Two-dimensional high-performance liquid chromatographic determination of day-night variation of D-alanine in mammals and factors controlling the circadian changes.

D-Alanine (D-Ala) is one of the naturally occurring D-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of D-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the D-Ala amounts, e.g., diet, D-amino acid oxidase (DAO) and intestinal bacteria, on the day-night changes in the intrinsic D-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking D-amino acid oxidase activity (ddY/DAO(-) mice), clear day-night changes were still observed, suggesting that the factors controlling the D-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of D-Ala were detected compared with those in the control mice, indicating that the main origin of D-Ala in the mice is intestinal bacteria. Because the D-Ala amounts in the digesta containing intestinal bacteria did not show the day-night changes, the controlling factor of the circadian changes of the D-Ala amount was suggested to be the intestinal absorption.