Three-dimensional high-performance liquid chromatographic determination of serine, threonine and allothreonine enantiomers in the d-amino acid oxidase deficient mice and rats.

The amounts of the serine (Ser), threonine (Thr) and allothreonine (aThr) enantiomers were determined in tissues (cerebrum, cerebellum, pancreas, liver and kidney) and physiological fluids (plasma and urine) of rats and mice with deficiency of d-amino acid oxidase (DAO). DAO is an enzyme metabolizing d-amino acids in mammals and has been implicated in the pathophysiology of several diseases via the alteration of d-amino acids. To determine trace levels of the amino acid enantiomers, a three-dimensional (3D) HPLC system composed of reversed-phase, anion-exchange and chiral separations was designed and utilized. Prior to the 3D-HPLC analysis, the analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to enhance the fluorescence detection sensitivity. By using the 3D-HPLC system, the tissues and physiological fluids of F344-Daoldao rats and B6DAO-/- mice (animals with the DAO deficiency) were analyzed. In both species, d-Ser levels were elevated in the absence of DAO activity except for the cerebrum. The amounts of d-Thr and d-aThr were increased in the cerebellum and kidney with the DAO deficiency while their amounts were almost the same in the other tissues and physiological fluids. These results indicated that the intrinsic d-Ser analogues were metabolized by DAO in mammals and further studies to clarify its physiological significance are expected.