Abstract
A new colorimetric method is presented for the assay of serum GPT activity on the basis of selective determination of pyruvic acid produced in the enzyme reaction under its optimal conditions by means of a modified procedure of the previously established method for the determination of the acid with p-dimethylaminobenzaldehyde. The method gives reliable results and is simple to be performed, and suitable to assay a large number of samples with a wide range of activity at the same time.
The outline of the standard assay procedure is as follows: 0.5ml of the substrate (10μ moles of 2-oxoglutaric acid and 100μ moles of DL-alanine) is mixed with 0.1ml of sample serum and incubated at 37° for 30 min. At the end of the incubation time, 1.0ml of 0.5% p-dimethylaminobenzaldehyde dissolved in an aqueous dimethylsulfoxide (60% by volume) is added to the reaction mixture to stop the enzyme reaction. The resulting mixture is mixed with 3.0ml of 12% potassium hydroxide solution with cooling, warmed at 37° for 45min. to develope the color, cooled and then diluted with 5.0ml of water. The absorbance of the final mixture at 420nm is read against the serum blank prepared in the same manner without the incubation. The enzyme activity expressed in the international units (μ moles of pyruvic acid formed/min,/1000ml of serum) is read on the calibration curve, which is prepared by treating 0.5ml portions of sodium pyruvate standard solutions and 0.1ml of 7.8w/w % bovine serum albumin (fraction No.V) solution or serum with very low GPT activity in the same way as described above without incubation.
