Simultaneous two-dimensional HPLC determination of free d-serine and d-alanine in the brain and periphery of mutant rats lacking d-amino-acid oxidase

A fully automated two-dimensional HPLC system combining a microbore-ODS column and a narrowbore-enantioselective column was designed and validated, and the amounts of d-serine (d-Ser) and d-alanine (d-Ala) in various tissues and physiological fluids of Long–Evans agouti/SENDAI (LEA/Sen) rats lacking d-amino-acid oxidase (DAO) were determined. Intra- and inter-day precision was less than 4.3% and accuracy ranged between 99.9 and 104%. LEA/Sen rats were reported to lack DAO in their kidneys and expected to be a novel mutant animal lacking DAO, however, the amounts of d-amino acids in the LEA/Sen rats have not been investigated. In the present study, the intrinsic amounts of d-Ser and d-Ala, which are neuromodulators of the N-methyl-d-aspartate (NMDA) receptors, were determined in seven brain tissues, four peripheral tissues, plasma and urine of the LEA/Sen rats and compared to those of the control (Wistar and SD) rats having normal DAO activity. The levels of d-Ser in the tissues and physiological fluids of the LEA/Sen rats were significantly higher than those of the Wistar and SD rats except for the frontal brain regions. Concerning d-Ala, the amounts in the tissues and physiological fluids of the LEA/Sen rats were drastically increased compared to those of the Wistar and SD rats. These results indicate that the intrinsic amounts of d-Ser and d-Ala in the tissues of rats are regulated by DAO, and that LEA/Sen rats would be useful for the study of NMDA receptor-related diseases in which DAO is implicated.