A simple enzymatic fluorimetric method for the determination of branched-chain l-amino acids in microlitre volumes of plasma

Abstract

Existing methods for the estimation of the branched-chain amino acids (BCAA) normally require sophisticated, expensive instrumentation. An alternative is an enzymatic spectrophotometric method but this requires relatively large volumes of blood and is rather time-consuming. A simple enzymatic fluorimetric method for the measurement of the BCAA in microlitre samples of plasma is described here. The method is based on the oxidative deamination of l-leucine, l-isoleucine and l-valine by leucine dehydrogenase from Bacillus species. The NADH which is formed in stoichiometric quantities is estimated fluorimetrically. In the presence of the ketone-trapping agent hydrazine the reaction goes to completion in an alkaline incubation medium at 37°C. By this method the combined BCAA can be measured routinely in a 20 μl sample of plasma. The test exhibits acceptable precision and reproducibility.