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WEEK 1
ENZYMOLOGY
Enzymes
• Enzymes are protein catalysts utilized by
essentially all mammalian cells in specific
biochemical reactions in different organs
of the body, which may also be physically
located in different organelles and
structures within a cell.
• Most chemical reactions in the body occur
at pH 7 and temperature of 37*C
Medical and Biological Importance
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
bores a tunnel through it so that passage
is far quicker and takes much less energy.
3. Enzymes make life on earth possible, all
biology from conception to the dissolution
that follows death depends on enzymes.
4. Enzymes regulates rate of physiological
process. So, defects in enzyme function
cause diseases.
5. When cells are injured enzymes leak into
plasma. Measurement of activity of such
enzymes in plasma is an integral part of
modern day medical diagnosis.
6. Enzymes are used as drugs.
7. Immobilized enzymes, which are enzymes
attached to solid supports are used in
clinical chemistry laboratories and in
industry. For example, glucose in blood or
urine is detected by using immobilized
glucose oxidase. In pharmaceutical
industry, glucose isomerase is used to
produce fructose from glucose.
8. Enzymes are used as biosensors.
9. AIDS detection involves use of enzyme
dependent ELISA technique.
10. Enzymes are used as cleansing agents in
detergent industry
Chemical Nature of Enzymes (Properties)
1. All the enzymes are proteins except
ribozymes and number of enzymes are
obtained in crystalline form.
2. In 1878, Kuhne, introduced term
‘Enzyme’ to indicate biological catalyst.
3. Enzymes cut big molecules apart and join
small molecules to form big molecules.
CLINICAL CHEMISTRY LECTURE WEEK 1
4. Most of the chemical reactions in the body
are enzyme catalyzed
5. The substance upon which an enzyme
acts is called as substrate. By the action
of enzyme, it is converted to product. An
enzyme-catalysed reaction consists of
substrate, enzyme and product
Enzymes
• These are proteins produced by living
cells that hastens chemical reaction in
organic matter.
• They are measured in terms of their
activity and not in terms of their absolute
values.
• They are large molecules and they are
normally confined within cells unless
increased membrane permeability allows
them to enter the blood.
• They frequently appear in the serum after
cellular injury, degradation of cells or
from storage areas.
• Abnormal large amount of enzymes in
serum are used clinically as evidence of
organ damage.
• Each enzyme catalyzes a single reaction
or a limited number of chemical reaction,
and it is specific for a substrate that it
converts to a defined product.
Factor Affecting Enzyme Reactions
1. Enzyme Concentration
o The higher the enzyme
concentration, the faster is the
reaction, because more enzyme is
present to bind with the substrate.
2. Substrate Concentration
o With the amount of enzyme
exceeding the amount of
substrate, the reaction rate
steadily increases as more
substrate is added.
o However, when the substrate
concentration reaches a maximal
value, higher concentration of
substrate no longer result in
increased rate of reaction
(saturation kinetics).
3. Cofactors
PRELIMS

o Nonprotein entities that must bind
to particular enzymes before a
reaction occurs.
a. Coenzymes – organic
compound (carbon containing)
–ex. NADP, cysteine, pyridoxal
phospate
- increasing coenzyme
concentration will increase the
velocity of an
enzymatic reaction.
b. Activators – inorganic ions
(non-carbon containing) –
It can be metallic: Calcium for
amylase, zinc for LDH, others:
Fe, Mn, Cu
It can be non-metallic:
Chloride for amylase
4. Inhibitors
o Enzymatic reactions may not
progress if an inhibitor interferes
with the reaction.
a. Competitive Inhibitor
- physically bind to the active
site of an enzyme.
- both the substrate and
inhibitor compete for the
same active site of the
enzyme.
- with a substrate
concentration significantly
higher than the
concentration of the
inhibitor, the inhibition is
reversible.
b. Non- Competitive Inhibitor
- binds an enzyme at a place
other than the active site
(allosteric site) of the enzyme.
- do not compete with the
substrate but look for areas
other than the active site.
- 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.
CLINICAL CHEMISTRY LECTURE WEEK 1
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c. Uncompetitive Inhibitor
- binds an enzyme at a place
other than the active site
(allosteric site) of the
enzyme.
- -do not compete with the
substrate but look for areas
other than the active site.
- 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.
- the inhibitor binds to the
enzyme-substrate
(ES)complex, so that
increasing the substrate
concentration results in
more ES complexes to
which the inhibitor binds
and thereby increases the
inhibition.
- increasing substrate
concentration, increases
inhibition.
5. Isoenzymes
o These are enzymes having the
same catalytic reaction but slightly
different molecular structures.
o The importance of the total
enzyme activity is enhanced by
fractionating the isoenzymes
6. Temperature
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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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS


 1.Emil Fisher’s/Lock and Key Theory
– the shape of the key (substrate) must fit
into the lock (enzyme).
 2. Kochland’s/Induced Fit Theory
- based on the substrate binding to the
active site of the enzyme.
Enzyme Kinetics
 A chemical reaction may occur
spontaneously if the energy or available
kinetic energy is higher for the substrate
then the product.
 Enzymes catalyze physiologic reaction by
lowering the activation energy level that
the substrate must reach for the reaction
to occur.
 An enzyme combines with only one
substrate and catalyzes only one reaction
absolute specificity.
 Enzymes combine with all the substrate
in a chemical group – group specificity.
 Enzymes reacting with specific chemical
bonds – bond specificity.
Enzymatic Reactions
 1. Zero – order reaction – rate does not
depend on substrate concentration
- reaction rate depends only on enzyme
concentration.
 2. First – order reaction – reaction rate is
directly proportional to substrate
concentration.
-rate depends on substrate concentration
 To measure the extent of enzymatic
reaction, 2 general methods may be used:
 1. Fixed – time – the reactants are
combined; the reaction proceeds for a
designated time.
The reaction is stopped and measurement
is made.
 2. Continuous monitoring/kinetic assay –
multiple measurements of absorbance
changed are made during the reaction;
more advantageous than fixed-time.
Enzyme Activity
 Enzymes are measured by means of:
CLINICAL CHEMISTRY LECTURE WEEK 1
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 1. Change in the substrate concentration.
 2. Change in the product concentration.
 3. Change in coenzyme concentration.
Units for Expressing Enzymatic Activity:
 1. International Unit (IU or U) – 1
micromole of substrate/minute, aka U/L
 2. Katal Unit (KU) – 1 mole of
substrate/second
 Enzymes are quantitated based on their
activity rather than absolute values.
 The units used to report enzyme levels are
activity units.
 The definition for activity unit must
consider change in pH, temperature,
substrate, etc
 Enzyme concentrations are expressed in
either.
Causes of Increased Serum Enzyme Levels:
 1. Impaired removal of enzyme from
plasma
 2. Tissue necrosis and degeneration.
 3. Increased permeability of cell
membrane.
 4. Increase in the number of cells or the
production of cells.
MAJOR CLINICAL ENZYMES
PHOSPHATASES
A. Alkaline Phosphate/Alkaline
Orthophosphoric Monoester
Phosphohydrolase (3.1.3.1)
• A nonspecific enzyme capable of
reacting with many different
substrates.
• It functions to liberate inorganic
phosphate from an organic
phosphate ester with the
concomitant production of an
alcohol at an alkaline pH 9-10
• In healthy sera, alkaline
phosphatase (ALP) levels are
derived from liver and bone
(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

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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS

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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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS

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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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS

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

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.

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS

 P a g e | 13

2. Tietz and Fiereck kidney

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

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,

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS


Isoenzymes of LDH
Origin Anode
LD5 ------- LD4 ------ LD3 ------ LD2 ------ LD1
Concentration in serum: LD 2>1>3>4>5
LD-1 > LD-2 = “flipped-pattern” – myocardial
infraction
LD1 – most negatively charged, fastest toward the
anode
LD5 – slowest
LD Isoenzyme as a Percentage of Total LD:
 LD-1 =17-27%
 LD-2 = 27-37%
 LD-3 =18-25%
 LD-4 = 3-8%
 LD-5 = 0-5%
 LD-2 = is the major Isoenzyme in the
sera of a healthy person.
 LD-2 > LD-1 in healthy sera.
 LD-6 = alcohol dehydrogenase
Isoenzyme; 6th band in electrophoresis;
elevated in drug hepatoxicity and
obstructive jaundice; it is responsible
for the metabolic coversion of
methanol and ethylene glycol to toxic
compounds; present in patients with
arteriosclerotic failure.
Methods:
1. Wacker- Method – (forward/direct
reaction) –pH8.8
 Is the most commonly used
method because it produces a
positive rate (NADH) and not
affected by product inhibition.
CLINICAL CHEMISTRY LECTURE WEEK 1
P a g e | 14
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.
Pyruvate + NADH (LD)-> Lactate +
NAD
3. Wroblluski Cabaud
4. Berger Broida
Increased LDH
• 1. Anemias – pernicious, hemolytic,
megaloblastic.
• 2. Myocardial infarction
• 3. Leukemia
• 4. Renal infarction
• 5. Hepatitis and hepatic cancer
• 6. Muscular dystrophy
• 7.Delirium tremens
• 8. Malignancy
VII. CREATINE KINASE/ATP-CREATINE-N-
PHOSPHOTRANSFERASE (CK) (2.7.3.2)
• It catalyzes the transfer of a phosphate
group between creatine phosphate and
adenosine diphosphate.
• CK requires MAGNESIUM and THIOL
source (cysteine)
• Inhibited by ZINC and MANGANESE;
excess Mg can also inhibit CK.
• Involved in the storage of high energy
creatine PO4 in the muscles.
PRELIMS

