
OH
O O O OH OH NH
O HO
OH OH NH O
HO NH C O
O N-acetylglucosamine-6- OH
NH C O CH3
OH sulphatase C O
C O CH3
CH3
Sanfilippo D
CH3
524
Mucopolysaccharidoses and sphingolipidoses 28.4.2
Figure 28.6.: Breakdown of chondroitin- and keratane sulphate in lysosomes

Proteoglycan
Aggrecan Ser
Xyl
Gal Galactosamine-
6-sulphatase
Chondroitin- Gal
Morquio A H2C O S
6-sulphate
GlcUA OH O
-
GalNAc COO O
n
O
S β1-3
H2C O S OH NH
OH O O C O
OH OH
β1-4
CH3
NH
β-glucuronidase
C O
β−hexosamidase Sly
CH3
GlcNAc-6-sulphatase
Aggrecan Ser Sanfilippo D H2C O S
β-galactosidase O
GalNAc Gal NANA
Morquio B H C OH OH
Keratan Gal OH2 O O
sulphate
S GlcNAc H2C O S O NH
n
O C O
β1-3 OH
H2C OH OH
OH O O CH3
OH β1-4 NH
C O N-acetylglucosamidase
OH
CH3 Sanfilippo B
525
Figure 28.7.: Spingolipidoses are caused by failure to break down sphingolipids and ce-
ramides in the lysosome
Sphingosine (C18) Name X=
C OH
H Ceramide H
N C
C
fatty acid C O X Sphingomyelin P Choline
O
head group P Ethanolamine
Sphingolipid
Glucosylcerebroside Glc (in non-neural tissue)
Galactocerebroside Gal (in neural tissue)
Globosides several neutral sugars (Glc, Gal, GalNAc)

H2C OH
H HC OH Gangliosides complex carbohydrates:
NANA
HN HC OH GM: single NANA
OC O GM1 Ceramide Glc Gal GalNAc Gal GD: two NANA
CH3 H H GT: three NANA
HO COO
-
GQ: four NANA
H β-galactosidase
H
OH Gal GM1 gangliosidosis
N-acetylneuraminic acid (NANA)
NANA
GM2 Ceramide Glc Gal GalNAc
Hexosaminidase A
GalNAc Tay-Sachs disease
NANA
GM3 Ceramide Glc Gal GalNAc Gal Gal Glc Ceramide
ganglioside neuraminidase Hexosaminidase A + B
Sandhoff´s disease Globos
NANA GalNAc
Globoside Ceramide Glc Gal Gal Gal Glc Ceramide
β-galactosidase α-galactosidase A
Gal Gal Fabry´s disease
Cerebroside Ceramide Glc

Glucocerebrosidase
Gaucher´s disease sphingomyelinase
Ceraminidase Glc
Niemann-Pick disease
Farber´s disease
Sphingosine Choline P Ceramide Sphing
Ceramide
Gal Gal-cerebroside- Phosphocholine
fatty acid β-galactosidase
Krabbe´s disease
Cerebroside Ceramide Gal
2-
SO4 cerebroside sulphatase
metachromatic leucodystrophy
Sulphatide Ceramide Gal S
526
Mucopolysaccharidoses and sphingolipidoses 28.4.2
he dy re ems
sh vio ard lex
m stos udi aly
bo st pro on
se
p
so clo eg
lo isea
or ral ati
ny atu bl
en is ng
ha et lti
dy al nom
ar lv a
be al r mu
he va lasi
in e d
ss
pa ce
rn ple
ar sp
he e fa
co tos
g
e
t
s
t
t
ar
Disease
co
Hunter
Hurler-Scheie
Maroteaux-Lamy
Sly
Sanfilippo A
Sanfilippo B
Sanfilippo C
Sanfilippo D
Morquio A
Morquio B
Table 28.1.: Problems in mucopolysaccharidoses. Dysostosis multiplex: thickened skull and

ribs, thick and short long bones, vertebral abnormalities
g
in
re
m ud
ilu
ki ras enc aly
n
la
to clo
fa
io
g
cu
in fic e
or e ia
at
a
in in t
A ea MΦ
sk de nom
ke ns
sk pa ear
C -lad hes
we res ma
bl lysi ard
bo tos le
lip ar s
io le
o p le
h
id est
pa sc
i
ne ple
n n
ra et
iz ed
ra
u
ng +
er s
jo ey +
dn h
he mu
in s
A nod
pa al r
ch nes
se y-r
d
t
ak
u
t
en
in
cr
Disease
m
GM1 -gangliosidosis
Tay-Sachs
Gaucher´s
Krabbe
metachrom. leukodystr.
Sandhoff´s
Fabry´s
Niemann-Pick
Farber´s
Table 28.2.: Signs in sphingolipidoses. These diseases are usually fatal at an early age. Treat-
ment: glycosylation inhibitors like nojirimycin, enzyme replacement therapy.
527
28.5. Mitochondria
Mitochondria are required for energy (Krebs-cycle, oxidative phosphorylation) and fatty
acid metabolism. Part of the urea cycle and heme synthesis also occur in mitochondria.
Most mitochondrial enzymes encoded in nucleus → autosomal recessive inheritance. How-

ever, the mitochondrial genome contains 37 genes:
• the genes for 13 subunits of OxPhos enzymes
• 2 rRNAs and 22 tRNAs
Mutations in those genes give maternal inheritance, as only very few (if any) of the mito-
chondria of the spermatozoon enter the egg during fertilization. Homoplasmy/heteroplasmy
and replicative segregation affect the distribution of defect and normal mitochondria in the
cells of the body, thus a mitochondrially inherited disease may break out at any time during
life, and in any organ. Organs with heavy respiratory activity are however most likely to
be affected: muscle, nervous system.
28.5.1. Pyruvate metabolism
Pyruvate is transported into the mitochondria and then metabolized by either of two en-
zymes:
Pyruvate dehydrogenase to enter the citric acid cycle
Pyruvate carboxylase to enter gluconeogenesis
Failure of either enzyme can lead to pyruvic and lactic acidosis, with significant sequelae
for the patient.
Pyruvate dehydrogenase Pyruvate dehydrogenase is a complex of 3 enzymes, a defect in

any of these causes serious problems: lactic and pyruvic acidemia, lethargy, poor feeding,
tachypnea, developmental delay, seizures, spasticity, ataxia, and sudden death. The disease
may present as Leigh syndrome (vide infra).
Defects in E1 α-subunit are the most frequent. This subunit is encoded on the X-chromosome,
but heterozygous ~ may be affected due to X-inactivation. E1 α is inactivated by PDH-
kinase and activated by PDH-phosphatase. Phosphatase deficiency presents similar to E1 -
deficiency. The kinase is inhibited by dichloroacetate, which may be used to get as much
activity as possible out of any remaining enzyme. Further management include a low carb
(“ketogenic”) diet and high thiamin.
528
Pyruvate metabolism 28.5.1
Figure 28.8.: Metabolism of pyruvate.

-
COO
CO2
C O P
GDP
CH2
PEP
ADP
GTP
PEPCK
PK
ATP
-
COO NADH +
+ NAD
C O COO
-
+H COO
-
CH2 C O HC OH
-
COO CH3 LDH CH3
Oxaloacetate
Pyruvate Lactate
Cytosol
Mitochondrium Pi
ADP CO2 -
ATP HS CoA HCO3
- -
COO COO S CoA
C O Pyruvate C O PDH C O
CH2 carboxylase CH3 CH3
-
COO Pyruvate Acetyl-CoA
Oxaloacetate
529
Figure 28.9.: Pyruvate dehydrogenase is a complex of 3 enzymes.

Mechanism of pyruvate decarboxylase complex:
O H
E1 T H + C C CH3 + E1 T C CH3
O
H2O + HCO3
O OH
Thiamine-PP on activated acetaldehyde
decarboxylase
H
E1 T C CH3 + E2 E1 T H + E2
OH S S S SH
liponamide on H3 C C O
acetyl transferase S-acetyl-dihydroliponamide
E2
+ CoA-SH E2 + H3 C C S CoA
S SH
SH SH O
H 3C C O
Dihydroliponamide Acetyl-CoA
E2 + E3 FAD E2
+ E3 FADH2
SH SH S S
Liponamide reduced dihydroliponamide

dehydrogenase
E3 FADH2 + NAD+ E3 FAD + NADH + H+
530
Pyruvate metabolism 28.5.1
Defects in E3 (chromosome 7) also affects α-ketoglutarate and branched chain amino de-
hydrogenase, they present clinically as maple syrup urine disease with lactic and pyruvic
acidemia.
Pyruvate carboxylase is encoded on chromosome 11q12-q13. Three types of PC-deficiency

have been described:
North America (group A) lactic, pyruvate + alanine acidemia. Severe mental, psychomo-
tor and developmental retardation. Inactive enzyme produced, amino acid substitu-
tions. Treatment with thiamine, lipoate + dichloroacetate.
France, UK (group B) respiratory distress, increased serum lactate, ammonia, citrulline,

proline and lysine; intracellular redox disturbance: cytosolic compartment more re-
duced and the mitochondrial compartment more oxidized. Usually do not survive
beyond 3 months of age. Cystic periventricular leukomalacia on cerebral ultrasound
at birth. No enzyme protein produced (nonsense-mutation).
‘Benign’ type (Group C) preservation of motor and mental abilities, episodes of metabolic
acidosis with elevated lactate, pyruvate, alanine, β-hydroxybutyrate, acetoacetate,
lysine, and proline, managed by rehydration and bicarbonate. PC activity a few % of
normal
Oxidative phosphorylation is performed by complexes I–V in the inner mitochondrial

membrane. Mutations in any of these complexes may cause Leigh syndrome:
• Frequency: I 33 %, II 4 %, III 7 %, IV 28 %, I + IV in 28 % of cases
• Early-onset progressive neurodegeneration with focal, bilateral lesions in CNS (brain-

stem, thalamus, basal ganglia, cerebellum, spinal cord): demyelination, gliosis, necro-
sis, spongiosis, or capillary proliferation.
• Clinical symptoms depend on which areas of the central nervous system are involved:
feeding difficulties, tachypnea, lactic acidosis, truncal hypotonia, growth retarda-
tion, cardiomyopathy, encephalopathy, myoclonic epilepsy (cave: valproate sensitiv-
ity), liver failure
• inheritance mitochondrial or autosomal recessive
Another disease caused by mutations in in mtDNA for complex I, III, and IV of oxidative
phosphorylation is Leber hereditary optic neuropathy with acute or subacute cen-
tral vision loss leading to central scotoma (blind-spot) or blindness in mid-age. Neurologic
manifestations may be seen in some cases. Alcohol or tobacco abusus accelerate disease
progression resulting in tobacco-alcohol amblyopia.
531
Figure 28.10.: β-oxidation of fatty acids.
S CoA
Acetyl-CoA
O
Carnitine
S CoA
S CoA
S CoA
S CoA
+
S CoA
O
O
O
O
NADH + H+
CoA SH
Carnitine
OH
FADH2
O
CoA-SH
FAD
+
NAD
H2O
trans-∆2-enoyl-CoA
L-β-hydroxyacyl-CoA
Acyl-CoA (-2 C)
β-ketoacyl-CoA
Acyl-carnitine
Acyl-CoA
Hydroxyacyl-CoA
dehydrogenase
dehydrogenase
hydratase
Thiolase
Carnitine acyl
Enoyl-CoA
transferase II
Acyl-CoA
Ragged red fibres are lumps of aggregated mitochondria in muscle biopsies which stain
red with Gomori’s trichrome. They are found in myopathies caused by either of two
mutations:
succinate dehydrogenase subunit A (SDHA) on chr. 5p highly variable phenotype: en-
cephalocardiomyopathy with leukodystrophy, dementia, myoclonic seizures, pigmen-
tary retinopathy, ataxia, cardiac conduction defects, short stature, generalized muscle
weakness and wasting with easy fatigability. Muscle biopsy: excessive lipid droplets in
muscle fibers, mitochondria with abnormal structure and paracrystalline inclusions
mtDNA for tRNALys Myoclonic Epilepsy with Ragged Red Fibers (MERRF) syndrome:
progressive myoclonic epilepsy, migrainous headache and vomiting, loss of vision
and/or hearing, dementia, short stature, exercise intolerance, Wolff-Parkinson-
White syndrome in EKG: circus movement of electrical signal through Kent-bundle,
resulting in sudden tachycardia. Pyruvate and lactic acidosis. Disease clinically vari-
able because of heteroplasmy. Other mitochondrial genes (e.g. tRNAHis , tRNASer )
may also be cause in about 10–20 % of cases. Management: CoQ, carnitine, anti-
convulsants.
28.5.2. β-oxidation of fatty acids
All steps of β-oxidation are performed by isoenzymes with overlapping specificity for fatty
acids of different chain lengths:
short < 6 C-atoms
532
Peroxisomes 28.6
medium 6–12 C-atoms

long 12–24 C-atoms
very long > 24 C-atoms (also oxidized in peroxisome)
A defect in acyl-CoA dehydrogenase leads to the accumulation of acyl-CoA of respective
lengths, ω-oxidation (with dicarboxylic aciduria) and Gly-conjugation increase but can not
fully compensate.
short chain (SCAD) generalized (infants with aciduria) or muscle only (middle aged with
myopathy). Metabolic acidosis, failure to thrive, developmental delay, seizures, my-
opathy, but no hypoglycemia. Autosomal recessive.
medium chain (MCAD) Mostly K304E, 1p31, remaining activity < 10 %. After fasting
or stress (cave: infection!) potentially fatal hypoglycemia (Glc consumption ↑, glu-
coneogenesis ↓ because Krebs-cycle ↓), structural changes in mitochondria. Long
term damage: slowed cognitive development, cerebral edema, encephalopathy, weak
muscles, exercise intolerance, fatty liver. Management: regular meals, carnitine, Glc
infusion in acute crisis.
long chain (LCAD) 2q34-q35, Q303K. Rare.
very long chain (VLCAD) 17p13.1-p11.2, various missense mutations or deletions, some
respond to bezafibrate. Membrane associated protein (other AD are soluble). Car-
diomyopathy, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy,
or sudden death in infancy with hepatic steatosis.
Mitochondrial tri-functional protein (TFP) is a complex containing long-chain enoyl-
CoA hydratase, hydroxyacyl-CoA dehydrogenase (LCHAD) and thiolase. Deficiency in
LCHAD is a serious disease resulting in fulminant neonatal liver failure or progressive
liver degeneration. Also affected are muscle, retina and peripheral nerves. ~ pregnant with
LCHAD defective fetus may get acute fatty liver of pregnancy or HELLP-syndrome
(hemolysis, elevated liver enzymes, low platelets). Intrauterine growth restriction of the
fetus may lead to pre-term delivery.
28.6. Peroxisomes
Peroxisome are formed by fission or de novo from the ER. They perform reactions that
involve ROS formation, which are then destroyed by enzymes. For example the FADH2 of
very long chain acyl-CoA dehydrogenase in peroxisomes transfers its hydrogens directly to
oxygen, forming hydrogen peroxide which is then taken care of by catalase.
The following key pathways occur in peroxisomes:
533
Plasmalogen synthesis defect in dihydroxyacetone phosphate acyltransferase in rhizomelic

chondrodysplasia punctata
β-oxidation of long and very long chain fa adrenoleukodystrophy because of transport de-
fect
phytanic acid oxidase infantile Refsum disease as phytol (from chlorophyll) can not be
digested
bile synthesis shortening of side chain by β-oxidation
degradation of pipecolic acid metabolite of a minor Lys-breakdown pathway
ABCD proteins are ABC-type half-transporters, that form homo- or hetero-dimers. They
transport long and very long chain fatty acids into peroxisome. A defect in ABCD1 leads
to adrenoleucodystrophy, inheritance is usually X-linked, with a milder form (adreno-
myeloneuropathy) affecting some heterozygous ~. Some rare autosomal forms have been
described (defects in the enzymes of peroxisomal β-oxidation). Failure to metabolize VLCFA
leads to accumulation in brain + adrenals → demyelinization of white matter → death in
adolescence. Patients present with loss of previously acquired neurologic abilities, seizures,
ataxia, loss of vision and hearing and Addison’s disease. Lorenzo’s oil is probably the only
film ever made in Hollywood about fatty acid metabolism. The mixture containing glycerol
esters of erucic C22:1 ω9 and oleic acid C18:1 ω9 normalizes serum fa profile, may slow down
disease progression in asymptomatic patients (jury is still out on that one) but does not
change final outcome.
Malformation of peroxisomes leads to Zellweger syndrome (cerebrohepatorenal syn-
drome), a severe disease usually fatal during the first half year of life. Signs include
facial features flattened facies, large anterior fontanelle, widely split sutures, and broad
nasal bridge
polycystic kidneys with adequate functional renal parenchyma
liver intrahepatic biliary dysgenesis, liver cysts, hepatomegaly, jaundice
CNS generalized hypotonia with absent Moro (startling) reflex, sudanophilic leukodystro-
phy, mental retardation, seizures, tapetoretinal degeneration, sensorineural hearing
loss
joints and bones chondral calcification
markers high serum iron + copper and high iron binding capacity, elevated pipecolic acid
in serum and cerebrospinal fluid
Peroxisomal proteins are nuclear encoded and translated on free ribosomes. The function
of proteins involved in peroxisomal enzyme import is still a matter of active research, the
following list is provisional:
534
Biotransformation 28.7.1
PEX3, PEX16, and PEX19 no peroxisomal membranes are formed without these proteins
PXR1 (PEX5) encodes a receptor that recognizes proteins containing peroxisomal target-
ing sequence 1 (PTS1), defined by the carboxy terminal consensus sequence serine-
lysine-leucine (SKL)
PEX7 encodes the PTS2 receptor and recognizes proteins with an N-terminal motif present
in fewer matrix proteins. Mutations in PEX7 are associated with the clinically distinct
disorder rhizomelic chondrodysplasia punctata (RCDP)
PEX14, PEX17, and PEX13 docking of the PTS1 and PTS2 receptors and their associ-
ated proteins
PEX10, PEX12, and PEX2 part of the translocation apparatus for matrix proteins
PEX 8 anchors the above import complexes at the lumenal aspect of the peroxisomal
membrane
PEX1 and PEX6 recycling of peroxisomal import receptors PEX5 and PEX7
PEX1, PEX6, PEX4 and PEX22 act late in the import pathway, perhaps after the translo-
cation process
For reasons poorly understood patients may show Mosaicism:
type 1 disparity between serum markers and cellular results from the same individual.
In some cases, cultured cells are immunohistochemically normal at 37 °C, but show
defective peroxisomes at 40 °C.
type 2 disparity in matrix protein import into peroxisomes in adjacent cells from the same
individual. Often corresponds to milder phenotype.
28.7. Endoplasmic reticulum
The ER is required for synthesis of membrane proteins (rough ER) and for lipid synthesis,
drug metabolism and sterol synthesis (smooth ER).
28.7.1. Biotransformation
Our food contains many substances that plants synthesize to protect them from parasitism.
Some such secondary metabolites are useful to us (e.g. as antioxidants), but many are
toxic or carcinogenic and have to be removed. Pharmaceuticals are often metabolized by
the same pathways. Together such “foreign” chemicals are called xenobiotics.
535
Table 28.3.: Affected genes in Zellweger syndrome. The frequencies were found in a recent study on 198 patients.
Gene Symbol OMIM Chromosome Protein Name Patients Freq. Exons Gene cDNA Protein
% (kb) (kb) (aa)
PEX1 602136 7q21-q22 Peroxisome biogenesis factor 1 134 68.0 24 41.5 3.9 1283
PEX10 602859 1p36.32 Peroxisome assembly protein 10 9 4.6 6 7.8 1 326 + 345
PEX12 601758 17q12 Peroxisome assembly protein 12 8 4.1 3 3.8 1.1 359
PEX13 601789 2p15 Peroxisomal membrane protein 13 2 1.0 4 31.2 1.2 403
PEX14 601791 1p36.2 Peroxisomal membrane protein 14 1 0.5 9 135.5 1.1 377
PEX16 603360 11p12-p11.2 Peroxisomal membrane protein 16 1 0.5 11 8.4 1 346
PEX19 600279 1q22 Peroxisomal biogenesis factor 19 1 0.5 8 5.7 0.9 299
PEX26 608666 22q11.2 Peroxisome assembly protein 26 13 6.6 5 10.7 0.9 305
PEX3 603164 6q23-q24 Peroxisomal biogenesis factor 3 3 1.5 12 39 1.1 373
PEX5 600414 2p13.3 Peroxisomal targeting signal 1 receptor 3 1.5 15 18.9 1.8 602 + 639
PEX6 601498 6p21.1 Peroxisome assembly factor 2 21 10.7 17 15.1 2.4 980
PXMP3 (PEX2) 170993 8q21.1 Peroxisome membrane protein 3 2 1.0 4 7.4 1.5 626
Total 198 100.0
28.7.1
536
Figure 28.11.: Phase I metabolism of xenobiotics makes the compounds more reactive and
more polar, hence water-soluble.
Phase I: Oxidation
+ +
X H + O2 + NADPH + H X OH + NADP + H2O
Cytochrome P-450
dependent
monooxygenase
During phase I metabolism, such compounds are made more reactive and more polar by
introduction of oxygen atoms into their structure. During phase II solubility is increased
by conjugating polar residues (e.g. sugars, amino acids, glutathione, mercapturic acid) to
them.
Phase I and II xenobiotic metabolism
Phase I drug metabolism makes compounds more reactive and more polar, hence water-
soluble, by introducing oxygen into their structure. This is performed by a group of enzymes
collectively known as cytochrome P450 (CYP450). These enzymes are monooxygenases
(“mixed functional oxygenase”) which introduce one oxygen atom of an O2 molecule into
the compound, the remaining atom is reduced to water with NAD(P)H.
CYP450 is located on the smooth ER of liver, lung, intestine and kidney. Apart from
drug metabolism it is required for ω-oxidation, fatty acid CoA desaturase, synthesis of
cholesterol, steroid hormones and leucotriens and for the degradation of heme. CYP450
absorbs light of 450 nm after reaction with CO (Soret-Band), which explains their name.
They can use either NADH or NADPH as co-substrate. Humans have 57 CYP450-genes (+
5 pseudo-genes), with broad and overlapping substrate specificity.
537
Figure 28.12.: Reaction mechanism of CYP450. The heme-Fe and the O2 are reduced by
electrons from NAD(P)H oxidation to a very reactive state (Fe+ does not
even exist as isolated ions!), that can attack the substrate. Note that both
binding of substrates and release of product are ordered.
+ + -
NAD(P)H:Cyp450 reductase NAD(P)H NAD(P) + H + 2e
(FMN + FAD-dependent)
Cytochrome P-450 (CYP450) 2+ +

E Fe H X E Fe H X
( ) (
H2O
) HO X
+
O2 O2 - O2 2 H
e
- 3+ 2+ 3+
X H e E Fe H X E Fe H X E Fe HO X 3+
-
E Fe HO X
oO2
-
o O2 H2O 3+
3+ 2+ E Fe
3+ E Fe H X E Fe H X
E Fe
2+
E Fe H X
CO
CO
absorbs light at 450 nm
538
Figure 28.13.: In some cases activation of compounds by CYP450 makes them carcinogenic
by producing highly reactive epoxides, that can introduce point mutations
into DNA by reacting with G-residues.
2 O2, 2 H+, H2O,
2 NADPH 2 NADP+
10 O
9
8 CYP450
HO
7
OH
Benzpyrene (+)-Benzpyrene-7,8-diol-9,10-epoxide
(in tobacco smoke) (causes lung cancer)
O O O O
O2, H+ H2O
O NADPH NADP+ O
O
CYP450
O O O O
Aflatoxin B1 Aflatoxin B1-2,3-epoxide
(in moldy food) (causes liver cancer and kwashiorkor)
+
H
O H O O
+
N O N C C
HN HN
+ C C
N spontaneous N
H2N N H2N N
R R
Guanine Point mutation
539
Figure 28.14.: Some harmless chemicals are activated by CYP450 into powerful poisons or
highly active pharmaceuticals. Can you think of a reason to give codeine
instead of morphine, if codeine needs to be activated to morphine before it
has any effect?
O +
N O
O + O2, H+ H2O O GSSG, O +

N O
+
N O N O AChE Ser OH Ser AChE
NADPH NADP+ 2 GSH H2S OH
O
H3C C O P O C CH3
CYP450 H2 H2
O
O O O
H3C C O P O C CH3 H3C C O P O C CH3 H3C C O P O C CH3
H2 H2 H2 H2 H2 H2
S S O O
Parathion (Thiophos, E605) Paraoxon (E600) inactivated enzyme
H3C O HO C O HO
O2, H+ H2O H2 H2C O
NADPH NADP+
O H O H O H
N CH3 CYP450 N CH3 N CH3
HO HO HO
Morphine
3-methoxymorphine
(Codeine)
540
Figure 28.15.: During phase II of xenobiotic metabolism the activated compounds resulting
from phase I are conjugated to polar or charged residues to increase water
solubility, so they may be excreted by the kidney.
Phase II: Conjugation
- -
COO COO
O O UDP O O X
OH + X OH OH + UDP
OH OH
OH OH
UDP-glucuronic acid glucuronic acid conjugate
O O H2
O S O P O C O Adenine
O
O O
+ X OH X O S O + 3-Phospho-AMP
O
O OH
O P O
O
Phosphoadenosine phosphosulfate Sulfate ester
(PAPS)
ATP AMP
CoA-SH PPi Gly CoA-SH
O O
- -
X COO X C S CoA X C N C COO
H H2
Glycine conjugate
Gln
O
C NH2
CoA-SH O (CH2)2
-
X C N C COO
H H
Glutamine conjugate
COOH HCl
H2N CH COOH Glu Gly
O OH
CH2 C
X Cl + CH2
O
H2N CH
O OH O
C
O
C N CH2 CH2 C
H O +
C N CH CH2 C N CH2 H3N CH
O H H
CH2 C N CH CH2
O H
SH CH2 S
S X
Glutathione X
Cysteine S-conjugate
(γ-Glu-Cys-Gly)
Glutathione S-conjugate
Acetyl-CoA
O O
O C
H3C C N CH
H CoA-SH
CH2
S
X
N-acetyl-cysteinyl derivative
(a mercapturic acid)
541
Pharmacogenetics
Humans vary in their
• ability to metabolize and excrete drugs:
– organ function (liver, kidney)
– drug metabolizing enzymes
• affinity of drug target (receptors, enzymes)
Therefore the same dose of the same drug will have different effects in different patients:
• intended effects: response rate 25–75 %
• side effects:
– severe in about 15 % of US approved drugs even when properly administered
– 2 × 106 people hospitalized per year for drug side effects
– leading to 1 × 105 deaths per year
→ No one-size-fits-all medicine! Look out for
Target selection Many diseases caused by problems in several different proteins. Pharma-
ceutical against target A may not work if defect is in B! Example: Cancer
Heightened or reduced target affinity
Increased sensitivity to side effects e.g. G6PDH deficiency
Rate of transformation e.g., CYP450 polymorphisms may affect both activation and in-
activation of drug
Drug interactions Alcohol is also metabolized by CYP450 (microsomal ethanol oxidizing

system, MEOS)→ induction → interference with barbiturate elimination. Alcohol in-
duces the synthesis of CYP450 in the liver, but also inhibits their activity acutely.
Barbiturates, for example, work in the intoxicated but not the sober alcoholic. Im-
portant in anesthesia!
542
Objectives 28.8
28.8. Objectives
After completion of this course unit students should be able to
• name the biochemical pathways occurring in the various organelles of a cell

• discuss defects, pathomechanism, symptoms management and probable outcome of
inherited diseases of metabolism
• describe how xenobiotics are modified by our metabolism and what the beneficial and
adverse consequences of these modifications may be
• explain, using examples, how drug-patient and drug-drug interactions require indi-
vidualized medicine
543
Part VI.
Semester two, Mini III

