ENZYMES

1. ALANINE AMINO TRANSFERASE

 It catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the
formation of glutamate and pyruvate.
 Serum Glutamic-pyruvic transaminase (SGPT, or GPT)
 The older terminology for ALT was?

 Tissue Source
 ALT is distributed in many tissues, with comparatively high concentrations in the liver. It is
considered the more liver-specific enzyme of the transferases.

 Diagnostic Significance
 Confined mainly to evaluation of hepatic disorders.
 In acute inflammatory conditions of the liver, ALT elevations are frequently higher than those
of AST and tend to remain elevated longer as a result of the longer half-life of ALT in serum
(16 and 24 hours, respectfully).

 Assay for Enzyme Activity

 The typical assay procedure for ALT consists of a coupled enzymatic reaction using LDH as
the indicator enzyme.
Alanine + a-ketoglutarate ALT pyruvate + glutamate
Pyruvate + NADH + H LD lactate + NAD
 The optimal pH is 7.3 to 7.8.

 Source of Error
 ALT is stable for 3 to 4 days at 4°C. It is relatively unaffected by hemolysis.

 Reference Range: 6–37 U/L (37°C)

2. ALKALINE PHOSPHATASE
 Alkaline phosphatase (ALP) belongs to a group of enzymes that catalyze the hydrolysis of
various phosphomonoesters at an alkaline pH.
 ALP functions to liberate inorganic phosphate from an organic phosphate ester with the
concomitant production of an alcohol.
 The optimal pH for the reaction is 9.0 to 10.0, but optimal pH varies with the substrate used.
The enzyme requires Mg2+ as an activator.

 Tissue Source
 ALP activity is present on cell surfaces in most human tissue. The highest concentrations are
found in the intestine, liver, bone, spleen, placenta, and kidney.
 In the liver, the enzyme is located on both sinusoidal and bile canalicular membranes;
activity in bone is confined to the osteoblasts, those cells involved in the production of bone
matrix.

 Diagnostic Significance
 Elevations of ALP are of most diagnostic significance in the evaluation of hepatobiliary and
bone disorders.
 In biliary tract obstruction, ALP levels range from 3 to 10 times ULN. Increases are primarily a
result of increased synthesis of the enzyme induced by cholestasis.
 Elevated ALP levels may be observed in various bone disorders. Perhaps the highest
elevations of ALP activity occur in Paget’s disease (osteitis deformans).
 In normal pregnancy, increased ALP activity, averaging approximately 1 1⁄2 times ULN, can
be detected between weeks 16 and 20. ALP activity increases and persists until the onset of
labor. Activity then returns to normal within 3 to 6 days.
 ALP levels are significantly decreased in the inherited condition of hypophosphatasia.
Subnormal activity is a result of the absence of the bone isoenzyme and results in inadequate
bone calcification.

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology
  Identifying ALP isoenzymes:
A. Electrophoresis
 Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis.
 The liver fraction migrates the fastest, followed by bone, placental, and intestinal
fractions.
B. Heat inactivation
 Difference in heat stability is the basis of a second approach used to identify the
isoenzyme source of an elevated ALP.
 ALP activity is measured before and after heating the serum at 56°C for 10 minutes.
 If the residual activity after heating is less than 20% of the total activity before heating,
then the ALP elevation is assumed to be a result of bone phosphatase.
 If greater than 20% of the activity remains, the elevation is probably a result of liver
phosphatase.
 These results are based on the finding that placental ALP is the most heat stable of the
four major fractions, followed by intestinal, liver, and bone fractions in decreasing order of
heat stability.
 Placental ALP will resist heat denaturation at 65°C for 30 minutes.
C. Selective chemical inhibition
 Phenylalanine inhibits intestinal and placental ALP to a much greater extent than liver
and bone ALP.
 3M urea inhibits bone ALP.
 Levamisole inhibits liver and bone ALP.
 Regan and Nagao isoenzymes - they have been referred to as carcinoplacental alkaline
phosphatases because of their similarities to the placental isoenzyme.

 Assay for Enzyme Activity

 A continuous-monitoring technique based on a method devised by Bowers and McComb
allows calculation of ALP activity based on the molar absorptivity of p-nitrophenol.
 p-Nitrophenylphosphate (colorless) is hydrolyzed to p-nitrophenol (yellow), and the increase
in absorbance at 405 nm, which is directly proportional to ALP activity, is measured.

 Reference Range = 30 to 90 U/L (30°C)

3. Y-Glutamyltransferase
 y-Glutamyltransferase (GGT) is an enzyme involved in the transfer of the y-glutamyl residue
from y-glutamyl peptides to amino acids, H2O, and other small peptides. In most biologic
systems, glutathione serves as the y-glutamyl donor.
 The specific physiologic function of GGT has not been clearly established, but it is suggested
that GGT is involved in peptide and protein synthesis, regulation of tissue glutathione levels,
and the transport of amino acids across cell membranes.

