Determination of Pyruvic Acid in Serum by the Enzyme Electrode Method

Abstract

Hyper-lactatemia and lactic acidosis are known to be an important clinical disorder. When blood levels of lactate/pyruvate ratio increased in lactic acidosis, it indicates the serious and poor prognosis of life, and the ratio reflects tissue hypoxia.
A rapid, specific, and simple enzyme electrode method for the determination of pyruvic acid was studied. The principle is as follows: The pyruvic acid is oxidized to acetylphosphate, carbon dioxide, and hydrogen peroxide with pyruvate oxidase in the presence of phosphate ion, thiamine pyrophosphate, and metal ion such as magnesium or manganese. The hydrogen peroxide generated by the enzymatic reaction is detectable using a polarographic electrode. The electrode current is directly proportional to concentration of pyruvic acid.
Pyruvate oxidase was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide in various oxidative materials. Properties of immobilized enzyme membrane, enzyme activity, apparent Km, optimum pH, optimum temperature, pH stability, and temperature stability, were 0.03 U/cm², 9.4 mmol/L, pH 6.5, 37°C, pH 5.0-8.0, and below 50°C, respectively in the given conditions.
The pyruvate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. The pyruvate electrode responded linearly to pyruvic acid over the concentration 0-1.0 mmol/L within 30s on the flow system. When the enzyme electrode was applied to the determination of pyruvic acid in control serum, within-assay precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.1% with mean value of 0.28 mmol, /L, 88.0%, and r=0.930 (Y=1.048X+0.0002, X: colorimetric method), respectively. The dried immobilized enzyme membrane retained 96% of its initial activity after storage at 4°Cduring 15 months. However the pyruvate electrode was unstable to assay the concentration of pyruvate continuously under the conditions at 30°C, pH 6.0 in sodium acetate buffer solution. The apparent output decreased by about 20% after a day.