CLINICAL ENZYMOLOGY

1. What are enzymes that are present in blood plasma?

Enzymes are found in all tissues, as well as in plasma En-
zymes in plasma can be separated into two groups:

Functional plasma Non-functional plasma

enzymes enzymes

Enzymes that are synthesized Substrates for these enzymes are NOT
by the liver predominantly, present in tissues (cellular enzymes)
present in plasma and have
functional role in plasma E.g. Pancreatic amylase
E.g. Lipoprotein lipase Clotting Creatine phosphokinase (muscles)
factors

2. What cause intracellular enzymes to be release into plasma?

3. In which clinical ways are enzymes used?

Enzymes are used clinically in three principal ways:

• in diagnosis and prognosis of various disease
• as analytical reagents in the measurement of activity of other enzyme or non-enzyme sub-
stances
• as therapeutic agents

4. How does enzyme activity assay?

Enzyme assays usually depend on the measurement of the catalytic activity of the enzyme
(more sensitive test), rather than it concentration.
Conditions of the assay are optimized and standardized E.g.

specific color
AMYLASE reagent

REDUCING
SURGARS * COLOUR
COMPLEX
optimal condition: time, (maltose, glucose)
buffer, t C
0
1
CALCULATE^_ READ OPTICAL
USING THE DENSITY
FORMULA

International Unit of Enzyme activity: (IU/L or m-IU/L)

• One IU is defined as the activity of the enzyme which transforms one micromole of sub-
strate per minute under optimal conditions.

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 5. What are isoenzymes?
Definition: Are the multiple forms of enzyme which catalyze the SAME REACTIONS and
differ from each other by:
-structurally
- electrophorecically
- Synthesis coded by different gene
-localized predominantly in the different organs/tissue -differ from each other by kinetic
properties (Km, Vmax or both)
6. What are common cellular enzymes and isoenzymes used for clinical diagnosis?
• AST/ALT (transaminases/aminotransferases)
• ALP (alkaline phosphatase)
• CK (creatine kinase) or CPK (creatine phosphokinase)
• LDH (lactate dehydrogenase)
• GGT (gamma- glytamyl transferase)
• ACP (acidic phosphatase)
• Amylase (AMS)
• Lipase (LPS)

Intracellular Distribution of
Diagnostic Enzymes

Liver Heart Pancreas Salivary Bone Muscle Biliary Prostate

Glands Tract

LD5 LD1 LPS AMS ALP CK ALP ACP

AMS GGT
ALT AST
AST CK

SOME ENZYMES OF CLINICAL INTEREST

1. SERUM CARDIAC MARKERS

Acute myocardial infarction (AMI).
• AMI generally occurs when blood flow increases abruptly after a
thrombotic occlusion of a coronary artery, previously narrowed by
atherosclerosis.
• AMI occurs when a coronary artery thrombus develops rapidly at
a site of vascular injury. This injury is produced by factors such as:
cigarette smoking, hypertension and lipid accumulation.

Cardiac markers
• Certain proteins, called serum cardiac markers, are released into the blood in large quantities
from necrotic muscles after AMI.
1. CK (Creatine phosphokinase).
• Total serum CK rise within 4 to 8 hours, reaches a peak level at 24 to 30 hours and generally
returns to normal by 48 to 72 hours.
• But total CK elevation may be due to:

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(i) Intramuscular injection of a narcotic
(ii) hypothyroidism
(iii) cardiac surgery
(iv) electric shock
(v) acute psyhosis
(vi) CNS trauma, extensive brain infarction
(vii) Progressive muscular dystrophy (levels may reach 300-400 times normal).
(viii) Chronic alcoholism

• The determination of CK isoenzymes may be helpful in a differential diagnosis.

• Structure: Dimer. Subunits: M (muscle) and B (brain) in different variation
• CK1 (CK-BB) Brain (trace)
• CK2 (CK-MB) Heart (0-4%)
• CK3(CK -MM) Skeletal muscles (96-100%)
• The elevation CK-MB (CK2), the cardiac isoenzymes, provides a more definitive indication of
myocardial cell damage than total CK.
• Appearance of this isoenzyme in plasma (> 20% of total CK) is considerably more specific in
AMI.
• But also cardiac surgery, myocarditis often results in elevated serum levels of the CK-MB.
• Diagnosis: Determination of relative index CK-MB/CK-BB.
• If CK-MB/CK-BB ratio > 1.5 is highly sensitive for diagnosis of AMI, particularly 4 to 6
hours after the coronary occlusion.
2. Lactate dehydrogenase □ LDH
• In AMI total serum LDH rises within 12 to 24 hours, reaches peak level at 48 hours and then
return to normal 8th to 14th day.
• But total serum LDH non specific for myocardial tissue.
• Elevated levels of total serum LDH are observed in various other conditions, such as
(i) Liver diseases (cirrhosis, alcoholism, acute viral hepatitis)
(ii) Cancer
(iii) Lung diseases (pulmonary infarction)
(iv) Skeletal muscles diseases
(v) (muscular dystrophy)
(vi) Megaloblastic and pernicious anemia
(vii) Sickle cell disease
Isoenzymes of LDH
Structure: Tetramer. Subunits: H (heart) and M (muscles) in different variation
• LDH1 (H4) cardiac, RBCs (17-27%)
• LDH2 (H3M) cardiac, brain, RBCs (29-39%)
• LDH3 (H2M2) lung, spleen, pancreas, placenta, brain, kidney (19-27%)
• LDH4 (HM3) - liver, skin, kidney (8-10%)
• LDH5 (M4) - liver, skeletal muscles (6-16%)
• The appearance of LDH flip (when LDHi is more than LDH 2) is extremely helpful in diagno-
sis of AMI.
• The presence of LDH flip a day following or with the detection of CK-MB is essentially diag-
nostic of MI.

