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.
Miyoshi
Yurika
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hamase
Kenji
K
Tojo
Yosuke
Y
Mita
Masashi
M
Konno
Ryuichi
R
Zaitsu
Kiyoshi
K
D000409:Alanine / Q000032:analysis* / Q000097:blood / Q000652:urine; D000818:Animals; D001826:Body Fluids / Q000737:chemistry* / Q000201:enzymology; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003605:D-Amino-Acid Oxidase / Q000032:analysis*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D012694:Serine / Q000032:analysis* / Q000097:blood / Q000652:urine
D012694:Serine; D003605:D-Amino-Acid Oxidase; D000409:Alanine