Determination of Serum Pyruvate Concentration Using Enzymatic Cycling

Abstract

We describe a simple enzymatic method based on an enzymatic cycling reaction using D-amino acid as substrate for the determination of serum pyruvate. The enzymatic cycling reaction is as follows: pyruvate in serum and D-glutamate are transformed to Dalanine and 2-oxoglutarate by the catalysis of D-alanine aminotransferase (EC. 1. 4. 3. 3). The D-alanine produced is then converted to pyruvate with D-amino acid oxidase, producing hydrogen peroxide and ammonia. From this enzymatic cycling reaction, the hydrogen peroxide produced is determined colorimetrically with 4-aminoantipyrine and phenol by the catalysis of peroxidase.
The standard curve was linear up to 4.0mg/dl and the mean recovery was 95.4%. The correlation coefficient between a method using deproteination and the present method was 0.955 (r) and the regression line was Y=1.12X-0.01 (mg/dl, Y: present method, X: deproteination method). The mean value of within-run reproducibility (C.V) was 4.9%. Thus the method based on enzymatic cycling using D-amino acid provides a simple, precise and accurate method. This method can be applied not only to the assay of pyruvate but also to that of other biological materials.