Quantification of Biological Thiols in the Plasma of a Homocystinuria Model with Cystathionine β-Synthase Deficiency Utilizing Hydrophilic Interaction Liquid Chromatography and Fluorescence Detection
Quantification of Biological Thiols in the Plasma of a Homocystinuria Model with Cystathionine β-Synthase Deficiency Utilizing Hydrophilic Interaction Liquid Chromatography and Fluorescence Detection
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DOI：
10.15583/jpchrom.2016.015
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发表时间：
2016-01-01
期刊：
影响因子：
1.7
通讯作者：
Tsunoda, Makoto
中科院分区：
文献类型：
--
作者：
Isokawa, Muneki;Kobayashi, Kentaro;Tsunoda, Makoto
采用亲水作用液相色谱(HILIC)和荧光检测法对半胱氨酸β-合成酶(CBS，EC 4.2.1.22)缺陷小鼠血浆中的生物硫醇--同型半胱氨酸(Hcy)、半胱氨酸(Cys)、γ-谷氨酰半胱氨酸(GluCys)、谷胱甘肽(GSH)和半胱氨酸甘氨酸(CysGly)进行定量检测。缺乏CBS的小鼠提供了同型半胱氨酸尿症的模型小鼠，同型半胱氨酸尿症是一种遗传性代谢性疾病，其特征是Hcy在尿液和血液中异常堆积。硫醇用7-氟-2，1，3-苯并二唑-4-磺酸铵(SBD-F)衍生化，然后用酰胺柱分离，流动相为乙腈-40 mm甲酸铵缓冲液(pH 3.0)(75/25，v/v)。CBS野生型(-WT)、杂合子(-Hetero)和基因敲除(-KO)小鼠血浆Hcy总浓度分别为24.7mU/L、29.3mU/L和237.7 mU/L，Cys分别为236.4、178.7和78.5mU/L，GluCys分别为5.8、4.87和0.75mU/L，谷胱甘肽分别为64.6mU/L、74.0和51.2mU/L；CysGly的产量分别为2.48、3.98和0.90 mU/L。与CBS-WT小鼠相比，CBS-KO小鼠Hcy浓度显著升高，Cys、GluCys和CysGly浓度显著降低。结果表明，除同型半胱氨酸外，其他生物硫醇也可用于同型半胱氨酸尿症的病理评估和研究。
Biological thiols-homocysteine (Hcy), cysteine (Cys), gamma-glutamylcysteine (gamma GluCys), glutathione (GSH), and cysteinylglycine (CysGly)-in plasma samples of cystathionine beta-synthase (CBS, EC 4.2.1.22)-deficient mice were quantified using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection. Mice deficient in CBS provide a model mouse of homocystinuria, an inherited metabolic disease characterized by the abnormal accumulation of Hcy in urine and blood. For quantification, the thiols were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by separation with an amide column containing a mobile phase of acetonitrile-40 mM ammonium formate buffer (pH 3.0) (75/25, v/v). The total concentrations of Hcy in the plasma samples of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were 24.7, 29.3, and 237.7 mu mol/L, respectively; 236.4, 178.7, and 78.5 mu mol/L for Cys; 5.80, 4.87, and 0.75 mu mol/L for gamma GluCys; 64.6, 74.0, and 51.2 mu mol/L for GSH; and 2.48, 3.98, and 0.90 mu mol/L for CysGly. The concentration of Hcy was significantly increased, while Cys, gamma GluCys, and CysGly concentrations were significantly decreased in CBS-KO mice compared to those in CBS-WT mice. The results indicated that biological thiols other than Hcy could also be utilized for the evaluation and investigation of homocystinuria pathologies.