The circadian profiles of D-alanine (Ala) amounts in human serum and urine have been clarified using a two-dimensional HPLC system combining a microbore reversed-phase C18 column and a Pirkle-type enantioselective column. The two-dimensional HPLC system enables the determination of trace amounts of D-Ala in the clinical samples without severe interference from intrinsic substances in the human physiological fluids. In the volunteers having normal sleep-awake patterns, the amounts of D-Ala in the serum were high in the late evening and low in the morning. On the other hand, in the volunteers having the reversed sleep-awake patterns, the amounts of D-Ala in the serum were high in the morning and low in the nighttime. The similar circadian changes of D-Ala amounts were observed in the urine. These 24-h profiles of D-Ala are almost the same as those observed in rats and mice, suggesting that D-Ala has fundamental physiological functions related to rest-active conditions in mammals, and could also be useful as the biomarker for sleep-awake profiles.
2017 The Society for Chromatographic Sciences