期刊
TOXICOLOGY MECHANISMS AND METHODS
卷 25, 期 8, 页码 589-595出版社
TAYLOR & FRANCIS LTD
DOI: 10.3109/15376516.2015.1094844
关键词
Glutathione; GSSG; N-ethylmaleimide; Tietze assay; 2-vinylpyridine; acetaminophen
类别
资金
- National Institutes of Health [R01 DK102142, R01 AA12916]
- National Center for Research Resources [5P20RR021940-07]
- National Institute of General Medical Sciences of the National Institutes of Health [8 P20 GM103549-07]
- National Institute of Environmental Health Sciences [T32 ES007079-26A2]
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.
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