14 Proteins

Amino acids

Organic compounds containing at least one amino

group (-NH2), one carboxyl group (-COOH) bonded to
the alpha-carbon and one R group.

Amino acid Pool

Sources
1. Dietary (Nutritionally essential)
e.g., valine, leucine, isoleucine, methionine,
tryptophan, phenylalanine, threonine, lysine
and histidine.
2. Endogenous synthesis
3. Normal protein turnover
4. Renal tubular reabsorption
Uses
1. Endogenous protein synthesis
2. Energy source
3. Formation of NPN compounds
E.g., creatine, purines, pyrimidine.
Proteins
Structure
Composed of up to 20 different primary L-alphaamino acids in characteristic combinations, numbers
and sequences determined by genetic code.
Covalently linked in a head to tail manner through
peptide bonds
Classification
1. Simple contain only amino acids
o may be globular (eg.,hemoglobin, enzymes)
or fibrous
2. Conjugated
o composed of protein (apoprotein) and a
non-protein substance
o Examples: lipoproteins, glycoproteins,
metalloproteins
Metabolism
1. Synthesis by hepatocytes and plasma cells as
specified by the DNA
2. Catabolism in the GIT, kidneys and liver (hydrolysis,
deamination).
Protein Catabolism

Laboratory Evaluation of Proteins

3 General Methods
1. Quantitation of TP and Alb by routine chemical methods
2. Semi-quantitation of serum and other body fluid proteins
by routine and/or special electrophoresis techniques
i.e., immunoelectrophoresis (IEP)
3. Quantitation of normal and abnormal specific protein by
immunoassay methods
i.e., nephelometry, turbidimetry and
radial immunodiffusion (RID).
Total Protein Methods
Serum
1.

2.

Biuret method
In alkaline medium, cupric ions
form a violet colored chelate with
peptide bonds. The color intensity
increases proportionately with the
number of peptide bonds present.
Refractometry
The velocity of light changes when it passes
between air and water, causing light to bend
(refract). The refractive index of water increases
proportionately with the concentration of solute
in the solution.

Urine and CSF

1. Turbidimetry / Nephelometry
Protein denaturing agent
precipitates proteins with trichloroacetic acid,
sulfosalicylic acid or benzethonium chlorides.
2. Dye Binding
Pyrogallol Red or Coomassie Brilliant Blue binds
to protonated amine groups of amino acids, with
absorption at 595 nm. Binding increases the
absorbance of the dye proportionately with
concentration.
Albumin Methods

Amino Acid

Serum
1.

Deamination
Ketoacids

NH3

Citric acid cycle

Urea cycle

Fats

Functions
1. Transport: lipids, metal, vitamins, toxic substances
2. Antibodies
3. Buffers
4. Coagulation factors
5. Regulation of metabolism
6. Hormones
7. Chromosomes
8. Plasma oncotic pressure
9. Nutrients

Glucose

Urea

Dye Binding

Serum, Urine and CSF

1. Nephelometry
2. Protein Electrophoresis
Protein separation based on their net electrical
charges, size, properties of the support medium
and temperature of operation.
Routine electrophoresis and High Resolution
electrophoresis (PAGE, 2-DE)
rainwater@mymelody.com || 1st semester, AY 2011-2012

Specific Protein Methods

Evaluating Test Results

Serum, CSF and urine

1. Immunochemical Methods
a. Nephelometry immunoglobulins react with a
specific antibody producing an
immunoprecipitate. Forward light scatter is
measured.
b. Immunodiffusion in an agarose gel saturated
with specific antibody solution, serum (antigen)
is applied. A precipition ring forms at the point
where antigen and concentrations are equal.
Ring diameter correlates with antigen
concentration.
2. Immunoassays
a. Immunoelectrophoresis Proteins are
electrophoresed into 5 zones similar to SPE.
Antibody is then added to produce precipitin
arcs that are visually interpreted.
b. Immunofixation electrophoresis Proteins are
electrophoresed into 5 zones similar to SPE.
Monospecific antiserum is then added. The
support medium is stained for visual
interpretation of the bands.

