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WEEK 1 4. Most of the chemical reactions in the body

are enzyme catalyzed
ENZYMOLOGY 5. The substance upon which an enzyme
Enzymes acts is called as substrate. By the action
of enzyme, it is converted to product. An
• Enzymes are protein catalysts utilized by enzyme-catalysed reaction consists of
essentially all mammalian cells in specific substrate, enzyme and product
biochemical reactions in different organs
of the body, which may also be physically Enzymes
located in different organelles and • These are proteins produced by living
structures within a cell. cells that hastens chemical reaction in
• Most chemical reactions in the body occur organic matter.
at pH 7 and temperature of 37*C • They are measured in terms of their
Medical and Biological Importance activity and not in terms of their absolute
values.
1. Enzymes are the chemical work horses of
the body. Enzymes are biological catalysts
that speed up the pace of chemical
reactions.
2. A chemical reaction without an enzyme is
like a drive over a mountain. The enzyme • They are large molecules and they are
bores a tunnel through it so that passage normally confined within cells unless
is far quicker and takes much less energy. increased membrane permeability allows
3. Enzymes make life on earth possible, all them to enter the blood.
biology from conception to the dissolution • They frequently appear in the serum after
that follows death depends on enzymes. cellular injury, degradation of cells or
4. Enzymes regulates rate of physiological from storage areas.
process. So, defects in enzyme function • Abnormal large amount of enzymes in
cause diseases. serum are used clinically as evidence of
5. When cells are injured enzymes leak into organ damage.
plasma. Measurement of activity of such • Each enzyme catalyzes a single reaction
enzymes in plasma is an integral part of or a limited number of chemical reaction,
modern day medical diagnosis. and it is specific for a substrate that it
6. Enzymes are used as drugs. converts to a defined product.
7. Immobilized enzymes, which are enzymes
attached to solid supports are used in Factor Affecting Enzyme Reactions
clinical chemistry laboratories and in
1. Enzyme Concentration
industry. For example, glucose in blood or
o The higher the enzyme
urine is detected by using immobilized
concentration, the faster is the
glucose oxidase. In pharmaceutical
reaction, because more enzyme is
industry, glucose isomerase is used to
present to bind with the substrate.
produce fructose from glucose.
2. Substrate Concentration
8. Enzymes are used as biosensors.
o With the amount of enzyme
9. AIDS detection involves use of enzyme
exceeding the amount of
dependent ELISA technique.
substrate, the reaction rate
10. Enzymes are used as cleansing agents in
steadily increases as more
detergent industry
substrate is added.
Chemical Nature of Enzymes (Properties) o However, when the substrate
concentration reaches a maximal
1. All the enzymes are proteins except value, higher concentration of
ribozymes and number of enzymes are substrate no longer result in
obtained in crystalline form. increased rate of reaction
2. In 1878, Kuhne, introduced term (saturation kinetics).
‘Enzyme’ to indicate biological catalyst. 3. Cofactors
3. Enzymes cut big molecules apart and join
small molecules to form big molecules.

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o Nonprotein entities that must bind c. Uncompetitive Inhibitor

to particular enzymes before a - binds an enzyme at a place
reaction occurs. other than the active site
a. Coenzymes – organic (allosteric site) of the
compound (carbon containing) enzyme.
–ex. NADP, cysteine, pyridoxal - -do not compete with the
phospate substrate but look for areas
- increasing coenzyme other than the active site.
concentration will increase the - because the inhibitor binds
velocity of an the enzyme independently
enzymatic reaction. from the substrate,
b. Activators – inorganic ions increasing substrate
(non-carbon containing) – concentration does not
It can be metallic: Calcium for reverse the inhibition.
amylase, zinc for LDH, others: - -the substrate and inhibitor
Fe, Mn, Cu (commonly metallic ion) may
It can be non-metallic: bind an enzyme
Chloride for amylase simultaneously.
4. Inhibitors - -the presence of the
o Enzymatic reactions may not inhibitor when it is bound
progress if an inhibitor interferes to the enzyme slows the rate
with the reaction. of the reaction.
a. Competitive Inhibitor - the inhibitor binds to the
- physically bind to the active enzyme-substrate
site of an enzyme. (ES)complex, so that
- both the substrate and increasing the substrate
inhibitor compete for the concentration results in
same active site of the more ES complexes to
enzyme. which the inhibitor binds
- with a substrate and thereby increases the
concentration significantly inhibition.
higher than the - increasing substrate
concentration of the concentration, increases
inhibitor, the inhibition is inhibition.
reversible. 5. Isoenzymes
b. Non- Competitive Inhibitor o These are enzymes having the
- binds an enzyme at a place same catalytic reaction but slightly
other than the active site different molecular structures.
(allosteric site) of the enzyme. o The importance of the total
- do not compete with the enzyme activity is enhanced by
substrate but look for areas fractionating the isoenzymes
other than the active site. 6. Temperature
- because the inhibitor binds
the enzyme independently
from the substrate,
increasing substrate
concentration does not
reverse the inhibition.
- the substrate and inhibitor
(commonly metallic ion) may
bind an enzyme
simultaneously.
- the presence of the inhibitor
when it is bound to the
enzyme slows the rate of the
reaction.

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o Increasing temperature usually o

2 -8℃= ideal for substrate and
increases the rate of a chemical coenzymes
reaction by increasing the o Room temperature = ideal for
movement of molecules. storage of LDH (LD4 and LD5)
9. Hemolysis
o mostly increases-enzyme
concentration.
10. Lactescent or Milky Specimen
o decreases enzyme concentration.

The effect of substrate concentration [S] on the

velocity (V) of an enzyme-catalyzed reaction.

Michaelis – Menten hypothesis

- THE HIGHER THE SUBSTRATE

CONCENTRATION, THE MORE SUBSTRATE
BOUND TO ENZYME AND THE GREATER THE
RATE/ VELOCITY OF THE REACTION

 Michaelis-Menten equation, accurately

describes virtually all single-substrate
enzyme-catalyzed reactions and many
bisubstrate reactions in which the
o Enzymes are active at 25℃, or concentration of one substrate is constant
37℃. throughout the course of the reaction.
o 37℃ - optimum temperature for Enzyme Cofactors
enzymatic activity.
o Most enzymes become denatured Coenzyme Reaction Type Deficiency
and insoluble within minutes of
Coenzyme A Acyl transfer
being subjected to temperatures of
60*C and above Thiamine Aldehyde Beriberi
o 60-65℃ - inactivation of enzymes pyrophosphat transfer
o Low temperatures render enzymes e
reversibly inactive – 0*C and below
at refrigerated/freezing Folic acid One-Carbon Megaloblastic
temperature. coenzymes transfer anemia
o Repeated freezing and thawing
Cobamide Alkylation Pernicious
tends to denature proteins &
(B12) anemia
should be avoided.
coenzymes
o Temperature Coefficient (Q10) -for
every 10℃ increase in Nicotinamide Oxidation- Pellagra
temperature, there will be a two- coenzymes reduction
fold increase in enzyme activity.
7. Hydrogen Ion Concentration or pH Flavin Oxidation-
o Extreme pH level may denature an coenzymes reduction
enzyme or influence its ionic state Biotin Carboxylation
resulting in structural change in
the charge of amino acid residue Lipoic Acid Acyl transfer
in the active site.
o Most physiologic reactions occur Pyridoxal Amino group
in the pH range of 7-8. phosphate transfer
8. Storage
Coenzyme Q Electron transfer
o -20℃ to 70℃= for longer period of
time (enzymes)

