ESTIMATION OF S.G.P.T

ESTIMATION OF S.G.P.T

(serum  glutamate – pyruvate transaminase)

REACTION:

                     G.O.T catalyzes the reaction...

alpha-ketoglutaric acid + aspartic acid
oxaloacetic acid +glutamic acid
          
                     G.P.T catalyzes the reaction

alpha-keto glutaric acid + alanine 
pyruvic acid + glutamic acid

the method used for the estimation of S.G.P.T is the Reitman and Frankel method, which is a colorimetric method

REITMAN FRANKEL METHOD
PRINCIPLE:

 the pyruvic acid produced by S.G.P.T reacts with 2,4-dinitrophenylhydrazine(DNPH) sol in an alkaline medium which is measured at 520nm

REAGENTS:

1. GPT substrate
2.2,4-DNPH sol
3. sodium hydroxide solution
std pyruvate solution

PROCEDURE:

(a) for the std curve:
ml. standard
pyruvate
solution    Concentration
(ug)    Water
(ml)    Unit
equivalent
0.0
0.1
0.2
0.3
0.4
0.5    2.2
250
500
750
1000
1250    000
2.1
2.0
1.9
1.8
1.7    2.20
20
55
95
148
216

To each of the above tubes add 1ml of 2,4-DNPH sol mix and let stand for 15min. Add 10ml of 0.4N sodium hydroxide soln and let stand for 20min. Read the absorbance at 520nm with a spectrophotometer. Plot a std curve with absorbance on the y-axis and the unit equivalent on the x-axis.

(b) FOR SAMPLE:
mix 0.1ml of serum and 0.5ml of GPT (glutamic-pyruvic transaminase) substrate and incubate at 37 degree C, for 1 hr. add 0.5ml of 2,4-DNPH sol and let stand for 15min at room temp.
add 5ml of 0.4N sodium hydroxide mix and let stand at room temp for 20min. Read the absorbance at 520nm with a spectrophotometer to calculate the concentration of S.G.P.T in the given serum sample from the standard graph.