Diagnostic Utility of Total Protein

1. Serum- Nutritional status and presence of severe
disease status (Liver, kidney, bone marrow).
2. Urine Glomerular integrity, renal tubular function
and presence of overflow of plasma proteins.
3. CSF BBB integrity, increased CNS protein synthesis
and CNS infection.
4. Other body fluids Transudate VS exudate

Alterations of Serum Total Protein

Hyperproteinemia
Dehydration
Monoclonal or polyclonal gammopathies

Renal and Nonrenal Proteinuria

Type of Proteinuria
Renal
Glomerular
Tubular
Non-Renal
Overload

Hypoproteinemia
Protein loss
Nephrotic syndrome, blood loss, burns
Protein catabolism
Inflammation
Malignancy
Protein synthesis
Liver disease
Amino acid intake

Defect

Diseases

Glomerular permeability
Defective reabsorption at proximal tubules

Glomerulonephritis
Nephrotoxicity
Metabolic disturbances
Chronic pyelonephritis

Overflow of abnormal protein from plasma

Multiple myeloma
Hemolytic disease
Functional: heavy exercise, fever, pregnancy
Orthostatic proteinuria
Inflammation
Malignancy

Physiological Nonpathologic

Glomerular permeability

Postrenal

Protein produced in urinary tract

Alterations of CSF Total Protein

CNS Defect
Conditions
Blood-Brain Barrier Intracranial pressure
Permeability
Brain tumor
Intracerebral hemorrhage/ trauma
Meningitis (especially high in bacterial meningitis)
Encephalitis
Obstruction of CSF circulation
Tumor above puncture site
CNS Synthesis (IgG) Demyelinating CNS diseases
Multiple Sclerosis
Chronic CNS inflammatory diseases
Guillain-Barr syndrome
Chronic meningoencephalitis
Neoplasia

Evaluation Method
CSF/serum albumin index
AlbuminCSF (mg/dL)/ AlbuminSER (g/dL
Evaluates the integrity of the blood-brain barrier
<9 = intact
9-14 = slight impairment
30-100 = severe impairment
<100 = complete breakdown
CSF-serum IgG-albumin index
IgG CSF (mg/dL) x albuminSER (g/dL)/
AlbuminCSF (mg/dL) x IgGSER (g/dL)
Increased index (>0.70) indicates CNS
synthesis

rainwater@mymelody.com || 1st semester, AY 2011-2012

Total Protein in Other Body Fluids

Fluid Type
Total Protein Concentration
Transudate
<3 mg/dL
Exudate
>3 mg/dL

Defect
Reflects changes in filtering membrane permeability
Usually the result of infection or malignancy and may contain WBCs or malignant
cells

Alterations of Serum Albumin

Hyperalbuminemia
Dehydration
Hypoalbuminemia
Albumin loss (Nephrotic syndrome, blood loss, burns)
Albumin catabolism (Inflammation, Malignancy)
Albumin synthesis (Liver disease, Amino acid intake)
Altered albumin distribution into extravascular compartment (Ascites fluid)
URINE ALBUMIN
Protein marker of glomerular permeability
CSF ALBUMIN
Protein marker of BBB permeability
Common SPE Patterns
Pattern
Normal SPE pattern
(Normal pattern is dotted)

Hypogammaglobulinemia

Comment
Used to compare with the patients pattern to detect
changes in peak slope or shape.

The gamma region is almost absent.

If severe, it is called agammaglobulinemia.

Monoclonal gammopathy

Marked, single spike in gamma region.

Caused by multiple myeloma, heavy chain disease, or
Waldenstrms macroglobulinemia.

