Serum Glutamic Oxalacetic Transaminase Activity: Revised Manual and Automated Methods Using Diazonium Dyes

Abstract

The diazonium dyes fast ponceau L and fast violet B react with oxalacetate, the product of glutamic oxalacetic transaminase (GOT), to produce colored products with absorbance peaks at 467 and 530 m μ , respectively. These products tend to precipitate, but colloidal and detergent agents solubilize them. Fast ponceau L in solution is unstable to light at room temperature; lactate buffer, 0.1 M, pH 3.0, stabilizes it. Oxalacetate colorimetry with both dyes obeys Beer’s law up to concentrations of 1 μ M per ml. Optimum serum GOT activities occur at L-aspartic acid and α -ketoglutaric acid concentrations of 50 to 90 mmol. per 1. and 16 mmol. per 1., respectively. Substrate serum blanks are minimal if the pH of the color reaction is controlled. Bilirubin and acetoacetate interfere in concentrations of more than 3 mg. per dl. and 5 mg. per dl. of serum, respectively. Fast ponceau L is 25% more sensitive to GOT than fast violet B and its serum–substrate blank is 20% less. The results of the proposed manual and automated methods usually correlate well with those of the kinetic malic dehydrogenase-coupled method of reference.