Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Jun 15;929:51-5. doi: 10.1016/j.jchromb.2013.04.004. Epub 2013 Apr 12.
Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-(13)C, (15)N-NEM and GSSG-(13)C,(15)N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CV's of 3.1-4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4μM for GSH and 0.1μM for GSSG and the lower limits of quantitation (LLOQ) were 1.5μM for GSH and 0.1μM for GSSG. Derivatized samples are stable for at least 3 years when stored at -80°C, and underivatized samples for at least 24h at either 4°C or room temperature. Reference intervals were determined for 59 control samples, and were (mean±SD): GSH 900±140μM; GSSG 1.17±0.43μM; GSH/GSSG 880±370.
谷胱甘肽（γ-谷氨酰半胱氨酸甘氨酸，GSH）水平和 GSH 与谷胱甘肽二硫化物（GSSG）的比例降低可作为氧化应激和疾病风险的重要指标。在不同的实验室中，GSH 和 GSSG 的测量浓度差异很大，主要是由于 GSH 在样品处理过程中的不稳定性以及不同分析方法带来的变量。我们开发了一种简单灵敏的液相色谱-串联质谱（LC-MS/MS）法来测量全血 GSH 和 GSSG，该方法最大限度地减少了分析前和分析中的变异性，可靠地消除了离子抑制的干扰，并且可以很容易地在临床实验室中实施。样品用磺基水杨酸（SSA）沉淀，在单一制备步骤中与 N-乙基马来酰亚胺（NEM）衍生化，所得上清液与稳定同位素内标（GSH-（13）C，（15）N-NEM 和 GSSG-（13）C，（15）N）合并，用 Hypercarb 柱进行色谱分离，然后在正离子模式下进行 MS/MS 分析。结果表明，GSH 和 GSSG 的线性范围均为生理正常范围，批内和批间 CV 分别为 3.1-4.3%，准确度在 95%-101%之间。GSH 的检测下限（LLOQ）为 0.4μM，GSSG 的检测下限（LLOQ）为 0.1μM，GSH 的定量下限（LLOQ）为 1.5μM，GSSG 的定量下限（LLOQ）为 0.1μM。衍生化样品在-80°C 下储存至少 3 年稳定，未衍生化样品在 4°C 或室温下至少 24 小时稳定。对 59 个对照样本进行了参考区间的确定，结果为：GSH 900±140μM；GSSG 1.17±0.43μM；GSH/GSSG 880±370。
