proportional to the enzyme activity which is measured at 540 nm and compared with
pyruvic acid standard.
C=O + (^) H
2 N−
NO 2
CH 2 NO 2
C=N−N
NO 2
- H 2 O
CH (^2) NO 2
H H
N
2,4-DNPH Oxaloacetate − 2,4-DNPH
Alkaline condition
Golden brown
colour
COO− COO−
COO− COO−
Oxaloacetate
CH 3
C=O + H 2 N−
NO 2
NO 2
C=N−N
CH 3
NO 2
NO 2
H H
N
Pyruvate 2,4-DNPH Pyruvate − 2,4-DNPH
Alkaline condition
Golden brown
colour
COO− COO−
25.4 Reagents........................................
1.Phosphate buffer (0.1 M, pH 7.4): Prepare phosphate buffer by dissolving
11.3 g Na 2 HPO 4 and 2.7 g KH 2 PO 4 infinal volume of 1 l distilled water. Adjust
pH to 7.4 using either KH 2 PO 4 or Na 2 HPO 4.
2.Buffer substrate for AST: Dissolve 1.33 g of L-aspartic acid and 30 mg
α-ketoglutarate in about 20 ml of phosphate buffer. Add 3–5 pellets of NaOH
to dissolve it and adjust pH to 7.4 by NaOH or HCl. Make volume up to 100 ml
with phosphate buffer and store at 4C.
3.Buffer substrate for ALT: Dissolve 0.9 g of alanine and 30 mgα-ketoglutarate
in about 20 ml of phosphate buffer (pellets of NaOH may be added to dissolve
alanine). Make the volume up to 100 ml with phosphate buffer, and adjust pH to
7.4 if required.
4.Pyruvate standard (10 mM, pH 7.4): Dissolve 110 mg of sodium pyruvate in
phosphate buffer, and make the volume up to 100 ml. Store working standard in
small aliquots in a freezer, and one aliquot of standard may be used for preparing
a calibration graph.
5.Color reagent: dissolve 20 mg of 2,4-DNPH in 100 ml of 1 N HCI, and store in
brown bottle.
6.0.4 M NaOH
104 25 To Determine Alanine and Aspartate Transaminase Activity in Serum