Simultaneous determination of d-amino acids by the coupling method of d-amino acid oxidase with high-performance liquid chromatography

Abstract

An enzymatic assay system of d-amino acids was established using the d-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the d-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of l-amino acids, which are generally present at much higher concentrations than d-amino acids in biological samples. With this method, d-Glu, d-Asn, d-Gln, d-Ala, d-Val, d-Leu, d-Phe, and d-Ile can be quantified in the order of micromolar, and other d-amino acids except d-Asp can be assayed within a sensitivity range of 50–100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of d-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of d-Asn. Both d-amino acids were difficult to be identified using the conventional method, because the large signals from l-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.