• It is a dimeric molecule with small
molecular size, composed of a pair of two
different monomers called M and B.
• In the sera of healthy persons, CK-MM is
the major Isoenzyme (95%).
• Physically well-trained individuals tend to
have elevated baseline levels.
• Intramuscular injections are known to
increase CK (< 5x URL).
• Bedridden patients may have decreased
CK activity.
• Major tissue sources: brain tissue,
smooth and skeletal muscle and cardiac
muscles
• Reference values: 15-160 U/L = male
15-130 U/L = female
< 6% of total CK = CK – MB
CK Isoenzymes
 CK1 or CK-BB (found
predominantly in the brain and
smooth muscles) half-life: 2-3
hours
 CK2 or CK-MB (normal muscle
contains 14% to 20% of CK-MB; in
skeletal muscle, CK-MB comprises
0% to 1% of total CK in type 1
fibers, and 2% to 6% of total CK
in type 2 fibers). half-life: 15 hours
 CK3 or CK-MM. (found in cardiac
and skeletal muscles) half-life: 12
hours
 Macro-CK (an oligomer present in
mitochondria and is seldom
released into circulation)
 Reference Value: 15-160 U/L Male
15-130 U/L Female
6% of total CK  CK-MB
 Electrophoresis is the method of
choice. All isoenzymes can be
measured at one time because of
technical difficulties, it has been
seldom used.
 CK-BB → most rapidly moving
isoenzyme
 CK-MB → hybrid
 CK-MM → slowest and most
common form
Isoenzymes
CLINICAL CHEMISTRY LECTURE WEEK 1
P a g e | 15
• CK-BB (brain type) half-life: 2-3 hours
• CK-MB (hybrid type) half-life: 15 hours
• CK-MM (muscle type) half-life: 12 hours
• Serum of adult rarely contains CK-BB of
brain origin due to its high molecular size;
it may be normally present in neonatal
sera.
• Cardiac tissues contain significant
amount of CK-MB (20%) – myocardium is
the only tissue from which CK-MB enters
the serum in significant quantities.
• CK-MM is both abundantly present in the
cardiac and skeletal muscles.
Diagnostic Significance;
• It is a very sensitive indicator of acute
myocardial infarction (AM) and Duchenne
disorder.
• Highest elevation of total CK is seen in
DUCHENNE’S MUSCULAR DYSTROPHY
(50x U/L).
• Total CK is markedly elevated after
trauma to skeletal muscle from crush
injury, convulsions, tetany, surgical
incision or intramuscular injections.
• Injury to both cardiac and skeletal muscle
accounts for the majority of CK-MM
elevations.
• Demonstration of elevated levels of CK-
MB; ≥ 6% of the total CK, is considered
the most specific indicator of myocardial
damage, particularly AMI.
• Following AMI, the CK-MB levels begin to
rise within 4-8 hours, peak at 12-24
hours and normalize within 48-72 hours.
• CK-MB is not elevated in angina.
Methods:
Tanzer-Gilbarg Assay (forward/direct method)-
pH 9.0; 340 nm
= CPK
Creatinine + ATP -> Creatinine PO4 + ADP
PK
PRELIMS
 P a g e | 16

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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS


• Increased levels: skeletal muscle disease,
leukemia, hemolytic anemia and hepatic
cancer, IM, muscular dystrophy
• Isoenzymes:
• Aldolase A = Skeletal muscle, RBC and
brain
• Aldolase B = WBC, liver, kidney
• Aldolase C = Brain Tissue
Methods:
1. Silbey- Lehninger Method
Subtrate: fructose 1, 6 diphosphate
Product measured: dihydroxyacetone-
phosphate
2. Coupled Enzymatic Rxn
RR: 2.5 to 10 U/L
OTHER CLINICALLY SIGNIFICANT ENZYMES:
1. 5’ Nucleotidase (5’N)/5’ribonucleotide
phosphohydrolase
 A phosphoric monoester hydrolase;
predominantly secreted from the
live.
 A marker for hepatobiliary diseas
and infiltrative lesions of the liver.
 Methods used: Dixon & Purdon,
Campbell, Belfield & Goldberg
 Reference values: 0-1.6 units
5’ Nucleotidase (5’ N)
 Used to differentiate obstructive from
hepatocellular jaundice and hepatobiliary
from bone metastasis (osseous disease)
 Increased → post-hepatic jaundice,
intrahepatic cholestasis and infiltrative
lesions of liver
 Slightly increased → hepatocellular
jaundice
 Normal → bone disease
2. Gamma glutamyl transamine
peptidase/transferase (GGT) (2.3.2.2)
It catalyzes the transfer of glutamyl groups
between peptides or amino acids through linkage
at a gammy carboxyl group.
CLINICAL CHEMISTRY LECTURE WEEK 1
P a g e | 17
• Located in the canaliculi of the hepatic
cells and particularly in the epithelial
cells lining the biliary ductules; also in
the kidney, prostate and pancreas.
• Useful in differentiating the source of an
elevated ALP level.
• Elevated in all hepatobiliary disorder –
biliary tract obstructions.
• Sensitive indicator of alcoholism (occult
alcoholism) – most sensitive marker of
acute alcoholic hepatitis.
• Useful in monitoring the effects of
abstention from alcohol.
• It affects the cell membrane and
microsomal fractions – elevated among
individuals undergoing warfarin,
Phenobarbital and pheytoin therapies.
• Also increased in pancreatitis and
prostatic disorder.
• Substrate: y-glutamyl-p-nitroanilide
• Methods used: Szass, Rosalki & Tarrow,
Orlowski
• Reference values: upto 25 U/L (F) / upto
40 U/L (M)
Uses of GGT
 Evaluation of liver injury (primary use)
 Test for alcoholic abuse (It is abnormal in
only 30%, 50% of those consuming
excessive amounts of alcohol. More often
elevated in maintenance drinkers rather
than alcohol drinkers)
 Half-life of GGT is about 7 to 10 days. In
alcoholic liver disease, half-life increases
to 28 days.
3. Cholinesaterase – (3.1.1.7) (CHS)
 Index of parenchymal function; secreted
by the liver
 Is used to monitor the effect of muscle
relaxants (succinylcholine) after surgery.
 Marker for insecticide/pesticide-poisoning
(organophosphate poisoning) – low CHS.
 Methods used: Ellman technique and
potentiometric
 Reference values: 0.5-1.3 pH units
(plasma)
PRELIMS

2 FORMS
 Pseudocholinesterase or acylcholine
acylhydrolase
o It is important in cleavage of
succinylcholine. (muscle relaxant
used during surgery)
o It is primarily produced in the
liver, but is also synthesized by
myocardium and pancreas.
o PRESENT IN PLASMA AND
LIVER
o ACTIVE AT BOTH HIGH AND LOW
SUBSTRATE CONCENTRATION
 TRUE CHOLINESTERASE
 PRESENT IN NERVE ENDINGS AND
ERYTHROCYTES, it has high activity in
CNS, red blood cells, lung, and spleen.
 IMPORTANT FOR TRANSMISSION OR
NERVE IMPULSES
CLINICAL SIGNIFICANCE
• ORGANOPHOSPHORUS INSECTICIDES
CAUSE A REDUCTION ON BOTH CHS.
• ACETYLCHOLINESTERASE CAN BE
DETECTED IN AMNIOTIC FLUID FOR
ASSESSMENT OF NEURAL TUBE
DEFECTS
METHODS
UV, MICHEL, ELLMAN – SENSITIVE, RAPID
AND RECOMMENDED METHOD
True Cholinesterase uses acetylcholine while
pseudocholinesterase uses butyrylthiocholin as a
substrate.
4. Angiotensin-Converting Enzyme (ACE)
(3.4.15.1)
• Also known as Peptidyl-dipeptidase A or
Kinase II.
• It converts angiotensin I to angiotensin II
within the lungs.
• Possible indicator of neuronal dysfunction
(Alzheimer’s disease - CSF).
CLINICAL CHEMISTRY LECTURE WEEK 1
P a g e | 18
• Increased: SARCOIDOSIS, GAUCHER’S
DISEASE, acute and chronic bronchitis
and leprosy
• Main source: macrophages and epithelioid
cells
5. Ceruloplasmin
 Copper-carrying protein and also an
enzyme.
 A marker for Wilson’s disease
(hepatolenticular diseases).
6. Ornithine Carbamoyl Transferase (OCT)
• For hepatobilliary diseases
• Reference values: 8-20mU/mL
o Found most exclusively in the liver
o Excellent marker for liver disease but it is
rarely used
7. Glucose-6-Phosphate Dehydrogenase (G-6-
PD) (1.1.1.27)
 It functions to maintain NADPH in the
reduced form in the erythrocytes.
 It is a newborn screening marker. FOR
ASSESSMENT OF X-LINKED DISORDER
G6PD DEFICIENCY
 It is found in adrenal cortex, spleen,
thymus, RBC and lymph nodes.
Deficiency of this enzyme can lead to
drug-induced hemolytic anemia
(primaquine,antimalarial drug).
 Increased levels: myocardial infarction,
megaloblastic anemia
 Specimen: red cell hemolysate, serum
 Reference values: 8-14 U/g hemoglobin in
RBC hemolysate
7. Leucine Aminopeptidase (LAP)
 Exhibits napthylamidase activity
→ enzyme attacks the free amino
end of the peptide chain
 Substrate: Acyl β napthylamide
 Rich in pancreas
 Goldbarg-Rutenburg method → β
napthylamide reacts with ethylene
diamine dihydrochloride → blue
end product
INCREASED LAP:
 Hepatobiliary disease
 Pancreatic Cancer
PRELIMS
 P a g e | 19