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Inorganic Cofactors for Enzyme Amylase E.C.3.2.1.1

 Mg++ Alanine E.C. 2.6.1.2

 Fe++/Fe+++ Aminotransferase
 Zn++
Aspartate E.C. 2.6.1.1
 Cu+/Cu++
Aminotransferase
 Ca++
 Mn++ Aldolase E.C. 4.1.2.13
 Co++
Angiotensin E.C. 3.4.15.1
Enzyme Nomenclature Converting enzyme

 To standardize enzyme nomenclature, the Creatine Kinase E.C. 2.7.3.2

Enzyme Commission (EC) adapted a
classification system in 1961 and revised True / Acetyl E.C. 3.1.1.7
the standards in 1972 and 1978. cholinesterase
 Enzymes are classified according to their E.C. 3.1.1.8
biochemical functions, indicating Pseudocholinesterase
substrate and class of reaction catalyzed,
and are designated by individual Gamma Glutamyl E.C. 2.3.2.2
identification numbers. Transferase
 The first digit, places the enzyme in its
classification (six classifications). G-6-PD E.C. 1.1.1.49
 The second and third digits, represents Lipase E.C. 3.1.1.3
the subclass to which the enzyme is
assigned. Lactic Dehydrogenase E.C. 1.1.1.27
 The final and fourth number’s, is a serial
5’ Nucleotidase E.C. 3.1.3.5
number that is specific to each enzyme in
a subclass.

Enzyme Classification and Nomenclature CLASS FUNCTIO EXAMPLES

1. Oxidoreductases. Catalyze an oxidation– N
reduction reaction between two Oxidoreduc Catalyze CO,
substrates tases the LDH,MDH,ICD,G-6-
2. Transferases. Catalyze the transfer of a removal or PD, GD, BHD
group other than hydrogen from one addition of
substrate to another electrons
3. Hydrolases. Catalyze hydrolysis of various (redox
bonds reaction)
4. . Lyases. Catalyze removal of groups from
substrates without hydrolysis; the
product contains double bonds
5. 5. Isomerases. Catalyze the Transferase Catalyze CK, AST, ALT, OCT
interconversion of geometric, optical, or s the
positional isomers transfer of
6. 6. Ligases. Catalyze the joining of two a chemical
substrate molecules, coupled with group
breaking of the pyrophosphate bond in other than
adenosine triphosphate (ATP) or a similar hydrogen
compound from one
substrate
Specific Subclass to
another.
Acid Phosphatase E.C.3.1.3.2

Alkaline Phosphatase E.C.3.1.3.1

Hydrolases Catalyze Esterases

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hydrolysis of the
or pyrophosp
splitting of - hate bond
a bond by ACP,ALP,Cholineste in ATP or
the rase,LPS similar
addition of compound
water .
(hydrolytic
reactions).

Peptidases  CO – Cytochrome Oxidase

- Trypsin, Pepsin,  MD – Malate Dehydrogenase

LAP
 ICD – Isocitrate Dehydrogenase
Glycosidase
 LAP – Leucine Aminopeptidase
- AMS, Glucosidase,
 GD – Glutamate dehydrogenase
Galactosidases, 5
nucleotidase,  BHD – Beta-hydroxybutyric
chymotrypsin, dehydrogenase
elastase
General Properties of Enzymes
Lyases Catalyze Aldolase, Glutamate
removal of decarboxylase,  Each enzymes contains:
group Pyruvate
 1. active site = water – free cavity,
from decarboxylase,
substance Tryptophan where the substrate interacts with
s w/o Decarboxylase particular charged amino acid residues.
hydrolysis  2. allosteric site = a cavity other than the
. The active site; may bind regulator molecules.
product
contains  As a protein, each enzyme is composed of
double a specific amino acid (primary structure).
bonds. Forming polypeptide chains (secondary
structure), which then folds (tertiary
structure) and result in structural
Isomerases Catalyze Glucose phosphate cavities.
the isomerase, ribose  Enzymes may exist in different form
intramolec phosphate within the same individual – isoenzyme.
ular isomerase,
arrangeme triosephosphate  Coenzyme is an organic factor necessary
nt of the isomerase for full enzymatic activity.
substrate
compound  When bound tightly to the enzyme, the
coenzyme is called a prosthetic group.

 Apoenzyme – protein portion of the

Ligases Catalyze Glutathione enzyme
the joining synthethase
of two  Holoenzyme – whole enzyme molecule
substrate (apoenzyme + cofactor = holoenzyme).
molecules,  Proenzyme or Zymogen – inactive forms of
coupled enzyme
with
breaking Enzyme Theories

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 1.Emil Fisher’s/Lock and Key Theory  1. Change in the substrate concentration.

– the shape of the key (substrate) must fit
into the lock (enzyme).  2. Change in the product concentration.

 2. Kochland’s/Induced Fit Theory  3. Change in coenzyme concentration.

- based on the substrate binding to the
active site of the enzyme.
Units for Expressing Enzymatic Activity:
Enzyme Kinetics
 1. International Unit (IU or U) – 1
 A chemical reaction may occur
micromole of substrate/minute, aka U/L
spontaneously if the energy or available
kinetic energy is higher for the substrate  2. Katal Unit (KU) – 1 mole of
then the product. substrate/second

 Enzymes catalyze physiologic reaction by  Enzymes are quantitated based on their

lowering the activation energy level that activity rather than absolute values.
the substrate must reach for the reaction
to occur.  The units used to report enzyme levels are
activity units.
 An enzyme combines with only one
substrate and catalyzes only one reaction  The definition for activity unit must
absolute specificity. consider change in pH, temperature,
substrate, etc
 Enzymes combine with all the substrate
in a chemical group – group specificity.  Enzyme concentrations are expressed in
either.
 Enzymes reacting with specific chemical
bonds – bond specificity. Causes of Increased Serum Enzyme Levels:

Enzymatic Reactions  1. Impaired removal of enzyme from

plasma
 1. Zero – order reaction – rate does not
depend on substrate concentration  2. Tissue necrosis and degeneration.

- reaction rate depends only on enzyme  3. Increased permeability of cell

concentration. membrane.

 2. First – order reaction – reaction rate is  4. Increase in the number of cells or the
directly proportional to substrate production of cells.
concentration.
MAJOR CLINICAL ENZYMES
-rate depends on substrate concentration
PHOSPHATASES
 To measure the extent of enzymatic
A. Alkaline Phosphate/Alkaline
reaction, 2 general methods may be used: Orthophosphoric Monoester
 1. Fixed – time – the reactants are Phosphohydrolase (3.1.3.1)
combined; the reaction proceeds for a • A nonspecific enzyme capable of
designated time. reacting with many different
The reaction is stopped and measurement substrates.
is made. • It functions to liberate inorganic
phosphate from an organic
 2. Continuous monitoring/kinetic assay – phosphate ester with the
multiple measurements of absorbance concomitant production of an
changed are made during the reaction; alcohol at an alkaline pH 9-10
more advantageous than fixed-time. • In healthy sera, alkaline
phosphatase (ALP) levels are
Enzyme Activity derived from liver and bone
 Enzymes are measured by means of: (osteoblasts).

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Tissue Source Heat stability is determined by heating serum at

56*C for 10-15 mins
• ALP activity is present on cell surfaces in
most human tissue. The highest Isonzyme Heat Chemical
concentrations are found in the intestine, inhibition test
liver, bone, spleen, placenta, and kidney. Fractionation
In the liver, the enzyme is located on both Phe ure Leva
sinusoidal and bile canalicular nyl- a miso
membranes; activity in bone is confined to le
Ani
the osteoblasts, those cells involved in the ne
production of bone matrix. The specific
location of the enzyme within this tissue rgt
accounts for the more predominant
elevations in certain disorders. 1 Liver ALP -- -- -- Inhi
bite
Phosphates d

• Bone isoenzyme increases due to 2 Bone Most heat -- inhi inhi

osteoblastic-activity and is normally ALP labile bite bite
elevated in children during periods of d d
growth and in adult older than age 50
years (geriatric).
3 Placental Most heat inhi -- --
• In normal pregnancy, increased ALP
ALP stable bite
activity can be detected between 16-20
d
weeks of pregnancy – PLACENTAL ALP
• The presence of intestinal ALP isoenzyme 4 Intestinal -- inhi -- --
in serum depends on the blood group and ALP bite
secretor status of the individual – B or O d
blood group increase intestinal ALP after
consumption of a fatty meal. 20% ethanol = Denatures liver ALP rapidly than
• Major tissue source: liver,bone,placenta, bone
intestinal,renal
Carcinoplacental ALP- Their characteristics
• reference values: 30-90U/L
resemble that of PLACENTAL ALP
• Diagnostic Significance:
• When total ALP levels are increased, it is  Regan ALP – lung, breast, ovarian and
the major liver fraction that is most gynecological cancers; bone ALP co-
frequently elevated – obstructive jaundice. migrator; most heat stable, inhibited by
• ALP is increased in obstructive jaundice phenylalanine reagent
due to greater rate of secretion.  Nagao ALP – adenocarcinoma of the
• For bone disorders, highest elevation pancreas and bile duct, pleural cancer,
occurs in Paget’s disease (osteitis variant of Regan; inhibited by L-leucine
deformans). and phenylalanine
 Kasahara ALP – hepatoma/hepatocellular
Phosphatases Electrophoresis
carcinoma
• Isoenzymes: +
Chemical Inhibition
• 1. Liver ALP – most anodal
• L-phenylalanine = inhibits placental,
• 2. Bone ALP intestinal, Regan and Nagao isoenzyme

• 3. Placental ALP • Levamisol = inhibits liver and bone

isoenzyme
• 4. Intestinal ALP – least anodal
• L-homoarginine = inhibits liver and bone
HEAT DENATURATION isoenzyme
(MOST HEAT STABLE TO MOST HEAT LABILE) • 2M Urea = inhibits bone isoenzyme
PLACENTA>>INTESTINAL>>LIVER>>BONE • L-leucine = inhibits Nagao isoenzyme

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• 20% ethanol = Denatures liver ALP • ALP is relatively stable at 4oC for up to
rapidly than bone one week.
• Optimum pH: 8.6-10
PHOSPHATASES
Increased ALP
• Methods:
• Bowers and Mc Combo (continuous- 1. Osteitis deformans
monitoring technique) – pH 10.15: 405nm 2. Obstructive jaundice

p-nitrophenylphosphate ALP p- nitrophenol + phosphate ion

METHODS SUBSTRATE END
PRODUCTS
3. Osteomalacia
4. Rickets
Bodonsky p-nitrophenylphosphate (ALP) -> p-nitrophenol +
phosphate ion
Shinowara Beta-glycero P04 Inorganic p04+
5. Osteoblastic bone tumors
Jones Glycerol
6. Spure
Reinhart 7. Hyperparathyroidism
8. Hepatitis and cirrhosis (slight increased)
King and Phenylphosphate Phenol 9. Bone cancer
Armstrong
Decreased ALP
Bessy,
• After blood transfusions or
lowry, &
cardiopulmonary bypass (transiently
Brock
decrease)
p-nitro phenyl p-nitrophenol or • Malnutrition
p04 yellow • Hypophosphatemia (prolonged, severely
low levels)
Bower and (PNPP) Nitrophenol ion • Zinc deficiency (prolonged, severely low
McComb levels.
• Zinc is a necessary cofactor in ALP
Huggins Phenolphthalein
activity.
and Talalay red
B. Acid Phosphatase/ Acid
Moss Alpha-n napthol Orthophosphoric Monoester
Phosphohydrolase (3.1.3.2)
Klein, Cc2major slide Free • It catalyzes the same reaction
Babson & 13 phenolphthalein made by ALP, expect that it is
Read active at pH 5 to 6.
• Useful in forensic clinical
chemistry, in the investigation of
rape cases – vaginal washing are
examined for seminal fluid-acid
Measurement phosphatase (ACP) activity, which
can persists for up to 4 days.
• Assays to measure ALP activity use p-
• Diagnostic Significance: detection
nitrophenyl phosphate substrate at an
of prostatic carcinoma
alkaline pH.
• Tissue source: prostate (major
• Activators for this enzyme are zinc,
source), RBC, platelets and bone,
magnesium. and other cations,
liver, spleen
• Chelators (such as EDTA, citrate,
• Reference Values: 2.5-11.7U/L
oxalate) can falsely lower activity.
(total ACP) – male
• Activity of enzyme increases slightly
0-3.5mg/mL (Prostatic ACP)
on storage due to loss of inhibitors.
* Trap – hairy cell leukemia

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Tissue Source Gutman

• ACP activity is found in the prostate, Shinowara PNPP p-nitrophenol

bone, liver, spleen, kidney, erythrocytes,
and platelets. The prostate is the richest Babson Read Alpha naphthyl Alpha-napthol
source, with many times the activity and Philips P04
found in other tissue.
Roy and Thymolpthalein Free
• CITRATE is the preferred anticoagulant Hillman MonoP04 thymolpthalein

ACP Isoenzymes ACP Elevations

• Band 1, the major source is prostate  Moderate elevation of Total ACP

gland. Prostatic ACP activity is inhibited
¤ Female Breast CA
by tartrate.
• Band 2 and 4 isoenzymes are from ¤ Paget’s disease
granulocytes.
• Band 3 is the major form present in ¤ Hyperparathyrodism
plasma. This isoenzyme (band 3) is  Non-prostatic ACP elevations
derived from platelets, erythrocytes, and
monocytes. ¤ Neimann-Pick disease
• Band 5 is found mainly in osteoclasts.
This isoenzyme, (band 5) is resistant to ¤ Gaucher’s disease
tartrate inhibition. ¤ Myelocytic leukemia
ISOENXXZYMES TRANSAMINASES/TRANSFERASES
ERYTHROCYTIC ACP PROSTATIC ACP A. Aspartate Aminotransferase (AST)
(2.6.1.1)
- INHIBITED BY: - INHIBITED BY
2% L-TARTRATE  Requires PYRIDOXAL PHOSPHATE
FORMALDEHY - SPECIFIC ACP as coenzyme
DE SOLUTION - Positive ACP is  Involved in the transfer of an
and 1mM evident in amino group between aspartate
CUPRIC vaginal swab if and A-keto acids with the
SULFATE semen is formation of oxaloacetate and
SOLUTION present for the glutamate.
- NON SPECIFIC first 12 hours  It has 2 isoenzyme fractions,
ACP up to four days cytoplasm and mitochondrial ASTs
from the
– the cytoplasmic isoenzyme is the
incident
predominant form in serum
 Major tissue sources: cardiac
Chemical Inhibition Technique tissue, liver and skeletal muscle
 Reference values: 5-37U/L
RBC ACP Prostatic ACP  Diagnostic Significance:
Copper + -  In the evaluation of myocardial
(Cu3+) infarction, hepatocellular
disorders and skeletal muscle
Tartrate - + involvement.
 In acute myocardial infarction
Copper Resistant: Prostatic ACP (AMI), AST levels begin to rise 6-8
Tartrate Resistant: RBC ACP hours, peak at 24 hours and
normalize within 5 days.
METHODS SUSTRATE END  It is released to a greater degree in
PRODUCTS chronic disorders of the liver with
progressive damage.
Gutman and Phenyl P04 Inorganic P04

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 Diagnosis of chronic alcohol abuse

and drug hepatotoxicity
 Pulmonary infarction, pericarditis,
acute hepatitis
 Decreased level is seen during
pregnancy

Karmen Method – pH 7.5; 340nm

 Uses malate dehydrogenase (MD)

and monitors the change in
absorbance at 340nm

Aspartate + A-ketoglutarate (AST) -> oxaloacetate

+ glutamate

Oxaloacetate + NADH + H (MD) -> malate + NAD+

B. Alanine Aminotransferase (ALT)

(2.6.1.2)
 It has enzymatic activity similar
(ALT)
 HIGHEST ELEVATION IS FOUND IN
ACUTE LIVER HEPATITIS
 It catalyzes the transfer of an amino
group from alanine to A-
ketoglutarate with the formation of
glutamate and pyruvate.
 The highest concentration is in the
liver – more liver-specific than AST.
 Other sources: kidney, pancreas,
RBC, heart, skeletal muscles, lungs
 Reference values: 6-37U/L
 Diagnostic Significance: Aspartate + a-Ketoglutarate (AST) -> Oxaloacetate
 Significant in the evaluation of + Glutamate
hepatic disorders – markedly
Alanine + A-ketoglutarate (ALT) -> Pyruvate +
increased concentration in acute
Glutamate
inflammatory condition than AST.
 It also monitors the course of Pyruvate + NADH + H + (LD) -> Lactate + NAD
hepatitis treatment and the effects of
drug therapy. SGOT/AST SGPT/ALT
 ALT measurement is a more
Major organ Heart Liver
sensitive and specific screening test
affected
for post transfusion hepatitis or
occupational toxic exposure. Substrate Alanine Alphe Alanine Alphe
 ALT levels are also used to screen Ketoglunatic Ketoglunatic
blood donors. Acid Acid

End products Glutamic Acid Glutamic Acid

+ Oxaloacetic + Pyruvic Acid
Acid

Color 2.4 DNPH 2.4 DNPH

developer

Color 0.4N NaOH 0.4N NaOH

intensifier

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methods Reitman and Reitman and  Major Tissue source: acinar cell of the
Frankel Frankel pancreas and the salivary glands.
 Other Tissue source: adipose tissue,
fallopian tubes, small intestine and
Increased Transaminases skeletal muscle.
 Reference – values: 60-180SU/dL
1. Toxic hepatitis (somogyi-units)
2. Acute-Myocardial Infraction – AST 95-290U/L
3. Wolff-Parkinson White Syndrome
4. Trichinosis Diagnostic Significance:
5. Chronic Alcoholism
 Increased AMS blood levels are
6. Dermatomyositis – AST
accompanied by increased urinary
7. Hepatic cancer
excretion – acute pancreatitis.
8. Reye’s syndrome
 In acute pancreatitis (AP), AMS levels rise
9. Viral hepatitis
2-12 hours after onset of attack, peak at
10. Muscular Dystrophy – AST
24 hours, and normalize within 3-5days.
11. Acute pancreatitis – AST
 AMS in urine (AP) remains elevated for up
 Hepatocyte injury (increase in AST, and
to 7 days.
ALT but to a lesser degree)
 In renal failure, increased blood level is
 Muscle injury (increase in both enzymes)
accompanied by decreased urine
 Kidney infarcts (increase in both enzymes)
concentration.
 Renal failure (falsely lowered)
 Salivary gland inflammation (parotitis)
Specimen Stability due to mumps can also release AMS into
 the circulation.
 The half-life of AST is 17+ 5 hours while
ALT has a half-life of 47 +10 hours. METHODS:
 Specimen
 Samples with high activity of AMS should
o AST is stable in serum at
be diluted with NaCL to prevent
refrigerator temperature for up to
inactivation.
three weeks, indefinitely if frozen.
 Many endogenous inhibitors of AMS such
ALT has the same stability but
as wheat germ are present in serum.
markedly decreases with freezing.
 Substrate: Starch
o Specimens for AST and ALT are
o Saccharogenic (SUGAR-
stable in whole blood for up to 12
GENERATING)– measures the
to 24 hours, but increase with
amount of reducing sugars
time due to release from red blood
produced by the hydrolysis of
cells.
starch by the usual glucose
 Optimum pH: 7.4
methods.
IV. AMYLASE/ALPHA-1-4 GLUCAN -4- o Amyloclastic (STARCH-CUTTING
GLUCOHYDROLASE (AMS) (3.2.1.1) OR IODOMETRIC METHOD)–
measures amylase activity of
 It catalyzes the breakdown of starch and following the decreases in
glycogen – an important enzyme in the substrate concentration
physiologic digestion of starch. (degradation of starch).
 Smallest enzyme in size – normally  3.Chromogenic - measures amylase
filtered by the renal glomerulus and also activity increase in color intensity of the
appears in the urine. soluble dye-substrate solution produced
 It is the earliest pancreatic marker. in the reaction.
 P3 is the most predominant pancreatic  4.Coupled enzyme – measures amylase
amylases isoenzyme in AP. activity by a continuous-monitoring
 Isoenzymes: S-type (ptyalin – MIGRATES technique.
FASTEST TO THE ANODE) and P-type
(amylopsin – MIGRATES SLOWEST TO
THE ANODE) – both present in normal
sera.

