Blood Glutathione Disulfide: In Vivo Factor or in Vitro Artifact?

Abstract

Background: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 μmol/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 μmol/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 μmol/L), and most published data on GSSG may be affected by artifacts.

Changes in free radical generation and antioxidant capacity during ultramarathon foot race

“…Red cell oxidized/reduced glutathione (GSSG and GSH, respectively) were assessed according to Rossi et al [28]. Whole blood was used for the above determination.…”

S‐Glutathionylation: from redox regulation of protein functions to human diseases

“…Total S-glutathionylated proteins, as well as GSH and GSSG, can be detected by reversed phase HPLC with fluorescence detection after thiol derivatisation with monobromobimane or 1-fluoro-2,4-dinitrobenzene [19][20][21]. Recently, the detection of PSSGs has been facilitated by the availability of specific monoclonal anti-GSH antibodies that allow detecting of S-glutathionylated proteins by slot immunoblotting or by 1D-or 2D-PAGE followed by Western blot [16,20,22,23].…”

“One-pot” ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC–MS analysis

“…In order to estimate glutathione in biological samples it is necessary to preserve the physiological balance between the redox couple GSH/GSSG during sample manipulation [10,12,13,[22][23][24][25].…”

“…Therefore, red blood cells (RBC), for their high availability seem to be the best blood matrix for glutathione determination. Free glutathione is mainly observed within the cells in its reduced form (GSH) at a concentration of about 3mM [20] , while only a minimal percentage, about 2 µM [20] is detected in its oxidized form as disulfide (GSSG). In case of oxidative stress, the redox couple GSH-GSSG acts to maintain homeostasis: GSH is promptly oxidized to glutathione disulfide, hence the concentration values of its reduced form and the molar ratio GSH/GSSG decrease.…”

“…There is evidence that changes in blood GSH concentration values are suitable representative of the redox status of other tissues. Values of GSSG and GSH/GSSG are reported to be less accurate indexes of the systemic oxidative balance since they are more affected by ex-vivo artifacts [23][24][25]. Even though over the last few years a large number of methods for GSH detection have been published, consisting in electrophoretic or chromatographic techniques with numerous different analytical detectors, no agreement has been already reached about the analytical protocol to be performed [26,27].…”