title: A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N-ethylmaleimide.
abstract: The ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH) in biological samples is a frequently used parameter of oxidative stress. As a result, many methods are developed to measure GSSG. The most popular and convenient of these relies on enzymatic cycling following the chemical masking of GSH in the sample using 2-vinylpyridine (2VP). However, 2VP is a slow reactant and its use may result in artificially high GSSG values due to oxidation of the sample over time. Fast-reacting reagents such as N-ethylmaleimide (NEM) may provide more accurate results. We performed a direct comparison of methods using 2VP and NEM. With 2VP, the percentage of total glutathione (GSH+GSSG) in the oxidized form was significantly higher in all tested tissues (kidney, lung and liver) compared to the same procedure performed using NEM. We conclude that NEM, when coupled with a simple solid-phase extraction procedure, is more accurate for the determination of GSSG. We also tested the effects of various handling and storage conditions on GSSG. A detailed description and a discussion of other methods are also included.
authors.affiliation: a Department of Pharmacology , Toxicology and Therapeutics, University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: Mcgill
authors.affiliation: a Department of Pharmacology , Toxicology and Therapeutics, University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Jaeschke
mesh_terms: D053000:Analytic Sample Preparation Methods; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007668:Kidney / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D055550:Protein Stability / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D004791:Enzyme Inhibitors; D011725:Pyridines; D013439:Sulfhydryl Reagents; C030953:2-vinylpyridine; D005980:Glutathione Reductase; D005978:Glutathione; D005033:Ethylmaleimide
keywords: 2-vinylpyridine; GSSG; Glutathione; N-ethylmaleimide; Tietze assay; acetaminophen