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AMS  Most specific pancreatic marker –

secreted exclusively in the pancreas; not
Maltopentose maltrotriose + maltose affected by renal disorders.
A-glucosidase  Concentrations are normal in conditions
of salivary gland involvement.
Maltrotriose + maltose 5-glucose  Major tissue source: pancreas
 Reference value: 0-1.0 U/mL
Hexokinase  Optimum pH: 7.8-8.0
5-glucose + 5 ATP 5-glucose-6-phosphate + 5NAD

G-6-Pd

5-glucose-6-phosphate + 5 NAD 5,6 phosphogluconolactone + 5 NADH Diagnostic significance:

 In acute pancreatitis (AP), LPS levels rise-

Maltopentose (AMS) -> maltotriose + maltose 6-hours after onset of attack, peak at 24
Maltrotriose + maltose (A-glucosidase) -> 5- hours, and remains elevated for 7days
glucose  In chronic AP, acinar cell degration occurs
resulting in loss of amylase and lipase
5-glucose + 5 ATP (Hexokinase) -> 5-glucose-6- production.
phosphate + 5NAD
Methods:
5-glucose-6-phosphate + 5NAD (G-6PD) -> 5/6
phosphogluconolactone + 5 NADH  Uses olive oil because other esterases can
hydrolyze TAG and synthetic diglycerides.
 Addition of colipase (protein secreted by
the pancreas) and bile salts will make
assay more sensitive and specific for AP
detection.
 Hemoglobin inhibits the activity of LPS
leading to falsely low rates.
 Triolein (purer form of TAG) is used also
as a substrate for LPS assay.
1. Cherry Crandal (reference method)
Formula for A/C ratio:  Principle: Hydrolysis of olive oil after
incubation for 24 hours at 37℃ and
Urine amylase x serum creatinine titration of fatty acids using NaOH.
 Substrate: 50% olive oil, TRIOLEIN is
Serumamylase x urine creatinine
now used as a substrate for purer
Increased Amylase form of TAG
 End product: Fatty acid
1. Acute pancreatitis
2. Ectopic pregnancy
3. Peptic ulcer
4. Alcoholism
5. Mumps

V.LIPASE/TRIACYGLYCEROL
ACYLHYDROLASE (LPS) (3.1.1.3)

 An enzyme that hydrolyzes the ester

linkages of fats to produce alcohol and
fatty acid.
 It catalyzes partial hydrolysis of dietary
TAG in the intestine to the 2-
monoglyceride intermediate, with the
production of long chain fatty acids.

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2. Tietz and Fiereck kidney

3. Peroxidase couping = most commonly
used method; does not use 50% olive oil. LD5 MMMM 0.5-1.5 Liver,
skeletal
Considerations in LPS Assays: muscle,
kidney
 Lipemic specimen → reduction lipase
activity
 Opiates and morphines → increases
LPS activity due to its spastic effect on Diagnostic significance:
the duodenal musculature and  Highest level is seen in MEGALOBLASTIC
Sphincter of Oddi ANEMIA (pernicious anemia) and
hemolytic disorders.
VI. LACTATE DEHYDROGENASE (LD)  In AMI, LD levels begin to rise within 12-
(1.1.1.27) 24 hours, peak levels within 48-72 hours
and remains elevated for 10-14days.
 Is an enzyme that catalyzes the  Hepatic carcinoma and toxic hepatitis –
interconversion of lactic and pyruvic 10 fold increased
acids.  Viral hepatitis and cirrhosis would give
 A hydrogen – transfer enzyme that uses LD slightly increased values (2-3x ULR).
the coenzyme nicotinamide dinucleotide  LD-1 > LD-2 = “flipped-pattern” –
(NAD+). myocardial infraction, hemolytic anemia
o LD is a tertramer of two active  LD-2, LD-3, LD-4 =LD cancer markers
subunits, H (for heart) and M (predominantly LD-3); acute leukemia,
(muscle). Combinations of the germ cell tumors, breast and lung
subunits produce five isoenzymes. cancers.
 It will be clinically significant if separated  LD-5 = is moderately increased in acute
into isoenzyme fractions. viral hepatitis and cirrhosis and markedly
 RBC and cardiac tissues contain high increased in hepatic carcinoma and toxic
levels of LD 1. hepatitis.
 An elevated total LD is a nonspecific
result because of its presence from LD-1 > LD-2 = “flipped-pattern” – myocardial
several tissues. infraction
 Reference values: 100-225U/L (forward LD1/LD2 flip: c/w
reaction) 80-280U/L (reverse reaction)
- AMI
LD Five isoenzymes - RBC hemolysis
Chain Approx. % Tissue rich - Renal cortical necrosis
composition of total in enzyme
normally
present in
SERUM

LD1 HHHH 29-37 Heart,

brain,
erythrocyte

LD2 HHHM 42-48 Heart,

brain,
erythrocyte

LD3 HHMM 16-20 Brain,

kidney

LD4 HMMM 2-4 Liver,

skeletal
muscle,

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Lactate + NAD (LD)-> pyruvate +NADH

@340 nm

2. Wrobleuski La Due (reverse/indirect

reaction) – pH 7.2
 It is about 2x faster as the forward
reaction.

Isoenzymes of LDH

Origin Anode

LD5 ------- LD4 ------ LD3 ------ LD2 ------ LD1

Pyruvate + NADH (LD)-> Lactate +
Concentration in serum: LD 2>1>3>4>5 NAD

LD-1 > LD-2 = “flipped-pattern” – myocardial 3. Wroblluski Cabaud

infraction 4. Berger Broida

LD1 – most negatively charged, fastest toward the Increased LDH

anode
• 1. Anemias – pernicious, hemolytic,
LD5 – slowest megaloblastic.

• 2. Myocardial infarction
LD Isoenzyme as a Percentage of Total LD:
• 3. Leukemia
 LD-1 =17-27%
• 4. Renal infarction
 LD-2 = 27-37%
 LD-3 =18-25% • 5. Hepatitis and hepatic cancer
 LD-4 = 3-8%
 LD-5 = 0-5% • 6. Muscular dystrophy
 LD-2 = is the major Isoenzyme in the • 7.Delirium tremens
sera of a healthy person.
 LD-2 > LD-1 in healthy sera. • 8. Malignancy
 LD-6 = alcohol dehydrogenase
Isoenzyme; 6th band in electrophoresis; VII. CREATINE KINASE/ATP-CREATINE-N-
elevated in drug hepatoxicity and PHOSPHOTRANSFERASE (CK) (2.7.3.2)
obstructive jaundice; it is responsible
for the metabolic coversion of • It catalyzes the transfer of a phosphate
methanol and ethylene glycol to toxic group between creatine phosphate and
compounds; present in patients with adenosine diphosphate.
arteriosclerotic failure.
• CK requires MAGNESIUM and THIOL
Methods: source (cysteine)

1. Wacker- Method – (forward/direct • Inhibited by ZINC and MANGANESE;

reaction) –pH8.8 excess Mg can also inhibit CK.
 Is the most commonly used
• Involved in the storage of high energy
method because it produces a
creatine PO4 in the muscles.
positive rate (NADH) and not
affected by product inhibition.