Nephrotic syndrome

These diseases are indistinguishable on SPE; further

laboratory testing by immunoelectrophoresis (IEP) or
immunofixation (IF) is needed to identify abnormal
immunoglobulins
in most serum proteins
A2 fraction
Nephrotic syndrome generally results in a loss of
most of the proteins except -macroglobulin
because it is a very large protein

Active hepatocellular
damage (cirrosis)

IgA
Beta-gamma bridging, or fusion of the beta and
gamma regions, is very characteristic for cirrhosis.
Pattern suggests IgA, which migrates in the
valley between beta and gamma.

rainwater@mymelody.com || 1st semester, AY 2011-2012

Characteristics and Clinical Significance of Selected Plasma Proteins

SPE Region
Proteins
MW
Function
(kD)

Acute
Phase
Reactant
(-)

Clinical Significance

Prealbumin

Prealbumin

55
21

Albumin

Retinol Binding
Protein (RBP)
Albumin

1-Antitrypsin
(AAT)

53

1-Acid
Glycoprotein
1-Lipoprotein
(HDL)
1-Fetoprotein
(AFP)

44

Haptoglobin
(HAP)

85 1000

Principal fetal protein,

no known adult
function
Binds and transports
free hemoglobin

2-Macroglobulin
(AMG)
Ceruloplasmin
(CER)
Transferrin (TFR)

800

Unknown

---

160

(+)

77

Involved in copper
metabolism
Transports iron

Hemopexin (HPX)
-lipoprotein
(Apoprotein B)
C4

57
~3000

Binds circulating heme

Transports lipids

-----

206

Immune system factor

(+)

May only be found on electrophoresis when

fresh serum is used.

Fibrinogen

340

Coagulation factor

(+)

C4

180

Immune system factor

(+)

2-Microglobulin
(BMG)

11.8

(+)

C-Reactive
Protein (CRP)

~120

Unknown, found on
cell surfaces of
nucleated cells,
particularly WBCs and
tumor cells
Immune function

Artifact on SPE when plasma specimen is used

instead of serum.
May only be found on electrophoresis when
fresh serum is used.
Indicator of renal tubular function, especially
useful for detection of kidney transplant
rejection. Also useful as a tumor marker to
monitor B-cell tumors.

IgG

160

IgA

170

IgM

9000

66

200
76

Transport protein:
T3, T4
Transport protein:
retinol (Vitamin A)
Transport protein,
maintains oncotic
pressure, source of
endogenous amino
acids

Antiprotease,
prevents tissue
breakdown by
neutrophil proteases
released during
inflammation
APR, exact function
unknown
Transports lipids

Immune function,
most abundant
immunoglobulin
Immune function,
found mainly in
secretions
Immune function,
early response

(-)
(-)

(+)

(+)
----(+)

(-)

(+) (+)

(+)

Band appears on high resolution electrophoresis

methods. Indicator of nutritional status or liver
dysfunction due to very short half-life (12 hr.)
in malnutrition, inflammation, liver cirrhosis.
Indicator of nutritional status or liver
dysfunction;
also a sensitive marker for glomerular and
blood-brain barrier integrity.
in malnutrition, nephrotic syndrome,
inflammation, liver cirrhosis, and many other
nonspecific disorders. may result in edema
and decreased albumin transport function, i.e.,
unexpected drug toxicity.
Makes up 90% of 1 region. Congenital decrease
may cause early onset emphysema or infantile
hepatitis, leading to cirrhosis.

Stains very weakly in SPE owing to

carbohydrate content.
Stains very weakly in SPE owing to lipid
content.
Fetal may indicate neural tube defect.
Adult may indicate hepatocellular tumor.
Monitor APR and hemolytic processes:
in intravascular hemolysis
inflammation (APR), nephrotic syndrome,
burns due to high molecular weight
One of the largest plasma proteins. Dramatic
in nephrotic syndrome
in Wilsons disease
in copper toxicity
in iron defiency anemia (correlates with TIBC)
inflammation (negative APR)
Hemolytic processes

Migrates in the beta-gamma region. Nonspecific,

but most sensitive acute phase reactant. in
inflammation, and used to monitor
inflammatory processes.
Monoclonal
B-cell tumors, i.e., myelomas

(+)

Monoclonal
Liver cirrhosis

(+)

Monoclonal
Waldenstrms macroglobulinemia

rainwater@mymelody.com || 1st semester, AY 2011-2012

10 Clinical Enzymology
Enzymes

Definition
Enzymes are proteins which catalyze
specific biochemical reactions in the
different cells, tissues and organs of
essentially all mammals and which may also
be physically located in the different
organelles and structures within the cell.

