Assay of quantitative determination of glutathione disulfide levels using enzymatic recycling methods

Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, New York, USA.
Nature Protocol (Impact Factor: 8.36). 02/2006; 1(6):3159-65. DOI: 10.1038/nprot.2006.378
Source: PubMed

ABSTRACT The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.

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    • "Reduced (GSH) and oxidized (GSSG) glutathione content were determined according to Rahman et al. (2007). For GSH the absorbance was read immediately at 412 nm. "
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