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• It is a dimeric molecule with small • CK-BB (brain type) half-life: 2-3 hours
molecular size, composed of a pair of two
different monomers called M and B. • CK-MB (hybrid type) half-life: 15 hours

• In the sera of healthy persons, CK-MM is • CK-MM (muscle type) half-life: 12 hours
the major Isoenzyme (95%). • Serum of adult rarely contains CK-BB of
• Physically well-trained individuals tend to brain origin due to its high molecular size;
have elevated baseline levels. it may be normally present in neonatal
sera.
• Intramuscular injections are known to
increase CK (< 5x URL). • Cardiac tissues contain significant
amount of CK-MB (20%) – myocardium is
• Bedridden patients may have decreased the only tissue from which CK-MB enters
CK activity. the serum in significant quantities.

• Major tissue sources: brain tissue, • CK-MM is both abundantly present in the
smooth and skeletal muscle and cardiac cardiac and skeletal muscles.
muscles
Diagnostic Significance;
• Reference values: 15-160 U/L = male
• It is a very sensitive indicator of acute
15-130 U/L = female myocardial infarction (AM) and Duchenne
disorder.
< 6% of total CK = CK – MB
• Highest elevation of total CK is seen in
CK Isoenzymes DUCHENNE’S MUSCULAR DYSTROPHY
 CK1 or CK-BB (found (50x U/L).
predominantly in the brain and • Total CK is markedly elevated after
smooth muscles) half-life: 2-3 trauma to skeletal muscle from crush
hours injury, convulsions, tetany, surgical
 CK2 or CK-MB (normal muscle incision or intramuscular injections.
contains 14% to 20% of CK-MB; in
skeletal muscle, CK-MB comprises • Injury to both cardiac and skeletal muscle
0% to 1% of total CK in type 1 accounts for the majority of CK-MM
fibers, and 2% to 6% of total CK elevations.
in type 2 fibers). half-life: 15 hours
• Demonstration of elevated levels of CK-
 CK3 or CK-MM. (found in cardiac
MB; ≥ 6% of the total CK, is considered
and skeletal muscles) half-life: 12
the most specific indicator of myocardial
hours
damage, particularly AMI.
 Macro-CK (an oligomer present in
mitochondria and is seldom • Following AMI, the CK-MB levels begin to
released into circulation) rise within 4-8 hours, peak at 12-24
 Reference Value: 15-160 U/L Male hours and normalize within 48-72 hours.
15-130 U/L Female
6% of total CK  CK-MB • CK-MB is not elevated in angina.
 Electrophoresis is the method of
choice. All isoenzymes can be Methods:
measured at one time because of
technical difficulties, it has been Tanzer-Gilbarg Assay (forward/direct method)-
seldom used. pH 9.0; 340 nm
 CK-BB → most rapidly moving
= CPK
isoenzyme
 CK-MB → hybrid Creatinine + ATP -> Creatinine PO4 + ADP
 CK-MM → slowest and most
common form
PK
Isoenzymes

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ADP + phosphoenolpyruvate -> pyruvate + ATP release of CK-MB from noncardiac

tissues when CK is very high.
LD
CK −MB µ g /L∨IU / L
Pyruvate + NADH -> Lactate + NAD • CK1 = x 100
Total CK IU / L
Increased Creatinine kinase

1. Ducheme’s muscular dystrophy

2. Myocardial infarction
3. Hypothyroidism
4. Pulmonary infarction
5. Reye’s syndrome
6. Strenuous exercise and intramuscular
injections
7. Cerebral vascular accident (occasional)
8. Rocky Mountain spotted fever – CK-MB
9. Carbon monoxide poisoning

Clinical Significance:

 Increases in CK MB may be due to:

2. Oliver-Rosalki (reverse/indirect method) o Cardiac or skeletal muscle
damage
• – most commonly used method; faster
reaction; pH 6.8; 340nm o Chronic myopathies

o Chronic renal failure

o Acute respiratory exertion

 CK-BB is increased in:

o Smooth muscle injury

(intestinal ischemia)

o Malignancies (prostate cancer,

small cell carcinoma of the
lung, and intestinal
malignancies).

Considerations in CK Assays:  Macro-CK2 is present in

 CK is light sensitive o Malignancies

 Anticoagulants (Oxalates and
o Myocardial infarction (when
Fluoride) → inhibits CK action
Macro-CK2 is present, it is
 CK in serum is very unstable and is
usually associated with poor
rapidly lost during storage → activity
prognosis)
can be regenerated by adding
substances with –SH groups (cysteine, o MI (less than 5% of the causes)
dithiothreitol, mercaptoethanol)
 Exercise and IM injections causes CK
elevations VIII. ALDOLASE/FRUCTOSE-1-6-
DIPHOSPHATE ALDOLASE (4.1.2.13)
CK
• Is a glycolytic enzyme that splits fructose
• CK relative index (CK) – is an -1,6-diphosphate into two triose
expression of the percentage of the phosphate molecules in the metabolism of
total CK that is attributed to CK – MB. glucose.
This is computed to know possible

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• Increased levels: skeletal muscle disease, • Located in the canaliculi of the hepatic
leukemia, hemolytic anemia and hepatic cells and particularly in the epithelial
cancer, IM, muscular dystrophy cells lining the biliary ductules; also in
the kidney, prostate and pancreas.
• Isoenzymes:
• Useful in differentiating the source of an
• Aldolase A = Skeletal muscle, RBC and elevated ALP level.
brain
• Elevated in all hepatobiliary disorder –
• Aldolase B = WBC, liver, kidney biliary tract obstructions.
• Aldolase C = Brain Tissue • Sensitive indicator of alcoholism (occult
Methods: alcoholism) – most sensitive marker of
acute alcoholic hepatitis.
1. Silbey- Lehninger Method
• Useful in monitoring the effects of
Subtrate: fructose 1, 6 diphosphate abstention from alcohol.
Product measured: dihydroxyacetone-
phosphate • It affects the cell membrane and
2. Coupled Enzymatic Rxn microsomal fractions – elevated among
RR: 2.5 to 10 U/L individuals undergoing warfarin,
Phenobarbital and pheytoin therapies.