 Tissue Source
 GGT activity is found primarily in tissue of the kidney, brain, prostate, pancreas, and liver.
 Clinical applications of assay, however, are confined mainly to evaluation of liver and biliary
system disorders.

 Diagnostic Significance
 In the liver, GGT is located in the canaliculi of the hepatic cells and particularly in the
epithelial cells lining the biliary ductules.
 Because of these locations, GGT is elevated in virtually all hepatobiliary disorders, making it
one of the most sensitive of enzyme assays in these conditions. Higher elevations are
generally observed in biliary tract obstruction.
 Because of the effects of alcohol on GGT activity, elevated GGT levels may indicate
alcoholism, particularly chronic alcoholism.
 GGT assays are useful in monitoring the effects of abstention from alcohol and are used as
such by alcohol treatment centers. Levels usually return to normal within 2 to 3 weeks after
cessation but can rise again if alcohol consumption is resumed.
 GGT activity is useful in differentiating the source of an elevated ALP level because GGT
levels are normal in skeletal disorders and during pregnancy.

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology
  Assay for Enzyme Activity
 The most widely accepted substrate for use in GGT analysis is y-glutamyl-p-nitroanilide.
 The y-glutamyl residue is transferred to glycylglycine, releasing p-nitroaniline, a chromogenic
product with a strong absorbance at 405 to 420 nm.

 Source of Error
 GGT activity is stable, with no loss of activity for 1 week at 4°C. Hemolysis does not interfere
with GGT levels because the enzyme is lacking in erythrocytes.

 Reference Range
 GGT: male, 6–45 U/L (37°C); female, 5–30 U/L (37°C).
 Values are lower in females, presumably because of suppression of enzyme activity resulting
from estrogenic or progestational hormones.

CARDIAC ENZYMES
 CK-MB
 AST
 LD (LDH) 1 & 2
 LD/HBD ratio
 MI Biomarkers

1. CREATININE KINASE
 Tissue Source
 CK is widely distributed in tissue, with highest activities found in skeletal muscle, heart
muscle, and brain tissue.
 CK is present in much smaller quantities in other tissue sources, including the bladder,
placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate, spleen, liver, and
pancreas.
 Has 3 isoenzymes:
CK-BB (CK1)= Brain
CK-MB (CK2)= Heart, muscle
CK-MM (CK3)= Muscle, heart
Cardiac muscle – CK-MM and CK-MB.
Skeletal muscle – CK-MM
Brain, GI, colon, prostate, uterus = CK=BB
 Trauma to skeletal muscle causes increase in total CK and MB isoenzyme, but % activity
of MB is <3% (>6% in MI)

2. Aspartate Aminotransferase
 It is commonly referred to as a transaminase and is involved in the transfer of an amino group
between aspartate and a-keto acids.
 The older terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT), may also
be used.
 Pyridoxal phosphate functions as a coenzyme

 Tissue Source
 AST is widely distributed in human tissue.
 The highest concentrations are found in cardiac tissue, liver, and skeletal muscle.
 With smaller amounts found in the kidney, pancreas, and erythrocytes.

 Diagnostic Significance
 The clinical use of AST is limited mainly to the evaluation of hepatocellular disorders and
skeletal muscle involvement.
 In AMI, AST levels begin to rise within 6 to 8 hours, peak at 24 hours, and generally return to
normal within 5 days. However, because of the wide tissue distribution, AST levels are not
useful in the diagnosis of AMI.

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology
  AST Isoenzyme
 AST exists as two isoenzyme fractions located in the cell cytoplasm and mitochondria.
1. Cytoplasmic AST – predominant in serum.
2. Mitochondria – cellular necrosis.

 Assay for Enzyme Activity

 Karmen method - which incorporates a coupled enzymatic reaction using malate
dehydrogenase (MD) as the indicator reaction and monitors the change in absorbance at
340 nm continuously as NADH is oxidized to NAD.
 The optimal pH is 7.3 to 7.8.

 Source of Error/C
 Hemolysis should be avoided because it can dramatically increase serum AST concentration.
 AST activity is stable in serum for 3 to 4 days at refrigerated temperatures.

