Analysis of GSH and GSSG after derivatization with N-ethylmaleimide
- 1 August 2013
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 8 (9), 1660-1669
- https://doi.org/10.1038/nprot.2013.095
Abstract
This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5–15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.Keywords
This publication has 36 references indexed in Scilit:
- Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: Influence of sample manipulation, oxidative stress and ageingMechanisms of Ageing and Development, 2011
- Differential thiol status in blood of different mouse strains exposed to cigarette smokeFree Radical Research, 2009
- Glutathione dysregulation and the etiology and progression of human diseasesBiological Chemistry, 2008
- Molecular Mechanisms and Potential Clinical Significance ofS-GlutathionylationAntioxidants and Redox Signaling, 2008
- Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling methodNature Protocols, 2006
- Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative StressClinical Chemistry, 2006
- Biomarkers of Oxidative Damage in Human DiseaseClinical Chemistry, 2006
- Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouseFree Radical Biology & Medicine, 2005
- Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studiesJCI Insight, 1969
- OXIDIZED GLUTATHIONE LEVELS IN ERYTHROCYTES OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE-DEFICIENT SUBJECTSThe Lancet, 1968