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3. AST: aspartate transaminases (aminotransferase) or sGOT □ serum glutamic-oxaloacetic
transaminase.
❖ AST is an enzyme present in tissues of high metabolic activity

heart, liver, skeletal muscle, kidney, brain, pancreas, spleen, lungs

decreasing concentration of enzyme

• The enzyme is released into the circulation following the injury or death of cells.
• The amount of AST in the blood is directly related to the number of damaged
cells and the amount of time that passes between injury to the tissue and the test.
• Following severe cell damage (AMI) the blood AST level rises sharply within
the first 12 hours with a peak level at 24 hours or over and returns to normal
within 3 to 5 days.
• AST is catalyzed the conversion of Aspartic acid to oxaloacetic acid

COOH COOH
COOH
I COOH |
o AST ^ = Q H2N-CH
II
H2N —CH
COOH CH2 CH2-o- f2
CH2 CH2 + CH2
COOH COOH COOH
aspartate alpha-keto- oxaloacetate glutamate
glutarate

3. Cardiac-specific troponins
cTnT and cTnl are regulatory proteins involved in myocardial contractility.
They are not normally detectable in the blood of healthy individuals, but may increase af-
ter AMI to levels over 20 times higher than the normal.
Levels of cTnl may remain elevated for 7 to 10 days after AMI, and cTnT levels may re-
main elevated for up to 10 to 14 days.

10- Creatine kinase MB fraction

Creatine kinase

f Aspartate \
transaminase
Lactate dehydrogenase

Time course of elevation (days)

Time course of myocardial enzymes appearing in the blood after myocardial in-
farction

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 2. SERUM ENZYMES IN LIVER DISEASES

• The liver contains thousands of enzymes, some of which are also present in the serum in
very low concentration.
• Liver serum enzyme tests can be classified into 2 groups:
(A) Enzyme whose elevation in serum reflects damage to hepatocytes (Hepatocellular dis-
eases). In hepatocellular diseases (such as viral hepatitis or alcoholic liver disease), fea-
tures of liver injury, inflammation, and necrosis predominant.)
(B) Enzymes whose elevation in serum reflects cholestasis (bile duct obstruction).
# 1 ENZYMES THAT REFLECT DAMAGE TO HEPATOCYTES

1. Serum transferases (transaminases) : AST, ALT

2. Y-GT

Serum aminoptransf erases (aminotransaminases)

• Aminotransferases (aminotransaminases) are sensitive markers of liver cell injury.

• They include the AST and ALT or another name: serum glutamic-pyruvic transaminases (s-
GPT)
• Alanine aminotransferase (ALT) occurs in high concentration in the liver, and relatively
low concentrations are found in the heart, muscle and kidney.
• This test is primarily used to diagnosis liver disease and to monitor the course of treatment
for hepatitis, and the effects of drug therapy.
ALP is catalyzed the following reaction:

COOH O
1 COOH II
C=O H,N—CHC—OH 2 1 COOH
1
1 ALT
CH2 +
CH-NH2
W
I
12 CH2
CH3 CH2 + C=O
I
C=O
1 CH3
1
2
pyruvate
CH2 1 2 COOH OH
glutamate
alpha-keto- alanine
glutarate

Normal values:
N g
A e
ALT (U/L) AST (U/L)

1 Adults 7-56 5-40

2 Children 10-35 10-50

3 Newborn 6-50 35-140

• Elevation of serum Dtotal aminotransferases > 1000 U/L occurs exclusively in disorders as-
sociated with extensive hepatocellular injury such as:
(i) viral hepatitis
(ii) toxin or drug-induced liver injury
(iii) Ischemic liver injury (acute heart failure).

