A simple enzymatic fluorimetric method for the determination of branched-chain L-amino acids in microlitre volumes of plasma.
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 microliter 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 degrees C. By this method the combined BCAA can be measured routinely in 20 microliter sample of plasma. The test exhibits acceptable precision and reproducibility.
D000328:Adult; D000594:Amino Acid Oxidoreductases; D000597:Amino Acids, Branched-Chain / Q000097:blood*; D001407:Bacillus / Q000201:enzymology; D006801:Humans; D050803:Leucine Dehydrogenase; D008297:Male; D008832:Microchemistry; D009249:NADP / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods
D000597:Amino Acids, Branched-Chain; D009249:NADP; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase