Determination of D-serine and D-alanine in the Tissues and Physiological Fluids of Mice with Various D-amino-acid Oxidase Activities Using Two-dimensional High-performance Liquid Chromatography with Fluorescence Detection

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of D-serine (D-Ser) and D-alanine (D-Ala), and their amounts in the tissues and physiological fluids of mice with various D-amino-acid oxidase (DAO) activities have been demonstrated. These two D-amino acids are modulators of the N-methyl-D-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the D-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm x 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm x 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of D-Ser and D-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of D-Ser and D-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these D-amino acids were drastically increased with a lowering of the DAO activity except for the cases of D-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of D-Ser and D-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.