BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. RESULTS: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30-150 micromol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. CONCLUSIONS: GSSG is present in healthy human blood at low concentrations (2-6 micromol/L), and most published data on GSSG may be affected by artifacts.
BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. RESULTS: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30-150 micromol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygendeprivation and incubation in an atmosphere of 5% carbon monoxide. CONCLUSIONS:GSSG is present in healthy human blood at low concentrations (2-6 micromol/L), and most published data on GSSG may be affected by artifacts.
Authors: Anatoly Samoylenko; Jubayer Al Hossain; Daniela Mennerich; Sakari Kellokumpu; Jukka Kalervo Hiltunen; Thomas Kietzmann Journal: Antioxid Redox Signal Date: 2013-04-15 Impact factor: 8.401
Authors: Khaled Khazim; Daniela Giustarini; Ranieri Rossi; Darlene Verkaik; John E Cornell; Sue E D Cunningham; Maryam Mohammad; Kara Trochta; Carlos Lorenzo; Franco Folli; Shweta Bansal; Paolo Fanti Journal: Transl Res Date: 2013-01-17 Impact factor: 7.012