Institute of Functional Genomics, University of Regensburg, Am BioPark 9, 93053, Regensburg, Germany.
Institute of Functional Genomics, University of Regensburg, Am BioPark 9, 93053, Regensburg, Germany.
Anal Chim Acta. 2019 Nov 8;1080:127-137. doi: 10.1016/j.aca.2019.07.001. Epub 2019 Jul 3.
Glutathione is an essential intra- and extracellular antioxidant. The level of glutathione in the body is highly related to different disease states and is a useful indicator of disease risk and oxidative stress status. We have developed a sensitive, selective, and comprehensive LC-MS/MS method for the absolute quantification and C-tracer analysis of total glutathione using dithiothreitol for the reduction of glutathione disulfide. The limit of detection (LOD) was 0.01 μM, while the lower limit of quantification (LLOQ) was 0.78 μM, with the linear (R = 0.9997) range extending up to 100 μM. The intra-run and inter-run coefficients of variation of 2.49% and 2.04%, respectively, attest to high repeatability. Mean (±SD) recoveries of three different concentrations (low, medium, high) of GSH spiked into aliquots of HCT116 cells prior to cell extraction were 108.9% (±2.1), 100.8% (±8.3), and 99.9% (±7.1), respectively. Finally, using a 20 Da wide Q1 window in MRM mode, we were able to detect and relatively quantify all isotopic labeling states of GSH extracted from HCT116 cells fed with either C-labeled glucose or glutamine.
谷胱甘肽是一种重要的细胞内和细胞外抗氧化剂。体内谷胱甘肽的水平与不同的疾病状态高度相关，是疾病风险和氧化应激状态的有用指标。我们开发了一种灵敏、选择性和全面的 LC-MS/MS 方法，用于使用二硫苏糖醇（DTT）还原谷胱甘肽二硫化物来绝对定量和 C 示踪分析总谷胱甘肽。检测限（LOD）为 0.01μM，定量下限（LLOQ）为 0.78μM，线性（R=0.9997）范围扩展至 100μM。日内和日间变异系数分别为 2.49%和 2.04%，表明具有很高的重复性。三种不同浓度（低、中、高）的 GSH 加入 HCT116 细胞等分试样中进行细胞提取前，GSH 的平均（±SD）回收率分别为 108.9%（±2.1）、100.8%（±8.3）和 99.9%（±7.1）。最后，在 MRM 模式下使用 20Da 宽的 Q1 窗口，我们能够检测和相对定量从用 C 标记的葡萄糖或谷氨酰胺喂养的 HCT116 细胞中提取的所有 GSH 的同位素标记状态。