• Also increased in pancreatitis and

prostatic disorder.
OTHER CLINICALLY SIGNIFICANT ENZYMES:
• Substrate: y-glutamyl-p-nitroanilide
1. 5’ Nucleotidase (5’N)/5’ribonucleotide
• Methods used: Szass, Rosalki & Tarrow,
phosphohydrolase
Orlowski
 A phosphoric monoester hydrolase;
predominantly secreted from the • Reference values: upto 25 U/L (F) / upto
live. 40 U/L (M)
 A marker for hepatobiliary diseas
and infiltrative lesions of the liver. Uses of GGT
 Methods used: Dixon & Purdon,  Evaluation of liver injury (primary use)
Campbell, Belfield & Goldberg
 Reference values: 0-1.6 units  Test for alcoholic abuse (It is abnormal in
only 30%, 50% of those consuming
5’ Nucleotidase (5’ N) excessive amounts of alcohol. More often
 Used to differentiate obstructive from elevated in maintenance drinkers rather
hepatocellular jaundice and hepatobiliary than alcohol drinkers)
from bone metastasis (osseous disease)  Half-life of GGT is about 7 to 10 days. In
 Increased → post-hepatic jaundice, alcoholic liver disease, half-life increases
intrahepatic cholestasis and infiltrative to 28 days.
lesions of liver

 Slightly increased → hepatocellular 3. Cholinesaterase – (3.1.1.7) (CHS)

jaundice  Index of parenchymal function; secreted
 Normal → bone disease by the liver
 Is used to monitor the effect of muscle
relaxants (succinylcholine) after surgery.
2. Gamma glutamyl transamine  Marker for insecticide/pesticide-poisoning
peptidase/transferase (GGT) (2.3.2.2) (organophosphate poisoning) – low CHS.
 Methods used: Ellman technique and
It catalyzes the transfer of glutamyl groups
potentiometric
between peptides or amino acids through linkage
 Reference values: 0.5-1.3 pH units
at a gammy carboxyl group.
(plasma)

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2 FORMS • Increased: SARCOIDOSIS, GAUCHER’S

DISEASE, acute and chronic bronchitis
 Pseudocholinesterase or acylcholine and leprosy
acylhydrolase
• Main source: macrophages and epithelioid
o It is important in cleavage of cells
succinylcholine. (muscle relaxant
used during surgery) 5. Ceruloplasmin
 Copper-carrying protein and also an
o It is primarily produced in the enzyme.
liver, but is also synthesized by  A marker for Wilson’s disease
myocardium and pancreas. (hepatolenticular diseases).
o PRESENT IN PLASMA AND
LIVER 6. Ornithine Carbamoyl Transferase (OCT)
o ACTIVE AT BOTH HIGH AND LOW • For hepatobilliary diseases
SUBSTRATE CONCENTRATION
• Reference values: 8-20mU/mL
 TRUE CHOLINESTERASE
o Found most exclusively in the liver
 PRESENT IN NERVE ENDINGS AND
ERYTHROCYTES, it has high activity in o Excellent marker for liver disease but it is
CNS, red blood cells, lung, and spleen. rarely used

 IMPORTANT FOR TRANSMISSION OR

NERVE IMPULSES 7. Glucose-6-Phosphate Dehydrogenase (G-6-
PD) (1.1.1.27)
CLINICAL SIGNIFICANCE
 It functions to maintain NADPH in the
• ORGANOPHOSPHORUS INSECTICIDES reduced form in the erythrocytes.
CAUSE A REDUCTION ON BOTH CHS.  It is a newborn screening marker. FOR
• ACETYLCHOLINESTERASE CAN BE ASSESSMENT OF X-LINKED DISORDER
DETECTED IN AMNIOTIC FLUID FOR G6PD DEFICIENCY
ASSESSMENT OF NEURAL TUBE  It is found in adrenal cortex, spleen,
DEFECTS thymus, RBC and lymph nodes.
Deficiency of this enzyme can lead to
METHODS drug-induced hemolytic anemia
(primaquine,antimalarial drug).
UV, MICHEL, ELLMAN – SENSITIVE, RAPID
 Increased levels: myocardial infarction,
AND RECOMMENDED METHOD
megaloblastic anemia
True Cholinesterase uses acetylcholine while  Specimen: red cell hemolysate, serum
pseudocholinesterase uses butyrylthiocholin as a  Reference values: 8-14 U/g hemoglobin in
substrate. RBC hemolysate
7. Leucine Aminopeptidase (LAP)
 Exhibits napthylamidase activity
4. Angiotensin-Converting Enzyme (ACE) → enzyme attacks the free amino
(3.4.15.1) end of the peptide chain
• Also known as Peptidyl-dipeptidase A or  Substrate: Acyl β napthylamide
Kinase II.  Rich in pancreas
 Goldbarg-Rutenburg method → β
• It converts angiotensin I to angiotensin II napthylamide reacts with ethylene
within the lungs. diamine dihydrochloride → blue
end product
• Possible indicator of neuronal dysfunction
(Alzheimer’s disease - CSF). INCREASED LAP:

 Hepatobiliary disease

 Pancreatic Cancer

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 Last trimester of pregnancy Glucose 6 phosphate Drug-induced

dehydrogenase (G6PD) hemolytic anemia
 Obstructive biliary disease
Glutamate Hepatic disorder
9. Myeloperoxidase dehydrogenase (GLD)
 Hydrogen peroxide oxidoreductase Gamma-glutamyl Hepatic disorder
 Stored in azurophilic granules of PMNs Transferase (GGT)
and monocytes
 Catalyzes the conversion of chloride anion Glutathione-S- Hepatic disorder
and hydrogen peroxide to hypochlorite transferase (GST)
 Clinical significance: MPO is released into
Glycogen Acute myocardial
the extracellular fluid and general
phosphorylase (GP) infarction
circulation during inflammatory
conditions Lactate Myocardial infarction
o Active mediator in the dehydrogenase (LDH)
atherosclerotic CV disease Hepatic disorder

Hemolysis

MAJOR ENZYMES OF CLINICAL SIGNIFICANCE Carcinoma

ENZYMES CLINICAL Lipase (LPS) Acute pancreatitis

SIGNIFICANCE
S’-Nucleotidase Hepatic disorder
Acid phosphatase Prostatic carcinoma
Pseudocholinesterase Organophosphate
(ACP)
(PChE) poisoning
Alanine Hepatic disorder Genetic variants
aminotransferase
(ALT) Hepatic disorder

Aldolase (ALD) Skeletal muscle Suxamethonium

disorder sensitivity

Alkaline phosphatase Hepatic disorder Pyruvate kinase (PK) Hemolytic anemia

(ALP)
Bone disorder Trypsin (TRY) Acute pancreatitis

Amylase (AMS) Acute pancreatitis

Angiotensin- Blood pressure HEPATOBILIARY DISORDERS – ALP AND GGT

converting enzyme regulation HEPATIC DISORDERS – AST AND ALT
(ACE)
Liver Disease Bone Disease
Aspartate amino- Myocardial infarction
transferase (AST) GGT Increase Normal
Hepatic disorder
LEUCINE
Skeletal muscle AMINOPPTIDASE
disorder
5’
Chymotrypsin (CHY) Chronic pancreatitis NUCLEOTIDASE
insufficiency

Creatinine kinase (CK) Myocardial infarction

Enzymes as Markers of Hepatic Disorders
Skeletal muscle
disorder Hepatic Disorders

