title: Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room.
abstract: Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, I-2033 Milan, Italy.
authors.forename: Graziano
authors.initials: G
authors.lastname: Colombo
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, I-2033 Milan, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Milzani
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, I-2033 Milan, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Dalle-Donne
authors.affiliation: Division of Nephrology, Department of Medicine, The University of Texas Health Science Center San Antonio, and Audie L. Murphy VA Hospital, San Antonio, TX 78229, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Fanti
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy. Electronic address: ranieri@unisi.it.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000032:analysis* / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction
chemical_list: D000975:Antioxidants; D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Glutathione; Glutathione disulfide; Oxidative stress; Protocols