Biochemistry

A. Introduction
Pharmaceutical Biochemistry
• The science concerned with the chemical basis of life, as applied to pharmacy practice and pharmaceutical,
biological science
• Science concerned with the chemical constituents of living and the reaction they undergo
• The application of chemistry to the study of biological processes at the cellular and molecular level

Cell - Basic unit of life

• Prokaryotic Cell
• No nucleus & true organelles
• Bacteria and archae
• Smaller than eukaryotic cell
• Contains only a single loop of stable chromosomal DNA
• Metabolic process occurs across the cell
• Eukaryotic Cell
• Have nucleus and organelles
• Plants, fungi, and animal cells
• DNA is found tightly bound and organized chromososme
• Larger than prokaryotic cells
• Mitochondria and chloroplast perform various metabolic processes

Human Eukaryotic Cell

• Plasma Membrane - Provides a barrier; contains transport and signalling system
• Nucleus - Contains chromosomes and the nucleus; site for RNA synthesis
• Mitochondria - Powerhouse of the cell. For ATP production. Has series of folds called “cristae” .
• Rough Endosplasmic Reticulum (RER) - Site for protein synthesis. Covered with Ribosome
• Ribosome - Protein and RNA complex responsible for protein synthesis (No Membrane) . Not a true organelle
• Smooth Endoplasmic Reticulum (SER) - Site of lipid synthesis and metabolism. Contains enzyme for
detoxifying xenobiotic
• Golgi Bodies - Process proteins to mature form
• Lysosome - Suicide bag. Degrades proteins and membrane in the cell and helps degrade material ingested by the
cell
• Peroxisome - Degrade hydrogen peroxide
• Cytoplasm - Houses organelles; liquid portion called cytosol
• Cytoskeleton - Protein filaments that provides shape and basis for movements
Microtubulues - Provides shape, basis for movement and locomotion. Support the
chromosome during division. Made up of tubulin
 Cellular Composition
• Biomolecules 9%
• Water 60-90%
• Inorganic Ions 1% (Essential Ions)
• Chemical properties of biomolecules are dependent o their functional group and most bimolecules have more
than one

Water and Cell - Biomolecules assumes shape in response to water

B. Protein
Proteins
• Foundation of the working cells
• Catalysis (enzyme)
• Chemical storage and transport (ion channel, pumps, plasma albumin)
• Structure (Structural protein)
• Mechanical work (Contractile protein)
• Information storage and retrieval
• Intracellular communication (hormones and neurotransmitter)
• Defense

Chirality of Amino Acids

Twenty Major Amino Acids

• Glycine - An inhibitory neurotransmitter , precursor of heme. Found in sharp protein folds

• Proline - Puts kinks in in polypeptide alpha helix breaker
• Tyrosine - Polar and a precursor of melanin, catecholamine and thyroid hormone. Catabolized to homogenntisate
• Alkaptonuria - Decrease homogentisate oxidase. Result: Dark colored urine
• Phenylalanine hydroxylase - Deficiency leads to phenylketonuria (Remedy: gives ↑ Tyr and ↓ Phe).
•Tryptophan - Precursor for Vit B3
•Methionine - Non-polar and initiator amino acid
•Cysteine - Polar amino acid

Zwitterions - Contains negative and positive charged groups at neutral point

Isoelectric Point (pI) - The pI is the pH at which the net charged of a molecule is zero
pI = (pK1 + pK2)/2

Peptide Bond Formation
 Structure of Protein
• Primary Structure of Protein
• Describes the linear sequence of amino acid in polypeptide chain
• Peptide bonds do not dissociate or does not ionize
• Has double bond characterristic (planar), resonance stabilized and polar
• Assumes trans configuration (more stable)

Chemical Test for Peptides and Amino Acids

• Biurets Test
• Test for peptide bond
• (+) Violet
• Ninhydrin/Triketohydrindene Test
• Test for ∝-amino aicd
• (+) Violet except Proline (yellow)
• Xanthoproteic Acid
• Test for amino acid with benzene ring
• (+) Yellow to orange due to nitration
• Millon Nasse Test
• Test for phenolic amino acid (Tyrosine)
• (+) Flesh ppt turning red/pink
• Hopkins Cole/Glyoxylic Acid Reaction
• Test for indole containing amino acid (Tryptophan)
• (+) Violet ring at the junction of two liquid
• Sakaguchi Test
• Test for guanidium containing amino acid (Argenine)
• (+) Red color
• Sodium Nitroprusside Test
• Test for amino acid with sulfhydryl group (Cysteine)
• (+) Red to purple color
• Basic Lead Acetate Test
• Test for sulfur containing amino acid (Met & Cys)
• (+) Black ppt
• Pauly Diazo Test
• Test for His & Try
• (+) Blue to Red Color

Purification of Proteins
1. Column Chromatography
2. Size Exclusion Chromatography
3. HPLC
4. SDS-PAGE (Sodium Dodecylsulfated Polycylamide Gel Electrophoresis)
5. Two Dimensional Electrophoresis
6. Partition Chromatography
7. Ion-exchange Chromatography
8. Absorption Chromatography
9. Hydrophobic Interaction Chromatography
10. Affinity Chromatography

Protein Structure Determination

1. X-Ray Crystallography
• Most Common
• Proteins can form highly defined crystals when highly purified then precipitated
• Advantage: Large proteins can be analyzed
• Disadvantage: Difficulty of crystallization

