Optimized Protocol for the In Situ Derivatization of Glutathione with N-Ethylmaleimide in Cultured Cells and the Simultaneous Determination of Glutathione/Glutathione Disulfide Ratio by HPLC-UV-QTOF-MS

URN to cite this document:

urn:nbn:de:bvb:355-epub-435190 Copy

DOI to cite this document:

10.5283/epub.43519 Copy

Sun, Xueni ; Berger, Raffaela ; Heinrich, Paul ; Marchiq, Ibtissam ; Pouyssegur, Jacques ; Renner, Kathrin ; Oefner, Peter J. ; Dettmer, Katja

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Date of publication of this fulltext: 30 Jul 2020 06:40

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Details

Item type:	Article
Open Access Type:	Gold (with APC)
Journal or Publication Title:	Metabolites
Publisher:	MDPI
Place of Publication:	BASEL
Volume:	10
Number of Issue or Book Chapter:	7
Page Range:	p. 292
Date:	17 July 2020
Institutions:	Medicine > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) Medicine > Lehrstuhl für Innere Medizin III (Hämatologie und Internistische Onkologie)
Projects (Historical):	Open Access Publizieren (DFG)
Identification Number:	Value Type 10.3390/metabo10070292 DOI 32709039 PubMed ID
Keywords:	OXIDIZED GLUTATHIONE; LIQUID-CHROMATOGRAPHY; QUANTIFICATION; PROTEINS; TISSUES; PLASMA; EXPORT; ASSAY; GSSG; MCT1; glutathione; glutathione disulfide; cell culture; liquid chromatography; UV detection; mass spectrometry
Dewey Decimal Classification:	600 Technology > 610 Medical sciences Medicine
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	43519

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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 withN-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio ...

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 withN-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 mu M and the linear range extended from 15.63 mu M to 1000 mu M with a squared correlation coefficientR(2)of 0.9997. The LOD for GSSG was 0.001 mu M, and the lower limit of quantification (LLOQ) was 0.01 mu M, with the linear (R-2= 0.9994) range extending up to 10 mu M. 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 transportersMCT1andMCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genesIDH1andIDH2.

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