Determination Of The Four Threonine Isomers And Isoleucine In Mammals

The presence of free D-amino acids has recently been shown in various living organisms including mammals, and their functions and origins have gradually been determined. Threonine and isoleucine have two chiral centers and four isomers (L-AA, D-AA, L-allo-AA and D-allo-AA) are present. The distribution of four threonine isomers and isoleucine isomers in the mammalian body has never been reported.A sensitive and selective method for the determination of four threonine (Thr) isomers (L-Thr, D-Thr, L-allo-Thr and D-allo-Thr) in mammalian tissues and physiological fluids has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of D-Thr, D-allo-Thr and L-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol - 5 pmol per injection, and that of L-Thr is 50 fmol - 50 pmol. Within-day and day-to-day precisions of the determination of four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of D-Thr, D-allo-Thr and L-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of D-Thr and D-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested,...