EXPERIMENT-

Aim: Assay of Serum transaminase SGPT/ALT activity in serum.

Materials required: Cuvettes, Eppendorf stand , Eppendorf tubes , spectrophotometer, wash bottle,
tissue paper, beaker, pipettes, etc.

Reagents: Serum sample, L1(enzyme reagent), L2(starter reagent)

Composition: Tris buffer (100mM), L-Alanine (500mM), LDH(2KU), ketoglutarate (15mM), NADH
0.18Mm, Good Buffer 100Mm, Non reactive stabilizers, preservatives.

Theory: SGPT/ ALT is an enzyme that catalyzes transfer of an amino group from L- alanine to alpha-
keto glutarate giving pyruvate and Glutamate. It is a reversible transamination reaction this transaminase
enzyme is mainly found in liver. Increased level of SGPT is found in hepatitis, cirrhosis, obstructive
jaundice and other hepatic disease. Slightly elevation of the enzyme is also seen in myocardial infarction
SGPT test is a blood test that checks for liver damage It basically checks functioning of liver.

Principle: SGPT/ALAT Catalyzes the transfer of amino group between L-Alanine and alpha
ketoglutarate to form pyruvate and glutamate. The pyruvate formed reacts with NADH in the presence of
lactate Dehydrogenase to form NAD. the rate of oxidation of NADH to NAD is measured as a decrease in
absorbance which is proportional to the SGPT (ALAT) Activity in the sample.

Procedure
1. Take two Eppendorf tubes and mark them as test (T) and blank (B).
2. In test Eppendorf tube, add 0.8ml of enzyme reagent (L1) and 0.1ml of sample.
3. Then incubate for 1min at Assay temperature which is 37°C.
4. Then add 0.2ml of starter reagent (L2) in test (T) Eppendorf tube and mix it well.
5. Blank tube was prepared by adding 1.2 ml of water.
6. After 1 minute of mixing L2 reagent, measure absorbance at 1,2 and 3 minutes at 340 nm.
7. Then calculate change in absorbance per minute.

Result:
The concentration of SGPT in the unknown sample was found to be 19.21 U/L.

Interpretation:
The normal serum range for SGPT is upto 40U/L for males and upto 31U/L for females. The
concentration of SGPT in the given serum sample which was taken from a male was found to be 19.21
U/L which lies in the normal range of SGPT. Hence, the person is healthy and has normal liver function.

Clinical significance:
The SGPT test is a blood test that is a part of a liver profile. SGPT test may be used by a doctor to
diagnose liver damage or liver disease in patients. The test may be used to evaluate and diagnose liver
health for people who are already known to have conditions that affect the liver.

Elevated levels are found in:

● Hepatitis
● Myocardial infarction
● Acute pancreatitis
● Cirrhosis
● Obstructive jaundice
● Cholestasis or congestion of bile ducts
● Liver damage due to alcoholism/ drugs

Precautions
1. Serum sample must be free from hemolysis.
2. Pipetting should be done accurately.
3. Proper incubation time is required for the reaction to be completed.