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Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method

Nature Protocols volume 1, pages 3159–3165 (2006)Cite this article

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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Figure 1: The enzymatic recycling of glutathione (GSH) from glutathione disulfide (GSSG) by glutathione reductase (GR) in the presence of NADPH.

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Authors and Affiliations

  1. Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, New York, USA

    Irfan Rahman & Aruna Kode

  2. Department of Biochemistry and Biotechnology, Dr. Ambedkar College, Nagpur, Maharashtra State, India

    Saibal K Biswas

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  1. Irfan Rahman
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  2. Aruna Kode
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  3. Saibal K Biswas
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Correspondence to Irfan Rahman.

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Rahman, I., Kode, A. & Biswas, S. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1, 3159–3165 (2006). https://doi.org/10.1038/nprot.2006.378

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