Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice
Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice
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离线二维 LC-MS/MS 测定哺乳动物尿液中的色氨酸对映体及其在 d-氨基酸氧化酶缺陷小鼠中的含量变化
DOI：
10.1016/j.jpba.2022.114919
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发表时间：
2022
影响因子：
3.4
通讯作者：
Hamase Kenji
中科院分区：
文献类型：
--
作者：
Ishii Chiharu;Takizawa Noburo;Akita Takeyuki;Mita Masashi;Ide Tomomi;Konno Ryuichi;Hamase Kenji
D-色氨酸（D-Trp）是在微生物和软体动物中发现的氨基酸的D-对映体之一。本研究设计并建立了一种高选择性的二维手性LC-MS/MS方法，用于色氨酸对映体的检测，以研究哺乳动物体内游离D-色氨酸的存在及其调控。所开发的系统包括第一维的反相分离，第二维的对映选择性分离，也使用三重四极杆质谱仪的第三/第四维的检测。应用本方法成功地测定了包括健康志愿者和小鼠在内的哺乳动物尿中D-色氨酸的含量。在健康志愿者的混合尿样中仅检测到lyl-Trp，而在C57 BL/6 J小鼠中检测到少量的D-Trp（n = 5，%D =6.18 ± 0.47）。在缺乏d-氨基酸氧化酶（DAO）活性的B6 DAO-小鼠中，观察到相对高水平的D-色氨酸（n = 6，% d =27.43 ± 3.26）。使用具有不同分离性质的各种对映选择性柱来确认C57 BL/6 J小鼠和B6 DAO-小鼠的尿中Trp的所获得的% d值。这些结果表明，尿中D-Trp水平受DAO的调节，并期待进一步研究内源性D-Trp的组织分布和生理意义。
D-Tryptophan (D-Trp) is one of the minorD-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of freeD-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinaryD-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although onlyl-Trp was observed in a mixed urine sample of healthy volunteers, small amounts ofD-Trp were detected in the C57BL/6J mice (n = 5,%D=6.18 ± 0.47). In B6DAO-mice lacking the activity ofd-amino acid oxidase (DAO), relatively high levels ofD-Trp were observed (n = 6,%d=27.43 ± 3.26). The obtained%dvalues of Trp in the urine of the C57BL/6J mice and B6DAO-mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinaryD-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsicD-Trp, are expected.