Title:

Chiral amino acid metabolomics using multi-dimensional HPLC for drug discovery and diagnosis

 

Speaker:

Kenji HAMASE

Graduate School of Pharmaceutical Sciences, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Phone: +81-92-642-6598, FAX: +81-92-642-6598
E-mail: hamase@phar.kyushu-u.ac.jp

 

 

Abstract:

D-Amino acids are the enantiomers of widely present L-amino acids, and are expected as novel candidates of physiologically active substances and biomarkers. The metabolomics studies focusing on chiral amino acids are thus currently the matters of high interest. However, the amounts of D-amino acids are less than 0.1-1% of the L-enantiomers in most cases, and an analytical method with sufficient sensitivity and selectivity is essential. In the present study, we have established multi-dimensional HPLC systems and applied to various clinical samples. For the sensitive analysis, all proteinogenic amino acids (plus allo forms of isoleucine and threonine) are derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) prior to the HPLC analysis. As the multi-dimensional separations of NBD-amino acids, on-line connected reversed-phase, anion-exchange and chiral separations are carried out. The elution of NBD-amino acids is monitored with fluorescence detectors (ex. 470 nm, em. 530 nm) and also by the MS/MS instruments. By using the present multi-dimensional HPLC, amino acid enantiomers in various biological matrices were successfully determined without interference by intrinsic substances in biological matrices. In human serum/plasma, trace amounts of D-Ala, D-Asn, D-Pro and D-Ser were found and their amounts were correlated with renal function. In the urine, D-forms of most of the proteinogenic amino acids were observed and their amounts were also changed by some diseases. Further clinical applications are in progress.

 

Representative Publications
1) Yurika MIYOSHI, Tsubasa OYAMA, Yusuke ITOH, Kenji HAMASE, Enantioselective two-dimensional high-performance liquid chromatographic determination of amino acids; analysis and physiological significance of D-amino acids in mammals, Chromatography, 35, 49-57 (2014). DOI:10.15583/jpchrom.2014.005
2) Jumpei SASABE, Yurika MIYOSHI, Masataka SUZUKI, Masashi MITA, Ryuichi KONNO, Masaaki MATSUOKA, Kenji HAMASE, Sadakazu AISO, D-Amino acid oxidase controls motoneuron degeneration through D-serine, Proceedings of the National Academy of Sciences of the United States of America, 109, 627-632 (2012). DOI:10.1073/pnas.1114639109
3) Tomonori KIMURA, Kenji HAMASE, Yurika MIYOSHI, Ryohei YAMAMOTO, Keiko YASUDA, Masashi MITA, Hiromi RAKUGI, Terumasa HAYASHI, Yoshitaka ISAKA, Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease, Scientific Reports, 6, 26137 (2016), DOI:10.1038/srep26137