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Final Estimation of SGOT

This document provides instructions for quantitatively measuring alanine aminotransferase (ALT) activity using a spectrophotometer. It involves a reaction where ALT catalyzes the conversion of L-alanine and α-ketoglutarate to pyruvate and L-glutamate. The pyruvate is then reduced to L-lactate by lactate dehydrogenase while oxidizing NADH to NAD+. The decrease in absorbance of NADH at 340nm over time is directly proportional to the ALT activity in the sample. The procedure mixes reagents with the sample and measures the absorbance at set time points to calculate the ALT level. Elevated ALT indicates potential liver injury.

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144 views3 pages

Final Estimation of SGOT

This document provides instructions for quantitatively measuring alanine aminotransferase (ALT) activity using a spectrophotometer. It involves a reaction where ALT catalyzes the conversion of L-alanine and α-ketoglutarate to pyruvate and L-glutamate. The pyruvate is then reduced to L-lactate by lactate dehydrogenase while oxidizing NADH to NAD+. The decrease in absorbance of NADH at 340nm over time is directly proportional to the ALT activity in the sample. The procedure mixes reagents with the sample and measures the absorbance at set time points to calculate the ALT level. Elevated ALT indicates potential liver injury.

Uploaded by

shibsankar rakshit
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Aim: In vitro quantitatively measurement of ALT/GPT activity.

Reference: Godkar Praful B.; “Clinical Biochemistry, principles and practice”; Published
by Bhalani Publishing house, Bombay, India; Page No: - 155-156

Requirement: AST/SGOT kit of synergy- bio A division of quantum biological Pvt.Ltd.


test tube, micropipette, UV spectrophotometer etc

Principle:

1) In this reaction L-Aspartate and alpha ketoglutarate react in the presence of GOT
in the sample to yield oxaloacetate and L glutamate.

L-Aspartate + alpha ketoglutarate GOT oxaloacetate + L- Glutamate.

2) The Oxaloacetate is reducing by malate dehydrogenase (MDH) to yield L- malate


with the oxidation of NADH to NAD. The reaction is monitored by measurement of the
decreased in the absorbance of NADH at 340 nm.

Oxaloacetate + NADH

MDH

Malate +NAD

The rate of reduction in absorbance is proportion to GOT activity in sample.

Procedure:

The kit contains RI- enzyme


and RII- substrate.

Working reagent was prepared by mixing 4 parts of RI and one part of RII
Working reagent 1000 µl
Sample 100 µl

The reagents were mixed immediately at 37 °C. After 1 minute record the initial
absorbance at 340 nm. And then at 1, 2, 3 minutes from the first reading. Determine
average change in the absorbance/min subtracting each reading from the previous one
and averaging these values. Use the ∆ A/min for the calculation.

Normal range: 10-50 U/L

Observation table:

Sr.No Time Absorbance of test ∆ A/min Mean


1 min
2 min
3 min

Calculation:

ST/ GOT (U/L) = ∆ A/min × 1746


A

Result:

---------------------------------------------------------------------------------------------------------------------------------

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Clinical Significance:

AST(Aspartate aminotransferase) or SGOT (Serum Glutamate Oxaloacetate Transaminase) is an


enzyme found mainly in heart muscle, liver cells, skeletal muscle and kidneys. Injury to these tissues
results in the release of the enzyme in the blood stream. Elevated levels are found in myocardial
infraction, Cardiac operation, hepatitis, cirrhosis, acute pancreatitis, acute renal diseases, primary
muscles disease. Decrease levels may be found in pregnancy, beri-beri and diabetic ketoacidosis.

The level is significantly elevated in Acute myocardial infarction (MI). In acute MI- Serum activity
rises sharply within the first 12 hours, with a peak level at 24 hours or over and returns to normal
within 3-5 days.

Extra:

Levels> 350 IU/L are due to massive infarction (Fatal), > 150 IU/L are associated with high mortality
and levels,  < 50 IU/L are associated with low mortality. Other diseases- The rise in activity is also
observed in muscle and hepatic diseases. These can be well differentiated from simultaneous
estimations of other enzyme activities like SGPT etc, which do not show and rise in activity in Acute
MI.

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