Composition
Each enzyme is composed of a specific
amino acid sequence (primary structure)
which results in a stearic arrangement
(secondary structure) that becomes folded
(tertiary structure).

Nomenclature
Each enzyme has 2 names, a practical or
trivial name, eg., Acid phosphatase, and a
systematic name (IUB group, EC no.), eg.
Hydrolase, EC 3.1.3.1
Classification
Class
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases

6. Ligases

Type of Reaction
Catalyzed
Oxidation-reduction
reactions
Transfer of
functional groups
other than hydrogen
Hydrolysis of ether
and ester
Group elimination to
form double bonds
Interconversion of
geometric or optical
isomers
Joining of two
substrate molecules

Examples
LDH, G6PD
AST, ALT
ALP, ACP
Loss of OH ion

Enzyme Specificity
1. Binding specificity
2. Reaction specificity

Factors Influencing Increased Plasma Enzyme Levels

Factor
Mechanism
Example
Enzyme
leakage from
cells

Enzyme
production

Cell stress, through

injury, through
necrosis
Results in varying
degrees of loss of
cell membrane
integrity (which
increases cell
membrane
permeability)
through complete
cell necrosis
Enzyme induction
Increased rate of
intracellular enzyme
production
Proliferation of a
cell type producing
a specific enzyme

AMI results in
hypoxia, leading to
cardiac cell death
and subsequent
necrosis. Cardiac
enzymes (e.g., CK,
LD, AST, are released
over time from the
damaged tissue.)

Drug administration,
e.g., GGT
production with
antiepileptics such
as phenytoin
Ethanol intake
Proliferation of PAPproducing cells in
prostatic carcinoma

Measurement of Enzyme Activity

1. Photometric Enzyme Reaction
e.g., ALP, GGT, LD
2. Coupled Enzyme Reaction
e.g., ALT, AST, AMS
3. Turbidimetric Enzyme Reaction
e.g., LPS
4. Immunoassay RIA, IRMA, IEMA
e.g., PAP, PSA
Measurement of Diagnostic Isoenzymes
1. Electrophoresis
e.g., CK-BB, CK-MB, CK-MM, LD 1 5
2. Immunoassay
e.g., AMS S and P types, LD-1
3. Selective Inhibition
e.g., AMS S type, ALP

Enzyme Cofactors
A group of heat-stable low molecular weight organic
molecules (coenzymes) or inorganic ions (activators)
required for catalysis.
Holoenzyme is the complete catalytic entity formed
by cofactors + protein portion (apoenzymes)
Examples of organic cofactors: Coenzyme A,
Nicotinamide coenzymes, Biotin
Examples of inorganic cofactors: Mg++, Ca++, Cu+
Enzyme Kinetics
Deals with the relationship between the enzyme, the
substrate, and the product
The rate at which substrate disappears as product
appears can be directly used to determine the
concentration of enzyme present

rainwater@mymelody.com || 1st semester, AY 2011-2012

Clinically Significant Enzymes

Enzyme
Acid Phosphatase (ACP)
Alanine Aminotransferase (ALT)

Principal Tissue Source(s)

See Prostatic Acid Phosphatases
Liver, kidney

Principal Clinical Applications

Alkaline Phosphatase (ALP)

Alkaline Phosphatase (ALP)
Isoenzymes
Liver
Bone
Intestinal
Placental
Regan
Amylase (AMS)
Amylase (AMS) Isoenzymes
P types
S types
Aspartate Aminotransferase (AST)

Liver, bone, placenta

Tissue-specific sources

Cholinesterase (SChe)
(Pseudocholinesterase)

Liver

Creatine Kinase (CK)

