title: NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes.
abstract: Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a 13C-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-13C]glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using 1H spin-echo NMR, while 13C[1H-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Australia.
authors.forename: Eleanor C
authors.initials: EC
authors.lastname: Kennett
authors.forename: William A
authors.initials: WA
authors.lastname: Bubb
authors.forename: Paramjit
authors.initials: P
authors.lastname: Bansal
authors.forename: Paul
authors.initials: P
authors.lastname: Alewood
authors.forename: Philip W
authors.initials: PW
authors.lastname: Kuchel
mesh_terms: D001671:Biochemistry / Q000379:methods*; D004151:Dipeptides / Q000737:chemistry; D004220:Disulfides; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D018384:Oxidative Stress
chemical_list: D004151:Dipeptides; D004220:Disulfides; D005978:Glutathione; D005947:Glucose; C017341:gamma-glutamylcysteine