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

¹ Department of Neuroscience, Pharmacology Unit, Via A. Moro 4, University of Siena, 53100 Siena, Italy.

² Laboratory of Biochemistry and Biophysics of Cytoskeleton, Department of Biology, Via Celoria 26, University of Milan, 20133 Milan, Italy.

^aAuthor for correspondence. Fax 39-057-723-4208; e-mail disimplicio{at}unisi.it.

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.

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