01.12.
2014
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 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.
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
Creatine Kinase (CK) Heart, liver, other Myocardial infarct Sometimes these enzymes are measured in
Lactate dehydro- Heart, liver, other Myocardial infarct, combination (panel).
genase (LDH) tissues Liver, muscle diseases
Alkaline Liver, bone Liver diseases, bone diseases Cardiac panel: LDH isoenzymes, CK
phosphatase (ALP)
isoenzymes, AST,
Acid phosphatase Prostate Prostate tumors
(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)

catalyses the following reaction:
• Acid phosphatase: hydrolase, marker for LDH
prostate tissue Lactate + NAD+  Pyruvate + NADH + H+
• Alkaline Phosphatase: there are isoenzymes
NAD(P)+
specific for liver, bone, intestine
Absorbance 

max= 260 nm

NAD(P)H
max= 340 nm

250 300  (nm) 350 400

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LDH, is widely distributed in human tissues.

Different isoenzymes are found in different tissues.
It is a tetramer with 140 kDa.
Two types of subunits H (heart) and M (muscle)
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

Creatine kinase (CK) reaction:

CK
Creatine + ATP  Creatine phosphate + ADP

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

each. Subunits: B (brain), M (muscle)
There are 3 CK isozymes consisting of B and/or M type
subunits.

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

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

CK-1 CK-2 CK-3

(BB) (MB) (MM)

Creatine kinase appear in the plasma after

cell death.
• In the circulation, CK isozymes lose their
activities by protease action.
enzyme activity of the serum

• In vitro conditions, light and high pH

Intracellular marker
inactivates CK.
Cell
anoxia death
• Half lifes of CK isozymes at +4C are as
follows:
• CK3 - 7 days
• CK2 - 3 days
0 Time (min) 30 • CK1 – less than 3 days

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CK Isoenzymes
CK Isozymes:
CK-MM (CK3) is mainly found in skeletal and
heart muscle. Almost all of the CK activity of a
healthy person is CK-MM.
CK-MB (CK2) is high in heart muscle, make up
24 - 46% of the total CK. It is low in skeletal
muscle (<1%).
Increase of CK-MB activity in serum most
probably indicates a myocardial infarct (MI).

Several Markers of Myocardial Infarction

MI
MI, after days
MI positive control
Liver disease
MI after 2 days
MI after 1 day
MI (cardiac insufficiency + liver
Normal sample congestion)

References
Erythrocyte enzymes in diagnosis
• McPherson RA, Pincus MR Henry’s Clinical Diagnosis

Glucose-6- Erythrocyte Anemias

by Laboratory Methods, 22nd Edition, 2011
phosphate • Berg, J.M., Tymoczko, J.L., and Stryer, L.,
dehydrogenase
Pyruvate kinase Erythrocyte Anemias Biochemistry, Fifth Edition, 2002
• Nelson, D.L., and Cox, M.M., Lehninger’s Principles
of Biochemistry, Third Edition, 2000.
• Mathews, C.K. And Van Holde, K.E., Biochemistry,
Second Edition,1996.