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 Determination of AST and ALT elevation can be helpful diagnostically
N Liver diseases Total AST ALT
aminotransferases
Acute hepatocellular necrosis Elevated, often > 500 ALT >>> AST
1.
(hepatitis) IU
2. Alcoholic liver disease AST/ALT ratio > 2.0- AST > 300 IU/L ALT - normal
3.0 or decreased due to alcohol
induced deficiency of Vit. B6,
requires for the activity of liver
ALT
3. Chronic hepatocellular disease Elevated, elevated elevated
But usually < 300
4. Intra- end extrahepatic cholestasis Normal to moderate Normal to Mild to
(obstructive jaundice) elevation mild moderate
elevation elevation

Serum y-glytamyl transferase (transpeptidase) -GGT or y-GT

GGT is present mainly in: liver, kidney, prostate, spleen.

GGT catalyses the transfer of the Y-glytamyl group from one peptide to another
peptide or an amino acid.
A B B A
GGT
'\y\S\S\s
+
CH2

CH2 CH2

CH2 CoO^

COOQ
Normal values:
Men: 5-85 U/L
Women: 5-55 U/L

• The Y-GT activity is elevated in all forms of liver disease

• The estimation of Y-GT used to determine liver cell dysfunction and the detect alcohol in-
duced liver disease (chronic alcoholism) and infiltrative diseases (metastasis, tumor).

# 2 ENZYMES THAT REFLECT CHOLESTASIS

The activities of three enzymes - ALP and 5'nucleotidase, Y-GT are usually elevated in
cholestasis.
5'nucleotidase hydrolyzes nucleotides with a phosphate group on carbon atom 5' of the ri-
bose:
NH

5'nucleotidase

HO V'A?
n—n
H2O OH OH
PI

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 • ALP and 5'nucleotidase are found in or near the bile canalicular membrane of hepatocytes,
while Y-GT is located in the endoplasmatic reticulum and in bile duct epithelial cells.
• 5'nucleotidase and Y-GT are rarely elevated in conditions other than liver disease.
5'nucleotidase:

Normal: 0 to 5 IU/L
N Liver diseases ALP total ALP-1 5'nucleotidase Y-GT
1. Acute hepatocellular Normal elevated Normal to slightElevated
necrosis elevated
(hepatitis)
2. Alcoholic liver disease Normal to < 3elevated normal Elevated
times normal (often > 4
elevation times
normal)
3. Chronic hepatocellularNormal to < 3elevated Normal to slightelevated
disease times normal elevated
elevation
4. Intra- end extrahepatic elevated elevated elevated
cholestasis (obstructiveElevated, often >4
jaundice) times normal
elevation
5 Infiltrative disease (tumor, Elevated, often >4 elevated elevated elevated
metastasis) times normal
elevation

3. SERUM ENZYMES IN BONE AND MUSCLES DISEASES

MYOPATHIES
• Skeletal muscles disorders or myopathies are defined as disorders with structural changes
or functional impairment of muscles.
• CK-MM is the muscle enzyme and its serum concentration is elevated in different types of
myopathies.
E.g. Hereditary myopathies - progressive muscular dystrophies (progressive weakness, progres-
sive kyphoscoliosis, impaired pulmonary function, acute gastric dilation, etc). Different types of
muscular dystrophies are exist: Duchenne, Emery-Dreifuss, Becker etc. with different types of
mutation of proteins, play important role in the muscular contractile, stability, etc.
In all these types serum CK-MM greatly elevated ( > 20 -100 times normal).
Different types of muscular dystrophies may be identified by new method: analysis of mutation of
DNA.
BONE DISEASES
ALP (alkaline phosphatase)
• The enzyme is a monomer and the isoenzymes are due to the difference in the carbohydrate
content:
• ALP 1 (Alpha1-ALP) liver, blood vessels
• ALP 2 (Beta 1- ALP) bone (osteoblasts)
• ALP 3 (Beta 2- ALP) intestinal epithelium, kidney
• ALP 4 (Gamma-ALP) placenta
Diagnostic importance of ALP:
• Increased ALP-2:
(i) Bone diseases: Paget' s disease, Osteoblastic bone tumors, Osteomalacia, Rickets, Skeletal dis-
ease

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 (ii) Obstruction of bile duct, diseases of bile duct

ALP-2 osteosarcoma
• ALP-2 of osteosarcoma cells binds with specific substrate (ELF - en-
dogenous light fluorescent substrate) forms a fluorescent precipitate at
the site of enzymatic activity (blue). For contrasting of these sites uses
green fluorescent precipitation of ELF with alcohol.

TUMOR MARKERS

Tumors markers- enzymes Cancer Non-Neoplastic condition

Acid phosphatase (pH=5.0)
Total LDH
Neuron-specific enolase
afetoprotein
Prostate cancer
Lymphoma
Small cell cancer of the lung
Hepatocellular carcinoma
Prostatitis, prostatic hypertrophy
Hepatitis, many other
Cirrhosis, hepatitis