25.11.
2015
Enzymes in Diagnosis
Prof. Dr. N. Leyla Açan
Hacettepe University
Faculty of Medicine
Department of Medical Biochemistry
Analytes which give information about an
organ or a tissue are classified as:
1. Synthesized in the tissue and secreted to
the plasma;
2. Released to the circulation due to a tissue
injury;
3. Removed from the circulation and
metabolized by the tissue;
4. Excreted into the urine or feces.
Enzymes constitute an important part
among these laboratory test.
• Either plasma or erythrocytes or tissue
homogenates are used depending on the
localization of the enzyme.
• Reference interval: the interval in which 95% of
the population fall into.
• The method of the activity measurement is
prefered to be rapid, easy to apply and
inexpensive.
Aim: To study the use of enzymes in diagnosis
of several diseases.
Outline:
• Tissue distribution of the enzymes
• Pathological conditions which cause a change in
serum enzyme levels
• Specific enzymes used for diagnosis
An enzyme or isoenzyme must fulfill at least
one of the following criteria, to be used in
diagnosis.
• It must be specific for a tissue.
• Change in the activity must be proportional
to the function of the tissue.
• Change in the activity must be proportional
to the injury of the tissue.
• Blood/serum level must correlate with the
stage of the disease.
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Activity units (SI = International system):
One unit of enzyme activity (U) = 1 micromole of
substrate converted to product per minute.
For plasma enzymes U/L is used.
For tissues, specific activity= U/mg of protein is
prefered.
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Measurement of the enzyme activity:

Rate of disappearance of S, or rate of formation • Enzymes may also be used as a tool to measure
of P. the concentration of a substrate.
Continuous, discontinuous, coupled assays.

Measurement of Isoenzymes: Examples:

Electrophoresis, chromatography, heat stability • Glucose oxidase is used to measure blood
test, inhibitors, etc. glucose.
• Urease is used to measure urea.
Detection of the enzyme: immunologic
methods, electrophoretic methods

Most common plasma enzymes and isozymes

in diagnosis
Enzyme Source Disease Sometimes these enzymes are measured in
Creatine Kinase (CK) Heart, liver, other Myocardial infarct
combination (panel).
Alkaline Liver, bone Liver diseases, bone diseases
phosphatase (ALP) Cardiac panel: CK isoenzymes, AST, (LDH
Acid phosphatase Prostate Prostate tumors isoenzymes)
(ACP)
Alanine amino- Liver Liver diseases
Liver panel: ALT, AST, GGT, ALP
transferase (ALT)
Aspartate amino- Heart, liver Heart, liver diseases
transferase (AST)
Gamma glutamyl Liver Liver diseases
transpeptidase
(GGT)
Amylase Pancreas Acute pancreatitis

Specific Enzymes
Lactate Dehydrogenase (LDH) is widely distributed
in human tissues. Different isoenzymes are found
• Acid phosphatase: hydrolase, marker for
in different tissues. It is a tetramer with 140 kDa.
prostate tissue
Two types of subunits H (heart) and M (muscle)
• Alkaline Phosphatase: there are isoenzymes
specific for liver, bone, intestine combine randomly in tetramers to make five types
of isozymes.

LDH1 LDH2 LDH3 LDH4 LDH5

H H H H H H H M M M
H H H M M M M M M M

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If LDH activity is used to diagnose MI, hemolysis should be

avoided, because erythrocytes are rich in heart isozyme (H 4).

Creatine kinase appear in the plasma after

Creatine kinase (CK) reaction: cell death.
CK
Creatine + ATP  Creatine phosphate + ADP
enzyme activity of the serum

CK is a dimer with identical or different subunits of 40 kDa

each. Subunits: B (brain), M (muscle) Intracellular marker

There are 3 CK isozymes consisting of B and/or M type Cell

subunits. anoxia death

CK-1 CK-2 CK-3 0 Time (min) 30

(BB) (MB) (MM)

CK Isozymes:
• In the circulation, CK isozymes lose their
CK-MM (CK3) is mainly found in skeletal and
activities by protease action.
heart muscle. Almost all of the CK activity of a
• In vitro conditions, light and high pH
inactivates CK. healthy person is CK-MM.
• Half lifes of CK isozymes at +4C are as CK-MB (CK2) is high in heart muscle, make up
follows: 24 - 46% of the total CK. It is low in skeletal
• CK3 - 7 days muscle (<1%).
• CK2 - 3 days Increase of CK-MB activity in serum most
• CK1 – less than 3 days probably indicates a myocardial infarct (MI).

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CK Isoenzymes
Erythrocyte enzymes in diagnosis

Glucose-6- Erythrocyte Anemias

phosphate
dehydrogenase
Pyruvate kinase Erythrocyte Anemias

References
• McPherson RA, Pincus MR Henry’s Clinical Diagnosis
by Laboratory Methods, 22nd Edition, 2011
• Berg, J.M., Tymoczko, J.L., and Stryer, L.,
Biochemistry, Fifth Edition, 2002
• Nelson DL, Cox MM, Lehninger Principles of
Biochemistry, 5. ED., WH Freeman & Co, New York,
2008
• Mathews, C.K. And Van Holde, K.E., Biochemistry,
Second Edition,1996.