Molecular Speciated Isotope Dilution Mass Spectrometric Methods for Accurate, Reproducible and Direct Quantification of Reduced, Oxidized and Total Glutathione in Biological Samples

Abstract

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.

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

“…Then samples are analyzed by tandem mass spectrometry (NEM was used as masking agent and added to collecting tubes) and the analytes are quantified using the stable-isotope labelled analogs. Oxidation of GSH to GSSG was corrected by using mathematical relationships in double-spiking SIDMS [44]. These LC-MS/MS methods may represent a good alternative to spectrophotometric and HPLC with UV absorbance detection assays but a good agreement with these techniques still lacks.…”

“…In whole blood of 9 subjects, Fahrenholz et al reported 1.46±0.07 μmol/g for GSH and 2.4±0.4 nmol/g for GSSG, yielding a mean GSH/GSSG ratio of 608. In RBCs of 9 healthy children, this group reported 1.69±0.83 μmol/g for GSH and 6.1±3.8 nmol/g GSSG, corresponding to a mean GSH/GSSG ratio of 277 [44]. These data indicate considerable discrepancies even between highly sophisticated LC-MS/MS methods and point out to additional not yet fully recognized pre-analytical and/or analytical problems.…”

“…This notwithstanding, the measured GSSG levels are very low, even if Steghens et al found a concentration seven-fold higher in RBC than in whole blood; these points would be worth to be further studied to understand the reason for this unexpected result. Conversely, blood samples in the work of Fahrenolz et al [44] were treated with NEM after sample collection and stored until analyses. These different pre-analytical treatments may be responsible for the observed discrepancies.…”

A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N -ethylmaleimide

“…Detailed reviews have been published elsewhere (Monostori et al, 2009; Iwasaki et al, 2009). The most popular of these techniques make use of high-performance liquid chromatography (HPLC) separation with UV absorbance (Reed et al, 1980; Katrusiak et al, 2001; Bald and Glowacki, 2005), chemiluminescence (McDermott et al, 2011a,b), fluorescence (Imai et al, 1983; Martin and White, 1991; Paroni et al, 1995; Yang et al, 1995; Kand’ar et al, 2007), electrochemical (Rodriguez-Ariza et al, 1994; Rose and Bode, 1995; Lakritz et al, 1997; Potesil et al, 2005; Sequellerio et al 2012), or mass spectrometry-based (Lafaye et al, 2005; Bouligard et al, 2006; Iwasaki et al, 2009; Squellerio et al, 2012; Moore et al, 2013; Zhang et al, 2015; Fahrenholz et al, 2015) detection. Even nanosensors for GSH and GSSG are now being developed (Xu et al, 2012; Moore et al, 2013; Ni et al, 2015).…”

Fluoreszenzsonden mit mehreren Bindungsstellen unterscheiden zwischen Cys, Hcy und GSH

“…So ist es einem Autor zufolge “erwiesen, dass die gemessenen Konzentrationen … zwischen verschiedenen Arbeitsgruppen stark abweichen. … Ergebnisse sollten erneut auswertet werden, sobald (es) eine allgemeine Einigung über die physiologischen Konzentrationen (gibt) …” [52] Diese Unstimmigkeit wurde der Variabilität der Analysemethoden, der Verwendung unterschiedlicher Matrizen (Plasma oder Blut), der Oxidation von Probenmaterial, inkonsistenten Aufbereitungsmethoden der Proben und allgemein dem Mangel an standardisierten Methoden zugeschrieben . Die Handhabung der Proben wurde als Hauptursache für die fehlende Reproduzierbarkeit und hohe Varianz in den GSH‐Bestimmungen beschrieben .…”