 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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS

 P a g e | 20

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

CLINICAL CHEMISTRY LECTURE WEEK 1 PRELIMS


• Clinical significance
• Increased in skeletal muscle injuries,
muscular dystrophy, and AMI
• Myoglobin is released early in cases of
AMI, rising in 1-3 hours and peaking in
5-12 hours, and returns to normal in 18-
30 hours.
• However, it is not tissue specific. It is
better used as a negative predictor in the
first 2-4 hours following chest pain,
• MYOGLOBIN IS AN IRON AND OXYGEN
BINDING PROTEIN!
Test methodology
1) Quantified by immunoassay
2) Reference ranges: Male, 30-90 ng/mL;
female, <50 ng/mL
Creatine kinase and CK-MB
• Tissue location:
• Highest concentrations: Skeletal
muscle, heart muscle, brain tissue
• CK isoenzymes
• a) CK isoenzymesconsist of two subunits:
M for muscle and B for brain.
• b) Each CK isoenzyme is a dimer with
three possible types: CK-MM (or CK-3),
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
amount of CK-MB (less than 6% of total
CK), whereas CK-BB is not normally
detectable.
• d) CK-MB increases are associated with
heart muscle damage, and elevations are
indicative of AMI when used in
conjunction with other markers, such as
troponin.
• However, CK-MB also increases in other
disorders, such as skeletal muscle
damage. CK-MM increases are associated
with skeletal muscle and heart muscle
disorders. CK-BB is elevated in central
nervous system disorders and tumors of
various organs, including the prostate
gland.
CK
CLINICAL CHEMISTRY LECTURE WEEK 1
P a g e | 21
Clinical significance
1) Elevations of total CK in serum are
associated with cardiac disorders, such as
AMI, and skeletal muscle disorders, such
as muscular dystrophy. Occasionally,
elevations are due to disorders of the
central nervous system, including
seizures and cerebral vascular accidents.
2) CK-MB values greater than 6% of total
CK are suggestive of AMI.
• When AMI is suspected, troponin is
assayed in conjunction with CKMB, and
sometimes myoglobin is assayed.
• Following AMI, CK-MB levels rise
within 4-6 hours, peak at 12-24 hours,
and return to normal within 2-3 days.
Test methodology
• CK isoenzymes are measured by
electrophoresis, ion-exchange
chromatography, and several types of
immunoassays. Immunoassays that
measure enzyme mass are more sensitive
than activity-based assays.
• Reference ranges:
• Total CK: male, 15-160 U/L; female, 15-
130 U/L at 37°C
• CK-MB: <6% of total CK; mass assay 0-5
ng/mL
Natriuretic Peptides: Polypeptide Hormones
• Tissue location and function
• ANP, CNP, BNP (Three Forms)
• they function to promote excretion of
sodium and water by increasing the
glomerular filtration rate and decreasing
the tubular reabsorption of sodium by the
kidneys.
• B-type (brain) natriuretic peptide (BNP)
is synthesized in and secreted from
myocardial ventricles in response to
ventricular volume expansion and
pressure overload
• BNP causes dilation of blood vessels and
promotes sodium and water loss, thus
reducing fluid load on the heart to
improve cardiac function
PRELIMS
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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