Parmentier C, Leroy P, Wellman M, Nicolas A
Laboratoire de Chimie Analytique, Faculté des Sciences Pharmaceutiques et Biologiques, Nancy, France.
J Chromatogr B Biomed Sci Appl. 1998 Nov 20;719(1-2):37-46. doi: 10.1016/s0378-4347(98)00414-9.
A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, gamma-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and gamma-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.
描述了一种用于测定最重要的细胞硫醇（还原型谷胱甘肽（GSH）、半胱氨酸、γ-谷氨酰半胱氨酸和半胱氨酰甘氨酸）的高效液相色谱（HPLC）方法。分离依赖于等度离子对反相色谱，检测通过荧光光谱法结合柱后衍生反应进行，使用N-（1-芘基）马来酰亚胺（NPM）或邻苯二甲醛（OPA）。当不使用共试剂的OPA时，仅检测到GSH和γ-谷氨酰半胱氨酸（异双功能反应）。然而，在流动相中存在甘氨酸的情况下进行OPA反应（硫醇选择性反应）或使用NPM均可检测到所有上述硫醇。该HPLC系统已在线性、准确度和精密度方面得到验证。所达到的低检测限（每种注入硫醇的皮摩尔范围内）允许使用异双功能OPA反应筛选和定量V79cl和V79HGGT细胞中的硫醇（作为NPM衍生物），以及测量与谷胱甘肽合成相关的两种胞质酶。
