CLINICAL ENZYMOLOGY

• Enzymes are biological molecules that catalyse a chemical

reaction without itself undergoing any change in the overall
process.
Diagnostic Enzymes and their clinical significance
• Normal blood serum contains several enzymes.
• Their levels are normally low.
• These enzymes ordinarily arise from the normal
destruction and wear and tear of RBC, WBC and
other tissues.
• If a particular tissue is rich in a certain enzyme, then in
the disease of that tissue more of that enzyme is
liberated.
• Thus an increased activity of a particular enzyme is
pointer to the disease of that organ which is rich in
that enzyme.
 ISOENZYMES
• Enzymes which exist in multiple molecular forms in the same species.

• It was first described by R.L.Hunter and Clement Market.

• Isoenzymes are the isoforms of the same enzyme, catalysing the same

chemical reactions at different locations at different times.

• These different forms of same enzyme are known as isoenzymes. They catalyse

the same chemical reaction but differ in many physical and chemical properties.

• An example of enzyme which exists in isoenzyme forms is lactate

Dehydrogenase (LDH). It has 5 different isoenzyme forms. They are : LDH1,

LDH2, LDH3, LDH4, LDH5

• Normal serum contains all the 5 isoenzymes in a certain proportion.

• Each isoenzyme is made up of 4 polypeptide subunits (It is a tetrarmer).

• There are two types of polypeptide subunits termed H and M (signifying tissue of

origin : H-heart, M-muscle).

They occur as H4, H3M, H2M2, HM3 and M4 corresponding to LDH1, LDH2, LDH3,

LDH4 and LDH5

• For example, heart is rich in H4 and during the normal wear and tear of heart muscle

H4 is released from heart into blood.

• In myocardial infarction (heart attack) where much damage

occurs in heart muscle much more H4 is released into blood.

• Similarly skeletal muscle and liver are rich in M4 and during the

normal wear and tear of these tissues M4 is released into blood. In

diseases of skeletal muscle and liver large amount of M4 is released.

• Other isoenzymes are derived from different tissues Eg: Alkaline

phosphatase, creatine phospho kinase (creatine kinase)

Properties:
1.Isoenzymes are coded by homologous genes.
2. They are formed during post translational modification of the protein.
3. They have the same specificity but differ from each other with respect to
their structure, amino acid sequence, electrophoretic mobility, immunological
properties and source.
4. They can be separated by electrophoresis.
 The isoenzymes can be separated by electrophoresis and their
percentage distributions in normal, in myocardial infarction and
liver disease are as follows:

H4(LDH1) H3M (LDH2) H2M2 (LDH3) HM3(LDH4) M4(LDH5)

Normal Serum 25% 35% 20% 10% 10%

Serum from 40 35 20 5 0
myocardial
infarction patient
Serum from 0 0 10 30 60
patient with
disease of liver or
skeletal muscle
Creatine kinase isoenzymes.
ENZYME MARKERS IN LIVER DISEASES

• Alkaline phosphatase (ALP)

• Aspartate transaminase (AST)
• Alanine transaminase (ALT)
• Gamma Glutamyl transferase (GGT)
Alkaline phosphatase (ALP)
• It is an enzyme found in the cells lining the biliary duct of the liver.
• It helps in the breakdown of proteins.
• Normal serum concentration of ALP is 45-150 IU/L.
• It increases in biliary obstruction, acute liver disease, hepatitis, liver cancer,
infiltrative liver granulomatous liver disease, and cirrhosis.

Aspartate transaminase (AST)/ SGOT

• It is an enzyme that helps in transamination.
• Normal serum concentration of AST is 5-40 IU/L
• It increases in primary hepatoma, liver cirrhosis and chronic liver diseases.
• An increase in AST may also determine pathology in other organs mainly
HEART.
Alanine transaminase (ALT)/SGPT:
• It is an enzyme found inside the liver cells and it helps in the transamination reaction.

• Normal serum concentration of ALT is 7-56 IU/L.

• It increases in hepatitis, ischemic liver injury, alcoholic liver disease, non-alcoholic fatty liver

disease, steatohepatitis, fat deposition in liver in childhood, and due to toxins that cause liver

damage.

• ALT is a more specific indicator of liver inflammation.

• Serum AST level, serum ALT level and their ratio are important biomarkers of liver

health.

• The AST/ALT ratio increases in liver functional impairment.

• Most causes of liver injury is associated with an increase in ALT than AST but in alcoholic

liver disease, the ratio of AST/ALT is 2:1 or greater.

• The ratio is also greater in hepatitis C and liver cirrhosis due to viral hepatitis.
 Gamma Glutamyl transferase (GGT)

• GGT is a microsomal enzyme found in hepatocytes, and biliary

epithelial cells that helps in glutathione metabolism by transporting
peptides across the cell membrane.

• An increase in GGT alone does not confirm liver disease but an

increase in alkaline phosphatase along with increase in GGT
is sure confirmation of liver disorder.

