Determination of phenylalanine enantiomers in the plasma and urine of mammals and D-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography
Determination of phenylalanine enantiomers in the plasma and urine of mammals and D-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography
复制标题
二维高效液相色谱法测定哺乳动物和 D-氨基酸氧化酶缺陷啮齿动物血浆和尿液中的苯丙氨酸对映体
DOI：
10.1016/j.bbapap.2020.140540
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Kenji HAMASE
中科院分区：
文献类型：
--
作者：
Sui-Wen HSIAO;Chiharu ISHII;Aogu FURUSHO;Chin-Ling HSIEH;Yukiko SHIMIZU;Takeyuki AKITA;Masashi MITA;Tadashi OKAMURA;Ryuichi KONNO;Tomomi IDE;Ching-Kuo LEE;Kenji HAMASE
建立了测定哺乳动物生理体液中苯丙氨酸对映体的二维高效液相色谱系统。表明β-Phe在几种神经元和代谢紊乱中具有作为疾病生物标志物和治疗分子的潜在价值，因此哺乳动物中β-Phe的调节是令人感兴趣的问题。然而，在复杂的生物样品中精确测定氨基酸对映体是困难的，并且期望开发具有实际可接受的灵敏度、选择性和通量的分析方法。本研究设计了一种二维高效液相色谱系统，该系统在第一维为反相柱，在第二维为对映体选择性柱，以4-氟-7-硝基-2，1，3-苯并恶二唑（NBD-F）为衍生试剂，对目标氨基酸对映体进行荧光衍生化。使用血浆和尿液样品验证了分析方法，并成功应用于人、大鼠和小鼠体液。在血浆中测定了痕量水平的α-苯丙氨酸，并且所有物种的% α-苯丙氨酸值约为0.1%。在尿液中，观察到相对大量的α-苯丙氨酸，人、大鼠和小鼠的% α-苯丙氨酸值分别为3.99、1.76和5.25%。β-氨基酸氧化酶（DAO）的酶活性与内源性β-Phe的量之间的关系也已阐明，并且在DAO缺陷的大鼠和小鼠中观察到高β-Phe量（血浆中约0.3%，尿液中约50%）。
A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.