2. NMR
• Examines the structure of protein in solution
• Advantage: Structures are determined in solution
• Disadvantage: Size of proteins that can be analyzed are limited to smaller protein
3. Edman Raction
• Method of determining primary sequence or structure of protein
• New methods of primary sequence determination includes mass spectrometry and genomics

• Secondary Structure of Protein

• Refers to the spatial arrangements of amino acids residue located near each other in the linear sequence of
the polypeptide chain
• Stabilized by extensive H-bonding between amidehydrogens and carbonyl oxygens within the protein
molecules: ∝helix - 3-6 amino acid/turn
• Proline & hyroxyproline will not provide hydrogen for H-bonding
• Presence of Amino Acid with bulky group at 𝛽-C atom can hinder the helix to form
• Presence of similarly charged amino acid can hinder because of charged formation
 • 𝛽-Sheet - Stabilized by H-bond between NH & C=O in different polypeptide strand the adjacent chain can run
in the same direction known as parallel or in different direction known as anti-parallel

• Tertiary Structure of Protein

• Overall arrangement and inter relationship of various region or domain and individual amino acid residue
• Stabilized by hydrophobic interaction and disulfide bonds
• Coiling and folding of protein chain into compact structure. Determines the 3D-structure of protein
• Other stabilized: 1. Ionic bonding, 2. H-Bonding 3. Hydrophobic Interaction 4. Covalent Interaction and 5. π-π
complex

• Quarternary Structure of Protein

• Arrangement of polypeptide chain in relation to one another in a stabilized by 1. H-Bonding, 2. Hydrophobic
interaction, 3. Ionic bonds and 4. Disulfide bonds

Protein Denaturation
• Unfolding and disorganization of protein structure without hydrolysis of polypeptide bond.
• Involves the dissociation of the weak interaction of the protein which causes the breakdown of secondary,
tertiary, quarternary structure

Methods of Isolating Protein

1. Based on Solubility
• IpH Precipitation
• Proteins are least soluble at their IpH value
• Salting Out
• Makes use of high concentration salt
• High concentration salt interacts with water so that the protein precipitates out
• Protein dissolvate ions, different protein precipitates at different concentration of salt

2. Based on Molecular Size and Weight

• Dialysis
• Uses a semi-permiable membrane to separate protein and small size of molecule will pass through the
pores of the semi-permiable membrane
• The process will be from a region of higher concentration to a lower concentration
• Ultrafiltration
• Pressure or vaccum in filtering solution of protein using small porous membrane

• Ultracentrifugation
• Useful in separating cellular organelles and biomolecules. It employees high speed centrifugal force
and gravitational force
• Gel Filtration Chromatography

3. Based on the Charge of the Protein

• Elecctrophoresis
• Movovement of the charge particles in an electric field
• Rate of migration depends on size, weight and charge
• Ion Exchange Chromatography
• Kind of chromatography defined as reversible exchange of ions in solution with ions electrostatically
bounded with an adsorbent called “ion exchanger”

4. Based on Binding Affinity

• Affinity Chromatography
• The high affinity of protein for specific chemical group called ligand and they bind to protein by non-
covalent forces, the ligand molecule is covalently bonded to porous large protein
• Precipitation by Antibodies
• Interaction of protein and specific antibodies
• Antibody-antigen complex is large enough to be separated by ultracentrifugation or filtration

Protein Function & Structure

1. Regulatory Protein
• Insulin
• Produced by the beta cells of the Islet of Langerhan
• Secreted in response to high blood glucose level
• Lowers the sugar level, promotes absorption of amino acid and sugar

• Glucagon
• Produced by the alpha cells of the pancreas
• It is secreted in response to low blood glucose level
• It increases the sugar level

2. Structural/ Fibrous Protein

• Simpler than globular protein but water insoluble
• Collagen
• Most abundant protein in vertebrates
• Present in skin, cartilage and tendons
• Basic unit: Tropocollagen
• Vitamin C is Pro-hydroxylation, key step in tropocollagen formation
 • Elastin
• Found in lungs, blood vessel walls and ligaments
• Keratin
• Found in harir, nails and horny tissue of the skin

3. Globular Protein
• Has more complex structure but water soluble
• Heme protein
• Fibrous and globular proteins are described by tertiary structure
• Myoglobulin
• Diffuses and stores oxygen in muscles, releases it in cases of oxygen deprivation
• Hemoglobulin
• Protein in erythrocytes
• Transports oxygen to tissues and returns CO2 to the lungs
• Biomedical Implication of Hemoglobin and Myoglobulin
• Prolonged exposure to high altitude elevates 2,3-BPG synthesis
• Methemoglobinemia
• Sickle cell anemia: Caused by mutations that exchange Val for Glu not Leu
• Thalassemia: Absences of one or more peptide chain in Hgb
• Myoglobinuria is indicative of muscle injury colors the urine dark brown
• Iron Deficiency or Impaired synthesis of RBC (due to B9 and B12 deficiency) causes anemia
• Hb1Ac indicates the mean blood glucose concentration during the past 6-8 weeks

4. Protective Protein
• Ig G - Major form of immunoglobulin, the only Ig that crosses the placenta
• Ig M - 1st Ig formed immunization but later gives way to Ig G. Responsible for A, B, AB and O blood
gropus. Larges Ig and is capable of opsonization
• Ig A - Found in exocrine gland secretion (Saliva, tears and sweat)
• Ig D - Least common
• Ig E - Participates in reactions that distorts mast cell and basophils

