title: Micro-method for the determination of glutathione in human blood.
abstract: A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy. Electronic address: giustarini@unisi.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.affiliation: Division of Nephrology, Department of Medicine, The University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA; Audie L. Murphy VA Hospital, San Antonio, TX 78229, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Fanti
authors.affiliation: Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56126 Pisa, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Matteucci
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry / Q000191:economics / Q000379:methods; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005260:Female; D005978:Glutathione / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D057230:Limit of Detection; D008297:Male; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D018401:Sample Size; D013053:Spectrophotometry / Q000191:economics / Q000379:methods*
chemical_list: D007202:Indicators and Reagents; D004228:Dithionitrobenzoic Acid; D005978:Glutathione
keywords: Blood; DTNB; Fingerstick; Glutathione; Oxidative stress