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.
"Total glutathione was quantified as described previously (Rahman et al., 2006) in a reaction mixture (1ml) containing 0.6 mM of the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), 0.4 mM NADPH and 1.8 U glutathione reductase, with minor modifications. In particular, sulfosalicylic acid was omitted to obtain a better signal to noise-ratio (own observations). "
"The colorimetric method described by Okawa et al. (1979) was used. Oxidized glutathione content (GSSG) was determined in liver and small intestine samples following the method of Rahman et al. (2006) and reduced glutathione (GSH) was measured following the glutathione-S-transferase assay described by Brigelius et al. (1983). Samples for glutathione analyses were obtained in the presence of N-ethylmaleimide and deproteinized (1:9, by vol.) using trichloroacetic acid (15% final concentration) as recommended by Asensi et al. (1994). "
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