Types of Allergic Reaction

• Type I (Immediate Hypersensitivity Reaction) - Mediated by IgE eg. Anaphylaxis
• Type II (Cytotoxic Reaction) - Mediated by IgE and IgM eg. Hemolytic anemia
• Type III (Immune Complex Mediated Reaction) - Mediated by IgG
• Type IV (Delayed Hypersensitivity) - Mediated by T cell and Macrophages eg. Contact dermatitis

Important Pharmaceutical Points on Protein

1. Albumin - Soluble in water and salt solution
2. Globulin - Sparingly soluble in water but soluble in salt solution
3. Histone - Soluble in salt solution
4. Protamine - Soluble in 70-80% ethanol; insoluble in water and absolute alcohol

General Classes of Proteins

1. Simple Protein - Yields amino acid or their derivatives on hydrolysis
• Albumin - Ovoalbumin in egg, serum albumin in blood
• Globulin - Serum globulin
• Glutein - Glutein in wheat
• Prolamine - Zein in corn; gliadin in wheat
• Albuminoid - Fibrous protein
• Histone - Hemoglobin
• Protamine - Antidote for heparin poisoning, salmin and sturin in fish sperm
2. Conjugated Protein - Contains non-protein groups or prosthetic group
• Phosphoprotein - Casein in milk, ovovitellin in egg yolk
• Nucleoprotein - Nuclein in cell nuclei
• Glycoprotein - Mucin in vitreous humor and saliva
• Chromoprotein - Hemoglobulin and flavoprotein
• Lipoprotein - Protein with sterol, fatty acids, lecithin and cephalin
• Metalloprotein - Enzyme such as tyrosinase,arginase and xanthine
3. Derived Protein - Formed by action of heat, alkali, acids , water, enzyme or mechanical shock on protein
• Primary Derived - From early stage of hydrolysis by water, acid and bases and enzyme
• Proteans - Fibrin from fibrinogen; myosan from myosin
• Metaprotein - Acid and alkali albuminate
• Coagulated Protein - Coagulated albumin; cooked meat
• Secondary Derived - Formed from progressive hydrolysis of protein
• Proteases - High MW
• Peptone - Lower MW
• Peptide - Small fragments

Protein Digestion
• Endopeptidase - First enzyme to act
• Pepsin in the gastric juice
• Trypsin, chymotrypsin and elastase secreted into the small intestine
• Exopeptidase
• Carboxypeptidase - Secreted into the small intestine by the pancreas
 • Aminopeptidase - Secreted by intestinal mucosal cell
• Dipeptidase - Hydrolyze ansorbed dipeptides in the brush border of the small intestine

Hormonal Control of Protein Digestion
• Gastrin
• Secreted by pancreas in response to entry of dietary protein
• Stimulates HCl secretion by parietal cell
• Stimulates pepsinogen secretion
• Secretin
• Secreted by duodenum during passage of the acidic contents with the partially digested proteins and fats
• Stimulates pancreas to secret water and carbonic acid
• Cholecytokinin
• Secreted by the duodenum
• Releases trypsin, chymotrypsin, elastase and carboxypeptidase from pancrease

Marasmus vs Kwashiorkor
• Marasmus - Is a protein calorie malnutrition due to chronic deficiency of calories despite adequate intake of
protein
• Kwashiorkor - Is a protein calorie malnutrition where there is sufficient calorie intake in the presence of a
negative protein consumption

C. Enzyme
Enzymes
• Biological catalyst
• Act best at 35-40 ºC
• All enzyme are protein except ribozyme

General Component
1. Haloenzyme
• Intact enzyme with a bound co-factor
• Active part of the enzyme
2. Apoenzyme
• Protein part that has no bound co-factor
• Inactive part of enzyme
• Proenzyme - The inactive form of the enzyme

General Classification of Enzyme

1. Oxidoreductase - Dehydrogenase, Reductase
2. Transferase - Kinases, Hydroxylases
3. Hydrolases - Proteases, Esterases
4. Lyases - Decarboxylase
5. Isomerase - Racemases, Mutases
6. Ligase - Synthetases

Enzyme Kinetic
• Study the rate of enzyme catalyzed reaction and the factors that affect these rate
• Clinically important for diagnosis based on changes in enzyme activity or amount
• Important in the study of potential therapeutic agent
Formula for Simple Enzyme Catalyzed Reaction
E+S ES ⇀ E + P E = Free Enzyme; S = Substrate; ES = Enzyme substrate complex; P = Product
Michaelis -Menten Equation

D. Nucleic Acid
Nucleic Acid
• Nitrogenous base is the most fundamental component
• Nucleoside: Base attached to pentose sugar
• Nucleotide: Nucleoside with one or more phosphate
• Nitrogenous Base ➝ Pentose Sugar ( 𝛽 -N- glycosidic bond)
• Pentose Sugar ➝ Pentose sugar (in polynucleotide ) Phosphodiester bond
• Additional phosphates are linked by acid anhydride bonds in mononucleotide
• Nucleic Acid are polymers of nucleotide
• Molecular repositories for genetic information
• Protein structure and cell constituents are products of information programmed into the nucleotide sequence of a
cell’s nucleic acid

Nitrogenous Bases
• Pyrimidine - Composed of a 6 membered ring only
• Uracil - Carbonyl group (C=O) at C2 and C4
• Cytosine - Amino (NH2)group at C4, Carbonyl group (C=O) at C2
• Thymine - Methyl (CH3)group at C5, Carbonyl group (C=O) at C2 and C4
 •Purine - Composed of a 6 membered and a 5 membered nitrogen containing ring fused together.
•Adenine - Amino (NH2) group at C2
•Guanine - Amino (NH2) group at C2, Carbonyl (C=O) group at C6

DNA
• The genetic information stored in the DNA’s nucleotide sequence serves as template for
• The information for synthesis of all proteins in cells and the entire organism (transcribed)
• The information inherited by the daughter cell or offspring (replicated)
• Replication occurs in semi-conservative manner
• DNA specifies the nucleotide sequence of the RNA which in turn dictates the amino acid sequence of every
protein
• Gene: DNA segment that contains the information required for biosynthesis of a specific RNA and a specific
protein
• Double stranded
• May be linear or circular
• Formed by nucleotides joined together by 3’-5’ phosphodiester linkage forming the backbone
• Strands are Anti-Parallel:
3’ ————————————-5’
5’ ————————————-3’
• Chargaff’s Rule: A-T (2 Bonds); G-C (3 Bonds)
• Number of Purines = Number of Pyrimidine
• Template or Non- Coding Strand - Where genetic information resides; transcribed
• Coding Strand - Opposing complementary strand
• Double helix structure was first proposed by James and Francis Crick
• Grooves serves as attachment for regular protein
• DNA occurs in 6 forms but the DNA within the human cells normally are in B-configuration
• B-DNA - Most stable
• Z-DNA the only left handed structure
• A-DNA
• C-DNA
• D-DNA
• E-DNA

DNA Central Dogma

Replication
• Duplication of DNA prior to cell division for inheritance of each daughter cell
• Helicases separate DNA strand
• Topaisomerase corrects for supercoiling
• Type
• Type I - Makes transient in one strand
• Type II - Makes transcient in both strand
• Primase
• DNA Polymerase
• DNA with 3’ end near replication fork is called the Leading strand
• Lagging strand/ Okazaki fragment has its end near the replication fork
• DNA polymerase remove primers
• DNA polymerase I has proof reading abilities
• DNA polymerase II has repair capabilities
• Ligase
 Transcription:
• Initiation
• The region of DNA that serves as a transcription initiation site is called a “Promoter”
• In bacteria, several genes are often co-transcribed from a single promoter (rarely happens in eukaryote)
• Prokaryotic structural genes which are transcribed as a unit along with their regulatory element called operon
• Actinomycin-D is a sample drug that intercalates between two GC base pair

• Elongation
• Sequential addition of ribonucleotides to the growing RNA strand via RNA polymerase
• Unlike the DNA polymerase, RNA polymerase do not need primer
• RNA is synthesized in 5’ to 3’ direction from the DNA template strand/ antisense/ nonsense/ non-coding strand

• Splicing
• shnRNA is composed of introns (inactive) and exon (active; required for translation)
• Introns are removed and the exons are linked together by the splicesome

• Relationship between Nucleic Acid & Protein Sequence

• Only one of two strands of DNA is copied into RNA by the RNA polymerase
• The protein is made in the N terminus to C terminus direction

DNA Coding Template 5’ - ATG CCA GTA GGC CAC - 3’

DNA Template Strand 3’ - TAC GGT CAT CCG GTG - 5’
mRNA 5’ - AUG CCA GUA GGC CAC - 3’
Protein N - Met- Pro- Val- Gly- His - C

• Genetic Code & Amino Acid

•The ribonucleotides code for each amino acid (each triplet is called Codon)
• Most of the amino acid can be encoded by more than one codon (The genetic code is degenerated)
• Three of the codon functions as translation stop signal

Types of Mutation
• Mutation affects the amino acid sequence and the function of the protein
• Point - A single base change
• Silent - A change that specifies the same amino acid (eg CGA➝ CGG)
• Missense - A change that specifies a different amino acid (eg CGA ➝CCA)
• Non-sense - A change that produces a stop codon (CGA ➝ UGA)
• Insertion Frameshift - An addition of one or more bases
• Deletion Frameshift - A loss of one or more bases
• Transitional - A purine replaces another purine or a pyramidine replaces another pyramidine
• Transversional - A purine replaces a pyramidine and vice versa.

E. Carbohydrates
Carbohydrates
• Hydrates of Carbon
• Polyhydroxy aldehyde or polyhydroxy ketone or substance that yields these compounds
• "Polyhydroxy" means it contains more than one hydroxyl group
• Polydroxy groups account for sweet taste

Chemistry of Carbohydrates
• Stereoisomer - Compounds with the same formula and attachment of atom but with different arrangement of
atom in space
• Enantiomer - Stereoisomer in which one isomer is the non-superimposable mirror image of the other
• L-Isomer - hydroxyl group (-OH) farthest from the carbonyl carbon is on the Left
• D-Isomer - Hydroxyl group (-OH) farthest from the carbonyl carbon is on the Right; Cells prefer this
configuration
• Epimer - Stereoisomer which differ in the arrangement of substituent in only one position. In monosaccharide
epimers, the position of -OH distinguishes the monosaccharide. The carbonyl group remains the same.
 • Epimerase - A type of enzyme responsible for inerconversion of epimer

Aldose & Ketose

• A monosaccharide can either be an aldose or a ketose based on the position of the carbonyl
• The carbonyl carbon is also known as most oxidized carbon
• Aldose - Carbonyl carbon is a position 1 (Aldehyde)
• Ketose - Carbonyl carbon is at any other position (Ketone)

Carbohydrate Formula
• Fischer Formula - Linear formula which can be depicted in the D or L form