Creatine Kinase (CK) Isoenzymes
CK-BB (CK-1)
CK-MB (CK-2)
CK-MM (CK-3)
-Glutamyltransferase (GGT)
Lactate Dehydrogenase (LDH)
Lactate Dehydrogenase (LDH)
Isoenzymes
LD-1 (HHHH)
LD-2 (HHHM)
LD-3 (HHMM)
LD-4 (HMMM)
LD-5 (MMMM)
Lipase (LPS)
Prostate-Specific Antigen (PSA)
Prostatic Acid Phosphatase (PAP)

Skeletal and cardiac muscle, brain

Liver
Bone
Intestine
Placenta
Regan-some neoplasia
Pancreas, salivary glands
P types: pancreas
S types: e.g., salivary glands,
ovaries, lung
Liver, skeletal, and cardiac muscle

CK-BB:
brain
CK-MB
cardiac, skeletal muscle
CK-MM: skeletal, cardiac muscle
Liver
Cardiac muscle, liver, skeletal muscle, RBCs
LD-1: cardiac muscle, RBCs, kidney
LD-2: cardiac muscle, kidney, RBCs
LD-3: spleen, kidney, lungs, RBCs
LD-4: spleen, lungs, kidney
LD-5: liver, skeletal muscle
Pancreas
Prostate
Prostate

Clinically Useful Enzyme and Isoenzyme Tumor Markers

Enzyme Isoenzyme
Principal Tissue Source(s)
Alkaline Phosphatase (ALP)
Liver
Bone
Placenta
Amylase (AMS)
Pancreas, salivary glands
Creatine Kinase Isoenzyme
(CK-BB)
Lactate Dehydrogenase (LD)

Hepatic parenchymal diseases, not usually in

renal diseases
Bone diseases, hepatobiliary disease
Identify likely tissue source causing ALP
levels; especially to diffentiate bone from liver
diseases

Pancreatic diseases
Identify likely tissue source causing AMS
levels, especially to rule out pancreatic source in
postoperative AMS.
Acute myocardial infarction (AMI), hepatic
parenchymal diseases, skeletal muscle diseases
Insecticide poisoning (organophosphorus),
suxamethonium sensitivity, rarely used for
hepatic parenchymal diseases
AMI, skeletal muscle diseases
Identify likely tissue source causing CK levels,
especially to rule out AMI
CK-MB isoenzyme quantitation is used for this
purpose.
Hepatobiliary disease, alcoholism
AMI, hemolysis, hepatic parenchymal diseases
Identify likely tissue source causing LD levels;
especially to rule out AMI
LD-1 isoenzyme quantification is used for
this purpose

Pancreatic diseases
Prostatic carcinoma
Prostate carcinoma

Tumor Marker Applications

Primary and metastatic liver, bone malignancies,
hematologic diseases, i.e., leukemia, lymphoma
Some lung and ovarian tumors

Brain, smooth muscle, many other minor

sources including lung, bladder, prostate,
uterus, liver, gut, breast
Cardiac muscle, liver, skeletal muscle, RBCs

Lactate Dehydrogenase (LD)

Isoenzymes

Cardiac muscle, liver, skeletal muscle, RBCs

Neuron-Specific Enolase (NSE)

Neurons, neuroendocrine cells

Prostatic Acid Phosphatase (PAP)

Prostate

Prostate-Specific Antigen (PSA)

Prostate

Many types of malignancies, particularly

prostate, lung (small cell) carcinoma, and
bladder and GI tract malignancies
Many types of primary and metastatic
carcinomas; see specific LD isoenzymes
LD-1: germ cell tumors
LD-3: leukemias
LD-5: breast, lung, stomach, colon tumors
Neuroendocrine system malignancies, such as
lung (small cell), neuroblastoma, carcinoid, and
pancreatic islet cells
Prostatic carcinoma. Not recommended for
screening purposes since it is usually not
significantly until tumor is metastasized. PSA
is preferred marker.
Prostatic carcinoma detection, monitoring, and
staging. Should be used in conjunction with
digital rectal examination for screening men
over 50 yr of age. Most useful for monitoring
therapy and tumor recurrence since PSA may
not be significantly until tumor is grown out of
prostate capsule.

rainwater@mymelody.com || 1st semester, AY 2011-2012