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

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.