• Haworth Formula - The cyclic form of the Fischer formula which can be depicted in the alpha and beta form
• Furanose - Five membered ring (eg Fructose)
• Pyranose - Six membered ring (eg Glucose); in biological solution, this is the most stable form of glucose
• The cyclic structure is a hemiacetal formed by a reaction between the aldehyde group and a hydroxyl group (R-
OC-OH)
• During cyclization, the carbonyl carbon transforms into a new stereocenter referred to as the anomeric center
• ocate the oxygen in the ring. There would be a carbon on either side of the oxygen. The carbon with the attached
-OH group is the anomeric carbon
• Depending on the position of the -OH group, anomeric carbon can either be in the alpha or beta form
• Alpha - OH group axial Down
• Beta - OH group axial Up

Classification of Carbohydrates
1. According to the Number of Monosaccaharide Unit Linked
• Monosaccharide - Cannot be hydrolyzed into simple carbohydrate unit. Basic carbohydrate unit of
cellular metabolism
• Disaccharide - Yields two monosaccharide units linked together. Often used by plant or animal to
transport monosaccharide from one cell to another
• Monosaccharide and Disaccharide generally end in "ose". They are water soluble carbohydrate which
characterize them as sweet taste and are called "sugar"
• Oligosaccharide - Two to six monosaccharide units linked together. The word "Oligo" means small or few.
Free oligosaccharide that contains more than 2 monosaccharide are rarely found in nature
• Polysaccharide - Macromolecule substance that can be hydrolyzed to yield monosaccharide unit.
Important for structural support, particularly in plant and also as storage depot for monosaccharide
which cells use for energy
2. According to the Number of Carbons
• Triose - C3H6O3
• Tetrose - C4H8O4
• Pentose - C5H10O5
3. According to the Functional Group
• Aldose - Aldehyde (-CHO) group is present
 • Ketose - Ketone ( C=O) group is present
4. According to the Spatial Orientation
• D-Isomer - Hydroxyl group (-OH) farthest from the carbonyl carbon is on the Right
• L-Isomer - Hydroxyl group (-OH) farthest from the carbonyl carbon is on the Left
5. According to Rotation in Plane of Polarized Light
• (+) - To the Right
• (-) - To the Left

Importance of Carbohydrates
1. Carbohydrates are very effective energy yielding nutrients
2. Carbohydrates can serve as very effective building material
3. Many carbohydrates are important water soluble molecules

Monosaccharide
• Glucose
• AKA Blood Sugar or Physiological Sugar
• Most abundant and important monosaccharide
• Occurs in the form of Beta D Glucose
• Synthesized by animals from glycerol and amino acid, but most is derived ultimately from plants
• Synthesized by plants from carbon dioxide and water (Photosynthesis)
• Stored as starch by plants or used to synthesize cellulose
• Stored as glycogen in the liver and muscle of animals
• Converted to sorbitol in vivo in diabetic patients causing cataract
• Precursor for all other carbohydrates in the body
• Produced from complete hydrolysis of starch and in hydrolysis of maltose, sucrose and lactose

• Fructose
• AKA Levulose; Fruit sugar
• Constituent of table sugar and inulin
• Sweetest monosaccharide
• Can be converted to glucose in the liver
• Fructose intolerance leads to hypogycemia

• Glyceraldehyde/Glycerose & Dihydroxyacetone

• Important intermediate of glycolysis
• Precursor for glycerol

• Ribose
• Component of nucleic acid and co-factor
• Co-Factor- Organic molecule that helps the enzyme

• Xylulose
• Found in cases of pentosuria
Xylulose (Xylose Reductase) --> Xylitol (Sugar alcohol)

• Galactose
• Produced from hydrolysis of lactose
• Can also be converted to glucose in the liver & metabolized
• Biosynthesized in the mammary glands
• Failure to metabolize causes galactosemia and further, cataract

• Arabinose
• Found In gum arabic, in vivo in glycoprotein

• Xylose
• Found in wood gum
• Obtained by boiling corn cobs
• Diagnostic aid for malabsorption studies treatment

Disaccharide
• Sugars which yield 2 same or different monosaccharide units/residue on hydrolysis
• Maltose
• Glucose + Glucose
• AKA Malt sugar, Beer Sugar
• Product of partial starch hydrolysis by amylase

• Cellobiose
• Product of partial cellulose hydrolysis

• Sucrose
• AKA Table sugar, Beet sugar, Saccharum
• Most common disaccharide
• Also called invert sugar since on hydrolysis optical activity is inverted
• Glucose + Fructose
• Non-reducing sugar
• Lactose
• AKA Milk sugar
 • Alkali rearrangement produces lactulose
• Deficiency both diarrhea & flatulence
• Lactase - Breaks down lactose
• Sucrase - Breaks down sucrose

Trisaccharide
• Maltotriose
• 2 Glucose units

• Raffinose/Melitose/Melitriose
• 1 glucose + 1 fructose + 1 galactose unit

• Gentianose
• 2 glucose + 1 fructose unit

Polysaccharide
• Agar
• Sulfated galactose units; from seaweed

• Inulin
• Polyfructan and used in the evaluation of GFR

• Dextrin
• Polyglucan intermediary product of starch hydrolysis

• Dextran
• Plasma expander
• Polyglucan synthesized from the action of non-pathogenic gram positive cocci on sucrose

• Chitin
• Principal component of the exoskeleton of arthropods, crustaceans and some mushroom

• Peptidoglycan/Murein
• N-acetylglucosamine + N-acetylmuramic acid

• Hyaluronic Acid
• N-acetylglucosamine + glucoronate unit; tissue barrier contributor, joint lubricant and shock absorbent

• Hyaluronidase
• Clinically used to increase absorption of solution administered by clysis (IM & ID)

• Heparid
• Anticoagulant mucopolysaccharide
• Activates antithrombin

• Chrondroitin
• N-acetylgalactosamine + glucoronate unit
• For osteoarthritis

Starch vs. Glycogen

Starch Glycogen

Storage for of CHO in plants Storage form of CHO in animal

Composed of 2 polymeric glucose unit Resemble amylopectin

(amylose & amylopectin)

Amylopectin branching Branching frequency 8-12 / 10-15 glucose

Freq. 25-30 glucose unit unit

Forms deep blue complex of starch - I2 Forms a deep red color with I2 solution
with Iodine solution due to amylose

Carbohydrate Digestion
• MoNosaccharide are directly absorbed
• Disaccharide requires enzyme of the small intestinal surface for hydrolysis into monosaccharide
• Polysaccharide depends on pancreatic amylase and intestinal surface enzyme for digestion
• Polysaccharide and disaccharide needs to be hydrolyzed before absorption can occur
 Chemical Test for Carbohydrates
• Molisch Test / Alpha Naphthol Reaction
• General test for CHO`s involving the formation of furfurals or hydroxyfurfurals
• Uses alpha-naphthol and concentrated sulfuric acid
• (+) Purple ring at the junction of the two liquids
• Anthrone`s Test
• Also a general test for carbohydrate
• (+) Green or Blue green colored solution
• Benedict`s Test
• Test for reducing sugar (mono & di except sucrose)
• Uses CuSO4, Sodium citrate (chelating/sequestering agent that prevents premature reduction of Cu+2)
• (+) Brick Red ppt
• Nylander`s Test
• Test for reducing sugar
• Bi(OH)2NO3
• (+) Black ppt
• Barfoed`s Test
• Test for reducing monosaccharide
• Uses Cu(CH3COO)2 , CH3COOH
• (+) Brick Red ppt
• Mucic Acid Test/ Galacturonic Acid
• Specific Test for Galactose
• Uses conc HNO3
• (+) Sandy Crystal
• Fehling`s Test
• Test for reducing sugar
• Uses CuSO4, KNaC4H4O6 (Chelating/Sequestering Agent)
• (+) Brick Red ppt
• Seliwanoff`s Test/Resorcinol Test
• Specific Test for pentose
• (+) Red Color
• Bial`s Test/ Bial`s Orcinol Test
• Specific test for Ketose
• (+) Green Color with ppt
• Osozone Formation/Kowarsky Reaction
• Means of producing precipitated sugar derivative especially mannose
• Tauber`s Test/ Aminoguanidine Reaction
• Also. A ketose test
• (+) Bright Reddish Purple Color
• All test for reducing sugar are affected by Vitamin-C, Glucoronidase

F. Lipids
Lipid
• Biopolymersrelated by their physical rather chemical properties
• Ester of long chain fatty acid and glycerol; including fats, waxes and fixed oil
• Principal stored form of energy

Simple Lipid
• Ester of fatty acid with various alcohol
• Fats - Ester of fatty acid with glycerol. Fixed oil are fats in liquid state
• Waxes - Ester of fatty acid with high molecular weight (HMW) monohydric alcohol

Complex Lipid
• Contains other groups in addition to the alcohol & the fatty acid
• Phospholipid
• Contains fatty acid, glycerol or sphingol/sphingosine, phosphate and nitrogenous compound
• Basic unit: Phosphatidic acid
• Major component of cell membrane (eg glycerophospholipid, sphingolipid)

• Lecithin
• Phosphatidyl choline
• Most abundant phospholipid of the cell membrane
• Dipalmitoyl lecithin - A major constituent of the surfactant that prevents the adherence of the inner
surface of the lungs together
• Essential for blood clotting
• Phospholipase - Enzyme that hydrolyzes phospholipid into fatty acid and other lipophilic substance
• PLA1, PLA2, - Gives lysophospholipid
• PLC - Removes glycerol-PO4, gives diacylglycerol
• PLD - Removes nitrogen containing compound, gives phosphatidate
 • Other Important Phospholipid
• Phosphatidylinositol - Precursor for secondary messenger
• Phosphatidylglycerol - Precursor for cardiolipins, the major lipid of the mitochondria
• Plasmalogen - Resembles cephalin and are found in the brain and muscle, the only difference is that instead
of an ester group is present and the alkyl group is unsaturated
• Sphingomyelin - Made up of fatty acid, sphingol, choline and phosphate. Building block of nervous tissue
membrane
• Sphingoside - Plus fatty acid gives ceramide

• Glycolipid/ Glycosphingolipid
• Contains fatty acid, sphingol and carbohydrates
• Ceramide - Is a basic unit
• Galactosylceramide - In neural tissue and glucosylceramide in extraneutral tissue
• Cerebroside
• Glucose/galactose + ceramide
• Lipids with glucose/galactose or both + 1 fatty acid + sphingosine with no PO4 and glycerol
• Gangliosides
• Glucose/galactose + ceramide + neuramic acid
• Lipid with glucose/galactose + 1 fatty acid + sphingosine with one or more molecule of neuraminic acid
• Cytolipins
• Glucose/galactose + fatty acid + sphingosine
• Globoside
• Contains N-acetylglucosamine or N-acetylgalactosamine

