Analytical and Bioanalytical Chemistry

, Volume 405, Issue 25, pp 8083–8091 | Cite as

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

  • Sachise Karakawa
  • Yurika Miyoshi
  • Ryuichi Konno
  • Satoru Koyanagi
  • Masashi Mita
  • Shigehiro Ohdo
  • Kenji HamaseEmail author
Research Paper
Part of the following topical collections:
  1. Amino Acid Analysis


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.


2D-HPLC Chiral separation d-Alanine Circadian changes 



This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Sachise Karakawa
    • 1
  • Yurika Miyoshi
    • 1
  • Ryuichi Konno
    • 2
  • Satoru Koyanagi
    • 1
  • Masashi Mita
    • 3
  • Shigehiro Ohdo
    • 1
  • Kenji Hamase
    • 1
    Email author
  1. 1.Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan
  2. 2.Department of Pharmaceutical SciencesInternational University of Health and WelfareOhtawaraJapan
  3. 3.Shiseido Co., Ltd.TokyoJapan

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