Elastase-1 (E1) Chronic pancreatitis • Acute injury (Acute hepatitis) and

insufficiency Necrosis

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o INCREASE ALT, AST, ALP, • Then the cardiac profile would be ordered
Bilirubin (B1 and B2), LD4 and for several samplings in 3- to 8-hour
LD5 intervals over a 12- to 24-hour period.

o Normal Total Protein and Albumin • Frequently blood is drawn every 3 hours
for analysis during the first 12-hour
• Cirrhosis period.
o INCREASE Bilirubin (B1 and B2), • Laboratory testing used to assess AMI
NH3 includes cardiac troponin T or I, CK-
MB, and sometimes myoglobin.
o Slightly increased/Normal – ALT,
AST, ALP and LD Troponin
o Decreased/Low total protein and • Tissue location: Troponins T, I, and C
albumin form a complex of three proteins that bind
to filaments of skeletal muscle and
o Increased Globulin
cardiac muscle to regulate muscle
• Biliary Tract Obstruction contraction.
• Clinical significance
o Increased ALP, Bilirubin (B2), • cTnT or cTnl (cardiac troponin T or
GGT, 5’-nucleotidase, LAP cardiac troponin I) is used as an AMI
indicator because of specificity and early
Enzymes as Cardiac Markers
rise in serum concentration following AMI.
Enzymes for Myocardial Infarction • In cases of AMI, cTnT increases in 3—4
hours following infarction, peaks in 10-24
Appear in peak Disappearance hours, and returns to normal in 10-14
serum days.
• cTnl increases in 3-6 hours following
CK 4-6 hrs 12-24 1-2 days
infarction, peaks in 14-20 hours, and
after hrs
returns to normal in 5-10 days.
AST 6-8 hrs 48 hrs 4-5 days
after

LD 8-10 hrs 72 hrs 7-12 days

aftre

Cardiac Profile

Enzymes for Myocardial Infarction

Appear in peak Disappearance

serum TROPONIN IS A COMPLEX REGULATORY
PROTEIN!
CK 4-6 hrs 12-24 1-2 days
after hrs • Test methodology
AST 6-8 hrs 48 hrs 4-5 days • Reference ranges: cTnT <0.03 ng/mL;
after cTnl <0.40 ng/mL (Values vary
considerably among laboratories and are
LD 8-10 hrs 72 hrs 7-12 days
dependent on the methodology employed.)
aftre

Myoglobin
• Upon arrival to the emergency
department, a cardiac profile would be • Tissue location: Found in skeletal and
ordered to establish baseline values. cardiac muscles

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• Clinical significance Clinical significance

• Increased in skeletal muscle injuries, 1) Elevations of total CK in serum are

muscular dystrophy, and AMI associated with cardiac disorders, such as
AMI, and skeletal muscle disorders, such
• Myoglobin is released early in cases of as muscular dystrophy. Occasionally,
AMI, rising in 1-3 hours and peaking in elevations are due to disorders of the
5-12 hours, and returns to normal in 18- central nervous system, including
30 hours. seizures and cerebral vascular accidents.
• However, it is not tissue specific. It is 2) CK-MB values greater than 6% of total
better used as a negative predictor in the CK are suggestive of AMI.
first 2-4 hours following chest pain,
• When AMI is suspected, troponin is
• MYOGLOBIN IS AN IRON AND OXYGEN assayed in conjunction with CKMB, and
BINDING PROTEIN! sometimes myoglobin is assayed.
Test methodology • Following AMI, CK-MB levels rise
1) Quantified by immunoassay within 4-6 hours, peak at 12-24 hours,
and return to normal within 2-3 days.
2) Reference ranges: Male, 30-90 ng/mL;
female, <50 ng/mL Test methodology

Creatine kinase and CK-MB • CK isoenzymes are measured by

electrophoresis, ion-exchange
• Tissue location: chromatography, and several types of
immunoassays. Immunoassays that
• Highest concentrations: Skeletal
measure enzyme mass are more sensitive
muscle, heart muscle, brain tissue
than activity-based assays.
• CK isoenzymes
• Reference ranges:
• a) CK isoenzymesconsist of two subunits:
• Total CK: male, 15-160 U/L; female, 15-
M for muscle and B for brain.
130 U/L at 37°C
• b) Each CK isoenzyme is a dimer with
• CK-MB: <6% of total CK; mass assay 0-5
three possible types: CK-MM (or CK-3),
ng/mL
CK-MB (or CK-2), and CK-BB (or CK-1).

• c) In serum, healthy individuals have CK-

MM as the major isoenzyme and a small Natriuretic Peptides: Polypeptide Hormones
amount of CK-MB (less than 6% of total
CK), whereas CK-BB is not normally • Tissue location and function
detectable. • ANP, CNP, BNP (Three Forms)
• d) CK-MB increases are associated with • they function to promote excretion of
heart muscle damage, and elevations are sodium and water by increasing the
indicative of AMI when used in glomerular filtration rate and decreasing
conjunction with other markers, such as the tubular reabsorption of sodium by the
troponin. kidneys.
• However, CK-MB also increases in other • B-type (brain) natriuretic peptide (BNP)
disorders, such as skeletal muscle is synthesized in and secreted from
damage. CK-MM increases are associated myocardial ventricles in response to
with skeletal muscle and heart muscle ventricular volume expansion and
disorders. CK-BB is elevated in central pressure overload
nervous system disorders and tumors of
various organs, including the prostate • BNP causes dilation of blood vessels and
gland. promotes sodium and water loss, thus
reducing fluid load on the heart to
CK improve cardiac function

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Clinical significance Homocysteine

• BNP increased in congestive heart Clinical significance:

failure (CHF)
• Elevated levels cause damage to arterial
Test methodology walls that precedes formation of plaques.
It is an indicator of arterial inflammation
• a. BNP quantified by fluorescence and
chemiluminescence immunoassays Test methodology

• b. Reference range: BNP < 100 pg/mL • a. Immunoassay, fluorometric,

chromatographic
• ProBNP assay measures N-terminal
proBNP (NT-proBNP), which is released • b. Reference range: 5-15 umol/L
when BNP is cleaved from precursor
proBNP.

• a. NT-proBNP has a longer half-life than

BNP.

• b. Measurement of NT-proBNP shows no

interference from Nesiritide (human
recombinant BNP) administration to treat
CHF.

• c. NT-proBNP is measured by
electrochemiluminesce

High-sensitivity CRP (hs-CRP)

• C-reactive protein (CRP): B-globulin that

is an acute-phase reactant

• High-sensitivity CRP refers to the

sensitivity of the assay to determine low
levels in serum.

Clinical significance

• Used as a predictor for cardiovascular

risk; increased levels seen in
inflammation, infection, stress, trauma,
and AMI

Test methodology

• a. Quantified by immunoassay; hs-CRP

detection limit 0.05 mg/L

• b. Reference ranges: Males, 0.3-8.6

mg/L; females, 0.2-9.1 mg/L

• Cardiovascular risk classification:

• Low risk <1.0 mg/L

• average risk 1.0-3.0 mg/L

• high risk >3.0 mg/L

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS