Principle:

The estimation of SGOT and SGPT is done by the Rietmen and Frankel's method.

In the method, the activity of transaminases (aminotransferases) is determined by measuring the colour of the hydrazone (brown) which is formed by the reaction between 2,4-dinitrophenyl hydrazine (DNPH) and the ketoacid, a products of transaminase reaction; and is highly coloured in alkaline medium.

 

DNPH reacts with all oxoacids. These include oxoglutarate and oxaloacetate, as well as pyruvate, which are on the two sides of the above equations.

DNPH gives more colour with oxaloacetate and pyruvate than with oxoglutarate, thus making the method feasible with an acceptable limit of error.

In both estimations - ALT and AST, the substrates are suboptimal, to reduce the background colour given by the alpha-ketoglutarate (or oxoglutarate) in the reaction with DNPH.

Though not as accurate as spectrophotometric method (UV) which measures decrease in the absorbance at 340 nm as NADH as it is consumed in the reaction (NAD has no absorbance), the colorimetric method is easily adaptable and is much faster to perform than the spectrophotometric method.

 

Requirements:

M/15 phosphate buffer pH 7.4,

GOT substrate (2.66 gm aspartic acid, 30 mg a-ketoglutaric acid, 20.5 ml 1N NaOH in phosphate buffer.

Adjust pH to 7.4 and make volume to 100 ml with phosphate buffer)

GPT substrate : (1.78 gm alanine. 30 mg a-ketoglutaric acid, 1.25 ml 0.4N NaOH in phosphate buffer.

Adjust pH to 7.4 and make volume to 100 ml with phosphate buffer),

DNPH reagent (200 mg of DNPH and 85 ml of cone. HCI in distilled water. Final volume to 1000 ml with distilled water).

0.4N NaOH,

22 mg/dl sodium pyruvate standard.