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

Mai Oyaide , Takeyuki Akita , Chiharu Ishii , Masashi Mita , Kenji Hamase
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Abstract

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.
三维高效液相色谱法测定小鼠组织和生理体液中丝氨酸、苏氨酸和异苏氨酸对映体
采用高选择性三维高效液相色谱(3D-HPLC)系统测定了小鼠6种组织(大脑、小脑、延髓、胰腺、肝脏和肾脏)和2种生理液体(血浆和尿液)中丝氨酸(Ser)、苏氨酸(Thr)和异素苏氨酸(aThr)对映体的含量。采用反相色谱(Singularity RP18)、阴离子交换色谱(Singularity AX)和手性分离色谱(Singularity CSP-013S)组成的3D-HPLC体系,测定了4-氟-7-硝基-2,1,3-苯并恶二唑衍生的氨基酸。为了分析小鼠组织和生理液,我们在之前的研究中重新研究了分离条件,并使用组织/生理液样本验证了该方法。对于Ser,可以在所有组织和生理液体中检测到d型的存在(1.77 ± 0.09-266.5 ± 11.3 nmol/g或mL)。d-Thr和d-aThr在大脑和尿液中的存在被清楚地证实(d-Thr为0.28 ± 0.01-1.44 ± 0.56,d-aThr为4.63 ± 1.44-6.44 ± 0.36 nmol/g或mL)。在其他组织和血浆中检测到微量的d-Thr和d-aThr。结果表明,目标3羟基d-氨基酸广泛存在于哺乳动物组织和生理体液中,值得进一步研究其生理意义。
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