Fahrenholz Timothy, Wolle Mesay Mulugeta, Kingston H M Skip, Faber Scott, Kern John C, Pamuku Matt, Miller Logan, Chatragadda Hemasudha, Kogelnik Andreas
Department of Chemistry and Biochemistry, Duquesne University , 600 Forbes Avenue, Pittsburgh, Pennsylvania 15282, United States.
Anal Chem. 2015 Jan 20;87(2):1232-40. doi: 10.1021/ac503933t. Epub 2015 Jan 8.
Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.
开发了新的方案，使用分子特异性同位素稀释质谱法（SIDMS）准确量化生物样品中还原型（GSH）、氧化型（GSSG）和总谷胱甘肽（tGSH）。对于GSH和GSSG的测量，向样品中加入分析物的同位素富集类似物（(310)GSH和(616)GSSG）以及N-乙基马来酰亚胺（NEM），并用乙腈处理，通过蛋白质沉淀使内源性分析物溶解，并使其与加入物达到平衡。通过液相色谱-串联质谱法（LC-MS/MS）分析上清液，同时跟踪并校正GSH自动氧化为GSSG，对分析物进行定量。对于tGSH测定，用二硫苏糖醇（DTT）处理加入(310)GSH的样品，将二硫键结合的谷胱甘肽转化为GSH。去除蛋白质后，通过LC-MS/MS分析上清液，并通过单加入同位素稀释质谱法（IDMS）对分析物进行定量。IDMS和SIDMS定量中的数学关系基于同位素比率，不涉及校准曲线。通过加入回收试验和分析合成标准溶液对方案进行了验证。分析了从健康受试者获得的红细胞（RBC）和唾液样品，以及从偏远地区采集并运送的全血样品。RBC和全血样品中tGSH的浓度比唾液中的高2个数量级。相应样品中GSSG的含量分别占游离谷胱甘肽（GSH + 2xGSSG）的0.2 - 2.2%（RBC和血液）和15 - 47%（唾液）。在样品处理过程中，高达3%的GSH自动氧化为GSSG；氧化程度最高（>1%）的是唾液样品。