29. Advanced DNA technology
29.1. Germline Gene Manipulations (analysis of gene

function)
Genes can be introduced into the germline, and normal genes can be artificially disrupted by
gene targeting. These methods are used to create transgenic mice and knockout mice.
• These animals provide models for human diseases, and the abnormalities of knockout
mice (if any) reveal the functions of the knocked-out gene.
• Transgenic animals are created to secrete valuable therapeutic proteins in their milk
or supply human-friendly organs for transplantation.
DNA constructs for producing transgenic mice should be devoid of viral sequences and
bacterial plasmid sequences, but they should carry flanking sequences that include the
promoter region which you wish to use to express the gene and normally, at least one
intron is needed for satisfactory expression. Techniques:
1. The gene construct is micro-injected into the male pronucleus of the zygote, or the
zygote is bathed in the DNA while an electrical pulse is applied to make the membrane
permeable (electroporation). The zygote is grown into an embryo, and the embryo
is implanted into a pseudo-pregnant female mouse.
2. The resulting mice are PCR analyzed to demonstrate insertion of the gene construct,
and the mouse that carry gene insertions are analyzed to firstly demonstrate expres-
sion of the gene and secondly to look for phenotypic effects.
Problems with the above described method are large, and include the following:
• Insertional mutagenesis: the insert may have disrupted an important function in the
recipient.
• Variable copy number of insertion: it is impossible to control how many copies are
inserted into a given mouse.
547
• Lack of expression: an inserted construct may have landed in a region of the genome
that is inactive (for example in heterochromatin) in the tissue of interest. However,
even insertion in a permissive location will not always result in expression, because
if a large copy number is inserted, the cell is likely to activate the protection against
virus which inactivate tandem repeats of DNA.
• With this mechanism, it is impossible to mimic loss of function mutations, as the only
outcome possible is addition of gene sequences, not replacement of gene sequences.
For these reasons, knock-in and knock-out methods are the currently preferred methods
in research labs. DNA constructs aimed at this use generally have a central area where a
difference is found relative to the native mouse sequence, flanked by long regions of perfect
match to the mouse sequences. These constructs can be incorporated into the genome by
double homologous recombination, one recombination event in each of the flanking regions
ensuring insertion of a single copy of the experimental, central DNA construct.
For knock-out:
• The central area of the construct will lead to replacement of at least one exon of the
gene with “junk” DNA, and the central area also will have a copy of a gene conferring
resistance to the drug neomycin.
• The DNA construct is electroporated into cultured embryonic stem (ES) cells. These
cells which are purified from an early embryo should be omnipotent, in other words
can become part of any tissue in the mouse including the Germline.
• ES cells are grown in the presence of neomycin to select for cells that have taken up
the DNA. Each colony of cells is thereafter analyzed to ensure that the construct has
been inserted in the appropriate position of the genome and with the wanted final
structure.
• Each colony of ES cells that have passed the test is inserted into the cavity of a blas-
tocyst, which is thereafter inserted into a pseudo-pregnant mouse. The mice produced
will therefore be chimeras, containing cells that originate in the ES cells as well as
cells from the blastocyst.
• Offspring mice are bred to produce pure mice originating from the ES cells. These mice
will be heterozygotes for the introduced mutation and will be analyzed for possible
phenotypic changes.
• The mice will be used in brother-sister matings to produce homozygote mutant mice,
which again will be used for phenotypic analysis.
548
Cre/LoxP system for recombination 29.1.1
For knock-in: This is a further development of the knock-out mechanism, where the main
difference is that the central area of the DNA construct carries something that is not junk
but instead is of interest. This could for example be the ∆F508 mutation of the cystic
fibrosis gene from a human CF patient inserted into the CF gene of a mouse to allow
further analysis of the mechanism by which the mutation alters the phenotype, or to allow
test of prospective drugs.
The above described germline gene manipulations all involve mice. It is widely recognized
that germline gene therapy in humans should be avoided because somatic gene therapy
carry fewer risks and are easier to reverse should the need arise. In the far future, one could
consider the introduction of useful genes to diversify the human gene pool, for example genes
for vitamin C synthesis or cobalamine synthesis, or additional copies of tumor suppressor
genes to reduce the cancer risk.
29.1.1. Cre/LoxP system for recombination
The neomycin resistance gene is employed in both knock-out and knock-in methods. Theo-
retically, this could result in phenotypic effects of its own, either directly due to the protein
produced or due to transcription from the neomycin promoter continuing into neighbor-
ing genes. Such considerations have led to use of the Cre/LoxP (or the similar FRT/Flip
system) to take out unwanted parts of the inserted DNA:
• LoxP is a DNA sequence that is included twice in the construct, flanking the region
that should be deleted.
• Cre is a recombinase that recognizes the specific sequence and catalyzes a reaction
whereby the area between the two DNA elements comes out as a circular DNA mole-
cule. One copy of the LoxP element is left behind, and one copy is included in the
excised circular DNA.
The usual setup is that the LoxP elements are inserted so that they flank the neomycin
resistance gene, while the Cre recombinase is found in a commercially available mouse.
When a promising knock-out/knock-in mouse has been identified, it will be bred with the
Cre-containing mouse and the offspring should loose the neomycin gene.
Conditional knock-out: Another interesting application of the Cre/LoxP system is based

on the availability of mice which harbor a Cre gene that is only expressed in a defined
tissue or at a regulated time during development. In this setup, the initial knock in replaces
an exon in the gene of interest with the same exon but now flanked with LoxP-elements.
Breeding the resulting mouse with the tissue-specific or temporally-specific Cre mouse leads
to loss of the exon but only in the tissue of interest, such that the effects of a mutation
549
in the liver can be studied independently of the effects of the same mutation in another
tissue.
29.2. RNAi (inhibitory RNA, Knock-down)
Some diseases are caused by the expression of undesirable genes; viral genes in virus diseases,
oncogenes in cancer, and regulatory genes in some autoimmune diseases. RNAi methods are
aimed at reducing expression of a specific gene in a cell, either a cell culture for analytical
purposes (what does the gene do) or as an experimental drug. The mechanism of action is a
little different, but this is an extension of the methods known as anti-sense technology. RNAi
is using a mechanism in the cell that was developed as a defense against double-stranded
RNA virus. It seems that a double stranded RNA molecule will first be cut into shorter
pieces by a protein called Dicer, that the resultant shorter fragment will be attached to
a complex known as RISC, and that it in that complex will be found in single stranded
form. Another single-stranded RNA molecule (e.g., an mRNA) can hybridize with the RNA
on the RISC complex; it will then be cut into shorter pieces and the hybridizing part will
be released while the RISC complex including the original single stranded RNA can be
recycled. Adding an RNA molecule that is complementary to an existing mRNA in a cell
can therefore produce the double stranded molecule that initiates the RNAi process, and
effectively eliminate translation of a given protein in the cell.
29.3. Microarray Technology (DNA Chips)
In traditional probing (dot blotting, Southern blotting), the probe is applied to the im-
mobilized target DNA. To test the target DNA for all theoretically possible mutations
(“mutation scanning”), a large number of probes would have to be applied successively; this
would therefore take prohibitively long time. Using many different probes immobilized in
a grid pattern on a solid support and simultaneously hybridize all of these with a chopped
up version of the target DNA is the principle in microarray technologies. The target DNA
will be labeled with fluorescent dye, left to interact with the probes for hours to days, non-
bound target DNA will be washed away, and the amount of hybridization will be quantified
by a laser-equipped reader.
Gene Chips: Gene chips are used for gene scanning, or the simultaneous analysis of many
mutations or polymorphisms: in this application, the probes each consist of one allele-
specific oligo (ASO - normally about 15 bases long with the central position differing
between the mutated and the normal form of the gene). Since up to 100 000 probes fit on a
550
Somatic Gene Therapy 29.4
single chip, all scanning and recording has to be computerized. Applications of gene chips,
or microarrays, include:
• Mutation scanning of individual genes in patients, to figure out whether a clinical

problem is caused by a defect in a specified gene.
• The genome-wide genotyping of polymorphisms, especially single-nucleotide polymor-

phism (SNPs), in the context of association studies aimed at finding disease causing
mutations (more on association studies in the section on Multifactorial Disease). There
are a few million SNPs in our genome, but only a few of them (several thousand?) are
medically important. Also, sequence differences between related species (e.g. human
and chimp) can be detected with gene chips.
In summary, rather than screening for one mutation at a time, people can be screened
for thousands of single-gene disorders, susceptibility genes for multifactorial diseases, and
normal polymorphisms, all in the same procedure. DNA chips are expected to become the
standard method for predictive genetic screening, for example in prenatal or preimplantation
diagnosis.
cDNA Chips: cDNA chips are used to study patterns of gene expression. The immobilized
probes are not ASOs but are either cDNAs or longer oligonucleotides corresponding to a
unique portion of a cDNA and they are not used to analyze genes, but mRNA. The mRNA
is extracted from the cells or tissue, usually simultaneously reverse transcribed into a cDNA
and labeled with a fluorescent group, and then hybridized to the cDNA chip. Since each
cDNA on the chip corresponds to one gene, you can quantify which genes are expressed
and how much. Most often, the experimental design calls for competitive hybridization with
samples coming from two different sources, e.g., the pattern of gene expression in a tumor
can be compared with gene expression in the normal cells from which the tumor originated.
This application is expected to soon be used in the clinic, as the expression pattern in the
tumor has prognostic value for the patient and has value for the oncologist in picking the
drug cocktail most likely to be effective on this particular tumor.
29.4. Somatic Gene Therapy
Many genetic diseases are caused by inactivating (“loss-of-function”) mutations. In theory,

these diseases can be treated by introducing an active, functional gene into those somatic
cells where it is needed. Gain-of-function mutation can theoretically be treated with a DNA
molecule that produces an mRNA which initiates an RNAi reaction and thereby hinders
expression of the damaging protein.
Problems:
551
1. Large DNA molecules are not easily taken up by cells.

2. Even if it enters the cell, the gene may be degraded by nucleases.
3. Exogenous genes rarely become integrated into the cellular genome, although this is
theoretically possible by homologous recombination.
4. The regulation of gene expression is problematic for artificially introduced genes.
Therefore, gene therapy is promising only if tightly-regulated gene expression is not
required.
5. The targeting of a gene to the relevant tissue is difficult.
6. Somatic gene therapy does not affect the Germline; the patient can still transmit
the defective gene to his children. Many consider this an advantage of somatic gene
therapy!
In the United States, some gene therapy trial participants have died as a result of the
therapy given. This has lead to a reconsideration of the safeguards necessary before an
experimental gene therapy can be tested in the treatment of human disease.
Integrating the therapeutic gene into viral vectors is probably the most active research field
in gene therapy. Retroviral vectors are attractive because they insert the gene directly
into the cellular genome. They contain retroviral long terminal repeats, but the gag, pol, and
env genes are replaced by the transferred gene, all nicely wrapped in a retroviral capsid
and envelope. The reverse transcriptase + integrase in the virus particle effect reverse
transcription and integration into the genome. Problems:
• Only low titers can be produced.
• Only dividing cells can be transfected by currently used retroviral vectors. Lentiviral
vectors (related to the AIDS virus) are promising because they can infect nondividing
cells.
Retroviral vectors and Adenoassociated Virus (AAV) suffer from limitation in size the
DNA that can be engineered into the virus, while Adenovirus can hold much more DNA.
Adenovirus can be produced in high titers and infect nondividing cells, but they rarely
integrate into the host cell genome. They also have the potential of damaging the infected
cells. Adenoassociated Virus (AAV) integrate into the genome of the host cell and are some
of the most promising viral vectors for therapy involving small genes.
In some contexts, repeated treatment would be necessary, and antibody produced against
the viral vector becomes a big problem. Physical methods of gene delivery (liposomes,
receptor-mediated endocytosis) could potentially be helpful in these circumstances.
Gene therapies are still experimental. They are not only promising for the treatment of
genetic diseases, but also for some other diseases, for example, bringing genes for anti-
inflammatory proteins into the joints of arthritis patients. Many gene therapy protocols
552
Objectives 29.6
are ex vivo techniques, and only a small proportion (Usually less than 1 %) of the cells are
stably transfected. Ex vivo means that for example bone marrow cells are aspirated, grown
in cell culture and transfected with the therapeutic gene; the gene insert is analyzed before
the cells are infused back into the patient. A similar process can be performed with liver
cells, but obviously not with muscle or nerve cells.
A single gene therapy protocol have moved beyond the experimental into the approved
protocols; the Chinese government has approved to inject a type of solid tumors with a
DNA construct containing the P53 protein. The expected outcome is to stop cell divisions
due to the sensing of DNA damage, and when combined with traditional chemotherapy, to
induce apoptosis in the cells that have taken up the gene construct.
29.5. Proteomics Methods
Proteomics entails the study of the complete complement of proteins encoded by an organ-
ism. Separation of proteins is usually accomplished using two-dimensional (2D) gel elec-
trophoresis. Firstly, proteins are separated by charge using isoelectric focusing (IEF); sec-
ondly, the separated proteins are then further purified by separation in a second dimension
based on size differences (using SDS in the gel). The 2D gel can then be used as a template
to analyze the proteome using antibody-based technology or chemical analysis. Mass spec-
trometry (MS) technology allows sensitive and accurate detection of proteins in 2D gels.
For large proteins, fragmentation with a specific protease may be necessary before using
MS, while smaller proteins can be analyzed directly. For instance, the slight mass difference
in the sickle-cell hemoglobin protein can be distinguished from the normal beta-hemoglobin
protein using a single spot on a 2D gel analyzed in a mass spectrometer.
29.6. Objectives
• Transgenic mice: understand methods, compare and contrast for especially what is
the outcome of
– Traditional
– Knock out
– Knock in
– Conditional
– Realize that the Cre-LoxP system can be used to delete a part of the inserted
construct in vivo
553
• Knock down technology: understand the differences from and similarity to knock out
• Describe the use of allele-specific oligonucleotides in a chip format to test for a persons
genotype
• Describe the use of longer oligonucleotides on a chip to measure expression level of a
gene
– Tumor as an example with the data improving diagnosis and choice of treatment
• Use of proteomics methods to identify completely unknown proteins
– Describe use in identification of new pathogens
– And in identification errors in protein modification
• Gene therapy
– Describe use of several different delivery systems
– The difficulty of making a system that is beyond “experimental”
• Recognize existence of therapies involving inhibitory RNA
554
ROSS UNIVERSITY SCHOOL OF MEDICINE
30. Population Genetics
BIOCHEMISTRY and Genetic
AND GENETICS II
Counseling
Handout 26
POPULATION GENETICS AND GENETIC COUNSELING

30.1. Genotype Frequencies
I. GENOTYPE FREQUENCIES
30.1.1. The Hardy-Weinberg equation
1. The Hardy-Weinberg equation
The Hardy-Weinberg equation describes the frequencies of genotypes for an autosomal
gene with
The two alleles. It reads:
Hardy-Weinberg equation describes the frequencies of genotypes for
2
2pq + q 2 = 1
an autosomal gene with two alleles.p It+reads: (30.1)
A a
p q
p = gene frequency of allele A
A p
2
p pq q = gene frequency of allele a
p2 = frequency of genotype AA
2pq = frequency of genotype Aa
a q pq q2
q2 = frequency of genotype aa
With With
two alleles A andAa and
two alleles in one locus,
a in one plocus,
+ q =p1. + q = 1.
Remember that gene frequency is synonymous with allele frequency.
Remember that gene frequency is synonymous with allele frequency
The Hardy-Weinberg equationequation
The Hardy-Weinberg is used toiscalculate
used tothe frequencies
calculate theoffrequencies
all genotypesof
when
all the
frequency of one genotype is already known. Example: You know
genotypes when the frequency of one genotype is already known. Example: the population incidence
of aknow
You recessive disease and incidence
the population want to know of athe carrier frequency.
recessive The want
disease and gene (allele)
to knowfrequency
the
can befrequency.
carrier calculated fromThethegene
disease frequency.can be calculated from the disease
frequency
frequency.
• For rare autosomal dominant traits, the gene frequency is about one half of the disease
-frequency.
For rare autosomal dominant traits, the gene frequency is about one half
of the disease frequency.
• -ForFor
rarerare
autosomal recessive
autosomal traits, the
recessive genethe
traits, frequency
gene is the squareisroot
frequency theofsquare
the disease
frequency.
root of of the disease frequency.
- For X-linked traits, the gene frequency is equal to the disease frequency
• For X-linked traits, the gene frequency is equal to the disease frequency in males.
in males.
The Hardy-Weinberg equation can be used only when:
The Hardy-Weinberg equation can be used only when:
a) The different genotypes have equal fertility.

555
b) The different genotypes have equal viability.
c) Mating is random for the trait.
d) There are no new mutations.
279
• The different genotypes have equal fertility.
• The different genotypes have equal viability.
• Mating is random for the trait.
• There are no new mutations.
Assortative mating Mating is positively assortative when carriers of the same trait have
an increased likelihood to mate with each other, and negatively assortative when they have
a decreased likelihood of mating with each other. Mating tends to be positively assortative
for most externally noticeable traits including height, education, beauty, deafness, obesity,
religion, money, etc. Positively assortative mating increases the incidence of homozygosity
for simple Mendelian traits. For quantitative traits, it favors the more extreme pheno-
types.
Balanced polymorphism A locus is considered polymorphic when the frequency of the

most common allele does not exceed 0.99. At least 30 % of all loci are polymorphic. Each
individual is heterozygous at about 7 % of his gene loci. In most cases, the variant alleles
do not cause disease.
Balanced polymorphism is a genetic polymorphism stabilized by selection. The most impor-

tant mechanism is heterozygote advantage, which favors the less common allele. It probably
important for the maintenance of many common polymorphisms: Blood groups, HLA anti-
gens. It also maintains certain recessive disease genes in the population, as in the case of
hemoglobin S. In the absence of heterozygote advantage, polymorphisms can be maintained
by random drift when there is no selection against them. Or they represent an evolutionary
transition from a more “primitive” allele to a more “modern” one.
Genetic drift The random variation of gene (allele) frequencies in small isolated popula-
tions is called genetic drift. Founder effect is the randomness associated with choosing the
few “founders” of a population, e.g., the few founders of one of the small genetic sects, or
the survivors of a natural catastrophe. Together these effects can lead to the unusually high
incidence of an otherwise rare genetic disease in a small or once-small population that is
descended from only a few founders. On the flip side, these effects also often lead to reduced
genetic variation in the population (genetic drift is most important for this).
556
Mutation and Selection 30.3
30.2. Inbreeding
Degrees of relatedness The percentage of shared genes is:

Monozygotic twins: 100 %
First-degree relatives (siblings, parent, child): 50 %
Second-degree relatives (half-sibs, uncle, grandparent...): 25 %
Third-degree relatives (grand-grandparent, first cousin): 12.5 %
Consequences of inbreeding The coefficient of inbreeding indicates the proportion of

gene loci at which a person is homozygous due to inbreeding. For the child of a first-cousin
marriage, the coefficient of inbreeding is 0.0625. For the child of a brother-sister mating, it
is 0.25.
Consequences:
• Inbreeding increases the frequency of homozygosity for rare recessive allele. This effect
is stronger for rare alleles than for common ones. It increases the incidence of rare
recessive diseases. About one-third of the children of incestuous matings between first-
degree relatives are physically or mentally abnormal, and many of these problems can
be diagnosed as recessive diseases. A different consequence of this is that for rare
recessive diseases, quite a few of the patients will have parents that are (sometimes
remotely) related.
• Multifactorial traits may be adversely affected. This includes slightly increased early
mortality and decreased IQ in children of first cousins. This inbreeding depression is
caused by homozygosity for mildly deleterious mutations and the loss of heterozygote
advantage.
30.3. Mutation and Selection
Mutation rate Mutations are either spontaneous (“basal mutation rate”) or induced by
chemicals or radiation. According to current estimates, each child is born with about 100
new mutations. Most of them are point mutations in the junk DNA and totally harmless,
but perhaps 2 or 3 on average are mildly unfavorable and can contribute to multifactorial
disease. About 1 in 200 children is born with a new mutation that is sufficiently serious
to cause a diagnosable disease. Among disease-causing mutations, the highest mutation
rate is for fragile X. Some genes with high mutation rates (neurofibromatosis, Duchenne
muscular dystrophy) are very large. Mutation rates can easily be determined for autosomal
557
dominant and X-linked diseases, but not for autosomal recessive. Advanced paternal age is
the most important risk factor for new point mutations (but not deletions).
Selection against mutations The fitness of a genotype is measured by the number of

offspring produced. A fitness of zero (no offspring) is a genetic lethal. A fitness of 1 means
normal fertility. Low fitness does not always imply poor health or early death. Selection
often works by differential reproduction rather than differential survival — especially in
countries with good medical care.
Selection against autosomal recessive disease genes is very inefficient because most of the
time they are hidden in unaffected heterozygotes. Inbreeding selects against bad recessive
mutations because it exposes them to selection in the homozygous state.
Selection and single-gene disorders
• Some “harmless” traits such as color blindness and pattern baldness are very common
because they do not compromise reproduction.
• Genes for some serious diseases, like Huntington’s disease and the dominantly in-
herited forms of Alzheimer’s disease are maintained because they strike after the
peak reproductive age. (Some studies show that male carriers of Huntington disease
on average have more children than their male non-carrier siblings).
• If the incidence of a disease is constant, then the bad alleles weeded out by selection
in the previous generation must have been replaced by new mutations. As this is the
case in most diseases we know of, autosomal recessive diseases must have a relatively
low mutation rate.
• There is an inverse relationship between the fitness of an autosomal dominant muta-

tion and the proportion of patients having a new mutation. If achondroplastics have
a fitness of 0.2 (20 %), then 80 % of them have a new mutation.
• Some recessive disease genes are maintained by heterozygote advantage: sickle cell,
thalassaemia and glucose-6-phosphate dehydrogenase deficiency for malaria protec-
tion, and cystic fibrosis for protection from diarrheal diseases. Also lipid storage dis-
eases in Jews may be due to heterozygote advantage.
Selection for quantitative traits
Stabilizing selection is present when persons at both tail ends of the normal distribution
have reduced fitness. It reduces the standard deviation for the trait without changing
the mean.
558
Mutation and Selection 30.4
Disruptive selection favors extreme phenotypes over average phenotypes. It increases the
standard deviation for the trait. This pattern is rare.
Directional selection favors phenotypes at one end of the normal distribution over those
at the other end. It changes the mean of the normal distribution.
Stabilizing selection is common both for health-related traits (example: blood pres-
sure) and for traits that are important for mate choice (example: height). Directional
selection can be strong for traits like socioeconomic status, education, intelligence,
and ethnic or religious background. The best-documented selection effect in mod-
ern societies is selection against female intelligence. Other examples: Catholics and
protestants in Northern Ireland, black and white South Africans, Anglos and Latinos
in the US. Selection for quantitative traits is the mechanism of evolution.
Selection and multifactorial disorders The risk of multifactorial diseases is influenced

both by freak mutations that increase disease risk but are not serious enough to be re-
moved speedily by selection (“genetic garbage”), and by common polymorphisms. Other
considerations:
• Senior citizens are not as healthy as young folks because there is no selection against
those polygenic diseases that strike after the reproductive age.
• Some multifactorial traits may have been adaptive in the past but lead to problems
in modern societies. Genes for obesity and type II diabetes can be adaptive under
conditions of limited food supply.
Medical intervention and selection Does medical intervention increase the incidence of
heritable (Mendelian or multifactorial) disease?
• In some multifactorial disorders, life-saving surgery increases the disease incidence in

the next generation. Parents who survived pyloric stenosis or congenital heart defects
thanks to surgery often produce children with the same defect. But only a small
percentage of children born with these defects have an affected parent, therefore the
effect is slight.
• Selection against some single-gene disorders is relaxed by medical intervention. This

can increase the disease incidence noticeably for some dominant and X-linked diseases,
but not for recessives.
• The incidence of diseases that are expressed after the reproductive age is not likely
to increase through medical intervention.
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30.4. Genetic Counseling
Aim Most genetic diseases are hard to treat. Therefore, extra attention should be given
to disease prevention. Typical situations:
• The prospective parents already have a child with a supposedly genetic disease. They
want to know if there is any risk for their future children.
• One of the prospective parents has a serious disease.
• A relative of the parents has a possibly genetic disease.
• The prospective parents are consanguineous.
• One of the prospective parents is very old.
The physician is expected to provide information about the nature of the disease, the risk
for the disease in future children, and available options for disease prevention.
Genetics clinics Genetic counseling is an integral part of medical practice. The primary
care physician is, for example, expected to inform an elderly pregnant patient about the risk
of Down’s syndrome and the possibility of prenatal diagnosis. And he is expected to inform
the mother of a cystic fibrosis child about the recurrence risk and possibility for prenatal
diagnosis. Failure to provide genetic counseling in such situations is malpractice!
More complicated cases should be referred to a genetics clinic. Genetics clinics exist in many
university hospitals and major medical centers. There are specialized clinics for the more
common diseases, such as sickle cell and cystic fibrosis.
Population risks Some relevant population risks for conditions, which may or may not
have a genetic background, are:
Infertile couple: 1 in 10
Pregnancy ending in spontaneous abortion: 1 in 8
Child born with serious handicap: 1 in 50
Perinatal death: 1 in 30 to 1 in 100
Death during first year of life, after first week: 1 in 50
Diagnosis Take a careful family history. Inquire about spontaneous abortions, stillbirths,
infant deaths, and consanguinity. If the condition may be either genetic or environmental
(examples: mental retardation, deafness), a careful evaluation of possible environmental
causes is essential.
560
Diagnostic Strategies 30.5
Risk estimate Mendelian ratios can be calculated for established single-gene disorders.
Otherwise you use empiric risks. Mendelian risks for an individual pregnancy are unaf-
fected by the birth of a previous affected child, in multifactorial traits the birth of another
affected child increases the empiric risk in future pregnancies.
Many disorders show genetic heterogeneity or phenocopies. When differential diagnosis is

not possible, an empiric risk may have to be applied to a supposedly Mendelian disorder.
Example: An isolated case of deafness may be caused by autosomal recessive inheritance or
by an unrecognized environmental agent. The current trend is that more and more genetic
etiologies are becoming diagnosable by molecular methods.
Options Frequently, a supposedly genetic condition turns out to be non-genetic, or the

recurrence risk of a multifactorial condition is so low that the prospective parents need not
worry. However, if there is a substantial risk of a severe disease, there are several options:
• No children. Or a lover with good genes. These are the old-fashioned recommenda-
tions. It is still a reasonable option in some cases, for example when a parent has
isochromosome 21 or the mother has PKU.
• Artificial insemination by donor (Nobel prize winner or Olympic gold medal winner,
if available). This makes sense in cases of male infertility and when the prospective
father may contribute a gene for a serious disease. Not acceptable for all couples.
• Prenatal diagnosis with selective abortion of any affected fetus. This method tends
to replace the others, but it is not acceptable for everyone or legal everywhere. Also,
not all diseases can be diagnosed prenatally.
• Preimplantation diagnosis with selective implantation of unaffected embryos. This

avoids the unpopular step of pregnancy termination, but it requires in-vitro fertiliza-
tion. Expensive!
Remember that counseling should be non-directive, the choice belongs to the patient, not
the doctor!
30.5. Diagnostic Strategies
Heterozygote detection Heterozygotes can be identified for many, but not all autosomal
recessive and X-linked recessive disorders. Traditionally, these tests are applied only in rel-
561
atives of a patient.
Disease Test Used
Cystic fibrosis Allele-specific probes
Sickle cell disease Sickledex, dot blotting
Thalassemias Hemoglobin electrophoresis, RBC morphology, DNA tests
Phenylketonuria Phenylalanine tolerance test
Mucopolysaccharidoses Enzyme activities
Lipid storage diseases “ “
Galactosemia “ “
Hemophilia A Linked markers
Duchenne muscular dystrophy Serum creatine kinase, PCR/Southern blot
G6PDH deficiency Enzyme assay, electrophoresis
Population screening Heterozygote screening makes sense only when:
• The disease is relatively common
• The disease is severe enough to justify prenatal testing and pregnancy termination.
• The screened population is sufficiently educated to understand what it’s all about.
Good candidates for population screening are sickle cell, cystic fibrosis, Tay-Sachs and
fragile X. Because most people don’t care much about genetic risks until they are expecting
a child, most screening programs are aimed at pregnant women.
Newborn screening Newborn screening is used for severe but treatable conditions. It is
often done for congenital hypothyroidism, PKU, other treatable metabolic diseases such as
galactosemia and biotinidase deficiency, hemochromatosis, and α1 -antiprotease deficiency.
30.6. Prenatal Diagnosis
30.7. Aim
Prenatal (=antenatal) diagnosis is performed when the fetus is at risk of a severe dis-
ease. The test is done as early as possible, and the pregnancy is terminated if the fetus is
affected.
562
Screening Tests 30.8
30.8. Screening Tests
Screening tests are noninvasive tests that are done routinely in every pregnancy.
Ultrasound is part of routine prenatal care. It detects most cases of twin pregnancy and
gross malformations. Can also detect e.g., Turner syndrome: should pregnancy termi-
nation be offered? There is at least 99 % risk of spontaneous abortion, but quite normal
phenotype after birth (increased risk of heart defects).
Maternal blood tests: Maternal serum α-fetoprotein is often elevated in neural tube de-
fects (NTD’s) and reduced in Down syndrome. Also popular: Triple test with α-fetoprotein,
human chorionic gonadotropin and estradiol. For Down and NTD’s. Suspicious cases are
referred for amniocentesis.
Amniocentesis In amniocentesis, performed at week 15 or 16, a small amount of amni-

otic fluid is removed in a transabdominal approach, under ultrasound guidance. The fluid
contains viable cells sloughed off from the fetal membranes. These cells, which have the
fetal genotype, can be cultured like fibroblasts. Culturing takes 1–3 weeks. Diagnostic tests
are done on the cultured cells. The risk of the procedure (fetal damage, infection, induced
abortion) is minimal. Indications:
Chromosome aberrations, Down syndrome in particular, are the most common indica-
tion. Pregnancies of women older than 35 a are routinely monitored by amniocentesis.
Mendelian disorders are tested when both parents are carriers of an autosomal recessive
disease gene, one parent has dominant disease, or the mother is a carrier for a X-linked
recessive disease gene.
Neural tube defects are tested in families who already had a child with a neural tube
defect. The level of α-fetoprotein is determined in the amniotic fluid. In many places,
α-fetoprotein is measured routinely with every amniocentesis, even if the primary
indication was different (usually advanced maternal age).
Chorionic villus sampling In this procedure, a biopsy is taken transvaginally from the
chorionic membrane. The risk may be slightly higher than for amniocentesis and you cannot
test for neural tube defects, but
• It can be done earlier, at week 8–10
• More cells are obtained. Therefore cell culturing is not always necessary. This saves
another 1–3 weeks.
563
Effects on disease incidence Even if the birth of children with recessive diseases is avoided
by prenatal diagnosis and selective abortion, the allele frequencies will not necessarily
decline because most parents keep trying until they have the desired number of children.
Two thirds of these children will be carriers. This is called reproductive compensation.
Ethical and legal concerns Attitudes toward prenatal diagnosis and pregnancy termi-
nation vary in different cultures. In most Western countries and ex-Communist countries,
prenatal diagnosis and the abortion of fetuses with severe diseases is legal. In Western
countries, the decision about pregnancy termination is made by the parents, not the doc-
tor. Because many diseases are now avoidable, the question arises of whether parents who
opt for a chronically ill child should be liable for the medical expenses. Sex-selective abortion
after ultrasound or amniocentesis is now considered acceptable in the US. Only a minute
proportion of pregnancy terminations worldwide are performed on diseased fetuses after
prenatal diagnosis.
30.9. Assisted Reproduction
Incidence of infertility Fertility problems occur in perhaps 10 % of younger couples and

