title: Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes.
abstract: Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.
authors.affiliation: Dept. of Internal Medicine, Slotervaart Hospital, Amsterdam, The Netherlands. e.nur@amc.uva.nl
authors.forename: Erfan
authors.initials: E
authors.lastname: Nur
authors.forename: Mirjam
authors.initials: M
authors.lastname: Verwijs
authors.forename: Dirk R
authors.initials: DR
authors.lastname: de Waart
authors.forename: John-John B
authors.initials: JJ
authors.lastname: Schnog
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Otten
authors.forename: Dees P
authors.initials: DP
authors.lastname: Brandjes
authors.forename: Bart J
authors.initials: BJ
authors.lastname: Biemond
authors.forename: Ronald P J Oude
authors.initials: RP
authors.lastname: Elferink
mesh_terms: D000328:Adult; D000755:Anemia, Sickle Cell / Q000378:metabolism* / Q000473:pathology*; D000975:Antioxidants / Q000378:metabolism*; D016022:Case-Control Studies; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D008297:Male; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D055815:Young Adult
chemical_list: D000975:Antioxidants; D005978:Glutathione; D019803:Glutathione Disulfide; D006454:Hemoglobins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009285:Naphthoquinones; D011422:Propionates; D011804:Quinolines; C059141:verlukast; C421109:multidrug resistance-associated protein 1