Fatty Acid
• Building blocks for triglyceride and phospholipid
• Obtained from hydrolysis of fats
• Biosynthesized from acetyl CoA
• Saturated Fatty Acid - Are regular carboxylic acid having even number of C atoms and as components of lipid,
they impart high melting point
• Unsaturated Fatty Acid - Can be monosaturated or polysaturated and as components of lipid they impart low
melting point
• Linoleic Acid - Dietary precursor for arachidonic acid
• Linolenic Acid - Omega 3
• Arachidonic Acid - Precursor for eicosanoid
• Timnodonic Acid - Important component of fish oil
• Cervonic Acid - Important component of fish oil

• Triglycerol
• Triester of fatty acid with glycerol
• Storage form of fatty acid

Basic Chemical Properties of Lipid

1. Hydrolysis
2. Saponification - Alkali hydrolysis of lipid forming alkali metal salt of the fatty acid
3. Oxidation to aldehyde & ketone
4. Rancidity - Production of unpleasant taste and odor due to liberation of free fatty acid usually due to
moisture, high temperature and hydrolytic agent

Fat Alcohols
1. Glycerol/ Glycerin
• Simplest trihydric alcohol
• ID Test: Acrolein Test (heating with KHSO4); (+) Pungent Odor
2. Sterol
• All have a cyclopentanoperhydrophenanthrene (CPPP) ring
Cholesterol
• Occurs as unsaponifiable matter in lipid sample
• Synthesized from Acetyl CoA
Other Sterol
• 7-Dehydrocholesterol (Pro-Vitamin D3)
• Present in skin; precursor for vitamin D3 (Cholecalciferol)
• Ergosterol (Pro-Vitamin D2)
• Present in fungi; precursor for vitamin D2 (Ergocalciferol)
• Stigmasterol/Phytosterol/Sitosterol
• Present in plants, no nutritional value but appears to decrease blood cholesterol
 Chemical Test for Sterol
• Liebermann-Burchard Test (Acetic Acid- H2SO4 Test)
• (+) Red →Blue→Bluish Green colored solution
• Salkowski Test (Sulfuric Acid Test)
• (+) Bluish Red → Cherry Red & Purple color of CHCl3 layer, green fluorescence in the acid layer

Digestion and Absorption

• Cholesterol
• Triglycerol → Monoacylglycerol + 2 fatty acid
• Phospholipid → Lysophospholipid + fatty acid
• Particles emulsified by bile acid & transported into the intestinal cell
• Chylomicron - TAG’s to peripheral tissues
• VLDL - TAG’s to peripheral tissues
• LDL - Cholesterol to tissues
• HDL - Cholesterol from tissue to the liver

G. Generation and Storage of Metabolic Energy

Introduction to Metabolism
• Sum total of all the reactions a compound undergoes from the time it enters the body until its product are
excreted, directed towards maintenance of life
• Catabolism Reaction
• Complex molecule are broken down
• Energy producing
• Makes NADH in support of ATP synthesis
• Makes NADPH for use in anabolism
• Anabolism Reaction
• Makes complex molecules
• Energy requiring
• Use NADPH as source of reducing equivalents
 Bioenergetics
• Study of energy transformation accompanying biochemical reaction in the living cell
• The ATP synthesis captures free energy from catabolic pathways and provides energy for anabolism movement,
ion gradient, etc
• Autotropic organism use simple exergonic procedure to produce energy (energy from sunlight)
• Heterotropic organism obtain energy by breakdown of complex organic molecules
Net Biological Synthesis of ATP
1. Substrate Level Phosphorylation
• Cytosolic (phosphoglycerate kinase & pyruvate kinase)
• Mitochondrial (succinyl CoA synthesis)
2. Oxidative Phosphorylation
• Greatest ATP source in aerobic organism
• Energy is generated by respiratory chain
Glycolysis
• Embden - Mayerhoff Pathway
• Occurs in the cytoplasm
• Major pathway for carbohydrates` catabolism in human tissue
• Functions:
• Converts energy stored in glucose to ATP
• Produce intermediate required for biosynthesis of: Glucose, Pentose, TAG`s, Amino Acid
• Produce pyruvate molecule which can be further oxidized to produce more ATP when Oxygen is available
while reduced to lactate in the absence of oxygen

• ATP Consumption:
• Hexokinase Reaction 1
• PFK Reaction 1
2
• ATP Generation
• Gly-3-PO4 Dehydrogenase Rxn 6
• PG kinase Reaction 2
• Pyruvate Kinase Reaction 2
10
 4 Fate of Pyruvate Anaerobic Conversion of Pyruvate to Lactate in Actively Contracting Muscle
• NADH Is normally re-oxidized to NAD+ in the mitochondrion, but in the absence of oxygen, it is re-oxidized by
reducing pyruvate to lactate
• Glycosis is erythrocytes, always leads to lactate formation both in aerobic and in anaerobic condition

Insulin vs Glucagon
Insulin
• Glucokinase, Phosphokinase-1 (PPK-1) and dependent kinases are induced by insulin
Glucagon
• Induces cAMP, which in turn activates a cAMP-dependent kinase which inhibits glucokinase and pyruvate
kinase
• Decrease F-2,6-bisphosphate synthesis and increases F-2,6 bisphosphate degradation
• Release from the pancreas into the blood when the blood glucose level are low, activating gluconeogenesis and
glycogenolysis
Clinical Aspect
• Aldose And pyruvate kinase deficiency leads to hemolytic anemia
• Muscle PFK-1 deficiency leads to low exercise tolerance particularly after high carbohydrate diet
• Glucosuria does not necessarily indicate DM