 Reference Range = 5 to 30 U/L (37°C)

3. Lactate DeHydrogenase 1 & 2

 LD 1 (Anodic), while LD 5 is (cathodic)
 LD 1 and LD2 (HEAT STABLE)
 LD5 (COLD LABILE)
 Used in evaluating Cardiac Disorders
 Normally LD1< LD2
 Normal Ratio is 0.5-0.75 or <1
 If LD1>LD2 it is called a flipped ratio
o Flipped ratio is seen in MI (provided sample is not hemolyzed)
o 50% MI: flipped ratio in 48hours
o 80% MI: flipped ratio in 72 hours
o Catalyzes:
o reversible lactate pyruvate using NAD+ as a cofactor.
 Forward method: Wacker (pH 8.8)
o Lactate  Pyruvate
 Reverse method: Wrobleuski La Due (pH 7.2)
o Pyruvate  Lactate

 CLINICAL SIGNIFICANCE
Increase in:
Anemia (pernicious, hemolytic, megaloblastic)
Myocardial infarction (MI)
Muscle trauma
Renal infarct

 4 SUBUNITS in each Isoenzyme

ISOENZYME SUBUNITS SHORTHAND SUBUNITS HIGH CONCENTRATION

LD1 HHHH LD H4 Heart, RBC, Brain

LD2 HHHM LD H3M Heart, RBC, Brain

LD3 HHMM LD H2M2 Brain, Kidney, Lung

LD4 HMMM LD HM3 Liver, Skeletal muscle, kidney

LD5 MMMM LD M4 Liver, Skeletal muscle, ileum

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology
NON-ENZYMATIC PROTEIN MARKERS

MYOGLOBIN
Major protein responsible for oxygen supply of striated muscle
TROPONIN
the troponin complex is a component of the thin filament of striated muscle linked to actin
Three Subunits:
Troponin I: an inhibitory subunit
Troponin T: tropomyosin-binding subunit
Troponin C: Calcium-binding subunit

ACUTE PANCREATITIS PROFILE

MYOGLOBIN TROP T TROP I CK-MB AST LD

Elevation after 2-4 hours 3-4 hours 3-12 hours 4-8 hours 6-8 hours 8-10 hours
chest pain (MI)
Peak activity 6-10 hours 48 hours 12-24 hours 12 - 24 hours 24-48 hours 72 hours
Duration of 2-5 days 2-5 days 5-10 days 2-3 days 4 days 10 days
elevation
Sensitivity Sensitive but More sensitive More sensitive Not entirely Not sensitive, Insensitive,
not specific and specific and specific specific for AMI not specific Nonspecific
than CK-MB than CK-MB
Usefulness Negative Eliminates Eliminates Gold standard Detect Detect
predictable need for LD need for LD infarction > 3 infarction > 5
marker isoenzyme isoenzyme days prior to days prior to
testing testing

1. Amylase
 found in the SALIVARY GLANDS and PANCREAS
 Breaks down starch to simple sugars
 Substrate: starch
 starch/ amylum maltose glucose
 Substrates:
o Pancreatic AMS: diastase
o Salivary AMS: ptyalin
 Serum AMS is usually pancreatic in origin
o microAMS: unbound, free. 50,000 dal. AMS found in urine
o macroAMS: bound to IgG, IgA. High MW. Measured in serum
Methods
METHODS PRINCIPLES

Saccharogenic Measures amount of maltose produced (glucose: Somogyi)

Iodometric/amyloclastic Measures starch remaining

Chromogenic Measures dye released from breakdown of polysaccharide

Kinetic Method Measures change of NAD to NADH at 340nm

2. Lipase
 Breaks down Triglyceride into fatty acids and glycerol
 Significance: Acute Pancreatitis
 Substrate: Olive Oil
 End Product: Fatty Acids
 Methods:
o Cherry-Crandall
o Sigma-Tietz
o Titration

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology
 PROSTATIC CANCER PROFILE
1. ACP
2. PSA

1. ACP (pACp)

 Optimum pH: 5
 Very Labile, Add 5M acetate buffer or citrate tablet to preserve
 ACP are found in Prostate, RBC, bone, liver, kidneys, platelets
 Substrate: organic Phosphate such as β-glyceroPO4 and ρ-nitrophenylPO4
Method
Chemical Inhibition Test
If Total ACP is normal: Stop test
If elevated: suggestive of prostatic CA
do p-ACP by Chemical Inhibition Test
Cu++ Tartrate C4H4O6-2

RBC-ACP Inactivated (+) Unaffected (-)

p-ACP Unaffected (-) Inactivated (+)

s
2. PSA (Prostate-Specific Antigen)
 Member of the kallikrein family of serine proteases uniquely produced form the epithelial cells of
the prostate gland
 Most useful TM for Prostate Cancer
 Drawback: weak in distinguishing prostate CA from nonmalignant prostate lesions
 Reference range: 0-4ng/mL

BRENT KENNETH T. LAGARTO BMLS-3

Clinical Chemistry 2 - Enzymology