> 30 % of couples beyond age 35 a. 40 % are caused by male infertility due to infections (ie.
Mumps), chromosomal abnormalities (most important: Klinefelter), immunological prob-
lems (sperm autoantibodies), anatomical problems, microdeletions on the Y chromosome
etc. The male partner is always evaluated first because the diagnostic workup is easier in
the male. The two most common causes of female infertility are ovulatory dysfunction and
tubal/pelvic pathology. Advanced age is the most important and least treatable cause of
female infertility!
Artificial insemination by donor (AID) AID is used for intractable male infertility, and
by single women. It can be used when there is a history of fetal loss due to rhesus incom-
patibility, and in cases of genetic diseases when the male partner may contribute a severe
disease gene. The success rate depends on the age of the patient, but typically 15–20 % per
cycle with fresh semen, 5 % with frozen semen. Technicalities and precautions:
• Semen can be quick-frozen in the presence of an antifreeze (10 % glycerol). It keeps
almost indefinitely in liquid nitrogen. Sperm banks express-mail their product in ni-
trogen tanks to the doctor’s office where the insemination is performed.
• Married women should be inseminated only after both partners have signed a consent
form. This is legally required in 20 states.
• Insemination should be timed to coincide with the LH surge. Take-home kits for LH
measurement are available.
564
Assisted Reproduction 30.9
• Practicing homosexuals, men with multiple sexual partners, and intravenous drug
users are less-than-optimal donors because of the risk of sexually transmitted disease.
• Frozen semen should be quarantined for 6 months, and repeat AIDS testing of the
donor performed before the batch is released. This is mandatory in most states.
• Most sperm banks use students as donors, and they apply an age limit (35 or 40 a)
because old sperm may have reduced sperm count or motility, and because of an
increased risk of new mutations. The typical pay for donors is about US$ 80.
• Most but not all sperm banks have only anonymous donors.
• Donor selection includes complete physical, semen analysis, testing for transmissi-
ble diseases (AIDS, syphilis, hepatitis), educational history, and family history for
Mendelian and multifactorial diseases. Carrier testing for sickle cell (Blacks), thalas-
saemia (Mediterraneans), Tay-Sachs (Jews) and cystic fibrosis (everyone) is increas-
ingly used. Some sperm banks are specialized on Nobel prize winners or homosexuals
(for lesbian customers).
• Sperm banks have catalogs for initial donor selection, and detailed donor profiles can
be obtained on request. Married couples often select donors with physical characters
similar to the husband’s. The physician should advise the patient about the heritabil-
ity of relevant traits. If the patient has a multifactorial disease, the use of a donor
with a family history of the same disease should be avoided.
• During routine insemination, semen is deposited at the entrance to the cervical canal
by means of a polyethylene catheter connected to a 1 ml syringe.
• Alternatively, intrauterine insemination (IUI) is performed with washed resuspended
sperm.
• Sperm enriched in X- or Y-bearing sperm cells is offered by many sperm banks.
Separation of the cells is always very incomplete, however.
In-vitro fertilization (IVF) IVF was first used in women with tubal disease, but is now
also used for male factor infertility, unexplained infertility, and infertility caused by en-
dometriosis or immunological factors. While AID is usually done in the doctor’s office (or
even by the patient herself at home), IVF is done in specialized clinics. Procedure:
• Hormonal stimulation is used to induce superovulation (maturation of multiple folli-
cles). These treatments raise the gonadotropin level during the follicular phase. It can
be done by exogenous human menopausal gonadotropin (HMG), human chorionic
gonadotropin (HCG), recombinant FSH, or clomiphene citrate, an estrogen partial
agonist/antagonist which blocks the negative feedback of estrogen on gonadotropin
secretion.
565
• Follicles are retrieved at mid-cycle under ultrasound guidance. Analgesics (fentanyl-

midazolam) are used. Up to a dozen follicles can be harvested (if you’re lucky).
• The oocytes are prepared under the dissection microscope, incubated for several hours,
and then exposed to washed, resuspended sperm.
• Embryos can be transferred any time between the pronuclear and blastocyst stages,
but transfer is usually done at the 4–10 cell stage (48–80 h after retrieval). About 3
embryos are placed in younger women, more in older women. Implantation rates are
low, but placement of too many embryos increases the risk of multiple pregnancy.
• Excess embryos are cryopreserved for later use. Freezing embryos is technically easier
than freezing oocytes.
• The delivery/retrieval pregnancy rate is about 20 % (10–15 % per cycle). The cost is
US$ 6000 to US$ 10 000 per treatment cycle.
• The incidence of multiple pregnancy after IVF is 30 %. Also ectopic pregnancies
are common (4–5 %). There is no increased incidence of congenital malformations.
There is no significant increase of developmental abnormalities in babies obtained
from frozen embryos.
• IVF can be done with embryos from donated sperm and ovum (intrauterine adoption).
This is an option for postmenopausal women and women with ovulatory failure, but
it is rarely done because donated oocytes are in short supply.
Intracytoplasmic sperm injection (ICSI) A sperm cell is injected directly into the cyto-
plasm of the ovum. This method can overcome problems with sperm motility or maturation,
and it can be used with sperm cells obtained by epididymal puncture in patients with atre-
sia of the vas deferens or post-vasectomy. The method seems fairly safe. Thanks to ICSI,
there are now cases of truly inherited male infertility.
Importance of assisted reproductive technology (ART) Between 1.5 and 2.0 % of chil-
dren in the US are born through ART, about half of them by donor insemination and the
other half by IVF. The procedures are not usually paid by the health insurance in the US,
but they are covered, with qualifications, by the health care systems of many other coun-
tries. About one-half of all married recipients tell their child of his/her origin. Divorce rates
are lower than usual in couples having an ART child, and parenting tends to be better.
There may be a tendency for children of well-screened donors to be healthier than naturally
conceived children, and smarter if the donor is a Nobel Prize Winner.
Lesbians and career women are now a major clientele. Children of single women and lesbians
conceived by ART do as well as those of married couples. Therefore there is no reason to
deny treatment to these customers.
566
Pre-implantation genetic diagnosis This is an advanced method for the diagnosis of

single-gene disorders (using PCR) and chromosome aberrations (using FISH), done only in
a few places. Procedure for single-gene disorders:
• Grow the IVF embryos to the 8- or 16-cell stage.
• Remove a single cell from each embryo. This does not interfere with embryonic de-
velopment. check – there was a recent paper (in Nature IIRC) stating the
opposite
• Use the DNA of this single cell to diagnose the disease with PCR. The test takes
about 8 hours.
• Discard the bad embryos and implant the good ones.
Pre-implantation diagnosis is a costly alternative to prenatal diagnosis for parents who

don’t like the idea of an abortion at all. Otherwise, it makes most sense in cases where IVF
is done anyway.
1. State the criteria for validity of the Hardy-Weinberg equation
2. Use the Hardy-Weinberg equation to predict the allele frequency and the frequency
of different genotypes for single gene disorders if the disease incidence is known.
3. Define the terms heterozygote advantage, genetic drift, founder effect and assortative
mating.
4. Predict the effects of inbreeding and incest on the incidence of autosomal recessive
and multifactorial disorders.
5. State the approximate mutation rates of genes for important Mendelian disorders,
including Huntington’s Disease, Achondroplasia, Neurofibromatosis, Hemophilias
A and B, and Duchenne’s Muscular Dystrophy.
6. Describe the expected effects of negative selection on allele frequencies of autosomal

recessive and dominant diseases, and explain why the lack of effects makes negative
eugenics unethical and scientifically wrong.
7. Define the term stabilizing selection, disruptive selection, and directional selection
and give examples for each.
8. Define the aims of genetic counseling and give the typical sequence of steps.
567
9. State the most likely causes for the observed population incidences of important ge-
netic conditions including Duchenne’s Muscular Dystrophy, Achondroplasia, Hunt-
ington’s Disease, Sickle Cell Disease, Thalassemias, Glucose-6-Phosphate Dehydro-
genase Deficiency, Cystic Fibrosis, Obesity, Type II Diabetes, Alzheimer’s Dis-
ease, Osteoporosis, Red-green Color Blindness, Pattern Baldness, Homosexuality,
Mild Mental Retardation, and Skin Color Differences.
10. Describe the procedure of amniocentesis, chorionic villus sampling and pre-implantation
diagnosis.
11. Specify the typical situations in which prenatal diagnosis for chromosome aberrations
and Mendelian disorders is indicated.
12. Compare the advantages and disadvantages of amniocentesis and chorionic villus sam-
pling for prenatal diagnosis.
13. Provide examples of congenital or hereditary diseases for which newborn screening is
currently practiced or proposed.
14. Describe the most important methods of assisted reproduction including artificial
insemination, in vitro fertilization and intracytoplasmic sperm injection, and list three
typical indications for each of these procedures.
15. State the ethical and legal implications of current procedures for prenatal diagnosis,
pre-implantation diagnosis and pre-symptomatic diagnosis.
16. State the effect of paternal age on rate of point mutations.
568
31. Integration of Metabolism
31.1. Regulation of Enzyme Activity
Mechanisms regulating key metabolic enzymes include:

• Allosteric regulation, usually by metabolic substrates, intermediates, or end products,
acts on many key enzymes in metabolic pathways. Also competitive inhibition by
metabolites is important. Action is immediate.
• Phosphorylation/dephosphorylation is regulated by hormones (glycogen phosphory-
lase, adipose tissue lipase, acetyl-CoA carboxylase) or metabolites (pyruvate dehydro-
genase). This is the most important mechanism by which second messengers (cAMP,
cGMP, diacylglycerol, Ca2+ ) induce their effects. Insulin induces the dephosphoryla-
tion of many metabolic enzymes although its initial effect is the phosphorylation of
regulatory proteins. Action is on an intermediate time-frame.
• Adaptive control, meaning adjustments in the synthesis or degradation of metabolic
enzymes, is a long-term (day-to-day) type of control. Enzyme synthesis is often under
hormonal control. Steroid and thyroid hormones affect transcription directly. Water-
soluble hormones achieve the same through their second messengers which induce the
phosphorylation of transcription factors and other nuclear proteins. Metabolites can
also regulate gene expression.
31.1.1. Hormonal Control
Hormones coordinate the activities of the metabolic pathways to adapt to the nutritional
state and physiological requirements.
Insulin satiety hormone: release stimulated by Glc and AA. Stimulates nutrient uptake
from blood into cells and synthesis of storage compounds (glycogen, fatty acids,
triglycerides, cholesterol) and inhibits the breakdown of stored nutrients, including
fat breakdown in adipose tissue and glycogen breakdown in liver and muscle.
Muscle, adipose tissue GluT-4 stimulated 10–20 fold.
Liver GluT-2 insulin-independent, but Glc-metabolism stimulated.
569
Brain, RBC insulin-independent Glc-consumption.
Glucagon is the most important functional antagonist of insulin in the liver. It has little
effect on other tissues. Its plasma level is elevated during fasting and in hypoglycemia.
It maintains an adequate blood glucose level during fasting.
Epinephrine and norepinephrine are released during acute physical and psychological stress,
including physical exercise, cold exposure, and biochemistry exams. They mobilize en-
ergy reserves (fat, glycogen) to cope with an increased demand. Although not involved
in glucose homeostasis, they are released in hypoglycemia → Stress-symptoms.
Glucocorticoids are elevated in chronic stress and act as gene-regulators. Adaption to life
in a dangerous world: mobilization of reserves by stimulation of lipolysis, protein
degradation, gluconeogenesis, glycogen synthesis. Glucagon, epinephrine, cortisol, and
growth hormone are functional insulin antagonists. The excessive release of these
hormones can cause hyperglycemia. Chronically ill patients: Muscle wasting, low Glc-
tolerance, insulin resistance.
Hormonal regulation of metabolism
Insulin Glucagon Epinephrine Cortisol Allosteric Allosteric

activator inhibitor
Liver
Glycolysis ↑↑ ↓↓ (↓) AMP, ADP ATP, citrate
Gluconeogenesis ↓↓ ↑↑ (↑) ↑↑ ATP, citrate AMP, ADP
Glycogen synthesis ↑↑ ↓↓ ↓ ↑ Glucose 6-P
Glycogenolysis ↓↓ ↑↑ ↑↑↑ Glucose
Fatty acid synthesis ↑↑ ↓↓ (↓) Citrate Acyl-CoA
Transaminases ↑ ↑↑
β-Oxidation ↑ Malonyl-CoA
Muscle
Glucose uptake ↑↑
Glycogen synthesis ↑↑ ↓↓ Glucose 6-P Glucose 6-P
Glycogenolysis ↓↓ ↑↑↑ AMP
Glycolysis ↑ ↑↑↑ AMP ADP ATP H+
Protein synthesis ↑ ↓
Adipose tissue
Glucose uptake ↑↑
Lipoprotein lipase ↑
Triglyceride synthesis ↑
Lipolysis ↓↓ ↑↑↑ (↑)
2. Glucagon is the most important functional antagonist of insulin in the

liver. It has little effect on other tissues. Its plasma level is elevated during
fasting and in hypoglycemia. It maintains an adequate blood glucose level during
31.1.2. Metabolic
fasting.
role of organs
3. Epinephrine and norepinephrine are released during acute physical
and psychological
Liver central organ of metabolism,stress, keeps
including physical
blood glucoseexercise, cold
constant: exposure,
Glycogen and gluco-
storage,
biochemistry exams. They mobilize energy reserves (fat, glycogen) to cope with
neogenesis, nitrogen
an increased metabolism. All other organs “extra-hepatic” or “peripheral”.
demand.
4. Glucocorticoids are elevated in chronic stress. They mobilize
reserves, including fat and protein, for energy and gluconeogenesis. Glucagon,
epinephrine, cortisol, and growth hormone are functional insulin antagonists.
570 The excessive release of these hormones can cause hyperglycemia.
III. THE STARVE-FEED CYCLE
Adaptations to an intermittent food intake include:

1) The storage of excess metabolic energy it is plentiful, and its
mobilization during fasting.
2) The maintenance of a stable blood glucose level, especially for the
brain which has only a limited ability to use alternative fuels.
The insulin/glucagon ratio is the most important hormonal factor for
The respiratory quotient 31.3
Kidney Considerable gluconeogenesis in kidney cortex during starvation (even short term),
exertion (Cori-cycle) and acidosis. Substrates: lactate, glutamine and glycerol.
Adipose tissue Storage of fat, fat synthesis from carbohydrate (limited).
Muscle storage of glycogen and creatine phosphate only for itself (no Glc-6Pase!). Cori-
and Ala-cycle.
Heart continuously active, aerobic metabolism only, Glc, fatty acids and ketone bodies
used as fuel. Stores only limited amount of phosphocreatine.
Brain uses O2 at constant rate, aerobic metabolism only, dependent on Glc, in starvation
partly ketone bodies as their higher concentration in the blood allows uptake. No
storage of glycogen. Fats can not cross blood/brain barrier.
Blood transporter of nutrients, hormones and waste products. Erythrocytes only anaerobic
Glc metabolism.
31.2. The respiratory quotient
RQ is defined as the ratio of CO2 formed by O2 consumed. Burning of different compounds

leads to different RQs:
Glucose C6 H12 O6 + 6O2 *

) 6CO2 + 6H2 O RQ = 6/6 = 1
Fat (Palmitic acid) C16 H32 O2 + 23O2 *

) 16CO2 + 16H2 O RQ = 16/23 = 0.7
Protein RQ = 0.8 − 0.9 (empirical)
RQ > 1 possible reasons
• Growth
• storage of fat
• anaerobic metabolism
At the same time different foodstuff contain different energy:

Nutrient ∆H
(kJ/g)
CHO 17
Protein 17
Fat 39
Ethanol 30
571
Figure 31.1.: Ergospirometers are now very small and portable. Their main use is in sports
and occupational medicine as well as in basic research (picture by the manu-
facturer).
572
The respiratory quotient 31.3
Figure 31.2.: Use of ergospirometry in sports medicine: The client is working out under a
constantly increasing load (green). As a consequence, O2 consumption (blue)
and CO2 production (magenta) increase up to a point where no more oxy-
gen can be delivered to tissues, therefore oxygen consumption stays constant.
The increased metabolic demand from the increasing load is met by anaer-
obic metabolism, resulting in a steeper increase of CO2 -production. Optimal
training results are achieved if the workload is adjusted so that 60 % of the
maximum oxygen consumption rate is used.
573
Table 31.1.: Energy stored in a well fed individual. Most of the energy is stored as fat, some
as protein. Other energy sources have (quantitatively) only a minor role.
Nutrient Organ Amount (kg) Energy (MJ)
Triglyceride Adipose tissue 10–15 377–586
Protein Muscle 6–8 126–167
Glycogen Muscle 0.3 5.1
Liver 0.08 1.3
small molecules Blood 0.023 0.42
31.3. The Starve-Feed Cycle
Adaptations to an intermittent food intake include:
• The storage of excess metabolic energy if it’s plentiful, and its mobilization during
fasting.
• The maintenance of a stable blood glucose level, especially for the brain which has
only a limited ability to use alternative fuels.
The insulin/glucagon ratio is the most important hormonal factor for metabolic adjustments
to the nutritional state.
Typical blood levels:
Analyte Very Post- Starved
well-fed absorptive 3 days 6 weeks
Insulin (U/mL) 40 15 8 6
Glucagon (pg/ml) 80 100 150 120
Insulin/glucagon 0.50 0.15 0.05 0.05
(ratio: U/pg)
Glucose (mg/dL) 113 89 70 67
Fatty acids (mM) 0.14 0.6 1.2 1.4
Acetoacetate (mM) 0.04 0.05 0.4 1.3
β-Hydroxybutyrate 0.03 0.10 1.4 6.0
(mM)
Glucose homeostasis:
Phase I (0Phases
31.3.1. – 4 hoursofafter
the meal):
starve-feed cycle
Blood glucose is derived from dietary sources. Blood glucose is derived
from dietary sources. Glucose uptake in muscle and adipose tissue and its
• Phase I (absorptive, 0–4 h after meal):
metabolism in most tissues are stimulated by insulin. Excess glucose is used for
the synthesis of glycogen
– blood (liver, from
glucose comes muscle) and fatty acids (liver). All tissues use
intestine.
glucose as fuel.
– glucose uptake in muscle, adipose tissue and liver (Km of glucokinase!)
Phase II (postabsorptive, 4 – 16 hours after meal):
Glycogen breakdown in the liver is the major source of blood glucose.
Hepatic gluconeogenesis starts.
Phase
574 III (16 – 48 hours after meal):
Liver glycogen is almost depleted. Blood glucose is from
gluconeogenesis.
Phase IV & V (>2 days after meal):
Blood glucose is from gluconeogenesis only.
During starvation, most tissues switch glucose to alternative energy

sources: In phase II, the liver no longer consumes glucose. In phase IV and V,
only brain, RBCs and renal medulla still use significant amounts of glucose. At
Phases of the starve-feed cycle 31.3.1
– glycogen synthesis in liver and muscle

– lipid synthesis in liver (glucose) and adipose tissue (glycerophosphate from glu-
cose, fatty acids from chylomicrons & VLDL)
– Lipoprotein lipase in adipocytes but not muscle stimulated by insulin: chylomi-
crons routed to adipocytes
– all tissues use glucose as fuel, liver, kidney and intestine also amino acids
– Brain consumes 90–120 g/d of glucose
– insulin/glucagon is high
– luxus consumption: increased metabolism (postprandial thermogenesis) ≈10 %.
Highest in unbalanced meals.
• Phase II (post-absorptive, 4–16 h after meal):
– blood glucose comes from liver glycogenolysis (about 50 %), and gluconeogenesis
in liver (30 %) and kidney (20 %).
– liver no longer consumes glucose
– lipid synthesis in adipose tissue drops from lack of glycerol phosphate
– glucagon ↑, insulin ↓ but still important
• Phase III (16–48 h after meal):
– adipose tissue lipoprotein lipase ↓, no uptake
– hormone sensitive lipase becomes active, lipids from adipocytes are major fuel
– liver glycogen almost depleted, blood glucose comes mostly from liver + kidney
gluconeogenesis, which is fully active (amino acids, glycerol, lactate)
– liver uses fatty acids from adipose tissue as energy source, glycerol for gluconeo-
genesis
– liver releases ketone bodies into serum, about 150 g/d. β-oxidation of FA in liver
generates energy for gluconeogensis
– various organs (e.g. heart) use ketone bodies as fuel (i.e. perform the TCA-cycle)
– respiratory quotient drops from 0.9 to 0.7
• Phase IV (starvation, 2–7 d after meal):
– adipose tissue main source of metabolic energy, loosing 180 g/d of triglyceride
– reduction of gluconeogenesis → saving of muscle mass
575
– blood glucose comes from gluconeogenesis only (150 g/d), muscle protein loss
75 g/d
– only brain (↓ to 100 g/d), RBC (20 g/d) and renal medulla use glucose as fuel
(uptake insulin-independent!). Consumption of ketone bodies by brain 50 g/d
(30 % of energy) as their higher concentration in the blood allows uptake over
the blood-brain barrier.
– moderate reduction in basal metabolic rate (15–25 %, privo conservation).
• Phase V (long-term starvation, several weeks after a meal):
– muscle and many other tissues stop using ketone bodies in favor of fatty acids
→ blood [KB] ↑
– brain uses mostly ketone bodies (about 100 g/d ≡ 75 % of energy consumption).
Glc consumption down to 30–40 g/d.
– muscle protein loss reduced to 20 g/d
– survival depends on fat reserves, not muscle protein (3–14 mo)
– after depletion of fat, proteins are used as fuel → heart failure, death
31.3.2. Role of organs during starve-feed cycles
During starvation, most tissues switch from glucose to alternative energy sources: In phase
II, the liver no longer consumes glucose. In phase IV and V, only brain, RBCs and renal
medulla still use significant amounts of glucose. At this point, the brain covers 50 % of its
energy needs from ketone bodies rather than glucose. The reduction of glucose utilization
spares protein, which would otherwise be degraded for gluconeogenesis. Glucose is redi-
rected to brain, kidney medulla and RBCs because glucose metabolism in these tissues is
independent of insulin.
Adipose tissue
Adipose tissue synthesizes fat after meals, using glycerol phosphate derived from glucose
and fatty acids from chylomicrons (after a fatty meal) or VLDL (after a carbohydrate
meal). Fat synthesis is inhibited during fasting, mainly for lack of glycerol phosphate, and
the hormone-sensitive lipase becomes active when the insulin level drops.
576
Role of organs during starve-feed cycles 31.3.2
Liver
The liver oxidizes mostly amino acids after a mixed meal. 20–25 % of the dietary carbohy-
drate is metabolized in the liver. Most of this is used for the synthesis of glycogen and fat.
Only a small fraction of the dietary fat is used by the liver because the liver doesn’t have
LPL. During fasting, fatty acids from adipose tissue are the only important energy source
for the liver.
Ketogenesis is stimulated during fasting because of the ample supply of fatty acids and be-
cause the carnitine shuttle and the ketogenic enzymes are induced. The acetyl-CoA formed
by β-oxidation is not used for biosynthesis because fatty acids synthesis and cholesterol
synthesis are inhibited. Also TCA cycle activity is reduced because β-oxidation supplies
enough NADH + H+ and FADH2 for the respiratory chain during fasting: the TCA cycle
is not required to produce reduced coenzymes.
Most people can survive for a few months without food, depending on fat stores. Re-
feeding of starved patients should be started slowly. There is severe glucose intolerance
during extended fasting because the levels of glycolytic enzymes are very low. Especially
in the case of kwashiorkor a protein-saving diet must be used, which contains enough
carbohydrate and fat to meet the catabolic needs of the patient. Protein degradation after a
protein-rich diet would overtax the capacity of an already damaged liver, possibly resulting
in liver failure and death! The diet also needs enough protein to meet the anabolic needs
of the patient, and this protein needs to be of high biological value. Otherwise the liver
would have to catabolize unused amino acid and... see above! Re-feeding of starving patients
is performed by the WHO 10-step scheme:
hypothermia blankets (low subcutaneous fat!)
hypoglycemia monitor blood [glucose], give oral or i.v. glucose
dehydration if conscious: oral rehydration with higher K+ , lower Na+ (i.v. if not). Cave:
heart failure!
micronutrients give vitamins and trace elements
infections give broad-band antibiotic (Cave: infections w/o fever!). Therapy against malaria
if endemic.
electrolytes K+ + Mg2+
starter nutrition SLOWLY! Protein-saving rather than protein-rich.
tissue building rich diet dense in energy, protein and all essential nutrients that is easy to
swallow and digest
stimulation prevent as far as possible permanent damage by psychological, intellectual and
motor activities
577
prevention of relapse identify cause of malnutrition, involve family and community in pre-
vention. Cave: HIV!
The basal metabolic rate decreases during extended fasting, but only to a modest extent.
The respiratory quotient decreases during fasting as the tissues switch from glucose oxida-
tion to fat oxidation.
Skeletal Muscle
Fatty acids (50 %) and glucose are the major fuels for resting muscle after a meal, fatty
acids and ketone bodies during fasting. Glycolysis, TCA cycle and oxidative phosphorylation
operate at 10 % of capacity in resting muscle.
Anaerobic exercise is heavy, short-term exercise like sprinting and weight lifting. Initially,
ATP is generated in the reversible creatine kinase reaction:
Creatine
Creatine-phosphate + ADP F GGGGGGGGGGGGGB
GG Creatine + ATP
kinase
Then, glycogenolysis and glycolysis are stimulated by low energy charge and cAMP (epinephrine).
Blood flow to the muscles is poor, and there is little inter-organ cooperation. Anaerobic gly-
colysis is limited by acidity (lactic acid!) which inhibits phosphofructokinase.
Aerobic exercise (swimming, long-distance running) relies on oxidative metabolism of:
• Locally stored glycogen
• Fatty acids from adipose tissue
• Glucose and ketone bodies from the liver.
Fat breakdown in adipose tissue and glycogen breakdown in muscle and liver are stimulated
by epinephrine. There is insulin-independent glucose uptake in contracting muscle.
The respiratory quotient may decrease during very mild physical activity, because mildly
active muscles burn a lot of fatty acids. But it increases during vigorous exercise, because
stored glycogen is oxidized. Lactic acid levels increase 5–10 fold during strenuous exercise.
Carbohydrate loading can be used to increase muscle glycogen before athletic contests. This
is important for endurance athletes, because muscle (and liver) glycogen lasts for 2 h during
a marathon.
578
Substrate cycles:
Substrate cycles .
Cori cycle (mostly during exercise):
Liver Muscle
Glucose Glucose
4 ATP 2 ATP
2 GTP
2 Lactate 2 Lactate
Alanine cycle (during fasting, also during exercise):
Liver Muscle
Glucose Glucose
4 ATP
2 GTP 2 ATP
2 NADH 2 NADH
2 Pyruvate 2 Pyruvate
Urea α- Amino acid
α- Ketoacid
2 Alanine 2 Alanine
In addition, there is the Gln cycle between muscle and kidney cortex. These cycles allow
the This cycle
muscles allowsamino
to oxidize the muscles to oxidize
acids and dispose of theamino acids
nitrogen in theand
formdispose of Gln.
of Ala and the
nitrogen
During fasting, any glucose taken up in spite of low insulin levels is recycled to the liver of
in the form of alanine. During dasting, any glucose taken up in spite as
low alanine.
insulin levels is recycled to the liver as alanine.
Anabolic
Anabolic effects
effects onon muscleprotein
muscle (more protein
protein(more protein synthesized
synthesizedthan degraded)
than degraded)
- Insulin
• Insulin - Growth hormone
- Androgens, anabolic steroids - Regular exercise
• Growth hormone
Catabolic effects:
- Glucocorticoids
• Androgens, anabolic steroids - Protein-calorie malnutrition
- Denervation - space travel (weightlessness)
• Regular exercise
The myocardium uses metabolic fuels similar to skeletal muscle, but
there is more oxidative metabolism and less lactate formation. Lactate from
skeletal muscle is oxidized in the heart during exercise. 579
V. DETOXIFICATION REACTIONS AND ALCOHOL METABOLISM
252
Catabolic effects (degradation exceeds synthesis)
• Glucocorticoids
• Protein-calorie malnutrition
• Denervation
• space travel (weightlessness)
The myocardium uses metabolic fuels similar to skeletal muscle, but there is more oxidative
metabolism and less lactate formation. Lactate from skeletal muscle is oxidized in the heart
during exercise.
Fuel use by the exercising muscle
sprint stored ATP (2–4 s) and creatine phosphate (6–20 s)
800 m anaerobic glycolysis, using glycogen stores: Glc + 3 ADP + 3 Pi → 2 lactate + 3

ATP + 2 H2 O. Limited by acidification after ≈ 2 min. Cori-, Gln- and Ala-cycle.
Fast-twitch, white muscle.
10 000 m aerobic oxidation of Glc (from muscle + liver glycogen stores), fatty acids (from
adipose tissue) and ketone bodies (from liver) to CO2 . Rate limited by oxygen supply.
Glc uptake no longer limited by insulin. Slow twitch, red muscle. 2–3 h.
Marathon Runner “hits the wall” as muscle + liver glycogen stores are used up. Aero-
bic oxidation of fuels (FA, KB) delivered from liver and adipose tissue, amino acid
catabolism.
Insulin ↓↓, Glucagon ↑, catecholamines ↑↑ during exercise. Training increases blood sup-
ply, mitochondria (number + size), GluT-4 and fatty acid transporter, glycolytic enzymes,
muscle mass per fibre.
Nutrition in athletes
• plenty of protein after training to replace lost AA and build muscle mass
• carbohydrate loading (70–80 % for 3–8 d) before competition to build up glycogen

stores
• no carbohydrates immediately before competition to lower insulin
• sweet drinks during endurance exercise to prevent hypoglycemia
580
Blood Cells
Mature RBCs have no mitochondria. Glucose is metabolized by anaerobic glycolysis (ATP

formation) and the pentose-phosphate pathway (NADPH + H+ ) formation. Only 20 g are
consumed per day.
Synthesis of 2,3-bisphosphoglycerate (BPG) .

O O Pi O
H2O
2-
C O PO3 C OH C OH
bisphosphoglycerate 2-
HC OH HC O PO3 HC OH
mutase
2- 2- 2-
C O PO3 C O PO3 C O PO3
H2 H2 H2
1,3-bisphosphoglycerate 2,3-bisphosphoglycerate 3-phosphoglycerate
2,3-bisphosphoglycerate is a potent inhibitor of bisphosphoglycerate mutase.

Methemoglobin reductase is a flavoprotein that uses either NADH + H+ or NADPH +
H+ to reduce methemoglobin back to hemoglobin:
2 Hb·Fe3+ + NAD(P)H → 2 Hb·Fe2+ + NAD(P)H + H+
Phagocytic cells rely on anaerobic glycolysis in the resting state but consume a lot of
oxygen during work (“respiratory burst”). The intracellular killing of phagocytosed microor-
ganisms can be achieved by:
• Low pH of the phagocytic vacuole (= phagosome)
• Lysosomal enzymes in the phagolysosome (created by fusion of phagosome and lyso-
some)
• Oxidative reactions which create toxic superoxide radicals, hydrogen peroxide, and
hypohalite:
NADPH
Superoxide radical NADPH + H+ + 2 O2 GGGGGGGGGGGGGA NADP+ + 2 O−
2 + 2 H
+
oxidase
Superoxide
Hydrogen peroxide 2 O−
2 + 2 H+G
GGGGGGGGGGGGGGGA O2 + H2 O2
Dismutase
Myelo
Hypohalite (hypochlorite, hypoiodite) Cl− + H2 O2 GGGGGGGGGGA OCl− + H2 O
peroxidase
An X-linked recessive deficiency of NADPH oxidase causes chronic granulomatous dis-
ease, with impaired intracellular killing and recurrent infections.
581
Brain
Composition
Water 70 % of white matter, 80 % of gray matter
Lipids 60 % of dry weight in white, 40 % in gray matter
Myelin contains 80 % of dry weight lipid. Galactocerebroside and -sulfatide are mostly in
myelin, gangliosides mostly in the gray matter.
Metabolism Glucose oxidation is the principal energy source except in prolonged fasting
when ketone bodies are also important. The brain accounts for 25 % of the basal metabolic
rate in adults and > 50 % in infants. Insulin has no effect on brain carbohydrate metabolism,
except in the ventromedial hypothalamus (satiety center!).
Brain activities such as perceiving, feeling, remembering, and reasoning cause moderate-
sized increases of brain metabolism in circumscribed brain regions. These metabolic changes
can be picked up in functional imaging studies (PET-scan).
Metabolic insults Oxygen deprivation causes loss of function within seconds, followed by
rapid cell death. In adults, irreversible brain damage occurs after about 7 min in drowning
victims. Newborns develop irreversible brain damage after 1 h of oxygen deprivation, with
cortical cell loss.
Hypoglycemia causes weakness, trembling, facial flushing, sweating, in severe cases pro-
gressing to confusion, seizures, and coma. The brain stores only very small amounts of
glycogen.
Hyperglycemia is probably not damaging itself, but is usually accompanied by acidosis
and dehydration which cause CNS depression in patients with “diabetic coma”.
Uremia, acidosis, hyperosmolarity, and hyperammonemia all result in encephalopathy.
The blood-brain barrier has carriers for the facilitated diffusion of glucose, amino acids
etc...
Cerebrospinal fluid, formed in the choroid plexuses, has:
• an electrolyte composition similar to plasma.
• a very low protein concentration (20 mg/dL).
• a glucose concentration that is 65 % of blood glucose.
Lumbar CSF is important for the diagnosis of CNS diseases.
582
Obesity 31.4
31.3.3. Unbalanced meals
A high carbohydrate/low fat meal stimulates lipogenesis in the liver. VLDL and plasma
triglycerides increase well above the post-absorptive level. In short-term experiments, su-
crose elevates plasma triglycerides more than complex carbohydrates, probably because
fructose is more rapidly degraded than glucose (no glucokinase and phosphofructokinase
required).
A high protein/low carbohydrate meal stimulates insulin release, but glucagon release is
also stimulated and this prevents hypoglycemia.
A high-fat/low carbohydrate/low protein meal leaves insulin low and glucagon high, with
very high plasma levels of free fatty acids and ketone bodies. This kind of diet (Atkins diet)
has been recommended for weight reduction and may indeed be “effective”: to maintain the
blood glucose level, a lot of muscle protein has to be degraded to supply amino acids for
gluconeogenesis. Ketone-bodies produced from ketogenic amino acids have a diuretic effect,
leading to loss of water.
Postprandial thermogenesis is highest for protein, lowest for fat and intermediate for car-
bohydrates. It is higher after a very unbalanced (carbohydrate only/no fat or fat only/no
carbohydrate) than after a well-balanced meal.
31.4. Obesity
Obesity is a chronic disease caused by imbalance between energy supplied with meals and
required by resting metabolism + physical activity. Body weight usually builds up over
long-terms:
• average | requires 12 000 kJ/d.
• said | exceeds requirements by 1% on average, 120 kJ/d (Lipostat theory)
• 1 kg of fat is equivalent to 32.3 MJ
• thus the | gains 1.36 kg/a in body fat
• over 20 years that adds up to 27 kg!
There are large differences in individual energy requirement, which make it difficult to
determine an appropriate caloric intake:
• physical activity
• resting metabolism (30 % individual difference after adjustment for age, sex, size)
583
Rapid weight gain in is observed in some serious diseases (hypothyroidism, Cushing dis-
ease) and always needs to be investigated!
Since obesity is caused by an imbalance between ingested and spend energy, it is, in princi-
ple, curable by reducing energy input and/or by increasing energy expenditure. In practice
however, this is much easier said than done. Several causes conspire:
• Until less than 50 a ago most people had to work hard physically to make a living.
Our digestive system is adapted to deliver 2–3 times more energy than required for a
modern sedentary lifestyle.
• During much of human evolution food supply was highly irregular. We evolved a
tendency to store energy for meagre times, details of which will be discussed in the
following sections. With a fast food store at every corner this ability has become a
liability.
• With fewer and fewer people having the ability, interest and time to prepare their
own meals dependency on industrially prepared food with a lot of protein, fat and
salt, but little fibre has increased.
• Portion size of prepared food has increased, “supersize”
• There is an effect of social contagion: The more people in a patients environment are
obese, the more likely the patient is to develop obesity. It just “seems ok”.
31.4.1. Adipose tissue in obesity
• Normal person 2 × 1010 fat cells each storing 0.3 µg of fat (6 kg total)
• Max. capacity 0.9 µg per fat cell, 18 kg total
• If this amount is exceeded, fat cells divide to accommodate extra fat → obesity,
“adipose tissue hyperplasia”
• Obese person, say, 8 × 1010 fat cells with 0.9 µg → 72 kg body fat
• after weight reduction still 8 × 1010 fat cells, but with, say, 0.2 µg of fat (16 kg body
fat)
• Underfilled fat cells signal: “Feed me!” → jojo-effect
• Reduction in BMR like in starvation
Thus it is the adipose tissue hyperplasia that defines obesity, BMI and waist/hip-ratio are
only tools to quickly assess a patient.
584
Adipose tissue in obesity 31.4.2
Figure 31.3.: In a healthy environment both people resistant and susceptible to a multi-
factorial disease do quite fine. Once the environment turns toxic, susceptible
people fare far worse then resistant ones.
585
31.4.2. Appetite control
• Stomach stretch receptor

• Blood glucose sensor
• Intestinal hormones
• Adipose tissue: Leptin (from Greek: thin, 167 aa protein)
– LepRb in ventromedial nucleus of the hypothalamus: satiety center, endo-cannabinoids
↓, vegetative NS ↑
– high serum [Leptin] prevents active transport of leptin across blood-brain barrier:
leptin insufficiency
– desensitization of LepRb by high [Leptin] in obesity: Leptin resistance, e.g. in
heart muscle
– inherited deficiency in leptin or its receptor in some obese people (see fig. 31.5)
– fertility regulation by leptin: amenorrhea in anorexic ~
– many other functions
31.4.3. Role of gut flora
• 1013 to 1014 symbiotic bacteria on humans, most of them in the end-gut.

• Digest fiber into acetate, propionate, butyrate → 10 % of total energy intake and
principle food for colonocytes.
• Synthesis of essential aa, vitamins (but recall that their uptake is mostly in the small
intestine!).
• detoxification of plant secondary metabolites (cancer protection?)
• Out of 70 eubacteria and 13 archaea divisions only 2/1: Bacteroidetes, Firmicutes and
Methanobrevibacter smithii make 93 % of gut biodiversity. Note: E. coli occurs in the
highest number, but is a γ-proteobacterium, a group that does not contribute much
to diversity.
• In obese people and ob/ob mice B/F ratio drops, but biodiversity stays constant.
• B/F ratio can be regenerated by weight reduction.
• Does bacterial fauna influence how well we use food?
586
Role of gut flora 31.4.4
Figure 31.4.: Model for the lipostat: If the amount of fat in adipose tissue increases, this
releases leptin into the blood stream. Leptin stimulates the release of various
hormones in the hypothalamus, which all reduce the production of neuropep-
tide Y. This reduces the inhibitory effect of NPY on the production of thyroid
hormone, the increased thyroid hormones lead to increased energy expendi-
ture and hence decrease fat stored in the adipose tissue. In addition, NPY
reduces feeding behavior, this too reduces fat stores. The system is set for a
slow weight gain, which made sense in a world with irregular food supply.
Leptin
Lipostat theory
Hypothalamus
propiomela- corticotropin cocaine and

nocortin releasing amphetamine
(POMC) hormone (CRH) regulated
transcript (CART)
melanocyte
stimulating
hormone ( MSH)
Thyroid
neuropeptide Y (NPY) hormone
feeding energy
behaviour expenditure
Thermogenesis
Adipose tissue mass
587
Figure 31.5.: Left: Inherited leptin deficiency in a 3 a old boy leads to morbid obesity. Such
patients show aggressive feeding behavior (stealing from table neighbors or
garbage cans, raiding fridges). Right: The same boy after 4 a treatment with
recombinant leptin. Figure from [O´Rahilly et al., 2003].
588
Epigenetics 31.4.5
31.4.4. Beneficial effects of dietary restriction
• life expectancy highest in people about 10 % underweight

• Class-III protein deacetylases (sirtuins) are NAD-dependent and measure NAD/NADH
ratio.
• act on: histones, p53,...
• In yeast silent mating type information regulator 2 (Sir-2) – human ortholog SIRT-1
– activated by polyphenols: beneficiary effect of red wine in low doses
31.4.5. Epigenetics
Until a few years ago evolution was assumed to be a slow process of accumulating mutations
in the DNA by a trial and error process. Recently it has turned out that the expression of
genes can be regulated by methylation of their DNA on the cytosine at CpG dinucleotides
or chromatin remodeling (mostly by histone acetylation, but also methylation, phospho-
rylation, ubiquitinylation, SUMOylation, ADP-ribosylation, biotinylation), and that these
changes are inherited. This allows rapid adaptation to a changing environment – and the
human environment has changed a lot over the last 100 a. In a way, this looks a bit like
theories of inheritance proposed by Lamarck. Information that is inherited other than
in nucleic acid sequences is called epigenetic, this includes for example the structural in-
formation of a living cell – which is why cells can originate only from cells, not from just
DNA.
Unfortunately our understanding of epigenetic phenomena is still rudimentary. Some ob-
servations may indicate how important this field will become:
• In a study on harvest records and death certificates in Överkalix, a remote village
in Sweden, researches found that if grandfathers had been exposed to famine dur-
ing adolescence, their grandchildren had longer life expectancy and a lower incidence
of type II diabetes. If the grandmothers had been exposed to famine during embry-
onal development the grandchildren had a higher incidence of low birth weight and
of type II diabetes. Think: Why is the sensitive period in males prepuberty and in
females embryonal development? This field is called transgenerational epigenetic
inheritance.
• Exposure to famine in utero seems to set a thrifty genotype by epigenetic mecha-
nism that is adapted to malnutrition. If later in life the nutrition is much richer, the
body is unable to handle it, resulting in metabolic disease. Similarly, if nutrition in
utero is rich, the person will find it more difficult to survive famine later in life. Low
birth weight has been shown to be a risk factor for glucose intolerance.
589
Figure 31.6.: Deacetylases come in 3 different classes. While class I and II use water, class
III deacetylases use NAD+ . Because the NAD+ /NADH + H+ ratio depends
on the nutritional state, class III deacetylases can regulate cellular processes
(like division) depending on food supply. Figure from [Buxbaum, 2007].
H +H2O + O CH3
N CH3
NH3 +

O
O
acetate
N-acetyl-lysyl-group
O NH2
NH2
N N
+
N N O O N
O O O O O
P P
O O

HO OH HO OH
NAD+
NH2
O NH2 N N
NH3
+ + +
N N
O O
O
O
O
O O OH
P P
N O O
HO OH HO O O
Nicotinamide
CH3
O-acetyl-ADP-ribose
(second messenger)
590
Systems biology 31.4.6
Figure 31.7.: Each gene gives rise to several mRNAs by alternative splicing, RNA editing
and the like. The collection of mRNAs present is called the transcriptome
of a cell. Each of these mRNAs gives rise to different protein species by post-
translational modification, the collection of proteins is called the proteome.
Interactions between proteins results in the interactome of a cell. By enzy-
matic activity these proteins change the composition of the cell with respect
to small molecules, the metabolome. This in turn has effects on the phys-
iological (and pathological) state of the cell, the phenome. Each of these
levels of organization can be investigated with specialized methods. Figure
from [Buxbaum, in press]
• Although the incidence of obesity in industrial nations is still increasing, the incidence
of coronary heart disease and type II diabetes is starting to decrease (“obese but
metabolically healthy”). In developing countries, where the obesity epidemic is just
starting, incidence of obesity and both diseases increase together.
• Rats neglected by their mothers during infancy become timid and nervous compared
to those raised by caring mothers. Drugs that facilitate the removal of methyl-groups
from DNA in the brain can normalize their behavior.
• Imprinting allows paternal and maternal genes to be distinguished and differentially

expressed (or silenced) during development. During germ cell development these im-
printing patterns are erased and reestablished.
• Since epigenetic regulation is influenced by environmental factors like nutrients (folic

acid and methionine, individual and maternal diet during pregnancy) the variable
penetrance of some genetic diseases can be explained. This offers a theoretical route
for the treatment of affected patients.
For a review on these topics see [Mariman, 2008].
591
Figure 31.8.: If genes are sorted by interaction of their products large clusters are revealed.
It is the proteins of these those clusters that work together for a particular
effect. Figure taken from [Sieberts and Schacht, 2007].
592
Systems biology 31.4.6
Figure 31.9.: A particular disease state like atherosclerosis involves several biochemical
pathways, each consisting of several proteins. Figure taken from [Ghazalpour
et al., 2004].
593
31.4.6. Systems biology
Originally biochemistry dealt with the working of a particular protein, say, an enzyme.
Later these enzymes were grouped into pathways, and we learned that in a pathway more
features could be observed that in a single enzyme (e.g. end product inhibition). The whole
is more than the sum of its parts. Today we try to understand the entire complexity of
the interaction of various pathways. This is the aim of systems biology. Long since the
complexity of the information exceeds human understanding, only with the help of ever-
more powerful computers can we hope to make head and tail of it. Only 100 a ago visitation
by god was an officially recognized diagnosis on death certificates. In 20 years a diagnosis
like metabolic syndrome will probably look equally quaint, having been recognized as a
hodgepodge of different diseases with quite different treatment.
31.4.7. Metabolic syndrome
Definition (International Diabetes Foundation 2005)
central obesity waist circumference > ethnic + sex specific limit
plus any two of
raised triglycerides > 150 mg/dl (1.7 mM)
reduced HDL cholesterol < 40 mg/dl (1.03 mM) in ♂, 50 mg/dl (1.29 mM) in ♀
raised blood pressure ≥ 130/85 mmHg
hyperglycemia fasting blood [glucose] ≥ 100 mg/dl (5.6 mM): Oral glucose tolerance test
recommended, but not required for diagnosis of metab. syndrome.
previously diagnosed diabetes
Waist circumference limits (cm):

Ethnic group ♂ ♀ Ethnic group use
Europids 94 80 South + Central Americans South Asian
South Asians 90 80 Sub-Saharan Africa European
Chinese 90 80 Eastern Mediterranean European
Japanese 85 90 Arab European
Insufficient data on some populations, use limits of comparable (!?) population for the time
being.
594
Diabetes 31.5
31.5. Diabetes
Type I insulin deficiency after destruction of pancreas β-cells by auto-immunity. Juvenile

onset. Insulin-dependent. Prevalence 1:400, not life-style dependent. Patients are thin
(wasting of fat and protein stores). Ketonemia and -uria, autoantibodies against islets.
Type II insulin resistance in obesity. Adult onset. Prevalence 7:100 in the USA, costing
1 × 1011 US$ (≈ 6 % of total health care spending). Associated with hypertension,
dyslipidemia, atherosclerosis (metabolic syndrome)
• lower number of insulin receptors
• lower affinity of receptors for insulin
• interference with signalling cascade
Therapy by weight reduction restores insulin responsiveness. Problem: Compliance.
MODY see later.
Patho-mechanism of Type-2 Diabetes

• insulin has anti-inflammatory effects, insulin resistance is pro-inflammatory
• food intake → steeper H+ -gradient across inner mito membrane → ROS production
in Complex II
• NADH/NAD+ ↑ + NADPH/NADP+ ↓ → polyol and hexosamine pathways ↑
→ glaucoma
• Adipose tissue generates hormones + pro-inflammatory cytokines (obesititis):
– leptin, adiponectin, visfatin
– tumor necrosis factors α
– transforming growth factor β, Insulin-like growth factor + binding protein
– Plasminogen activator inhibitor 1, angiotensinogen
– complement C3, IL-1, IL-6, IFN-γ
– serum amyloid
Consequences (“chronic acute phase response”):
Adipocytes insulin resistance, free fatty acid release
Liver release of acute phase proteins and lipids
595
Figure 31.10.: Uterus and ovaries of a normal (left) and a type I diabetic (right) rat. Note
the absence of adipose tissue and the reduction of the organs. Figure from
the archives of E.B.
596
Diabetes 31.5
Brain somnolescence, stimulation of Hypothalamus-Pituitary-Adrenal axis: corticos-

teroids and catecholamines, leucocytosis, sympathetic activation
vascular endothelia activation by cytokines, steroids and catecholamines → hyper-
tension. Microvascular damage → complications.
muscle insulin resistance by Ser-phosphorylation of IRS-1 and -2 (via NFκB, IKK,
JNK1?)
gonads lowered sex hormone production, impotency in ♂
pancreas reduced β-cell function
Note that in “obese but metabolically healthy” persons the production of pro-inflammatory
hormones is not increased!
Maturity onset diabetes of the young (MODY, #606391, monogenic diabetes)

• insulin resistant diabetes with early onset, usually < 25 a
• no obesity or metabolic syndrome
• high frequencies in Romania and African Americans
• autosomal mutations of
– hepatocyte nuclear factor HNF-4α (MODY-I, #125850), chromosome 20 and
HNF-1α (MODY-III, #600496). Mild diabetes, progressive decrease of insulin
secretion.
– glucokinase (MODY-II, #125851), 130 different mutations, chromosome 7, mild
glucose intolerance in heterozygotes with risk of gestational diabetes
– insulin promotor factor (IPF-1). Pancreatic agenesis in homozygotes, MODY-IV
(#606392) in heterozygotes.
– β-cell transcription factor neuroD1 (MODY-VI, #606394)
– carboxyl-ester lipase gene (MODY-VIII, #609812): diabetes with dysfunction of
exocrine pancreas
– phosphoenolpyruvate carboxykinase (PEPCK)1 (+261680): no down-regulation
by insulin
– mito DNA (OxPhos ↓, mitochondrial diabetes)
– insulin receptor, GluT4, hexokinase II, SUR-1, insulin, IRS-1, PC-1, glycogen
synthase
597
Figure 31.11.: The glucose tolerance test. After ingestion of 75 g of glucose on an empty
stomach the blood glucose concentration is measured repeatedly over 2 h.
In normal persons blood glucose concentration raises slightly and returns
to normal quickly. In diabetics, the raise is much higher and the return to
normal slower.
20
(mM)
diabetic
[blood glucose] (mg/dl)

300
15
200
10
normal range
100
5
0 0
0 30 60 90 120
time after glucose feeding (min)
redrawn from Meisenberg & Simmons, 2006
Diagnostic
• random blood glucose ≥ 11.1 mM (200 mg/dl) plus symptoms
• fasting blood glucose level > 7.0 mM (126 mg/dl) on two occasions
• Kidney threshold for glucose 10 mM (180 mg/dl), no threshold for ketone bodies
• If 6.1 mM (110 mg/dl) ≤ fasting [glucose] ≤ 7.0 mM (126 mg/dl): Oral glucose (75 g)
tolerance test: →≥ 11.1 mM (200 mg/dl) after 2 h, impaired glucose tolerance if [glu-
cose] ≥ 7.8 mM (140 mg/dl)
• Glycated hemoglobin (HbA1c ) 2.8–3.7 % in normal, > 6 % in diabetic patients. Inte-

gration of [glucose] over life time of erythrocytes (≈ 120 d). Note: Value is method-
dependent, Labs need to report reference ranges with the result. The ranges here are
for measurement according to current standard of the Int. Fed. of Clin. Chem. & Lab.
Med.
• Further info: Clin. Chem. Lab. Med. 41 (2003) Reviews on Diabetes (available in the
library).
598
random
Yes Yes
blood glucose Symptoms? Diabetes
no > 11.1 mM
Diabetes
no
blood sample
take blood determine
taken after > 8 h
sample fasting BGL
fasting? > 11.1 mM
Yes 7.8..11.1 mM
no no Reduced
fasting blood fasting blood oral glucose glucose
2 h blood glucose
Diabetes
glucose < 6.1 mM glucose > 7 mM tolerance test tolerance
Yes Yes
< 7.8 mM
no diabetes Diabetes no diabetes

31.5
Figure 31.12.: Flow-diagram on the diagnosis of diabetes and reduced glucose tolerance.
599
Figure 31.13.: Glucation (non-enzymatic! ) on N-terminal Val of Hb-β. Initially an unsta-

ble Schiff-base is produced, which slowly turns into the relatively stable
ketosamine. Upon heating this turns into caramel, which gives roast food its
aroma. In the body, the ketosamine may be further processed into AGEs via
Strecker degradation.
+ H2O
COOH OH COOH H H COOH
O
HC + H2N CH HC N CH HC N CH
+
H
HC OH R' HC OH R HC OH R'
R R R
Schiff base Immonium
CH2OH COOH
O COOH HC N CH
H + H
N CH H C OH R'
OH R' R
OH
Amadori-Rearrangement
Glucosylamine OH
H2O
COOH
O
C H2C N CH
H Strecker degradation
H2C N C C O R'
H
C O R' !
R
ketosamine
R
Maillard-Reaction
Reductone (Caramel)
600
Diabetes 31.5
Glycation, AGE, ALE There are several modified hemoglobins in our blood:
HbA1a glucose phosphate + fructose phosphate
HbA1b pyruvate
HbA1c glucose, ≈ 80 % of HbA1
Of those, only HbA1c is of diagnostic importance. From its concentration the average blood
glucose level over the last 3 months can be calculated:
[glukose](mM) = 1.84 × HbA1c (%) − 0.01
Again, this equation works only if the HbA1c was determined according to Int. Fed. of Clin.
Chem. & Lab. Med. guidelines.
The following advanced glycation + lipoxydation end-products (AGE/ALE) are impor-

tant:
Amadori-reaction aldose + amine → ketosamine (see fig. 31.13).
ALE by similar reactions with lipid peroxides
Lys + Arg adducts CML, CEL, GALA, LOMA, Pyrraline, CMA, Argpyrimidine, ...
Lys-Lys crosslinks GOLD, MOLD, Crossline, Fluorolink, Vesperlysine
Lys-Arg crosslinks Glucosepane, Pentosidine, GODIC, MODIC
Modification of nucleophilic aa His, Trp, Ser, Cys
Analytical procedures (ELISA) have been developed for total AGE/ALE and for CML and
pentosidine. However, the interpretation of these values is currently unclear, the assays are
used only in research. For that reason you may forget about all the acronyms above.
• Concentration of AGE/ALE too low to directly affect enzymatic function
• AGE-receptor (RAGE) on macrophages → inflammatory mediators →ROS-formation

→ damage
• Correlation between collagen-AGEs and secondary diabetic damage, uremia, liver

cirrhosis, arthritis, coronary artery disease, smoking.
• Autofluorescence of connective tissue.
601
Catalogue of investigations for diabetics

• First visit:
– Complete physical exam (chronic complications?)
– Neurological and angiological exam
– Exam of feet
– EKG
– Eye exam (referral to ophthalmologist)
– Blood sugar, HbA1c, Cholesterol (total, HDL, LDL, TG), creatinine, electrolytes
– Urine sample (Glucose, Albumin (microalbumiuria 30–300 mg/d), Ketone etc.)
– Start of:
∗ Agreement on short- and long term aims
∗ Structured training of patient / family
∗ Training in self exam (see below)
∗ Individual nutrition advise (nutritionist)
∗ Advise on healthy life style
∗ Pharmacological management
∗ Treatment of acute problems
∗ Contraceptives / pregnancy
∗ Next appointment
• Each visit (follow up):
– weight
– blood pressure
– blood glucose
– Present condition and therapy (results, measures, aims)
– Continue training
• Every 3 months:
– weight
– blood pressure
602
Diabetes 31.5
– blood sugar
– HbA1c
– Lipids (only when elevated)
– Urine for albumin (only when pathologic)
– training with diabetes team
• Annually:
– complete physical (see first visit)
– complete lab (see first visit)
– check self-exam techniques
Patient self exam for diabetics (daily):

• Blood glucose
• Acetone (if BG >250 mg/ dl / 13,8 mM)
• Urine glucose
• Weight
• Blood pressure
• Foot exam
• Documentation in a log book!
Complications of diabetes and their management

• Hyperglycemia + ketoacidosis (rapid breathing, breath smells of acetone). Treatment:
– Fluid replacement
– correction of acidosis
– insulin injection
• Hypoglycemic shock in insulin-injecting patients if they fail to eat regularly.
• long term damage:
microvascular damage
neuropathy
603
impaired wound healing monitor feet to prevent amputation
retinopathy monitor retina, laser surgery where needed
glaucoma monitor eye pressure, medications, (laser-)surgery
nephropathy monitor microalbuminuria, use angiotensin converting enzyme (ACE)

inhibitors or angiotensin receptor blocker (ARB) to treat
cardiovascular disease tight blood pressure control
Wasting in terminal illness X.Y. is in the end-stage of cancer. Upon a routine exam you
find that he shows muscle and adipose tissue wasting. Following up on this finding you diag-
nose a reduced glucose tolerance combined with insulin resistance. Increased concentrations
of which of the following hormones is responsible for the complication?
A) Glucagon
B) Insulin
C) Epinephrin
D) Thyroid hormone
E) Cortisol
Obesity Which of the following is not usually associated with obesity?
A High levels of leptin
B Low levels of leptin
C Changes in gut microbial fauna
D Amenorrhea in females
E Reduced sirtuin activity
604
Metabolic syndrome A male patient comes to you with a waist circumference of 150 cm.
Which of the following signs is he least likely to show?
A) raised triglycerides (≥ 150 mg/dl or 1.7 mM)
B) raised HDL cholesterol (≥ 100 mg/dl or 2.6 mM)
C) raised blood pressure (≥ 130/80 mm Hg)
D) raised fasting blood glucose (≥ 100 mg/dl or 5.6 mM)
E) previously diagnosed diabetes
At the end of this lecture students should be able to

• describe the role of insulin, glucagon, epinephrin and glucocorticoids in the control of
metabolism.
• discuss the use of macronutrients and their flow between various organs in the well-fed
and fasting state.
• describe how nutrients are used during physical exercise.
• define RQ and describe how it can be used clinically
• discuss the epidemiology, patho-mechanism, health consequences and treatment op-
tions of obesity. Describe obesity as chronic disease and state the lipostat theory of
weight maintenance.
• critically evaluate modern theories on mechanism and treatment of obesity.
• define “metabolic syndrome”
• describe the causes, clinical manifestations (especially in emergency room setting)
and treatment options of diabetes type 1 and 2.
• name secondary diabetic complications, their detection and treatment.
• critically discuss the role of AGE/ALE in the development of diabetic complications.
605
32. Multifactorial Inheritance
32.1. Definitions
Multifactorial traits are caused by “many factors”, both genetic and environmental. Poly-
genic means almost the same, implying the action of “many genes” but no environmental
influence. Most of the contributing genes are still unknown. A trait is assumed to be poly-
genic or multifactorial if:
• It is more common in close relatives of the index case than in the general population
even if they were raised in uncorrelated environments, but
• A Mendelian pattern of inheritance cannot be identified and
• There is no recognizable chromosome aberration.
Multifactorial traits include:
• Most differences between normal people, including physical traits (height, weight,
blood pressure) and psychometric traits (intelligence, personality). These traits show
continuous variation.
• Many common diseases, including diabetes mellitus, allergies, autoimmune diseases,
cardiovascular diseases, neuropsychiatric disorders, and even infections.
• Most congenital malformations.
Our models for explaining multifactorial traits assume that the gene effects take on one of
the following forms:
Additive gene effects are effects of individual alleles. Additive inheritance assumes co-
dominance. Also the phenotypic effects of alleles in different loci are assumed to be
additive. This mode of inheritance predicts that close relatives should have similar
phenotypes, in proportion to the genes they share.
Non-additive gene effects are the effects of gene combinations, rather than individual al-
leles. Nonadditive effects within a locus include dominance, recessivity, and het-
erozygote advantage. Nonadditive interactions between genotypes in different loci
are called epistasis and would for example include effects of mutations in a transcrip-
tion factor that led to changes in expression of one allele in the target gene, but no
607
changes in expression of another allele in the same target locus. Non-additive inheri-
tance does not necessarily predict similarity between close relatives, except identical
twins who share not only individual alleles but all gene combinations.
32.2. Quantitative Traits
If many loci determine the phenotype, the effects are additive, and each locus contributes
only a small proportion of the total variability, we get a smooth bell-shaped distribution of
phenotypes. This is called a normal distribution or a Gaussian distribution.
The standard deviation σ is a measure of the variability of the observed phenotype. In

a distribution, 50 % of individuals are within ± 0.68 σ of the mean (x), 95 % are within
1.95 σ. The variance (V ) is the square of the standard deviation (V = σ 2 ).
A bell-shaped distribution can be produced by many loci with 2 co-dominant alleles each,
or by multiple alleles in one or a few loci. Broad heritability (H 2 ) is the proportion of the
total phenotypic variance that is due to heritable factors. It can range from zero (only genes
are important). Narrow-sense heritability (h2 ) is the proportion of the total variability
that is caused by additive gene effects. The environmental variance can be partitioned
into shared environment (factors that make members of the same family similar) and
non-shared environment (factors that make members of the same family different).
Examples of shared environmental effects are the education and economic conditions of the
parents; most non-shared environmental effects originate outside the home. Most or all of
608
Quantitative Traits 32.2
the environmental variance is non-shared in adults. The various genetic and environmental
effects are assumed to add up:
VPhenotypic = VGadd + VGnonadd + VEshared + VEnonshared (32.1)
Measurement error appears as part of the non-shared environmental variance. Therefore

accurate measuring instruments have to be available, otherwise you over-estimate the im-
portance of non-shared environment and under-estimate everything else. Assortative mating
artificially increases the shared environmental variance.
Heritability differs in different populations because the genetic and environmental variability
are not the same in different populations.
For quantitative traits, the average phenotype of a child is between the “midparent” and the
population mean. This tendency for children to have less extreme phenotypes than their
parents is called regression to the population mean. This effect is really only notable
for the most extreme parents, and the explanation is that the parents have their extreme
phenotype due to environmental rather than genetic factors. The greater the additive genetic
+ shared environmental variance, the lower is the regression to the mean. In adoption
studies, regression from the phenotype of the biological parents to the population mean can
be used to estimate the additive genetic variance.
Note that the “population mean” differs in different populations. The children of unusually
tall pygmy parents, for example, regress to the mean for pygmies, but children of equally
sized (unusually small) non-pygmy parents regress to their own population mean.
Height (stature) shows a normal distribution with a slight hump at the lower end, which
is due to pathological conditions reducing height. Heritability is high (≈ 90 % in most
studies). In Western countries, height increased by 5–10 cm during the past century, due to
environmental changes (nutrition?). Differences between populations in different parts of
the world are in part genetic and in part environmental.
Body weight has a high heritability, but less than stature: ≈ 70 % in most studies. As
in the case of height, the high heritabilities are obtained with reasonably homogeneous
populations where environmental inequalities are not exceedingly large.
Skin color differences between races are determined by about 3–5 major genes. Most of the
gene products are unknown. Skin color differences evolved for protection from UV damage
and the regulation of vitamin D synthesis.
Test intelligence has a bell-shaped distribution with a hump at the lower end. In IQ
tests, the population mean is defined as 100, and 15 points are one standard deviation.
Broad heritability is typically about 40 % in children and 70 % in adults. The average IQ
has increased by about 30 points during the past century because of environmental changes
(better nutrition or education).
609
Mental retardation has heterogeneous causes:
• Most cases of “mild” mental retardation (IQ 50–70) are multifactorial. This means
that many relatives of mildly retarded individuals are mentally subnormal.
• Mental retardation with IQ < 50 usually has a specific cause, such as an infection,
perinatal hypoxia, fetal alcohol syndrome, kernicterus, trauma, severe epilepsy, a chro-
mosome aberration, or a Mendelian disorder (most important: fragile X). In about
half of retardates, however, no specific cause can be identified. Most etiologies are
one-time accidents, therefore close relatives are usually normal.
• Other multifactorial traits with continuous variation include personality traits with
moderately high heritabilities (happiness, divorce liability...) or rather low heritability
(love styles, self esteem...). Also athletic performance, age at first menstruation, age
at first sexual intercourse, life span etc. are multifactorial traits.
32.3. Common Diseases
Most diseases encountered in medical practice are “heritable” in the sense that they are more
common in close relatives of a patient than in the general population. Typical features of
multifactorial diseases:
• The concordance between monozygotic twins is higher than between dizygotic twins
of the same sex. But even concordance between monozygotic twins is rarely 100 %.
• The disease incidence in first-degree relatives of a patient is close to the square root
of the population incidence.
• The risk for close relatives increases with increased severity of the disease in the
index patient, and with the presence of more than one affected family member. These
features suggest an especially bad accumulation of predisposing genes in the family.
Note, however, that many diagnostic labels (“schizophrenia”, “diabetes”) are not a
single disease. In these cases, the more severe forms do not always have a higher
recurrence risk.
• The recurrence risk decreases rapidly if the relationship is less close.
The cutoff between “normal” and “affected” is often arbitrary. Therefore, a “disease” may
simply be the extreme end of a normal distribution.
Genetic counseling for multifactorial diseases is based on the empiric risk that is known
from observations of a large number of families in the same situation.
Diabetes Mellitus is a typical example:
610
Congenital Malformations (“Birth Defects”) 32.4.1
Type I diabetes is probably an autoimmune disease that can, most likely, be triggered
by viral infection. Concordance in monozygotic twins is 30–50 %. It is associated
with certain HLA-haplotypes (haplotype = combination of linked genes). The HLA
antigens regulate immune responses.
Type II diabetes has no HLA-associations, but the heritability is higher than for type I,
with almost 100 % concordance in monozygotic twins. The risk in first-degree relatives
of those patients who develop type II diabetes late in life (> 60 years) is not much
increased above the population mean, but relatives of those who develop the disease
early (< 40 years) are not high risk. One of the important risk factors for this type
of diabetes is obesity. Genes predisposing for this type of diabetes seem to have been
selected for in populations living in regions with a high risk for frequent starvation.
When people from these populations move to areas where they live at “westernized”
standards they have a high incidence of diabetes type II.
Psychiatric disorders are usually multifactorial. In schizophrenia, with a population inci-

dence of 1 %, the empiric risk for first-degree relatives is about 10 %. The empiric
risk in children of two schizophrenic parents is about 30 %, and concordance between
monozygotic twins is about 50 %. In cases with a negative family history, advanced
paternal age is a risk factor. This suggests that new mutations are important. Also, for
manic-depressive disease, primary epilepsy, Alzheimer’s disease, autism, attention
deficit disorder, alcoholism, and personality disorders are multifactorial.
Infections have substantial heritability in those cases where most or all members of the
population are exposed to the disease agent. This has been shown for malaria, hook-
worm, tuberculosis, and other infections. The immune system is highly polymorphic
by design, and therefore people vary greatly in their susceptibility to different infec-
tions.
32.4. Congenital Malformations (“Birth Defects”)
About 1 in 40 babies are born with a congenital malformation (congenital = present at

birth). Some of these are caused by a recognized single-gene disorder, chromosome aberra-
tion, or exposure to a teratogen; but most cases are of unknown origin and can be classified
as multifactorial. Many chemicals, including many drugs, are known or suspected terato-
gens.
611
32.4.1. Nongenetic Causes Of Congenital Malformations
Teratogenic agent Typical malformations

a) Maternal conditions
Advanced age
Diabetes Cardiovascular and CNS malformations
Phenylketonuria Microcephaly, mental retardation
High fever Microphthalmia, microcephaly, neural
tube defects
Systemic lupus erythematosus
b) Intrauterine infections
Rubella Cataracts, deafness, patent ductus
Cytomegalovirus Various malformations in 5 % of infected
pregnancies
Toxoplasmosis
Syphilis Sabre shin, Hutchinson teeth etc.
c) Chemicals
Alcohol Mental deficiency, growth retardation, fa-
cial abnormalities, joint anomalies, heart
defects
Phenytoin Craniofacial malformations, growth re-
tardation, limb defects, mental deficiency
Valproic acid Neural tube defects, heart defects
Cocaine Cerebral infarction, low birth weight, mi-
crocephaly
Amphetamine
Retinoic acid Ear, brain and heart malformations
Lithium
Mercury
Warfarin Chondrodysplasia
Thalidomide Phocomelia (a limb reduction defect)
d) Radiation Microcephaly, ocular defects
e) Cigarette smoke Low birth weight, incr. rate spontaneous
abortion
Most teratogens act only during a specific period of gestation (examples: rubella before 16
weeks, thalidomide 20–36 d post-conception). But “fetal alcohol syndrome” can be caused by
alcohol consumption at any time during pregnancy. In general, the most vulnerable period
is in the first trimester. This is the time when most pregnant women develop pregnancy
sickness, with nausea and food aversions. Pregnancy sickness is considered a protective
612
Mercury
Warfarin Chondrodysplasia
Thalidomide Phocomelia (a limb reduction defect)
d) Radiation Microcephaly, ocular defects
e) Cigarette smoke Low birth weight, incr. rate spontaneous abortion
Nongenetic Causes Of Congenital Malformations
Most teratogens act only during a specific period of gestation (examples:
32.4.1
rubella before 16 weeks, thalidomide 20-36 days post-conception). But “fetal
alcohol syndrome” can be caused by alcohol consumption at any time during
mechanism
pregnancy.that limitsthe
In general, themost
ingestion
vulnerableofperiod
food-borne teratogens.
is in the first trimester. This is
the time when most pregnant women develop pregnancy sickness, with nausea
Theand food aversions. Pregnancy sickness is considered a protective mechanism
multifactorial malformations are threshold traits, with a clear distinction between nor-
that limits the ingestion of food-borne teratogens.
mal and abnormal phenotype.
The multifactorial Empiric
malformations arerisks can be
threshold applied.
traits, with aWeclear
assume that the “liability”
for the defectbetween
distinction shows normal
continuous variation
and abnormal in the population,
phenotype. Empiric risks but only individuals beyond a
can be
applied. We assume that the “liability” for the defect shows continuous variation
certain
in thethreshold show
populaiton, but onlythe defect:beyond a certain threshold show the defect:
individuals
Threshold
Frequency
Liability
Cleft lip and palate Some cases 273 are caused by single-gene disorders, chromosome aberra-
tions, teratogens, or rubella, but most are multifactorial. Cleft palate is genetically different
from cleft lip (with or without cleft palate).
Risk of cleft lip:
• 0.1 % total population
• 4 % sibs of one affected child
• 9 % sibs of two affected children
• 30 % concordance in monozygotic twins
The empiric risk increases with increased severity of the defect in affected family members.
Pyloric stenosis Abnormal narrowing of the pylorus is caused by hyperplasia of the pyloric
muscle. This results in propulsive vomiting. Surgical correction is often required.
Frequency: 0.55 in males, 0.1 % in females
Recurrence risk: Higher in relatives of affected females than in relatives of affected males.
613
According to the multifactorial threshold model, the risk is higher for close relatives of the
less commonly affected sex than for close relatives of the more commonly affected sex.
Neural tube defects

Anencephaly is agenesis of the midbrain and forebrain, with death shortly before or after
birth.
Spina bifida is failure of fusion of the vertebral arches in the lower part of the spinal
column. In severe cases, there are protruding meninges (meningocele) or meninges +
neural elements (meningomyelocele). This is an important cause of paraplegia!
Incidence: About 1:500 in North America
Cause: Some cases have a specific cause (teratogens, trisomy 13), but the large ma-
jority are multifactorial.
Recurrence risk after the birth of an affected child: 4 %. A previous child with anen-
cephaly means an increased risk not only for anencephaly but also for spinal
bifida in a next child, and vice versa.
Environmental factors: Periconceptional folic acid reduces the risk of neural tube
defects. Because in the US, one half of all pregnancies are unplanned and folic
acid supplementation at the time an unplanned pregnancy can will detected
comes too late, the current recommendation is 400 µg folic acid/day for all women
of reproductive age.
Prenatal diagnosis: Ultrasound, screening of maternal serum α-fetoprotein, followed
by determination of α-fetoprotein in amniotic fluid obtained with amniocentesis.
Heart defects
Types: There are many types of congenital heart defect: septal defects, aortic stenosis,
pulmonary stenosis, patent ductus, tetralogy...
Incidence: 1 in 150 overall, with all degrees of severity from minimal impairment to lethal.
Causes: Sometimes a cause can be found, but 80 % are “multifactorial”. The known causes
include several single gene disorders, contiguous gene disorders like DiGeorge syn-
drome, as well as infections.
Recurrence risk: For individual defects, the recurrence risk after the birth of an affected
child is close to the square root of the population incidence. The risk is increased if
two family members are affected. For heart defects overall, the recurrence risk after
the birth of an affected child is 2 %, for the child of an affected parent 4 %, and 10 %
if two first-degree relatives are affected.
614
Quantitative Trait Loci (QTLs) 32.5
32.5. Quantitative Trait Loci (QTLs)
The identification of QTLs for continuously variable traits or for multifactorial disorders
(“susceptibility genes”) is a major aim of current genetic research. The possibilities are:
1. A common polymorphism modifies the disease risk. Such polymorphisms can be main-
tained by balancing selection, either through simple heterozygote advantage or be-
cause the disease-promoting allele does something useful for those who carry it without
developing the disease.
2. Disease susceptibility is caused by the accumulation of mildly deleterious mutations.

Although a single mutation is insufficient to cause the disease, a constellation of
several mutations along with an unfavorable environment puts people at risk. The
mutations do nothing useful and can be considered “genetic garbage”. It is estimated
that the average child is born with 2 or 3 mildly unfavorable new mutations on top
of those inherited from the parents.
3. The disease is caused by a single major gene in a small subpopulation of patients.
Also normal variation for traits such as blood pressure and intelligence may be due either
to common polymorphisms that are maintained by balancing selection, or rare gene defects
that are continuously created by mutation and removed by selection.
A susceptibility gene that is important in one population may be unimportant in another.

Example: A polymorphic aldehyde dehydrogenase is an important genetic determinant of
alcoholism in Orientals, but not in Westerners. For this reason, geneticists searching for
QTLs like to work with genetically homogeneous populations in places such as Iceland or
Finland. Methods:
Linkage studies These studies require families with multiple affected members. To make a
genome-wide scan, a large number (> 100) of highly polymorphic VNTRs have to be
used. In one popular design, affected sib pairs are studied under the assumption that
there is more than the otherwise expected 50 % sharing of alleles near the QTLs.
With hundreds of VNTRs, there is always a high chance that one of them reaches a
moderately high Lod score even in the complete absence of linkage. Therefore only
Lod scores higher than 4 or 5 are strong evidence for linkage. A genome map, but
further gene mapping is needed to identify the gene itself.
Extremely discordant sib pairs can be used to find genes for continuously variable
traits. Linkage studies are very costly, and they require sophisticated statistical meth-
ods. Also, undisclosed nonpaternity can bias the results, especially in studies of ex-
tremely discordant sibpairs.
615
Candidate genes In some multifactorial conditions, known genes are suspected to be in-
volved, for example genes for HLA antigens in autoimmune diseases. You have to
know the genetic polymorphism first. Then you determine the allele frequencies in a
group of patients and a matched control group. If an allele is over-represented in the
patients, it is a bona-fide risk factor for the disease. This method is easier and more
powerful than linkage studies, but it cannot detect “unexpected” susceptibility genes.
This method is known as an association study. As with all epidemiological methods,
choosing a control group that is truly matching is the most important and difficult
step.
single-nucleotide polymorphism (SNP) account for the majority of sequence
variants in our genome. There are a few million SNPs, at least 100 000 of them in the
coding and regulatory sequences of genes. They will be used for association studies
on a genome-wide basis, using DNA chips to test for thousands of them at a time.
Problem: An SNP variant that is associated with a particular condition (such as
asthma, hypertension, or high intelligence) may either be the cause of the trait, or it
may be in linkage disequilibrium with another polymorphism that causes the trait.
Association does not prove causation!
Linkage disequilibrium is defined as the occurrence together on the same chromosome
of specific alleles at two closely linked loci more often than would be expected from
the allele frequencies of the alleles involved.
32.6. Examples Of Susceptibility Genes
Type I diabetes is associated with the DR3 and DR4 alleles in the major histocompatibil-
ity complex (the genes encoding the HLA antigens). An allele of the HLA-DQ gene
reduces the risk of type I diabetes one hundred-fold. Also most other autoimmune
diseases, such as ankylosing spondylitis, multiple sclerosis, and autoimmune thyroidi-
tis, are associated with HLA antigens. In addition to HLA antigens, variations at a
closely linked VNTR near the proinsulin locus contribute to the disease risk for type
I diabetes.
Type II diabetes is usually multifactorial; some chromosomal regions but no genes have
been implicated in this, the more common type of diabetes. The heritability of this
subtype is higher than in type I diabetes. One subtype, “maturity onset diabetes
of the young” (MODY), is caused by a dominant gene (glucokinase deficiency in
some patients; 6 different genes identified). This entity should probably be known as
diabetes mellitus type III.
Alzheimer’s disease is associated both with common polymorphisms and some rare mu-
tations. The disease is more likely, and begins earlier, in individuals carrying the
616
apolipoprotein E4, variant. This apoE variant is deposited along with β-amyloid in
senile plaques. Some other genomic locations have been pinpointed in linkage studies.
In a few families with early-onset Alzheimer (< 60 a), the disease is caused by a dom-
inantly expressed point mutation in the gene for amyloid precursor protein (APP),
the precursor of β-amyloid. Mutations in at least two other genes are known to cause
early-onset Alzheimer. The APP gene is positioned on chromosome 21, and over-
expression due to the extra gene copy is probably the reason why Down syndrome
patients often develop Alzheimer.
Osteoporosis is related to a common polymorphism in the gene for the calcitriol (vitamin
D) receptor.
Asthma risk is affected by a polymorphism in the gene for a protein in bronchial mucus
(Clara cell secretory protein, CC16). In addition, several chromosomal regions have
been identified by linkage.
Atherosclerosis is usually multifactorial with rather low heritability (bad habits are more
important than bad genes). But some patients have a single-gene defect, for example
familial hypercholesterolemia (defective LDL receptor); high level of Lp(a); or “familial
combined hyperlipoproteinemia” (presumably causing increased synthesis of apoB-
100).
1. Explain why multifactorial traits usually show a bell-shaped phenotypic distribution.
2. Define the term “heritability” and state its limitations for analyzing the nature-nurture
problem.
3. Make semi-quantitative statements about the hereditability of some multifactorial

traits, including height, body weight, life-span, athletic performance, intelligence, al-
coholism and criminal behavior.
4. State the approximate population incidence of multifactorial conditions including

Type I and Type II diabetes, schizophrenia, manic-depressive disorder, cleft lip, neural
tube defects and congenital heart defects, and the approximate incidence of these dis-
orders in first degree relatives of a patient.
5. Specify the typical effects of the number of affected relatives and of the severity of
the disease in an affected relative on the risk of multifactorial disorders.
6. State the roles of teratogens and intrauterine infections in the pathogenesis of con-
genital malformations.
617
7. State which methods can be used for the prenatal diagnosis of congenital malforma-
tions.
8. Apply the multifactorial threshold model to predict the relative risk to first-degree
relatives of patients with a sex-influenced multifactorial disorder.
9. Describe the importance of identifiable single-gene effects in multifactorial disorders
including autoimmune diseases, types I and II diabetes.
10. Describe the effect that major gene and minor gene alleles have for the risk assessment
in diseases where they occur, and state examples of each in Hirschsprung’s and
Alzheimer disease.
11. State the possible impact of pre-symptomatic testing for susceptibility genes for mul-
tifactorial disorders on disease incidence and on the use of prenatal diagnosis with
elective abortion.
618
Part VII.
Appendix
33. Answers to the example questions
33.1. Thermodynamics
1) Entropy, driving force of a reaction
• ∆G = ∆H − T × ∆S
• Boiling point: Equilibrium liquid/gas → ∆G = 0 J/mol
• 0 J/mol = 40 700 J/mol − 373.14 K × ∆S
• 373.14 K × ∆S = 40 700 J/mol

40 700 J mol−1
• ∆S = 373.14 K ≈ 100 J mol−1 K−1
2) Ligand binding to receptor X + R *

) RX
[R][X] ([R]t − [RX])[X]

Kd = = | ×[RX]
[RX] [RX]
Kd [RX] = ([R]t − [RX])[X] | : ([R]t − [RX])
Kd [RX]
[X] =
([R]t − [RX])
1nM × 1000 1 1
= = nM = nM
10000 − 1000 10 − 1 9
≈ 0.11nM
3) Virus capsid stability The high activation energy of the dissociation makes virus
metastable. Note that most of the other options would actually decrease virus stability,
rather than increasing it. The difference between medicine and shamanism or quackery is
the belief that the rules inside a living organism are the same as those in the test tube and
that therefore disease can be cured by applications of the laws of science.
621
4) Elimination of drugs from the body Since the blood sample was taken 3 half life
periods after injection, the concentration after injection must have been 2 × 2 × 2 = 8 times
that found in the experiment, hence 8 × 12.5 nM = 100 nM.
The amount of the drug is concentration times volume, hence 100 nmol/l × 50 l distribution
volume = 5000 nmol = 5 µmol.
Since 1000 g : 1 mol = x g : 5 µmol. Separating known and unknown variables yields x =
1 × 103 g × 5 × 10−6 mol : 1 mol = 5 × 10−3 g = 5 mg.
pKa
5) Effect of pKa on membrane diffusion BH+ E
GGGGGGGGC
GG B + H+
The higher the pKa is above the environmental pH (here blood pH, 7.4), the less of the drug
is in the uncharged form which can cross the membrane. According to Fick’s diffusion law,
this lowers transport velocity.
The Henderson-Hasselbalch-equation allows

us to calculate the ratio of charged over
[BH + ]
uncharged drug at the given pH: log [B] = pKa − pH blood pH = 7.4
[BH + ]
Compound pKa [B]
Lidocaine 7.7 2.00

Bupivacaine 8.1 5.01
Procaine 8.9 15.84
6) Follow-up: Bacterial metabolism inside an abscess leads to the formation of lactic acid,
which drops the pH. As a consequence lidocain can no longer enter the nerve cells, exposing
the patient to a considerable amount of pain.
7) pH-dependent drug trapping :
622
Proteins 33.2
[HA]
log = pK a - pH = 3.4 - 7.4 = -4
[A- ] [H + ]
[HA]
= 10 -4
[A- ] [H + ]
1 10 000
HA H ++ A-
Plasma, pH = 7.4
10 001
Plasma membrane Ratio = = 9902
1.01
Stomach juice, pH = 1.4

HA H ++ A-
1 0.01
[HA]
log = pK a - pH = 3.4 - 1.4 = 2
[A- ] [H + ]
[HA]
= 10 2
[A- ] [H + ]
33.2. Proteins
pKa
GGGGGGGGB
1) Effect of pH on enzyme activity HA F GG H+ + A−
Glu-35 (pKa = 5.9): pH ≤ pKa : more HA → higher activity
Asp-52 (pKa = 4.5): pH ≤ pKa : more HA → lower activity
2) Structure of alpha-amino acids, radioactivity

A β-radiation is emission of electrons by decay of a neutron of the nucleus into a proton
and an electron.
B Decarboxylation of an amino acid produces CO2 from the carboxy-group. Since that
C-atom is radioactive, the CO2 will also be.
C The carboxy-group is attached to the rest of the molecule by a very stable c−C-bond.
Sparging with radioactive CO2 will therefore not exchange this group.
D For most intents and purposes, the physico-chemical properties of radioactively labelled
compounts are identical with the unlabelled ones. Indeed, on this identity their use
in tracer studies rests.
623
E see above.
+G
2.18
3) pI-value of amino acid HOOC CHNH+ GGGGGGGB
3 CH2 NH3 F GG − OOC CHNH+ +
3 CH2 NH3
8.95 − 10.53
GGGGGGGGB
F
+ GGGGGGGGGB −
GG OOC CHNH2 CH2 NH3 F GG OOC CHNH2 CH2 NH2
1/2 × (8.95 + 10.53) = 1/2 × 19.48 = 9.74
4) Properties of amino acids At pH 8.0 Glu is negatively charged, while Val is not. A
Glu→Val mutation will reduce movement towards the positive pole.
At pH 1.0 Glu is uncharged, as is Val. Hence the mutation has no effect on electrophoretic
mobility at this pH.
5) Functional replacement of amino acids Ala is Ser minus the −OH group which is the
catalytically active part of Ser.
6) pH dependence of solubility of amino acids Glu + Asp acidic residues, give additional
charges at alkaline pH.
7) Determination of protein concentration from UV-absorption Light absorption A

as a function
of concentration c and path length l is described by Lambert-Beer’s law
A = − log I = λ c l, where λ is the extinction coefficient at wavelength λ. In our case
I
0
the sample was diluted 100-fold, as Lambert-Beer’s law is valid for most substances only
if A ≤ 1.0.
Hence the molar protein concentration c = 100× Al = 100× 4 ×0.410mol cm
4 ×1 l cm =
40
4 ×10
−4 mol/l =
λ
1.0 × 10−3 mol/l.
Since the average molecular weight is 65 kDa, and hence 65 × 103 g is equivalent to 1 mol,
we can convert the molar concentration into a weight concentration: 1.0 × 10−3 mol/l ×
65 × 103 g/mol = 1.0 × 65 g/l = 65 g/l = 6.5 g/dl, which is in the normal range.
8) Strange disease The description is typical for a case of FFI.
9) Collagen related inherited disease The case description is typical for chondrodysplasia,
the molecular genetics confirms this.
624
Enzymes 33.3
10) CAG-length variation Huntington’s disease caused by CAG-expansion in huntingtin

→ poly-Glu amyloid formation
33.3. Enzymes
1) Enzyme turnover rate: There are two ways to answer this question, both are equally
valid:
the industrious student
Vmax ∗ [S]
v= (33.1)
(Km + [S])
V ∗ 3 Km
= max (33.2)
(Km + 3 Km )
= Vmax ∗ 3/4 (33.3)
the bright student no calculation required:

• [S] > Km → v > 1/2 Vmax eliminates A–C
• [S] < ∞ → v < Vmax eliminates E
• D is the only possible option!
2) Enzyme reaction velocity When the substrate concentration is (nearly) saturating,

v ≈ Vmax . However, Vmax = kcat × [E], thus doubling [E] doubles the velocity.
3) Enzyme turnover number

First Calculate molar amount of enzyme
1 mol × 5 × 10−6 g
x=
5 × 104 g
= 10−6 × 10−4 mol
= 1 × 10−10 mol
Now turnover number

1 × 10−5 mol/min
kcat =
1 × 10−10 mol
= 10−5 × 1010 min−1
= 1 × 105 min−1
625
4) Enzyme activity, HMM-equation An enzyme with the higher activity will turn over
more substrate. An enzyme with lower Km can do so even at low [S].
5) Action of pharmaceutical When interpreting plots always look at the labels of the axis
first.This is a time-dependent inactivation by a suicide substrate. Inhibitions are reversible,
diffusion controlled reactions that happen on a ms time scale.
6) Metabolic and thermodynamic direction Le Chatelier’s principle: removal of the

product stimulates its formation.
7) Henri-Michaelis-Menten law, Lineweaver-Burk plots The parallel lines indicate an

uncompetitive inhibition. To get Km we locate the line in the absence of inhibitor (red).
Its x-intercept (−1/Km ) is −0.5 mM−1 , thus Km = 2 mM.
8) Hill-equation, co-operative binding
θmax × [O2 ]h
θ=
K0.5 + [O2 ]h
100% × 12.9
=
13 + 12.9
100%
=
14
≈ 7%
9) difference between inhibition and inactivation Covalent modification usually results

in irreversible loss of activity → inactivation
10) Enzyme nomenclature, enzyme classes Transfer of a phosphate group from ATP to
glycerol is catalyzed by a transferase, specifically a phosphotransferase or kinase.
33.4. Amino acid metabolism
1. Protein consumption
• 56 g/d recommended - 34 g/d plant protein uptake = 25 g/d additional plant protein
required
626
Digestion 33.5
• 75 g/d animal protein x 41 Mt / 7 Mt = 439.3 g/d of plant protein equivalent
• - 25 g/d additional plant protein = 439.3 g/d wasted per US citizen
• / 56 g/d recommended uptake = 7.4 daily protein doses wasted per US citizen
• x 3 × 108 US citizens = 2.2 × 109 daily doses
• That is about 1/3 of the worlds population!
2. Biological value of proteins The ratio of the mixture 2 parts rice, 1 part soy is calcu-
lated as weighted average of the ratios for the individual food stuffs, weighting factor is the
proportion in the mixture. For Ile that yields:
2 × 4.08 + 1 × 3.15
= 3.46 (33.4)
2+1
amino acid ratio rice ratio soy ratio mix

Ile 3.15 4.08 3.46
Leu 4.41 3.95 4.26
Lys 2.67 4.43 3.26
Met 1.58 0.69 1.28
Phe 2.72 2.51 2.65
Thr 4.29 4.29 4.29
Trp 0.67 3.33 1.56
Val 4.33 3.56 4.07
After filling the table for all amino acids you see that Met is now limiting, with a ratio of
1.28. This determines the biological value of the protein. Note that while both rice and soy
proteins individually lack certain essential amino acids (ratio < 1.0), the mixture provides
all of them in super-optimal amounts, that is, has a high biological value. Such calculations
are important for making food mixtures for parenteral nutrition.
33.5. Digestion
1) Weight loss on 0 J diet: Under those circumstances, patient would not be very active,
energy consumption would be limited to resting metabolism, on average 7500 kJ/d in |,
5400 kJ/d in ~. Fat yields 38.9 kJ/g of energy when it is metabolized. So | would loose
just under 200 g/d, ~ just under 140 g/d, if only body fat were metabolized.
627
2) Loss of lean muscle mass during diets: To maintain a constant blood [glucose] the
body needs to perform gluconeogenesis. Fatty acids can not be used for that (why?), glycerol
from fat catabolism can, but amount is insufficient. Amino acids from muscle protein is used
instead.
3) Brain glucose metabolism during fasting: The brain uses energy at a fairly constant
rate, irrespective of the metabolic situation of the body and of intellectual activity. There-
fore, brain glucose consumption is not hormone regulated. As we will see later (see chapter
31 on page 569), in long term fasting ketone body concentrations in blood become high
enough to supply part of the energy the brain needs, sparing glucose and hence lean muscle
mass.
4) Athlete in distress: The patient has used up his entire glycogen stores during his
strenuous activity. The only way to maintain his blood glucose level is gluconeogenesis,
which however is inhibited by alcohol. Thus hypoglycemia results in loss of consciousness,
and has to be corrected by infusion of glucose to save his life. Note that Ringer/Lactate
would not work under these conditions! (Why?)
33.6. Integration of metabolism
1) Wasting in terminal illness: Glucocorticoids like cortisol are released as consequence

of chronic stress.
2) Obesity: Low levels of leptin may cause obesity, while high levels may be the result.
Both reduced sirtuin activity and changes in B/F ratio in the gut are regularly observed in
obesity. However, amenorrhea in ~ is more a consequence of anorexia (albeit it may occur
secondary to long-term damage in severe obesity).
3) Metabolic syndrome: In a patient with increased hip circumference one would expect
a reduction of HDL (good cholesterol), not an increase.
628
34. Tables
629
34.1. Conversion from non-metric to metric units
non-metric metric
1 cal 4.1868 J
1 cup 236.6 ml
1 inch 2.54 cm
1 lb 453.59 g
1 oz 28.350 g
1 tablespoon 15 ml
1 teaspoon 5 ml
34.2. Symbols used
C heat capacity W/K

q heat
T Temperature K or °C
630
Greek alphabet 34.3
34.3. Greek alphabet
alpha α A
beta β B
gamma γ Γ
delta δ ∆
epsilon , ε E
zeta ζ Z
eta η H
theta θ, ϑ Θ
iota ι I
kappa κ K
lambda λ Λ
mu µ M
nu ν N
xi ξ Ξ
o o O
pi π, $ Π
rho ρ, % R
sigma σ, ς Σ
tau τ T
upsilon υ Υ
phi φ, ϕ Φ
chi χ X
psi ψ Ψ
omega ω Ω
631
34.4. The genetic code
mRNA-codons and the corresponding amino acids. Alternative uses of codons are marked
on the exterior. The colors used to symbolize different compounds are known as “Shapely
color set”, a quasi-standard in molecular modeling. Figure from [Buxbaum, 2007].
632
The genetic code 34.5
633
34.5. Periodic system of the elements
634
35. Acronyms
AAV adeno-associated virus, used in gene therapy

ABC ATP-binding cassette, largest class of primary active trans-membrane transporters
ACAT Acyl-CoA-cholesterol-acyl transferase
ACE angiotensin converting enzyme, involved in blood pressure regulation
ACP Acid phosphatase, enzyme elevated in late stages of prostrate cancer
ACTH adrenocorticotrophic hormone
ADA adenosine desaminase
ADH alcohol dehydrogenase, enzyme found mostly in liver and involved in ethanol detox-
ification
ADHS attention deficit hyperactivity syndrome, disease in children of controversial exis-
tence, causation and treatment
ADP adenosine diphosphate
AGE advanced glycation end-products, formed from proteins and aldoses (esp. glucose)
AGT aspartate:glutamate aminotransferase, liver enzyme
AI adequate intake, dose recommendation for nutrients in those cases where scientific evi-
dence is insufficient to set RDA-values
AICD APP intracellular domain, cytosolic fragment of APP produced by secretases
AID artificial insemination by donor
AIDS acquired immuno-deficiency syndrome, disease cause by infection with a retro-virus,
HIV
Akt AKR mouse directly transforming retrovirus associated oncogene, also known as pro-
tein kinase B
ALA δ-aminolevulinate, intermediate of heme synthesis
ALE advanced lipoxydation endproducts, formed from proteins and lipoperoxides
635
35 Biochemistry and Genetics
ALP Alkaline phosphatase, enzyme that appears in serum after bone and liver damage
ALT Alanine transaminase, enzyme that appears in serum after liver damage
AMP adenosine monophosphate
AMPA 3-amino-3-hydroxy-5-methyl-4-isoxacol propionate, agonist of certain glutamate-
receptors
ANF Atrial natriuretic factor
APC adenomatous polyposis coli gene, gene involved in colon cancer formation
apoA apolipoprotein A, major apolipoproteins of HDL
apoB apolipoprotein B
apoC apolipoprotein C
apoE apolipoprotein E
APP β-amyloid precursor protein, involved in plaque formation in Alzheimers disease
APTT Activated partial thromboplastin time
ARB angiotensin receptor blocker, class of pharmaceuticals used to lower blood pressure
and manage diabetic nephropathy
ASO application specific oligo, DNA segment used as probe on gene chips
AST aspartate transaminase, enzyme that appears in serum after liver damage
ATM ataxia telangiectasia mutated kinase
ATP adenosine triphosphate
BARK β-adrenergic receptor kinase
BioH2 dihydrobiopterine
BioH4 tetrahydrobiopterine
BMI body mass index, calculated by dividing body weight (kg) by the square of the height
(m), should be 18–25
BMR basal metabolic rate, energy expenditure in the absence of activity
BPG 2,3-Bisphosphoglycerate, regulator of oxygen affinity of hemoglobin
BRCA breast cancer, genes that increase the likelihood of breast cancer development
CAM cell adhesion molecule
CATH class architecture topology homology at http://www.cathdb.info/latest/index.html
636
Acronyms 35
cAMP cyclic AMP, second messenger

CBB Coomassie brilliant blue, group of dyes used to stain proteins in gels
Cdk cyclin dependent protein kinase
cDNA complementary DNA, DNA generated by action of a reverse transcriptase upon a
mRNA
CDP cytidine diphosphate
CF cystic fibrosis, inherited disease
cGMP cyclic GMP, second messenger
CETP cholesterol ester transfer protein
CFTR cystic fibrosis transmembrane regulator, protein defect in cystic fibrosis
CHD coronary heart disease
CHO carbohydrates
CJD Creutzfeldt-Jakob-disease, a prion disease
CK creatine kinase, enzyme which appears in serum after damage to muscle, heart or brain.
Source can be determined by iso-enzyme analysis
cMOAT canalicular multiple organic anion transporter, defect in Dubin-Johnson-syndrome
CNS central nervous system, brain and spinal cord
CoA coenzyme A, carrier of acyl residues in metabolism
CRABP cellular retinoic acid binding protein, receptor for retinoic acid
CRBP cellular retinol binding protein, receptor for retinol
Cre , recombinase used in the production of knock-out and knock-in mice
CREB cAMP response element binding, family of transcription factors regulated by cAMP
CRP C-reactive protein, acute-phase serum protein
CSF cerebrospinal fluid, fluid surrounding the CNS and separated from serum by the
blood/brain barrier
CTAB cetyl trimetylammonium bromide, positively charged (cationic) detergent
CTL cytotoxic lymphocyte
CTP cytidine triphosphate
dATP deoxy-ATP, used for making DNA
637
DCCD dicyclohexylcarbodiimide, substance used to activate carboxy-groups in chemical

synthesis
dCTP deoxy-CTP
dGTP deoxy-GTP
DHA docosahexaenoic acid, polyunsaturated fatty acid, possibly a vitamin
DHF dihydrofolate
DHFR dihydrofolate reductase
DMD Duchenne muscular dystrophy
DNA desoxyribonucleic acid
l -DOPA 3,4-dihydroxy phenylalanine
DRI Dietary reference intake, EAR, RDA and UL of a nutrient
DTPA , chelator used against iron poisoning
dTTP deoxy-Thymidine triphosphate
EAR estimated average requirement, dose of a nutrient required by an average member of
the group studied
EDTA ethylene diamine tetraacetic acid, chelator
EGF Epidermal Growth Factor
ELISA enzyme linked immunosorbent assay, method to determine the concentration of an
antigen or antibody
env envelope, transmembrane proteins in the envelope of virus, required for binding to the
target cell
EPA eicosapentaenoic acid, polyunsaturated fatty acid, possibly a vitamin
EPO erythropoietin, hormone produced by the kidney that stimulates erythrocyte forma-
tion in bone marrow
ER endoplasmic reticulum, intracellular membrane system
ERAD ER associated protein destruction, mechanism that destroys incorrectly folded pro-
teins during their maturation in the ER
erb ?
Erk extracellular signal regulated kinase, protein in the MAP-kinase pathway
ES embryonic stem cells, pluripotent stem cells extracted from embryos
638
Acronyms 35
EST expressed sequence tags, short pieces of cDNA that identify genes expressed in a cell
or tissue
FAD flavin adenine dinucleotide, oxidized
FADH2 flavin adenine dinucleotide, reduced, cofactor of flavo-proteins
FFI fatal familial insomnia, a prion disease
FGF Fibroblast Growth Factor
FIGLU N-Formiminoglutamate, intermediate in His-breakdown
FISH fluorescent in-situ hybridization, method to localize genetic information on micro-
scopic slides
Flip , recombinase recognizing the FRT DNA sequence. Used in a similar fashion as Cre/LoxP
FMN flavin mononucleotide, cofactor in some oxidoreductases
Fmoc N-α-9-fluorenylmethoxycarbonyl, protective group during Merrifield protein syn-
thesis
fos Finkel-Biskis-Jinkins osteosarcoma oncogene
FRT , DNA element recognized by the Flip recombinase
FSH follicle stimulating hormone
FSSP families of structurally similar proteins at http://www.chem.admu.edu.ph/ nina/rosby/fssp.htm
G6PDH glucose-6-phosphate dehydrogenase, enzyme of the pentose phosphate pathway,
defective in favism
GABA γ-amino butyric acid, a neurotransmitter
gag group-specific antigen, protein in retro-virus capsid
Gal galactose, a hexose
GDP guanosine diphosphate
GFR glomerular filtration rate, volume of blood filtered by the glomerulus per unit time
GFP green fluorescent protein, marker in molecular biology studies
GI gastrointestinal
Glc glucose, a hexose, essential component of food
GlcNAc N-acetylglucosamine, sugar derivative found in glycoproteins and -lipids
GM-CSF granulocyte/macrophage colony stimulating factor, cytokine
639
GMP guanosine monophosphate

GOT glutamate:oxoglutarate transaminase, liver enzyme
GPI glycosyl phosphatidylinositol
GPT glutamate:pyruvate transaminase, liver enzyme
GSS Gerstmann-Stäussler-Scheinker-disease, a prion disease
GTP guanosine triphosphate
GuHCl guanidinium hydrochloride, substance that destroys protein secondary, tertiary and
quaternary structure
HbA adult hemoglobin,(α2 β2 )
HbF fetal hemoglobin,(α2 γ2 )
HbS sickle cell hemoglobin, Glu-6→Val in β
HCG human chorionic gonadotropin
HDL high density lipoprotein, “good cholesterol”
HDN late-onset haemorrhagic disease of the newborn, treated with vitamin K
HFE hemochromatosis
HGPRT hypoxanthine:guanine phosphoribosyl transferase
HIC hydrophobic interaction chromatography, method to separate proteins
HIF hypoxia inducible transcription factor, transcription factors which regulate gene ex-
pression depending on available oxygen
HIV Human immuno-deficiency virus, causative agent for AIDS
HLA Hauptlymphozyten-Antigen, synonym for MHC
HMG hydroxymethyl glutaryl-, also: human menopausal gonadotropin
HMM Henri-Michaelis-Menten
HPLC high performance liquid chromatography, chromatography on fine-grained, homoge-
nous stationary phases to increase separation power
HRE hypoxia response element, segments in the promotor of a gene, which bind HIF
Hsc70 70 kDa heat shock cognate, constitutively expressed isoform of Hsp70
Hsp heat shock protein, group of chaperons or chaperonins amplified under stress conditions
ICSI Intracytoplasmic sperm injection
640
Acronyms 35
IDL intermediate density lipoprotein

IDoM inherited disease of metabolism
IEC ion exchange chromatography, method to purify proteins based on their charge
IEF isoelectric focussing, method to separate proteins based on their different pI
IFN interferon
Ig immunoglobulin
IgA immuno-globulin A
IgD immuno-globulin D
IgE immuno-globulin E
IGF Insulin-like Growth Factor
IgG immuno-globulin G
IgM immuno-globulin M
IMP inosine monophosphate
INH isonicotinic acid hydrazide, isoniacid, tuberculostaticum
INK4 inhibitor of kinase 4
IP3 inositol-1,4,5-trisphosphate, second messenger
IQ intelligence quotient, result of an intelligence test
IUBMB International Union for Biochemistry and Molecular Biology
IUPAC International Union of Pure and Applied Chemistry
IVF In-vitro fertilization
jun Junin virus oncoprotein
lac lactose
LWB low weight at birth
LCAT lecithin-cholesterol acyl transferase
LDH Lactate dehydrogenase, enzyme which appears in serum after tissue damage. Source
can be determined by iso-enzyme analysis
LDL low density lipoprotein, “bad cholesterol”
LoxP , DNA element recognized by the Cre recombinase
641
LPL lipoprotein lipase, enzyme that cleaves triglycerides

LH luteinizing hormone
LT leukotriene
MAO monoamine oxidase
MAP mitogen activated protein
[MCAD medium chain acyl-CoA dehydrogenase, enzyme in fatty acid catabolism
MCD multiple carboxylase deficiency, inherited disease
Mdm Murine double minute, oncogene
MEK MAP kinase/Erk kinase, protein in the MAP pathway
MHC major histocompatibility complex
mRNA messenger ribonucleic acid, RNA that gets translated into proteins
MS mass spectrometry, sensitive analytical technique that identifies molecules by molecular
weight
MSUD maple syrup urine disease, inborn error of amino acid metabolism
MTX Methotrexate, dihydrofolate reductase antagonist used against cancer and auto-
immune diseases
MW molecular weight (more correctly: -mass)
myc myelocytomatosis proto-oncogene
NAD+ nicotinamide adenine dinucleotide, oxidized
NADH + H+ nicotinamide adenine dinucleotide, reduced, soluble carrier of activated hy-
drogen in catabolic reactions
NADP+ nicotinamide adenine dinucleotide phosphate, oxidized
NADPH + H+ nicotinamide adenine dinucleotide phosphate, reduced, soluble carrier of
activated hydrogen in anabolic reactions
NAD(P)H either NADH or NADPH
N.C.E.P. National cholesterol education program
neor neomycin resistance, genetic marker used in DNA technology
neu ?
NF-κB nuclear factor κB
642
Acronyms 35
NGF Nerve Growth Factor

NMDA N-methyl-D-aspartate, agonist of inotropic glutamate receptors in the nervous sys-
tem
NMR nuclear magnetic resonance, method to determine the 3D-structure of molecules
NO· nitric oxide, paracrine hormone
NPU net protein utilization, measures biological value of proteins
NPY neuropeptide Y
OMIM online Mendelian inheritance in man, catalogue of inherited diseases at http://www.ncbi.nlm.nih.gov/sites
OPA ortho-phtaldialdehyde, chemical that gives fluorescent compounds with primary amines
PAGE polyacrylamide gel electrophorese, separation method for proteins and nucleic acids
PAH phenylalanine hydroxylase, protein defect in PKU
PCNA proliferating cell nuclear antigen
PCR polymerase chain reaction, method to amplify a particular stretch of DNA
PDB protein data base, database of protein structure at http://www.rcsb.org/pdb/home/home.do
PDI protein disulphide isomerase, enzymes responsible for the formation of correct disul-
phide bonds in the ER
PDGF Platelet-derived Growth Factor
PEP phosphoenol pyruvate
PEPCK phosphoenolpyruvate carboxykinase
PER protein efficiency ratio, used to express the biological value of proteins
PET positron emission tomography, non-invasive method to measure metabolic activity of
an organ, especially the brain
PI3 phosphatidylinositol trisphosphate
PKA protein kinase A, mediator of cAMP responses
PKC protein kinase C, mediator of Ca responses
PKU phenylketonuria
PLC phospholipase C
pol RNA-dependent DNA-polymerase, reverse transcriptase of retro-virus
pRb retinoblastoma protein
643
PrP prion protein, prion stands for proteinaceous infectious agent

PRPP phosphoribosyl pyrophosphate
PTH parathyroid hormone
PUFA poly-unsaturated fatty acids, fatty acids with several double bonds
QTL Quantitative trait locus
RA rheumatoid arthritis
RAGE receptor for advanced glycation end-products of proteins
Raf receptor associated factor, protein in the MAP-kinase pathway
Ras rat sarcoma viral antigen, protein in the MAP-kinase pathway
RBC red blood cell, erythrocyte
RDA recommended dietary allowance
RFLP restriction fragment length polymorphism
RIA radioimmuno assay, method to determine the concentration of an antigen or antibody,
largely superseded by ELISA
RNA ribonucleic acid
RNAi RNA interference, technique used to prevent expression of a specific gene product
RISC RNA-induced silencing complex, involved in RNAi
ROS reactive oxygen species, compounds derived from partially reduced oxygen molecules
rRNA ribosomal ribonucleic acid, structural component of ribosomes with ribozyme activ-
ity
RPC reversed phase chromatography, separation method for small organic molecules
RQ respiratory quotient, ratio CO2 produced by O2 consumed
SAH S-adenosyl homocysteine, demethylation product of SAM
SAM S-adenosyl methionine, carrier of C1-bodies in metabolism
SCOP structural classification of proteins at http://scop.mrc-lmb.cam.ac.uk/scop/
SCID severe combined immunodeficiency disease, inherited immuno-deficiency
SDH succinate dehydrogenase, enzyme of Krebs-cycle
SDS sodium dodecylsulphate, detergent used to destroy tertiary and quaternary structure
in proteins
644
Acronyms 35
SEC size exclusion chromatography, synonym for gel filtration

SLE systemic lupus erythematosus, autoimmune disease
sis simian sarcoma, oncogene
SNP single-nucleotide polymorphism
snurp small nuclear ribonucleoprotein
src Rous sarcoma virus oncogene
STAT signal transducer and activator of transcription
SUMO small ubiquitin-like modifiers, peptides transferred to Lys -amino groups in pro-
teins for regulatory purposes
TAP transporter associated with antigen presentation, transport antigenic peptides into
the ER, encoded by the ABCB2 and ABCB3 gene
Taq Thermus aquaticus, thermophile archaebacterium found in deep-sea hydrothermal
vents (“black smokers”)
TCA tricarboxylic acid, summary name for the intermediates of the Krebs-cycle
TCR T-cell receptor, protein that binds to the antigen-loaded MHC on antigen-presenting
cells
TGF Transforming Growth Factor
THF 5,6,7,8-tetrahydrofolate, active form of the vitamin folic acid
TIBC total iron binding capacity
LMP latent infection membrane protein
TNF tumor necrosis factor
TOBEC total body electric conductivity, method to determine body fat content
TPP thiamin pyrophosphate, active form of vitamin B1
tRNA transfer RNA
TSH thyroid-stimulating hormone
UDP uridine diphosphate
UL tolerated upper intake level, amount of a substance that does not cause negative effects
in the majority of the population even when taken regularly over a long time
US United States (of America)
645
US-RDA US recommended daily allowance, short version of the RDA, not differentiated
by gender nor age, and not regularly updated.
UTP uridine triphosphate
UV ultraviolet, light with wavelength shorter than 400 nm
vCJD variant Creutzfeldt-Jakob-disease, “mad cow disease”
VLDL very low density lipoprotein, transports cholesterol from the liver to the tissues
VNTR variable number of tandem repeats
vWF von Willebrand factor, protein involved in platelet activation
waf ?
WHO World Health Organization
YAC yeast artificial chromosome, used to clone large segments of DNA
646
Bibliography
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E. Buxbaum. Biophysical chemistry of proteins: An introduction to laboratory methods.

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Drake, and A.J. Lusis. Toward a biological network for atherosclerosis. J. Lipid Res., 45:
1793–1805, 2004. URL http://www.jlr.org/cgi/reprint/R400006-JLR200v1.pdf.
R.S. Gibson. Principles of Nutritional Assessment. Oxford University Press, Oxford, 2nd
edition, 2005. ISBN 978-0-195-17169-3.
E.C.M. Mariman. Epigenetic manifestations in diet-related diorders. J. Epigenetics Epige-

nomics, 1:232–239, 2008.
C.C. Metges and C.A. Barth. Metabolic consequences of a high dietary-protein intake in
adulthood: Assessment of the available evidence. J. Nutrition, 130:886–889, 2000.
D.L. Nelson, M.M. Cox, and A.L. Lehninger. Principles of Biochemistry. W.H.Freeman,
New York, 5th edition, 2008. ISBN 978-1-429-20892-5.
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648
Index
α2 -macroglobulin, 336, 343 acute fatty liver of pregnancy, 533

γ-carboxy-glutamic acid, 298 acute phase reactant, 206
Na+ /K+ -ATPase, 341 acyl transferase, 369
acyl-CoA dehydrogenase, 533
A/B toxin, 63 deficiency, 367
ab initio, 52 Acyl-CoA-cholesterol-acyl transferase, 373
ABC-R transporter, 291 adaptation, 304
ABC-transporter, 534 adenoassociated virus, 552
abetalipoproteinemia, 261, 375 adenomatous polyposis coli, 191, 495
ABO-system, 219–220 adenosine desaminase, 509
abortion, 564, 612 adenosine triphosphate, see ATP
acetal, 7 adenosyl
acetaminophen, 462 S-adenosyl homocysteine, 422
acetazolamide, 156 S-adenosyl methionine, 422
acetoacetate, 366 adenovirus, 111, 552
acetone, 366 adenylate cyclase, 274
in protein precipitation, 68 adenylate kinase, 167
acetyl-CoA, 61 adenylation, 63
acetyl-CoA carboxylase, 367 adhesion
acetylation, 61, 117 focal, 488
acetylcholine, 275–277 adiabatic, 16
achondroplasia, 236, 258 adipocyte, 289, 296
acid, 9–10 adiponectin, 595
maltase deficiency, 354 adipose tissue, 356, 363–364, 402, 576
phosphatase, 209 hyperplasia, 381
acid maltase, 519 hypertrophy, 381
acidosis, 174, 435, 582 ADP
lactic, 201 ribosylation, 275
acne, 287, 291 adrenal, 289, 334, 342, 356, 393
aconitase, 245 adrenocortical, 494
acridine, 114 adrenoleucodystrophy, 534
acrodermatitis enteropathica, 338 adrenomyeloneuropathy, 534
actinomycin A, 99 aflatoxin, 347
Index
AGE, 57, 601 amenorrhea, 123, 130

aggregation post-partum, 477
protein, 55 Ames test, 114
aging, 57, 254 amide, 7
AIDS, 104, 190, 193, 336, 565 amine oxidase, 333
Akt, 490 amino acid, 27–34
alanine essential, 406
cycle, 435, 579 metabolism, 405–438
transaminase, 209 oxidase, 410
albinism, 263, 336 amino sugar, 358
occulocutaneous, 425 aminoacyl-tRNA synthetase, 98
albumin, 204–205, 208, 289, 301, 308, 334, δ-aminolevulinate synthase, 457
336, 364, 461 aminophylline, 274, 276
alcohol, 394, 398, 452–454, 471, 473, 476, ammonia, 10, 399
531, 611, 612 amphetamine, 612
dehydrogenase, 453 amphipatic, 41, 371
alcoholism, 376 amphophilic, 72
aldehyde, 12 AMPylation, 63
dehydrogenase, 453 amylase, 210, 316, 439, 442
aldehyde oxidase, 342 amyloid, 74, 125, 617
aldolase, 354 serum, 595
deficiency, 354 Amytal, 252
aldolase B, 518 anabolic, 447
aldosterone, 277, 323, 393, 402 analbuminemia, 205
ALE, 601 anaplerotic reaction, 245
aleuron, 300 Anderson disease, 519
alkali disease, 347 androgen, 130, 513
alkaline phosphatase, 209 anemia, 201, 207, 296, 299, 302, 308, 311,
alkalosis, 174 318, 334, 343
alkaptonuria, 427 Heinz-body, 517
allele, 233 hemolytic, 469
allergy, 294, 313 iron deficiency, 479, 481
allopurinol, 471 megaloblastic, 464
allostery, 200, 222, 448, 569 sickle cell, 226, 558
Alport-syndrome, 46 anencephaly, 337, 474, 614
Alu-sequence, 110, 111 aneuploidy, 123
Alzheimer’s disease, 616 Anfinsen-hypothesis, 52
Alzheimer’s disease, 80–82, 125, 558, 611 Angelman syndrome, 268
Amadori-rearrangement, 59 angina, 208
Amanita phalloides, 116 angiotensin, 275, 276, 393
α-amanitin, 116, 462 angiotensinogen, 595
amaurotic idiocy, 369 anhydride, 7
650
Index
phospho-, 7 arsenite, 243

ankylosing spondylitis, 508 arteriosclerosis, 338
annealing, 93 artery
anomer, 12 intima, 374, 387
anoxia, 253 arthritis, 334, 347
antabuse, 453 rheumatoid, 508
antacid, 321, 481 artificial insemination, 564
antagonist, 286 ascites, 398
anti-sense, 550 ascorbate, 46, 255, 294, 313–314
anti-vitamin, 286 Ashkenazi Jews, 511
antibiotic, 99, 298, 300, 309, 311 asparaginase, 418
antibody, see immunoglobulin asparagine
catalytic, 163 synthetase, 418
anticipation, 235 aspartate
anticodon, 98 pKa, 28
anticonvulsant, 293, 311, 316, 317, 321, transaminase, 209
336, 481 aspirin, 481
antimycin A, 252 asthma, 276, 476, 617
antioxidant, 294, 355 ataxia, 261, 268
antiport, 172 Friedreich’s, 261
antitrypsin, 206 ataxia-telangiectasia, 116, 261
antivitamin, 303 atherosclerosis, 374, 387, 617
aorta, 257 Atkins-diet, 415, 583
Apert syndrome, 258 ATP, 165–167
Apicomplexa, 51 citrate lyase, 368
apoA synthase, 252
I, 374, 375 atresia, 264
apoB atrial natriuretic factor, 277
48, 372 attention deficit disorder, 322, 611
100, 372, 373, 376 autism, 611
apoE, 373, 374, 376, 617 autocrine, 271
in Alzheimer’s disease, 82 autosomal, 233
apoptosis, 485, 487 autosomal macular dystrophy, 291
aquaporin, 237 autosome, 109
arachidonic acid, 307 autosplenectomy, 226
archaea avidin, 316
genome, 109 azaserine, 468
arginase azide, 252
inhibition by Lys, 429
arginine, 429 bacteria
pKa, 28 genome, 109
arsenate, 201 bacteriophage
651
Index
lambda, 103 blood group, 58

T4, 102 blood pressure, 322, 324, 337
baldness, 266 Bloom syndrome, 116
barbiturate, 542 blot
BARK, 277 dot, 192–194
baroreceptor, 393 eastern, 183
Barr-body, 512 northern, 183
Barr body, 123 Southern, 182, 191, 237
basal metabolism, 382, 405, 448 western, 183
base, 9–10, 91 Bohr-effect, 224
basement membrane, 401 bond
Bayes theorem, 240 acetal-, 12
bean covalent, 3
broad or fava, 516 disulfide, 47
Beckwith-Wiedemann syndrome, 269 glycosidic-, 12
benzene, 114 hydrogen, 4, 47
benzoate, 413 hydrophobic, 47
Beri-Beri, 285, 300 ketal-, 12
bicarbonate, 174 peptide-, 34–89
biguanides, 481 polar, 5
bile, 287, 289, 293, 298, 302, 311, 334, 337, salt, 4, 47
343, 363, 370, 397, 459 van der Waals, 47
acid, 370 van der Waals, 5
bilirubin, 255, 397, 459 bone, 209, 257, 287, 291, 294, 298, 319,
direct, 461 332, 333, 336
indirect, 461 Bordetella pertussis, 275
biliverdin, 459 Bordetella pertussis, 63
biotin, 245, 304, 316–317 bowel, 210, 264
biotransformation, 535 Bowman’s capsule, 400
birth defect, 611 bradykinin, 277
2,3-bisphosphoglycerate, 223, 581 brain, 210, 299–302, 308, 309, 311, 314,
biuret, 66 316, 318, 323, 324, 334, 337, 341,
blindness 343, 347, 375, 582
color, 265 branching enzyme, 351, 519
congenital, 265 BRCA, 494
blood, 289, 293, 301, 308, 309, 311, 317, breast feeding, 319
318, 323, 333, 334, 336, 343 Briggs, G.E., 140, 142
brain barrier, 582 Bruton agammaglobulinemia, 509
coagulation, 213–217 Buchner, E., 138
group, 219–221 Buchner, H., 138
pressure, 393 buffer, 140, 173
blood clotting, 296, 320, 333 burns, 208
652
Index
Ca-ATPase, 173 caries, 479

cadaverine, 432 carnitine, 364
caffeine, 274, 476 acyl transferase deficiency, 367
calciferol, 291–294 deficiency, 367
calcitonin, 275, 293, 321 carnosine, 406
calcitriol, 291, 399 caroten, 392
calcium, 275, 291, 298, 309, 313, 319–322, carotene, 289
353, 475, 476, 480, 488 carotenodermia, 289
channel blocker, 276 carpal tunnel syndrome, 309
calmodulin, 276, 277 carrier, 234, 238, 512
cAMP, 101, 274–275, 351, 353, 364, 368, casein, 476
488 catabolic, 447
cancer, 112, 206, 208, 209, 254, 267, 269, catalase, 162, 255
287, 296, 313, 317, 318, 344, 347, catalysis, 22
469, 490–495 acid/base-, 162
bladder, 494 catalytic perfection, 144
bone, 209 catalytic triad, 165
brain, 494 cataract, 265, 355, 517, 612
breast, 493, 494 catecholamine, 425
colon, 493, 494 CD4, 500
esophagus, 494 CD8, 500
ovarian, 494 cDNA, 189
pancreas, 494 chip, 551
prostate, 494 CDP, 368
throat, 494 Cech, T., 137
virus-induced, 493 cell
capping, 116 adhesion molecule, 488, 500
capsid, 102 antigen-presenting, 500
carbohydrate, 11–12, 323, 338 culture, 486
energy content, 447 cycle, 485–491
metabolism, 349–358, 449–450 motility, 488
carbon natural killer, 503
monoxide, 222, 252, 459 phagocytic, 500
tetrachloride, 398, 462 shape, 488
carbonic anhydrase, 156, 225 cellulose, 12, 445
carboxylase, 316 centromere, 110
carboxylic acid, 5 ceramide, 369
carboxypeptidase, 442 cerebrohepatorenal syndrome, 534
carcinogen, 112, 490 cerebroside, 582
carcinoma, 490 cerebrospinal fluid, 582
colorectal, 495 ceruloplasmin, 204, 207, 329, 333, 334
cardiomegalia glycogenica, 519 cerumen, 266
653
Index
cGMP, 277 inversion, 133

channel iso-, 132
voltage-gated, 276 metacentric, 121
chaperonin, 55 murder, 127
chaperons, 55 painting, 122
Charcot-Marie-Tooth syndrome, 260 rearrangement, 131–135
checkpoint, 485 ring, 132
G1, 486, 487, 492 submetacentric, 121
cheese, 137 telocentric, 121
chenodeoxycholic acid, 370 translocation, 133
chimera, 548 X, 121, 123, 512
chip Y, 121
cDNA, 551 chronic granulomatous disease, 581
DNA, 550 chylomicron, 363, 373, 375, 396, 576
chloramphenicol, 100 chylomicrons, 289, 293, 296, 451
chloride, 324 chymotrypsin, 60, 442
chloride shift, 324 circular dichroism, 74
cholecystitis, 397 cirrhosis, 505
cholecystokinin, 397 citrate synthase, 244
cholera, 275 citric acid cycle, see Krebs-cycle
cholestasis, 397, 462 cleft palate, 291, 302, 613
cholesterol, 306, 307, 313, 325, 333, 334, clotting, 203, 207
370–376, 390–393, 397, 476 co-dominant, 233
ester transfer protein, 374 co-lipase, 442
cholic acid, 370 co-substrate, 165
cholinesterase, 208 co-transport, 172
choroid plexus, 582 cobalamine, 309–311, 317, 320
Christmas disease, 216 cocaine, 208, 473, 612
chromatin, 110 Cockayne syndrome, 115
chromatography code
affinity, 70 genetic, 98
ion exchange, 70 Codex Hamurabi, 137
reversed phase, 70 codon
size exclusion, 70 stop-, 29
chromium, 268 coenzyme, 165–169
chromosome, 109 A, 168
abberation, 121–135 Q, 250
acrocentric, 121 coenzyme A, 306
banding, 122 colchicine, 121, 469
breakage, 131 Coley’s anemia, 227
breakage syndrome, 116 colic, 397
deletion, 131 collagen, 45, 257, 313, 333, 334
654
Index
colon, 445 Cutis laxa, 334

colostrum, 476 cyanide, 252
coma cyanosis, 225
hepatic, 399 cyclin, 60, 485–487
common variable immunodeficiency, 508 A, 492
complement, 207, 497, 505, 595 D, 490, 492
complex dependent protein kinase, 487, 492
initiation, 99 cycloheximide, 100
condensation, 6 cysteine, 47
conjugation, 105 pKa, 28
constant cystic fibrosis, 193, 264, 296, 558, 560, 565
equilibrium, 19 cystinuria, 435
contact inhibition, 486 cytidine triphosphate, see CTP
contraceptives, 308, 317 cytochrome, 250
cooperativity, 146 c oxidase, 251
copper, 168, 207, 250, 255, 333–336, 338, oxidase, 254
343, 473 P-450, 457
Cori-Forbe-disease, 519 cytochrome c oxidase, 333, 336
cornea, 356 cytochrome P450, 537
coronary heart disease, 387–393 cytokine, 490, 595
corpus luteum, 474 cytomegalovirus, 612
corticosteroid, 130, 205
Counseling deacetylase, 589
genetic, 560 deaf-mutism, 265
CRABP, 289 deafness, 612
craniosynostosis, 235 congenital, 265
CRBP, 289 deaminase, 307
Cre/LoxP, 549 debranching enzyme, 352, 519
creatine, 432 decarboxylase, 307
kinase, 210, 259, 432 dehydration, 207
creatinine, 399, 432 dehydrogenase, 299, 303
CREB, 274 deletion, 113
Cri-du-cat syndrome, 131 deoxycholic acid, 370
Crigler-Najjar syndrome, 462 deoxyribonucleotides, 467
Crohns disease, 336 dermatomyositis, 259
crossing-over, 105, 111 desoxynojirimycin, 57
croton oil, 276 detergent, 55, 72
Cruzons craniosynostosis, 258 deuteranopia, 265
cryoprecipitate, 208 Dewar, Sir James, 16
crystallins, 55 dextran, 479
CTAB, 70 diabetes, 57, 337, 401, 518, 595–604, 611,
CTP, 167 612, 616
655
Index
bronze, 267, 332 chip, 550

gestational, 384, 387 cloning, 187–189
insipidus, 237 complementary, 189
mellitus, 374, 376, 383–388, 401, 508 damage, 487
MODY, 597 depurination, 115
type 2, 277, 382 diagnostics, 190
diacylglycerol, 275, 368 fingerprinting, 186
dialysis, 68 junk, 110
diarrhea, 291, 296, 307, 323–325, 334, 336, ligase, 115, 181
338, 343, 347, 476, 517 methylation, 115
diastereomer, 8, 11 mobile, 111
Dicer, 550 polymerase, 115, 183, 485, 486
dicoumarol, 216 probe, 181
diet repair, 114–116
protein-saving, 398 nucleotide excision, 114
diffusion, 170 repetitive, 110
facilitated, 171 replication, 485
difluoromethylornithine, 435 satelite, 110
DiGeorge syndrome, 509 sequencing, 185
DiGeorge syndrome, 614 structure, 91–94
digestion, 439–447 DNase, 102
digoxin, 394 Domagk, G.J.P., 421
dihydrobiopterine domain, 47
reductase, 425 dominant, 233
dihydrofolate reductase, 421 dominant negative, 512
dihydrotestosterone, 130, 513 Down syndrome, 124
dimethylallyl-pyrophosphate, 370 Down syndrome, 124, 135, 560, 617
2,4-dinitrophenol, 253 Down’s syndrome, 336
diphtheria toxin, 117 Dubin-Johnson syndrome, 463
dipole, 4 Dumas, 285
disaccharide, 12 dwarfism, 235, 257, 258
disease dysbetalipoproteinemia, 376
genetic, 112 dysosteogenesis, 258
sex-influenced, 513 dysostosis, 258
sex-limited, 513 dysplasia
X-linked, 512 skeletal, 258
Disse dystrophy
space of, 373 muscular, 169
disulfiram, 453
diuretic, 156, 323–325 EC code, 138
DNA, 91–105 Escherichia coli, 275
amplification, 183 eczema, 476
656
Index
edema, 203, 205, 208, 300, 398, 400, 401 envelope, 102, 103
Edman-sequencing, 74 enzyme, 137–179
Edward syndrome, 124 class, 138
Edward syndrome, 126 commission, 139
Eflornithine® , 435 diagnostic, 208
egg, 289, 298, 301, 308, 316, 336 inactivation, 157–159
Ehlers-Danlos-syndrome, 45, 257 inhibition, 150–157
elastase, 442 competitive, 150
elastin, 307, 333, 334 mixed, 157
electrolyte, 323, 399 noncompetitive, 154
electronegativity, 4 uncompetitive, 154
electrophoresis, 70, 204, 206, 208, 209, 226 kinetics, 140–150
2D, 553 naming of, 138
immuno-, 205 epidermolysis bullosa, 46, 264
electroporation, 547 epigenetics, 589
element epilepsy, 156, 611
chemical, 3 epimerase, 355
ELISA, 501 epinephrine, 275, 277, 353, 364, 402, 570
elliptocytosis, 263 equilibrium, 15
elongation factor, 117 ER, 275, 370
emphysema, 206 erb B, 492
enantimer, 8 erythroblastosis fetalis, 220
end product inhibition, 146 erythrocyte, 57, 201, 209, 294, 296, 308,
endocrine, 271 318, 323, 324, 342, 343, 347, 356,
endocytosis, 206, 277 375, 517, 581
of virus, 103 erythromycin, 100
endonuclease erythropoietin, 189, 399, 489, 490
restriction, 181 ester, 11
endopeptidase, 442 carboxylic, 6
endoplasmic reticulum, 535 phosphate, 6, 10
energy, 15 thio-, 7
activation, 19, 22 ethanol, 300, 301, 304, 308, 311, 317, 325,
free, 18, 249 336
of reaction, 15 forensic determination, 143
requirement, 448 in protein precipitation, 68
Engelhart, J.F., 137 ether, 7
enhancer, 117, 131 ethidium bromide, 114
enrichment, 285 ethylene oxide, 114
entero-hepatic circulation, 370 euchromatin, 110
enteropathy, 207 eukaryota
enteropeptidase, 447 genome, 109
enthalpy, 17 evolution
657
Index
convergent, 518 fetal alcohol syndrome, 473

exercise, 321 fetoprotein, 207
exocytosis, 276 fetuin A, 322
exoglycosidase, 369 fever, 402
exon, 109 fibrillin, 258
exonuclease, 94 fibrin, 213, 214
exopeptidase, 442 fibrinogen, 203, 204
expressivity, 234 fibrocystic breast disease, 294
extein, 60 fibronectin, 501
eye, 287, 289, 302, 334, 337 ficin, 137
Fischer, E., 140
Fab -fragment, 497 FISH, 122, 131, 186
factor fish, 293, 300, 304, 306, 309, 320, 333, 336,
elongation, 99 338, 345
initiation, 99 flatulence, 445
factor VIII, 189 flavin adenine dinucleotide, see FAD
FAD, 47, 167, 301 flavin mononucleotide, see FMN
Fanconi anemia, 116 flavonoid, 393
Fanconi-Bickel-disease, 520 fluoride, 201
fat, 11, 364 fluorine, 332–333, 342
energy content, 447 fluoroacetate, 245
metabolism, 450 fluorouracil, 468
fatty acid, 11, 311, 316 Flynn effect, 479
biosynthesis, 367–368 FMN, 167, 301
branched-chain, 366 foam cell, 374
essential, 367 folate, 313, 317–318, 336, 421, 422, 468,
mono-unsaturated, 11 473, 474, 481, 614
odd-chain, 366 folliculosis, 291
poly-unsaturated, 11 fortification, 285
saturated, 11, 361 fos, 492
trans-unsaturated, 389 founder effect, 556
unsaturated, 361, 476 fragile X syndrome, 261
favism, 357 frequency
Fc fragment, 497 allele, 555
feces, 293, 306 gene, 555
feedback inhibition, 455 fructoaldolase, 518
feedforward stimulation, 455 fructokinase, 354, 518
Fenton-reaction, 314 deficiency, 354
ferment, 137 fructose, 449, 518
ferritin, 255 intolerance, 354
ferrochelatase, 459 metabolism, 354
ferroxidase, 333 fructose-1,6-bisphosphatase, 349
658
Index
deficiency, 355 thrifty, 589

fructose-1,6-bisphosphate, 351 geophagy, 337
fructose-2,6-bisphosphate, 351 Giardia, 337
fructosuria, 354, 518 Gibbs-Helmholtz-equation, 18
fruit, 298, 308, 314, 317, 323 glaucoma, 265
Funk, 285 in diabetes, 595
globulin, 204
G-protein, 272, 273, 490 glomerulonephritis, 400, 401
galactitol, 355, 517 glomerulus, 400, 401
galactocerebroside, 582 glucagon, 200, 275, 350, 353, 570
galactonate, 517 glucocerebrosidase, 369
galactosaemia, 355 glucocorticoid, 364, 402, 570
galactose, 11, 100, 449 receptor, 93
metabolism, 355 glucokinase, 349, 355
galactosemia, 517 gluconeogenesis, 245, 349–351, 356, 402,
galactosidase, 100 450, 452, 454
gall stone, 371, 382, 397 glucose, 11, 100, 101, 306, 316, 321, 323,
ganglioside, 369, 582 334, 355, 449, 582
gastroenteritis, 477 glucose transporter
Gaucher’s disease, 369 2, 520
Gaussian distribution, 608 glucose-6-phosphatase, 349, 519
gel filtration, 70 deficiency, 353
gene, 91–105, 109, 111–112 glucose-6-phosphate dehydrogenase, 356,
candidate, 187, 616 368
duplicated, 111 deficiency, 558
mapping, 185–187 glucuronic acid, 358
pseudo-, 111 GluT2, 520
regulation, 100 glutamate
SRY, 123 dehydrogenase, 410
gene manipulation pKa, 28
germline, 549 glutaminase, 418
gene therapy glutamine
somatic, 551 cycle, 435, 579
genetic drift, 556 in the kidney, 435
genome synthetase, 418
archaea, 109 glutathione, 357
bacteria, 109 peroxidase, 357
eukaryota, 109 reductase, 357
human, 109 glutathione peroxidase, 344
organelle, 109 glyceraldehyde-3-phosphate dehydrogenase,
virus, 109 201
genotype, 233 glycerol, 11
659
Index
phosphate, 364 sulfhydryl-, 6, 10

shuttle, 246 growth, 405
glycine, 370, 418 growth factor, 488
cleavage enzyme, 418 epidermal, 486, 489
in collagen, 257 fibroblast, 489
glycogen, 12, 351–354, 402, 449 insulin-like, 489
degradation, 352 nerve, 489
phosphorylase, 352, 355 platelet-derived, 486, 489, 492
deficiency, 353 receptor, 492
storage disease, 353 transforming, 489
storage diseases, 519 growth hormone, 189, 364
synthase, 519 GTP, 99, 167
regulation of, 352 guanosine triphosphate, see GTP
synthesis, 351 guanylate cyclase, 277
glycogenoses, 519 gyrase, 95
glycolysis, 199–201, 356, 449
anaerobic, 449 Hageman factor, 214
glycoprotein, 203, 220 hair, 291, 337
glycosidic bonds, 12 Haldane, J.B.S., 140, 142
glycosphingolipid, 369 haploinsufficiency, 236
goiter, 341 haplotype, 237
gonad, 336 haptoglobin, 204–206
gout, 343, 469 Hardy-Weinberg equation, 555–556
grain, 296, 300, 301, 306, 308, 316, 317, Hartnup’s disease, 261
320, 333, 336, 343, 345 Hartnup’s disease, 436
GroES/GroEL, 55 hawkinsinuria, 427
groove HbA1c , 57
major, 92 HDL, 451
minor, 92 heart, 209, 210, 267, 300, 324, 325, 333,
group 334, 347, 393
aldehyde-, 11 defect, 614
alkyl-, 5 heat, 15
amino-, 6, 10, 27, 34 capacity, 15
carbonyl-, 6, 12 of evaporation, 18
carboxy-, 5, 9, 10, 27, 34 Heinz-bodies, 517
guanidino-, 28 helicase, 95
hemiacetal-, 6, 12 helix
hemiketal-, 12 alpha-, 39
hydroxy-, 5, 12 amphipatic, 39
imidazole-, 28 DNA, 92
keto-, 11 helix-loop-helix, 118
selenol-, 28 helix-turn-helix, 118
660
Index
HELLP-syndrome, 533 female, 129

hematocrit, 203 male, 130
heme, 47, 168, 206, 250 true, 129
degradation, 459–463 Hers disease, 520
oxygenase, 459 heterochromatin, 110
synthesis, 457–459 heterogeneity
hemiacetal, 6, see group,hemiacetal- allelic, 235, 237
hemicellulose, 445 locus, 234
hemiketal, see group,hemiketal- heteroplasmy, 234
hemizygote, 512 heterotropic, 146
hemochromatosis, 207, 267 heterozygote
classic, 267 advantage, 556
juvenile, 268 compound, 233
hemoglobin, 137, 146, 206, 221–227, 333, hexokinase, 200
517 hexosamidase A, 369
-C, 226 hexose, 11
adult, 225 Hill-coefficient, 146
carbamino-, 225 hippurate, 413
degradation, 459 histamine, 275–277
embryonal, 225 histidine, 429
fetal, 221, 225 pKa, 28
glucated, 57 histone, 110, 117
H disease, 227 HIV, 193
Lepore, 269 HMG-CoA, 370
sickle cell, 226 reductase, 373
hemolysis, 205, 206, 462 Homer, 137
hemopexin, 204–206 homocysteine, 422
hemophilia, 263 homocysteinuria, 422
A, 216, 512 homogentisate, 427
B, 216 homotropic, 146
hemostasis, 213 homozygous, 233
Henri, V., 140 hookworm, 337, 611
heparin, 214, 216, 363 hormone, 271–277
hepatitis, 208, 398, 462, 565 receptor, 271–273
hepatoblastoma, 269 tyroid, 272
hepatocyte, see liver Hsc70, 55, 323
hepatomegaly, 517 Hsp70, 55
heptose, 11 human
hereditary fructose intolerance, 518 genome, 109
Hereditary non-polyposis colon cancer, 115 Hunter disease, 527
hermaphrodism Huntington’s disease, 83, 236, 262, 558
pseudo- Hurler-Scheie disease, 527
661
Index
hybridization, 96 I-cell disease, 57, 521

hydrocephalus, 291 icterus, see jaundice
hydrogen peroxide, 253, 581 IDL, 451
hydrogen sulfide, 252 IEF, 553
hydrolysis, 6 IgA, 476
hydroperoxide radical, 253 ileum, 370
hydrops fetalis, 220 Iliad, 137
hydroxy radical, 253 immune system, 291, 307, 316, 318, 333,
hydroxyapatite, 319, 332 337, 338
hydroxybutyrate, 366 immunoglobulin, 497–501, 506–507
7α-hydroxylase, 370 A, 499
hydroxymethylcytosine, 102 D, 499
hyperammonemia, 582 E, 294, 499
hyperbilirubinemia, 302, 461 G, 497, 499
conjugated, 462 M, 499
mixed, 462 subclass deficiency, 508
unconjugated, 462 imprinting, 235, 268
hypercholesterolemia, 374, 375
in-vitro fertilization, 565
familial, 236, 375
inactivation
hyperchylomicronemia, 375
X-, 512
hyperglycemia, 582
inbreeding, 511, 557
hyperglycinemia
induced fit hypothesis, 163
non-ketotic, 418
infant, 319
hyperlipidemia, 401
infarct, 169, 208, 209, 226
hyperlipoproteinemia, 375
hyperlysinemia/uria, 429 myocardial, 387
hyperparathyroidism, 209 infertility, 123
hypertension, 382, 393–396, 400, 401 inflammation, 206
portal, 398 INK4a, 492
hypertriglyceridemia, 376 inositol-1,4,5-trisphosphate, 275
hyperuricemia, 469 insertion, 113
hypoalbuminemia, 401 insulin, 189, 200, 268, 276, 320, 336, 350,
hypochlorite, 253, 581 363, 364, 368, 383, 569
hypochondroplasia, 258 resistance, 277
hypoglycemia, 518, 582 insulin receptor substrate, 597
hypoketotic, 367 integrase, 103, 104
hypophosphatemia, 259 integrin, 488
hypothalamus, 341, 477 intein, 60
hypotonia, 517 interactome, 145, 591
hypoxia, 253 interferon, 189, 490, 505, 595
induced transcription factors, 61 interleukin, 294, 490
response element, 61 intermittent claudication, 294
662
Index
intestine, 209, 287, 289, 293, 296, 298, 300, ketone, 12

301, 304, 308, 309, 314, 317, 321, ketone bodies, 366, 577
323, 325, 334, 336, 337, 341, 343, kidney, 207, 209, 226, 237, 289, 291, 293,
442 301, 302, 314, 316, 317, 321, 324,
brush border, 443 325, 333, 341, 342, 393, 399–402
intrinsic factor, 309, 311 disease polycystic, 264, 401
intron, 109–111 stone, 471
iodine, 338–342 kinetics, 15
ion channel reaction, 19–21
ligand gated, 272 Klinefelter syndrome, 127
IQ, 479, 609 knock
iron, 168, 250, 255, 311, 313, 325–333, 338, down, 550
473, 475, 480 in, 549
deficiency, 207 out, 548
poisoning, see hemochromatosis conditional, 549
iron-sulfur protein, 168, 250 Koshland, D.E., 163
ischemia, 201, 253, 449 Krabbe’s disease, 370
isocitrate dehydrogenase, 244 Krebs-cycle, 199
isoelectric focussing, 72 Krebs-cycle, 244–247, 449
isoelectric point, 30 Kuppfer cell, 397
isoenzyme, 37, 169, 208 kwashiorkor, 577
isomer, 8, 11
cis-trans, 8 β-lactamase, 105
geometric, 8 lactate, 325
optical, 8 lactate dehydrogenase, 201, 209
positional, 8 lactation, 293, 314, 338, 356, 405, 476–479
isoniazid, 303, 304, 308 lactoferrin, 476
isopentenyl-pyrophosphate, 370 lactoglobulin, 476
isoprene, 286 lactose, 12, 100, 321, 476
isoprenoid, 370 intolerance, 518
intollerance, 445
janus kinase, 490 permease, 100
jaundice, 398, 461, 517 lamin, 485
obstructive, 208, 209 Lavoisier, 285
jun, 492 LCHAD, 533
LDL, 451
karyotype, 121–123, 131 Le Chatelier’s principle, 21
Kashin-Beck-disease, 347 lead
katal, 143 poisoning, 459
kernicterus, 220, 461 leavening (of bread), 337
Keshan disease, 347 Leber hereditary optic neuropathy, 531
ketoglutarate dehydrogenase, 244 lecithin-cholesterol acyl transferase, 374
663
Index
legume, 300, 304, 316, 333, 336, 343 VLDL, 364, 368, 373, 375
Leigh-syndrome, 528, 531 liposome, 73, 552
leptin, 490, 595 Lisch nodules, 495
Lesch-Nyhan syndrome, 469 lithium, 612
leucine lithocholic acid, 370
zipper, 118 liver, 169, 203, 206, 209, 215, 267, 287,
leucocytes, 477 289, 291, 293, 296, 298, 301, 308,
leukemia, 125, 494 309, 311, 313, 314, 316, 317, 333,
lymphoblastic, 125 334, 338, 342–344, 352, 355, 356,
myelogenous, 125 370, 396–399, 402, 452, 577
Lewis acid, 168 cancer, 207
Lewy-bodies, 83 cirrhosis, 207, 208, 398
Li-Fraumeni syndrome, 494 infantile, 206
library enzymes, 415
genomic, 188 fatty, 398
Limbic, 285 locus, 233
light, 291 heterogeneity, 234
polarized, 9 lod score, 187
lignin, 445 lordosis, 258
likelihood ratio, 187 low birth weight, 473, 612
Lind, 314 Lowry, 66
LINE-1, 110–112 lung, 206, 264, 294
Lineweaver-Burk-transformation, 143 Lunin, 285
linkage, 186 lymph, 289, 363
linoleic acid, 307, 361, 476 lymphocyte, 207
linolenic acid, 361 B, 507
lipase, 210, 320, 442 T, 507
adipose tissue, 364 lymphocytes
hepatic, 373 T-, 294
pancreatic, 363 Lyons hypothesis, 123
lipid, 320 Lyons-hypothesis, 512
metabolism, 361–376 lysine, 429
lipid storage disease, 369 pKa, 28
lipofuscin, 296 lysis, 103
lipoic acid, 243 lysogenic, 103
lipoprotein, 363, 364, 371–376, 451 lysosome, 277, 354, 369, 506, 519
HDL, 373–375 lysozyme, 102, 439, 477
high density, 338 lysyl oxidase, 333, 334
IDL, 373
LDL, 373–375 macroglobulin, 206
lipase, 363, 373 macrophage, 373, 374, 477
low density, 296 macula degeneration, 265
664
Index
magnesium, 99, 324–325, 473 fragile X, 261

Maillard-reaction, 59 Menten, M.L., 140
malaria, 51, 226, 558, 611 mercury, 612
G6PDH deficiency in, 516 MERRF-syndrome, 532
malate aspartate shuttle, 246 metabolic syndrome, 594
malic enzyme, 368 metabolome, 591
malnutrition, 207 metachromatic leukodystrophy, 370
malonyl-CoA, 367 metallothionein, 255, 334, 336
maltose, 12 metaphase, 121
mammary gland, 356 metastable, 22, 53
manganese, 168 metastasis, 491
manic-depressive, 611 methanol, 151
manifestation, 512 methemoglobin, 221, 225
mannose, 11 reductase, 221, 581
maple syrup urine disease, 422 methionine, 422
Marfan syndrome, 257 methotrexate, 151, 421, 468
marker
methyl bromide, 114
linked, 237
methylation, 61, 117
Maroteaux-Lamy disease, 527
RNA-, 97
mass
methylene blue, 222
molecular, 3
methylmalonic aciduria, 425
mass action
methylmalonyl-CoA mutase, 310
law of, 19
mevalonate, 370
mass element, 319–325
MHC, 501
mass spectrometry, 74, 553
matrix protease, 60 class I region, 503
Maxam-Gilbert method, 185 class II region, 504
McArdle disease, 520 class III region, 505
McArdle’s disease, 353 polymorphism, 508
Mdm2, 493 micelle, 371
meat, 298, 300, 304, 306, 308, 309, 320, Michaelis, L., 140
323, 336, 343, 345 microarray, 550
megadose, 285 microcephaly, 612
meiosis, 105, 111, 121, 124 microsatellite, 110
MEK, 490 milk, 289, 293, 298, 300, 301, 309, 314,
melanin, 334, 336 319, 333
membrane allergy, 476
protein, 39 composition, 476
Mendel, Gregor, 233 millet, 304
Menke’s disease, 334 mineral, 318–347
Mennonites, 511 mineralocorticoid, 130
mental retardation minisatellite, 110
665
Index
mitochondria, 41, 60, 109, 199, 234, 243, loss of function, 236
364, 366, 449 missense, 113
inherited diseases, 528–533 nonsense, 113
mitogen, 486, 488–491 point, 113
mitosis, 121, 131, 485 rate, 557
S-phase, 114 recessive, 236
mitral valve, 257 silent, 113
molten globule, 53 site directed, 190
molybdenum, 342–344 myasthenia gravis, 259
monosaccharide, 11 myc, 492, 493
monosomy, 124 myelin, 582
Montezuma’s revenge, 275 myeloma
Morgan, T.H., 186 multiple, 509
Morquio disease myoglobin, 221
A, 527 myokinase, 167
B, 527 myopathy, 336
mosaicism, 512 ragged red fibre, 532
motive, 49 myositis, 208, 259
mountain sickness, 156 myxedema, 341
mRNA
processing, 116 Na+ /K+ -ATPase, 322
mucous membranes, 287 Na/K-ATPase, 172
Muenke’s craniosynostosis, 258 NAD, 167, 303, 304
Mulder,G.J., 137 NADH
muscle, 201, 209, 210, 276, 294, 296, 300, oxidase, 254, 581
314, 320, 323, 324, 333, 352, 375, Q reductase, 251
402, 449, 578 NADP, 303, 304
muscular dystrophy, 208, 259, 260, 296 necrosis, 206, 208
Becker, 259 neoplasia, 490
Duchenne, 259, 557 nephritis
Duchenne, 192 glomerular, 401
limp-girdle, 260 nephron, 400
myotonic, 260 nephrotic syndrome, 207, 401
mutagen, 112, 254, 490 neu, 492
mutagenesis neural tube, 474, 612, 614
insertional, 547 neurofibromatosis, 495
mutation, 109, 112 neuropathy, 260
dominant, 236 neuropeptide Y, 587
dominant negative, 236 neurotransmitter, 271
frameshift, 113 niacin equivalent, 304
gain of function, 236 nicotinamide, 169, 304–307, 316
haploinsufficient, 236 production from Trp, 427
666
Index
Nicotinamide-adenine dinucleotide, see NAD organophosphate, 208

nicotine, 303 Ori-c, 95
Niemann-Pick disease, 370 oriental flush, 453
ninhydrin, 67 ornithine
nitric oxide, 277 decarboxylase, 435
synthase, 277 orotic aciduria, 464
nitroglycerin, 277 osteitis deformans, 209
nitrous acid, 114 osteoarthritis, 382
nojirimycin, 57 osteoblast, 209, 287
norepinephrine, 364, 402, 570 osteocalcin, 298
normal distribution, 608 osteoclast, 287
nuclear magnetic resonance, 74 osteogenesis imperfecta, 45, 237, 257
nuclease, 97, 443 osteomalacia, 209, 294, 322
nucleoside, 91 osteoporosis, 322, 332, 334, 617
nucleosome, 110 ouabain, 394
nucleotide, 91 ovary, 517
nut, 300, 304, 316, 333 oxalate, 321
nutrient pool, 283 oxaloacetate, 454
oxanion hole, 165
oasthouse disease, 436 oxidative phosphorylation, 249–253, 449
obesititis, 595 oxidoreductase, 301
obesity, 376, 381–384, 394, 397, 479 oxygen, 250, 285
occur, 287, 291, 296 reactive, 253–255
ochronosis, 427 toxicity, 254
Okazaki-fragment, 95 oxytocin, 477
oligo
allele-specific, 550 p53, 487, 492, 493, 553
oligomycin, 253 Paget’s disease, 208
oligosaccharide, 12 palindrome, 97, 181
oncogene, 491 pancreas, 169, 264, 276, 287, 289, 298, 311,
cellular, 491 316, 337, 442
proto-, 491, 493 pancreatitis, 208, 210, 447
viral, 493 pantothenate, 306–307
oncotic pressure, 203 pantothenic acid, 169
oogonia, 124 papain, 497
operator, 101 paracrine, 271
operon paraprotein, 205
lac-, 100–101 paraproteinemia, 509
Trp-, 101 parathormone, 291, 293, 321
order of a reaction, 19 parathyroid, 325
organelle Parkinson’s disease, 83
genome, 109 Patau syndrome, 124
667
Index
Patau syndrome, 126 phosphatidic acid, 368

PBX2, 505 phosphatidyl
PCR, 183–184, 193 inosititol, 275
pedigree, 238 inositol, 213
pellagra, 436 serine, 213
penetrance, 234, 238, 512 phosphoanhydride, 165
penicillin, 105 phosphodiesterase, 274, 277
penicillinase, 105 phosphofructokinase, 146, 200, 349, 351
pentachlorophenol, 253 M isoform, 520
pentose, 11 phosphoglyceride, 368
phosphate pathway, 355 phospholipase, 102, 368, 443
PEP-carboxykinase, 349 C, 275, 488, 489
pepsin, 442, 447 phospholipid, 333
peptide, 34 phosphorus, 291, 319–322
signal, 60 phosphorylase, 520
permanent wave, 41 phosphorylation, 61, 117, 351, 364, 448,
pernicious anemia, 311, 318 485, 487, 489, 569
peroxidase, 255 phototherapy, 302, 462
peroxisome, 366 phylloquinone, 296
peroxisomes phytanic acid, 367
inherited diseases, 533–535 phytate, 321, 325, 336, 337
pesticide, 83 phytohemagglutinin, 121, 486
petechia, 314 pI, 70
pH, 9 pica, 306
in protein precipitation, 68 pinocytosis, 203
phage, 188 pituitary, 341
phagocytosis, 497 placenta, 209, 474, 497
phenobarbital, 459, 462 plaque, 374
phenocopy, 235, 511 atheromatous, 387
phenol, 10 senile, 617
phenome, 591 plasma, 203, 207
phenotype, 233 plasma cell, 203
phenylacetate, 413 dyscrasia, 509
phenylalanine, 425 plasmid, 104, 188
hydroxylase, 425 plasmin, 214
phenylketonuria, 266, 612 plasminogen, 214
phenytoin, 612 plasminogen activator inhibitor 1, 595
phocomelia, 612 platelet, 207, 213, 216
phorbol ester, 276 pleiotropy, 234
phosphatase polyadenylation, 116
I, 353 polyamines, 432
phosphate, 173, 259 polycistronic, 101
668
Index
polycythemia, 225 prostate

polymerase cancer, 209
DNA, 94, 112 prosthetic group, 165
RNA-, 97 protanopia, 265
polymorphism, 556 protease
polyol, 358 inhibitor, 206
polyphenol, 589 serine, 164, 206, 214
polyphenolic, 393 proteasome, 504
polyploidy, 123 protein, 34–89, 137, 307, 323
polysaccharide, 12 -denaturation, 55
Pompe disease, 519 acetylation, 61
Pompe’s disease, 354 as buffer, 174
porphyria, 457 band 3, 263
acute intermittent, 236, 457 Bence-Jones, 509
cutanea tarda, 459 biological value of, 406
porphyrin, 457 C-reactive, 206
porphyrinogen, 457 concentration, 66
potassium, 322–324 constitutive, 100
Prader-Willi syndrome, 268 energy content, 447
pregnancy, 209, 287, 293, 300, 309, 314, heat shock, 506
318, 337, 341, 405, 473–476 hydroxylation, 60
primaquine, 357 inducible, 100
primase, 95 intrinsically disordered, 50, 74
primer, 183, 184 kinase, 61, 274
prion, 77–80 A, 276
proband, 233 C, 276
probenicid, 471 G, 277
processing mitogen activated, 490
RNA-, 97 membrane
proenzyme, 60 purification, 72
progesterone, 376 metabolism, 452
progestin, 130 methylation, 61
prolactin, 477, 490 phosphatase, 61
proline, 429 phosphorylation, 61
prolyl hydroxylase, 61 RDA, 405
promotor, 101, 117, 131, 276 splicing, 60
Prontosil, 421 synthesis, 98–100
proofreading, 94 tyrosine kinase, 489
prophage, 103 proteinuria, 400, 401
prophase, 111, 122 proteolysis, 60
propositus, 233 proteome, 591
prostaglandin, 271, 316 proteomics, 72, 553
669
Index
prothrombin 0th-order, 20
time, 215 1st order, 21
protomer, 51 2nd order, 21
provitamin, 286 order, 19
pseudocholinesterase, 208 velocity, 19
Pseudomonas aeruginosa, 264 receptor
pseudouridine, 97 7-transmembrane segment, 273
psoriasis, 208, 469 acetylcholine, 272
purine muscarinic, 277
biosynthesis, 463 desensitisation, 277
degradation, 464 down-regulation, 277
puromycin, 100 G-protein linked, 272
putrescine, 432 GABA-A, 272
pyelonephritis, 401 glutamate
pyloric stenosis, 613 NMDA, 276
pyridoxalphosphate, 410 muscarinic, 276
pyridoxin, 307–309 phosphorylation, 277
pyrimidin scavenger, 373, 374
metabolism, 464 tyrosine kinase, 272
pyrophosphatase, 166, 418 recessive, 233, 511
pyrrolysine, 29 recombination, 105
pyruvate, 199, 454 homologous, 105, 111
carboxylase, 245, 261, 349 redox potential, 249
dehydrogenase, 243, 244, 261 redox-reaction, 167–168
kinase, 200, 349 reductionism, 138
inherited defect, 201 Refsum’s disease, 367
regression toward mean, 609
QH2 - Cytochrome c reductase, 251 renal disease
quantitative trait loci, 615 end-stage, 401
renin, 393, 399
R-factor, 105 replicase
radiation, 612 RNA, 104
ionizing, 113 replication, 94–95, 112
radical, 253, 294 -fork, 94
Raf, 490, 492 resistance
RAGE, 505 antibiotic-, 104
ragged red fibre, 532 respiratory burst, 581
Ras, 490, 492, 495 response element, 118
rate constant, 20 restriction enzyme, 181
RDA, 281, 283 reticuloendothelial, 206
in pregnancy, 474 retinal, 287–291
reaction retinitis pigmentosa, 265, 291
670
Index
retinoblastoma, 494 S-adenosylmethionine, see SAM

protein, 486–487, 492, 493 Sørensen, P.L., 140
retinoic acid, 272, 287, 288, 612 saccharose, 518
retinoid, 336, 338 saliva, 338, 341
retinol, 255, 294 salivary glands, 439
binding protein, 205 salt, 323
retinol equivalent, 287 in protein precipitation, 68
retinopathy SAM, 169
diabetic, 265 Sanfilippo disease
retrolental fibroplasia, 296 A, 527
retroposon, 111, 112 B, 527
viral, 112 C, 527
retrovirus, 493, 552 D, 527
Rett-syndrome, 513 Sanger dideoxy method, 185
reverse transcriptase, 111 sarcoma, 490, 494
RFLP, 186, 237 Kaposi, 493
rhabdomyoma, 266 Schiff-base, 59
rhabdomyosarcoma, 269 Schilling-disease, 311
rhesus schizophrenia, 306, 611
factor, 220–221 scleroderma, 505
incompatibility, 462 scurvy, 314
rhizomelia, 258 SDS, 70
rhodopsin, 265, 277, 287, 288 secretor, 220
RhoGAM, 221 seed, 298
riboflavin, 169, 301–302, 304 seizures, 156
ribose-5-phosphate, 355 selection, 558
ribosome, 96, 99 selenium, 255, 294, 344–347
ribozyme, 137 selenocysteine, 29, 255
rickets, 209, 259, 294 pKa, 28
rifampicin, 99 senescence, 486
rigor, 322 serotonin, 275, 425
RISC, 550 serum, 169
RNA, 96–97 sex hormone
editing, 119 binding globulin, 205
messenger, 96 SH3-domain, 46
polymerase, 116, 118 sheet,beta-, 41
ribosomal, 96, 99 Shine-Delgarno sequence, 99
transfer, 96, 98 shock, 208
RNAi, 550 sialidase, 506
Robertsonian translocation, 134 silencer, 117
rotenone, 252 silk, 41
rubella, 612 sirtuin, 61, 589
671
Index
skin, 291, 293, 296, 306, 308, 314, 316, 337 Streptococcus mutans, 479
sleeping sickness, 435 streptomycin, 100
Sly disease, 527 stringency, 182
small for date birth, 318 structure
smoking, 374, 388, 394, 476, 612 primary, 74
SNP, 616 quaternary, 51, 74
snurp, 116 secondary, 37, 74
sodium, 322–324, 394 tertiary, 47, 74
soil, 319 substitution, 113
solubilization, 72 subunit, 51
sorbitol, 518 succinate dehydrogenase, 244
sorghum, 304 succinyl-CoA, 418
spectrin, 263 succinylcholine, 209
spectroscopy sucrose, 12, 479
infrared, 74 suicide substrate, 159
spermatogonia, 124 sulfanilamide, 421
spermidine, 432 sulfatase, 369
spermine, 432 sulfonamide, 469
spherocytosis, 263 sulphite oxidase, 342
sphingolipid, 220, 369 sulphonamide, 151, 298, 300, 421
sphingolipidosis, 369 Sumner, J.B., 137
sphingosine, 369 SUMO, 63
spina bifida, 337, 474, 614 supercoiling, 93
spleen, 216, 459 superfemale, 128
spliceosomes, 116 superoxide, 253, 314
splicing, 116 superoxide dismutase, 254, 333
alternative, 119 superoxide radical, 581
protein, 60 superoxide themselves, 336
spongiform encephalopathy, 77 surgery, 402
sprue, 317, 336 symport, 172
src, 492 syndactyly, 258
Staphylococcus aureus, 264 syndrome
starch, 12 metabolic, 594
Stargardt-disease, 291 synuclein, 83
starvation, 576 syphilis, 565, 612
steatorrhea, 264 system, 16
stem cells systemic lupus erythematosus, 401, 505,
embryonic, 548 612
steroid, 272, 569
stomach, 304, 309, 311, 333, 334, 338, 343, T-cell
442 receptor, 500
Strecker-degradation, 59 tandem repeat, 110
672
Index
Tangier disease, 375 thymidylate kinase, 485

TAP1/2, 504 thymidylate synthase, 468
tapeworm, 311 thyroid, 293, 301, 311, 325, 338, 341, 344
Taq, 183 hormone, 364, 569
Tarui disease, 520 thyroxin, 321, 338
taurine, 370, 476 thyroxine, 205, 402
tautomer, 113 tissue
Tay-Sachs disease, 369 factor, 214
Tay-Sachs disease, 565 plasminogen activator, 189, 214
telomerase, 112 TNF
telomere, 110, 112 α, 595
temperature, 15 tobacco, 314, 531
teratogen, 611 tobacco-alcohol amblyopia, 531
teratogenesis, 291, 294, 302, 318, 337, 343 tocopherol, 294–296
tetany, 322, 325 equivalent, 294
tetracycline, 100, 481 tooth, 291, 294, 314, 319, 332, 342
tetrose, 11 tophi, 469
TGF topoisomerase, 94, 95
β, 595 toxin, 104
thalassemia, 192, 227, 558, 565 toxoplasmosis, 612
major, 227 trace element, 319, 325–338
minor, 227 training, 405
thalidomide, 612 tranquilizer, 293
thanatophoric dysplasia, 236, 258 transaldolase, 356
theophylline, 274, 276 transaminase, 307
thermodynamics, 15–19 transamination, 408
first law of, 16 transcobalamin, 309, 311
second law of, 18 transcortin, 204, 205
third law of, 18 transcriptase
thermogenesis, 382, 448 reverse, 104
Thermus aquaticus, 183 transcription, 96–97, 116
thiamin, 299–301, 304 factor, 118
thiamine, 243, 356 transcriptome, 591
thiazide, 481 transcuprin, 334
thioglycolic acid, 41 transducin, 277
thiol, 6 transduction, 105
thoracic duct, 363 transferrin, 205, 207, 336, 476
threonine, 418 transformation, 105
thrombin, 214 transfusion, 219
time, 215 exchange, 462
thrombomodulin, 214 transgenic mice, 547
thrombosis, 213, 401 transglutaminase
673
Index
tissue, 83 tyrosinemia
transition, 113 I, 427
state, 22 II, 427
transketolase, 299, 356 III, 427
translation, 98–100, 117 tyroxine
translocation, 113 binding globulin, 205
Robertsonian, 124
transport ubiquinone, see coenzyme Q
membrane-, 170–173 ubiquitin, 63
active, 172 UDP-glucuronyl transferase, 462
passive, 170 ultratrace element, 319, 338–347
transposon, 111 uncoupler, 253
transthyretrin, 204, 205 uniport, 171
transversion, 113 urea, 399, 452
Traube, M., 137 cycle, 261
trauma, 206, 402 in protein denaturation, 56
tri-functional protein, 533 urease, 137
trichloroethylene, 83 uremia, 401, 582
triglyceride, see fat uric acid, 255
Trimethoprim, 421 uridilylation, 63
triose, 11 uridine triphosphate, see UTP
trisomy, 124 urine, 298, 300, 302, 304, 306, 308, 309,
-13, 124, 126 311, 314, 316, 337, 343, 345
-18, 124, 126 blood in, 206
-21, 124 urobilin, 459
troponin, 275, 276 urobilinogen, 459
Trypanosoma brucei, 435 uroporphyrinogen decarboxylase, 459
trypsin, 60, 442, 447 uroporphyrinogen I synthase, 457
tryptophan, 427 US-RDA, 283, 285
tuber, 298, 308 uterus, 477
tuberculosis, 209, 611 UTP, 167
tuberous sclerosis, 266 UV, 113
tumor
progression, 491 valinomycin, 253
supressor, 491 valproic acid, 612
tumor necrosis factor, 506 vampire, 306
tungsten, 342–344 vas deferens, 264
Turner syndrome, 128 vasopressin, 237, 275, 276, 402
Turner syndrome, 128, 131, 132 vector
tyrosinase, 263 retroviral, 552
tyrosine, 425, 427 vegetable, 289, 298, 300, 301, 314, 316,
pKa, 28 317, 323, 338
674
Index
velocity, 19 intoxication, 323

Vibrio cholerae, 63, 275 Watson-Crick structure, 92
Vibrio parahaemolyticus, 63 weaning, 478–479
Vicia faba, 516 weight, 338
virion, 102, 103 whooping cough, 275
virus, 102–104, 114, 181, 493 Williams syndrome, 132
adeno-, 552 Wilm’s tumor, 269
adenoassociated, 552 Wilson’s disease, 334
animal, 103 Wilson’s disease, 207
genome, 109 Wolff-Chaikoff-effect, 342
papilloma, 493 work, 15
retro-, 104, 493, 552
RNA, 104 X-inactivation, 512
Rous sarcoma, 493 X-ray crystallography, 74
visfatin, 595 xanthin oxidase, 342
vitalism, 138 xanthine oxidase, 471
vitamin, 169, 285–318, 475 xantoma, 375
A, 362, 392, 473 xenobiotics, 535, 537
B3 , 427 xeroderma
B6 , 410, 422, 474 pigmentosum, 115
B12 , 422, 442, 481 YAC, 189
C, 46, 362, 392, 474, 480, 481 yeast, 293, 316, 317
D, 272, 481 yellow fever, 398, 462
E, 255, 362, 375
fat soluble, 286–299 Zellweger syndrome, 534
K, 216 Zellweger-syndrome, 366
M, 421 zinc, 168, 255, 289, 313, 318, 334, 336–338,
water soluble, 299–318 442, 453, 473, 475, 476
vitamin K, 296–299 finger, 93
VLDL, 451, 452 Zn
VNTR, 186, 237 finger, 118
von Gierke’s disease, 353 zymogen, 276, 447
von Gierke-disease, 519
von Recklinghausen’s disease, 495
von Willebrand factor, 216
von Willebrand factor, 213
VopS, 63
Waldenstroms macroglobulinemia, 509

warfarin, 216, 612
water, 323, 332
anomaly of, 4
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Ross University School of Medicine

Biochemistry and Genetics

© DeVry Inc. 2007–2009

I. Semester one, Mini I 1

2. Energy changes and rates of chemical reactions 15

3. Amino acids and proteins 27

3.2.2. Protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

4. DNA and Gene Expression 91

4.8. Types of Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

5. The Human Genome and Mutations 109

6. Chromosome Aberrations 121

6.7. Abnormal Sexual Development With Normal Chromosomes . . . . . . . . . 129

8. Methods in Molecular Medicine 181

II. Semester one, Mini II 197

9. Glycolysis: Splitting glucose in half 199

10.Plasma Proteins 203

10.5. Objectives in summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

11.Blood Coagulation 213

III. Semester one, Mini III 231

13.Mendelian Inheritance 233

14.Krebs- (TCA-) cycle: Burning the carbon skeleton 243

15.Respiratory Chain, Oxidative Phosphorylation and Reactive Oxygen Species 249

16.Single-Gene Disorders and Traits 257

16.5. Blood Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

17.Hormone Biochemistry 271

IV. Semester two, Mini I 279

18.Vitamins and minerals 281

18.3.3. Ultratrace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

19.Carbohydrate Metabolism 349

20.Lipid Metabolism 361

20.9. Sphingolipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

V. Semester two, Mini II 379

21.Nutritional Management of Disease 381

22.Amino Acid Metabolism 405

22.3.6. Phe and Tyr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

23.Biochemistry of Digestion 439

24.Heme, Purines and Pyrimidines 457

25.Nutrition During the Life Cycle 473

26.Cell Cycle Control and Cancer 485

27.Immunoglobulins and Immunogenetics 497

28.Inherited diseases of metabolism 511

28.1.3. Newborn screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

VI. Semester two, Mini III 545

29.Advanced DNA technology 547

30.Population Genetics and Genetic Counseling 555

30.10.Objectives in Brief . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

31.Integration of Metabolism 569

32.Multifactorial Inheritance 607

VII. Appendix 619

33.Answers to the example questions 621

33.6. Integration of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628

General Biochemistry Courses:

Database searching for journal articles, and biochemical information:

Semester one, Mini I

1.1. Elements and molecules

1.2. Covalent Bonds And Non-covalent Interactions

1.2.1. Covalent bonds

Table 1.1.: Important elements

Table 1.2.: Composition of the human body

Electronegativity is the tendency of an atom to attract electrons. The order of electroneg-

1.2.2. Non-covalent interactions

H2N NH2 H2N CH3 H3C CH3 H3C CH3