Gluconeogenesis
• Synthesis of glucose from non-carbohydrate precursor
• Alanine & other glucogenic Amino Acid (except Leu & Lys)
• Lactate & Pyruvate
• Glycerol

Citric Acid Cycle

• Enzymes of the reaction are either free or attached to the inner mitochondrial membrane
• Oxidized acetyl residues and residue coenzymes that can be re-oxidized to the formation of ATP
• Glucose, fatty acids and most amino acids are metabolized to acetyl CoA or intermediates of the cycle
• It also has a central role in gluconeogenesis, lipogenesis & interconversion of amino acid
• RBC`s are the only cell that lacks mitochodria
 Bioenergetics
• Isocitrate Dehydrogenate Reaction 6 ATP
• Alpha ketoglutanate Dehydrogenase Reaction 6 ATP
• Succinyl CoA Synthetase Reaction 2 ATP
• Malate Dehydrogenase Reaction 6 ATP
24 ATP
Shuttle System
• Utilized by cells to transfer NADH from glycolysis into mitochondria for ETC
• Glycerophosphate Shuttle
• Generates 2ATPs per FADH2 oxidized in ETC
• Maleate Aspartate Shuttle/Malate Shuttle
• Generates 3ATPs per NADH oxidized in ETC
Anaplerotic Reaction
• Replenish citric acid cycle intermediate
• Pyruvate carboxylase is a major anaplerotic reaction in mammalian tissue
Pyruvate + CO2 + ATP +H2O → Oxaloacetate + ADP + P1
Electron Transport Chain (ETC & Oxidative Phosphorylation)
• A pair of electrons passes from NADH to O2 through complexes I, III & IV (Enough protons are pumped to
make 3 ATPs
• A pair of electron passes from FADH2 to O2 through complexes II, III & IV (Enough protons are pumped to
make 2 ATPs
• Involves 4 protein complexes linked by mobile electron carrier CoQ/Q and cytochrome C
• Complex I: NADH CoQ oxidoreductase (Inhibited by Barbiturate)
• Complex II: Succinate CoQ oxidoreductase
• Complex III: CoQ-Cytochrome C (Oxidoreductase/Cytochrome Reductase)
• Complex IV: Cytochrome C-O2 (Oxidoreductase/ Cytochrome Reductase)
• H2S, CO & CN- inhibits complex IV’s function
• Oxidative Phosphorylation
• Complex V: ATP Synthase - ATP synthase uses the proton gradient energy for the synthesis of ATP.
Composed of a “Knob & Stalk” structure

Pentose Phosphate Pathway

• Alternative pathway of glucose metabolism but is not ATP generating
• Important Product of PPP
1. Interconversion of Pentose & Hexoses
2. Production of NADPH for reductive biosynthesis of Fatty Acid, cholesterol & other steroid, nucleic acid
3. Formation of Ribose 5-phosphate for ribonucleotide
• Enzyme of the pathway are cytosolic
 Oxidative Stage
• Three molecules of G-6-P are converted to 3 molecules of ribulose-5-phosphate with NADPH product
• G6PD deficiency causes low level of reduced GSH stores, essential for removing H2O2 from erythrocytes

Non-Oxidative Stage
• 3 molecules of ribulose-5-phosphate are converted to 2 molecule of fructose-6-phosphate and one
molecule of glyceraldehyde-3-phosphate

The Cori Cycle

• Interaction of glycolysis & gluconeogenesis
• Lactate from from peripheral tissues goes to liver & is converted into glucose
• Glucose can go back to the peripheral tissue
• Liver uses lipid for energy

Glycogenesis
• Pathway consist of :
• Glucose transporter
• Hexokinase (Muscle) or Glucokinase (Liver)
• Phosphoglucomutase
• UDP Glucose Phyrophosphorylase
• Glycogen Synthase
• Branching Enzyme
Glycogenolysis
• Consist of:
• Glycogen Phosphorylase
• Debranching Enzyme
• Phosphoglucomutase

Regulation of Glycogen Metabolism

Glycogen Synthase
• Inhibited by epinephrine, norepinephrine, glucagon via cAMP
• Insulin inhibits Phosphodioesterase
Glycogen Phosphorylation
• Activated by epinephrine, norepinephrine, glucagon via cAMP
• Insulin inhibits activation of Glycogen Phosphorylase

Beta-oxidation of Fatty Acid

• Occurs in heart, skeletal muscles, liver
• Enzymes are located in the mitochondria
• Two carbon atoms at a time are removed from the fatty acid
• Involves 3 steps: 1. Activation, 2. Transport in mitochondria 3. Oxidation
 ATP Generation from Beta Oxidation
Palmityl CoA + 7 HS CoA + 7FAD + 7NAD+ + 7H2O → 8 Acetyl CoA + 7FADH2 + 7NADH+ + 7H+
Acetyl CoA (TCA Cycle) 8 x 12 =96
In Oxidative Phosphorylation
FADH2 = 2 ATP’s 7 x 2 = 14
NADH = 3 ATP’s 7 x 3 = 21
- 2 (for activation)
129 ATP / Palmitic acid

Beta Oxidation of Odd Chain Fatty Acid

• Odd chain fatty acid occurs in bacteria and microorganism
• Final cleavage product is propionyl CoA rather than acetyl CoA
• Propionyl CoA is converted to succinyl CoA