• Normal serum concentration of GGT is 0-30 IU/L.

• It increases in cholestasis, acute viral hepatitis, hepatitis C,

obstructive liver disease, alcoholic liver disease, and liver cancer.
ENZYME MARKERS IN
MYOCARDIAL INFARCTION

• Creatine phosphokinase
• Aspartate transaminase
• Cardiac troponins
• Lactate dehydrogenase
Creatine phosphokinase (CPK) / creatine kinase
• Normal serum conc.

• Males is 60-400 IU/L and in females it is 40-150 IU/L.

• The isoenzyme CPK 2 or CPK MB is found in the myocardium and a

rise in serum concentration indicates myocardial cell wall injury.

• Normal serum concentration of CPK 2 is 5-25 IU/L.

• During myocardial infarction, creatine phosphokinase (CPK MB) is

released into the circulation.

• The level starts to rise in 4-6 hours of onset of chest pain,

peak within 24-30 hours

returns to normal after 48-72 hours.
Aspartate transaminase (AST)

• Normal serum concentration of AST is 5-40 IU/L.

• The AST level starts to rise within 6-12 hours

of MI

peak within 48 hours.

returns to normal in 4 -5 days.

Lactate dehydrogenase (LDH)

• Normal serum concentration of LDH is 60-250

1U/L.
• The main source of this enzyme is the heart
and red blood cells.
• LDH level starts to increase from the 2nd day
after Ml
peak within 3-4 days.
returns back to normal in 10-15 days.

• LDH is the last enzyme to rise during MI and the

last enzyme to return back to normal.
 Cardiac troponins
• Troponins (Troponin I and Troponin T) are the preferred markers of Ml as it is

known to have the highest sensitivity.

• It is a protein released from myocytes when irreversible myocardial damage

occur.

• This protein stays in blood for a longer time period when compared to other

biomarkers.

• It enters the bloodstream immediately after an MI and the level increases in

3-12 hours from the onset of chest pain,

• Peak at 24-48 hours and return to normal in 5-14 days.

• The normal serum concentration of troponin l in young and healthy adults is less

than 0.12ng/ml and troponin T levels are less than 0.01ng/ml.

Enzyme markers in muscle disease
• The enzyme markers in muscle disease are creatine kinase and aldolase.

Creatine phosphokinase (CPK)/creatine kinase (CK)

• The normal serum concentration of CPK in males is 60-400 IU/L and

females is 40-150 IU/L

• The isoenzyme CPK3 (CPK MM) is found in skeletal muscle and so its

increase indicates muscular dystrophy, muscle injury, dermatomyositis

(inflammatory disease that affects skin and muscles), polymyositis

(inflammatory disease that cause muscle weakness), malignant hyperthermia

(inherited disorder that cause muscle contractions), over exercising or

seizures.
Aldolase

• The normal serum concentration of aldolase is 1-7.5 IU/L.

• The main source of the enzyme aldolase

is the muscle tissue.

• Elevated levels of aldolase indicates a muscle or liver damage.

• A muscle damage from a heart attack releases aldolase in large

quantities.

• A high level indicates muscular dystrophy, polymyositis,

dermatomyositis, and muscle damage.

• A low level indicates muscle wasting disease or late stage muscular

dystrophy.
Enzyme markers in bone disease
The main enzyme marker in bone disease is alkaline phosphatase as the main
source of this enzyme is the bones.

Alkaline phosphatase
• The normal serum concentration of ALP is 20-140 U/L.

• Children usually have a higher level ALP than adults as their bones are still

growing.

• A person recovering from bone injury may also have a raised ALP Ievel in the 3

months after injury while their bone heals.

• The ALP level increases in bone diseases like osteoblastic tumours,

osteomalacia, rickets, Paget's disease, malnutrition, especially deficiency in

vitamin D, calcium, protein, magnesium and zinc.

 Enzyme markers in prostate cancer
• The enzyme that helps in the diagnosis of prostate cancer are acid phosphatase
and Prostate specific antigen (PSA) as they are secreted by prostate gland.

Acid phosphatase
• The normal serum concentration of acid phosphatase is 0.8 IU/L.
• The main source of origin of this enzyme is post pubertal prostatic
epithelial cells.
• It is also found in bone, spleen, kidney, liver, intestine and blood.
• The rise in this enzyme (prostate acid phosphatase -PAP) helps to diagnose
prostatic cancer. The level rises as the malignant tissue proliferates.
Prostate Specific Antigen

• PSA is the most specific and sensitive marker available for

prostate cancer.

• PSA is a protein produced by normal and malignant cells

of prostate gland.

• It is usually found in the semen and in the serum it is found in

very minute quantity (0-4ng/ml)

• A PSA level between 4-10ng/ml may indicate prostatic

cancer, but a prostate biopsy will be needed to confirm
the diagnosis.