Abstract
Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.
Highlights
Glutathione is the major non-protein thiol in mammals, which is present at millimolar concentrations within cells, especially in the liver [1]
An enzymatic recycling method has been reported for the quantitative assay of GSH and GSSG. It involves the reaction of GSH with 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB) to form UV detectable 5’-thio-2-nitrobenzoic acid (TNB) and the reduction of GSSG to GSH by glutathione reductase and NADPH [4]
GS-NEM yielded two separate peaks of equal peak area ratio with an RSD of 3.13% at 6.7 min and 7.8 min due to the generation of diastereomers that are separated under the given chromatographic conditions (Supplementary Figure S2)
Summary
Glutathione is the major non-protein thiol in mammals, which is present at millimolar concentrations within cells, especially in the liver [1]. An enzymatic recycling method has been reported for the quantitative assay of GSH and GSSG It involves the reaction of GSH with 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB) to form UV detectable 5’-thio-2-nitrobenzoic acid (TNB) and the reduction of GSSG to GSH by glutathione reductase and NADPH [4]. This method can be used to determine total glutathione (GSH + GSSG). Giustarini et al reported a protocol for GSH and GSSG determination with NEM derivatization in blood and solid tissues [3] and cultured cells [15]. We tested the applicability of the method by demonstrating the impact of genetic ablation of the monocarboxylate transporters MCT1 and MCT4 on the GSH/GSSG ratio of colon cancer cells under oxidative stress as well as the effect that NADPH consuming neomorphic mutations in the isocitrate dehydrogenases IDH1/2 have on the reduction of GSSG to GSH
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