Three-dimensional high-performance liquid chromatographic determination of serine, threonine and allothreonine enantiomers in mouse tissues and physiological fluids

The amounts of serine (Ser), threonine (Thr) and allothreonine (aThr) enantiomers in 6 tissues (cerebrum, cerebellum, medulla oblongata, pancreas, liver and kidney) and 2 physiological fluids (plasma and urine) of mice were determined using a highly-selective three-dimensional high-performance liquid chromatographic (3D-HPLC) system. The 3D-HPLC system was composed of reversed-phase (Singularity RP18), anion-exchange (Singularity AX) and chiral separation (Singularity CSP-013S) columns, and the amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole were determined. To analyze the mouse tissues and physiological fluids, the separation conditions were re-investigated from those reported in our previous study, and the method was validated using the tissue/physiological fluid samples. For Ser, the presence of the D-form could be determined in all the tissues and physiological fluids (1.77 ± 0.09–266.5 ± 11.3 nmol/g or mL). For D-Thr and D-aThr, the presence in the cerebrum and urine was clearly demonstrated (0.28 ± 0.01–1.44 ± 0.56 for D-Thr and 4.63 ± 1.44–6.44 ± 0.36 nmol/g or mL for D-aThr). Trace levels of D-Thr and D-aThr were detected in the other tissues and plasma. The results indicate that the target 3 hydroxy D-amino acids are widely present in mammalian tissues and physiological fluids, and further investigations focusing on their physiological significance are expected.