ESTIMATION OF S.G.P.T(serum glutamate – pyruvate transaminase)
REACTION:
G.O.T catalyses the reaction....alpha-ketoglutaric acid + aspartic acid
oxaloacetic acid +glutamic acid
G.P.T catalyses the reaction
alpha-keto glutaric acid + alanine
pyruvic acid + glutamic acid
the method used for the estimation of S.G.P.T is reitman and frankel method, which is colorimetric method
REITMAN FRANKEL METHOD
PRINCPLE:
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 substrate2.2,4-DNPH sol
3.sodium hydroxide solution
std pyruvate solution
PROCEDURE:
(a) for 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 tube and 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 y-axis and the unit equivalent on 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 calculate the concentration of S.G.P.T in the given serum sample from the standard graph.
Puvvukonvict
0 Comments
Thanks for your feedback, i'll get back to you soon