Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 1130033, Japan.
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 8128582, Japan.
Molecules. 2020 Apr 9;25(7):1735. doi: 10.3390/molecules25071735.
Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine β-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.
采用亲水相互作用色谱法结合荧光检测法对胱硫醚β-合酶（CBS）缺陷型小鼠模型尿液中的同型半胱氨酸和相关硫醇（半胱氨酸、半胱氨酰甘氨酸和谷胱甘肽）进行定量分析。将尿液样品与三（2-羧乙基）膦孵育，以将二硫键还原为硫醇。在进行蛋白质沉淀去除后，用铵 7-氟-2,1,3-苯并恶二唑-4-磺酸盐（SBD-F）将硫醇衍生为荧光物质。采用酰胺型色谱柱，以乙腈/120mM 甲酸铵缓冲液（pH3.0）（81:19）作为流动相，对小鼠尿中的同型半胱氨酸、半胱氨酸、半胱氨酰甘氨酸和谷胱甘肽进行分析。所建立的方法得到了很好的验证。采用该方法对 CBS-野生型（-WT）、-杂合型（-Hetero）和-敲除型（-KO）小鼠尿中的硫醇浓度进行了定量分析。正如预期的那样，CBS-KO 小鼠的总同型半胱氨酸浓度明显高于 CBS-WT 和 CBS-Hetero 小鼠。该方法有望应用于临床前和临床研究中的诊断。
