The multiple forms of an enzyme catalyzing the same chemical reaction are called isoenzmyes. They, however, differ in their physical and chemical properties. Examples: Isozymes of numerous dehydrogenases, and several oxidases, transaminases, phosphatases, transphosphorylases, proteolytic enzymes, aldolases.

1. ISOENZYMES
MISS AMANJOT KAUR SIDHU
MSN, CCN.

2. ENZYMES
•Enzymes are organic compounds produced in
living organisms to speed up and regulate the
rate of spontaneous biochemical reactions in
and outside the cells, without themselves being
used.
•They are also known as bio-catalysts.

3. CHARACTERISTICS OF ENZYMES
•The enzymes remain unchanged in amount and
chemical composition at the end of reaction.
•Only a small quantity of enzyme is generally
needed.
•Enzyme does not initiate or begin the reaction.
•Enzyme is specific in its action.

4. Contd….
• Enzyme usually cannot alter the nature of the product
of reaction.
• Enzyme may be poisoned by certain substances
(inhibitors).
• In enzyme catalyzed reactions, the substances on
which enzyme acts, is called substrate. During the
reaction, the substrate gets converted into products.

5. ISOENZYMES
• The multiple forms of an enzyme catalyzing the
same chemical reaction are called isoenzmyes.
They, however, differ in their physical and
chemical properties.
• Examples: Isozymes of numerous
dehydrogenases, and several oxidases,
transaminases, phosphatases,
transphosphorylases, proteolytic enzymes,
aldolases.

6. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Liver Diseases: Liver cells contain several
enzymes which may be released into the
circulation in liver damage. Measurement of
selected enzymes in serum is often used to
assess the liver function.
• There is no single enzyme that is absolutely
specific to liver alone. Despite this fact, serum
enzymes provide valuable information for LFT.
• Some of these enzymes are discussed below:

7. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
1. Aminotransferases or Transaminases: The activities of two
enzymes, i.e. ALT (alanine transaminase; also called SGPT or
serum glutamate pyruvate transaminase) and AST (aspartate
transaminase; also called SGOT or serum glutamate oxaloacetate
transaminase), are widely used to assess liver function.
• ALT is a cytoplasmic enzyme while AST is found in both cytoplasm
and mitochondria. The activity of these enzymes is low in normal
serum (ALT, 5-40 IU/L; AST, 5 - 45 IU/L).
• Serum ALT and AST levels are increased in liver damage. However,
ALT is more sensitive and reliable for the assessment of LFT.

8. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
1. Estimation of Aspartate Aminotransferase (AST):
• AST (or SGOT) effects the conversion of a-
ketoglutarate (2-oxoglutarate) and aspartate to L-
glutamate and oxaloacetate respectively by amino
group transfer.
• The oxaloacetate thus formed is coupled with 2,4-
dinitro-phenylhydrazine to produce a coloured
complex whose absorbance in alkaline solution is
measured at 505 nm.

9. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
2. Estimation of Alanine Aminotransferase (ALT):
•ALT or SGPT effects the conversion of α-
ketoglutarate and alanine to 2-oxoglutarate and
pyruvate respectively by amino group transfer.
•Pyruvate formed is coupled with 2,4-
dinitrophenyl hydrazine to produce a coloured
complex. Its absorbance in alkaline solution is
measured at 505 nm.

10. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
II. Estimation of Alkaline Phosphatase (ALP):
• ALP is mainly derived from bone and liver (from the cells lining the bile
canaliculi).
• Normal level of serum ALP in males (20-60 years) and females (15-60 years)
is 20-90 IU/L while in children (1-2 years) is up to 350 IU/L.
• A rise in serum ALP usually associated with elevated serum bilirubin is an
indicator of biliary obstruction. ALP is also elevated in cirrhosis of liver and
hepatic tumors.
• A liver is not the sole source of ALP; its measurement has to be carefully
viewed (along with others) before arriving at any conclusion.
• The liver and bone isoenzymes of ALP can be separated by electrophoresis.

11. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
•Principle of the ALP Test:
•In the presence of magnesium and zinc ions, p-
nitrophenyl phosphatase is cleaved by
phosphatases into phosphate and p-nitrophenol.
The p-nitrophenol released is directly
proportional to the catalytic ALP activity. It is
determined by measuring the increase in
absorbance at 409 nm (violet filter), setting the
spectrometer to zero with the blank.

12. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Gamma Glutamyl Transpeptidase (GGT) Test:
• This microsomal enzyme is widely distributed in body tissues
including liver. Measurement of GGT activity provides a
sensitive index to assess liver abnormality. The activity of this
enzyme almost parallels that of transminases in hepatic
damage.
• Normal serum GGT level is 5-40 IU/L. Serum GGT is highly
elevated in biliary obstruction and alcoholism. Several drugs
(eg, phenytoin) induce (liver synthesis) and increase this
enzyme in circulation.

13. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Principle of the GGT Test: The DxC800 uses an
enzymatic rate method to determine the GGT activity
in serum or plasma. In the reaction, the GGT
catalyzes the transfer of a gamma-glutamyl group
from the colorless substrate (gamma-glutamyl-p-
nitroaniline) to the acceptor (glycylglycine) with
production of colored product, p-nitroaniline. The
system monitors the rate of change in absorbance at
410 nm over a fixed time interval. The rate of change
in absorbance is directly proportional to the activity of
GGT in the sample.

14. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
Myocardial Infarction:
Following enzymes are commonly Kits are cardiac
infarction).
1. Creatine kinase (CK),
2. Cardiac troponins
3. Aspartate amino transferase (AST)
4. Lactate dehydrogenase (LDH)

15. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
1. Creatine Kinase (CK): CK is also released from damaged skeletal
muscle (CK-MM), heart cells contain another CK isoenzyme, CK-
MB. This isoenzyme is a more specific indicator of cardiac muscle
damage, and it increasingly used (rather than total CK) in the
investigation of MI.
2. Cardiac Troponins: Troponins are the most widely recognized and
important cardiac enzyme used in the diagnosis of acute
myocardial ischaemia in modern medicine. Troponin T and
troponin I levels in the blood rise as early as 4 hours from the
onset of acute MI symptoms, peaks in 24 to 48 hours, and remain
elevated for multiple days thereby making them useful for detecting
initial ischaemic events but not reliable to detect re-infarction.

16. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
•Lactate Dehydrogenase: LDH is useful for
the diagnosis of myocardial infarction,
infective hepatitis, leukemia and muscular
dystrophy (serum LDH normal 50-200 IU/L).

17. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Creatine kinase (CK): CK is the most widely used
enzyme to diagnose and follow muscle disease. Its
concentration rise in serum in response to muscle injury
and is the best measure of the course of muscle injury.
• Aldolase: An increase in serum aldolase is found in
myotonic muscular disease, such as progressive
muscular dystrophy. The enzyme rises in myocardial
infarction, reaches a maximum within 24-48 hours and
returns to normal in the course of 5 days.

18. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Bone Diseases:
• ALP (Alkaline Phosphatase) Enzyme: Raised activity of ALP
enzyme is especially useful in diagnosis of bone and liver
pathology, since these are the major sources of the enzyme.
ALP in bone probably plays a role in calcium phosphate
deposition. This enzyme is often elevated in several bone
disorders where osteoblastic activity increases. In addition,
malignant disorders, primary or metastatic, may raise the
enzyme activity.

19. ENZYMES OF DIAGNOSTIC
IMPORTANCE IN VARIOUS DISEASES
• Prostate Cancer:
1. Acid Phosphatase (ACP): It is increased in the cancer of prostate gland (normal 0.5- 4 KA units/dl). The
tartarate labile ACP (normal<1KA units/dl) is useful for the diagosis and prognosis of prostate cancers, i.e,
ACP is a good tumor marker.
2. Prostatic Specific Antigen (PSA): Serum PAP increases only when the tumor has penetrated the prostate
capsule due to its large molecular mass (100kDa). While specimen handling is extremely important for PAP
because of its instability.
• PSA is stable at room temperature up to 48 hours. It was also found to be stable at 20°C for as long as 6
months.
• Several studies have shown PSA to be a more sensitive tumor marker for prostate cancer than PAP. PSA
increased directly with increased clinical stage, where PAP failed to distinguish among the early clinical stages.
• With respect to monitoring treatment, serum PSA reflects more accurately the course of the disease than does
PAP.
• PSA was found to be more sensitive than PAP in monitoring the effect of radiotherapy in prostate cancer
patients.
• As an immunohistochemical marker, PSA appears to be more specific marker for prostate than PAP.