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

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. We measured GS...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2002-05, Vol.48 (5), p.742-753
Main Authors: Rossi, Ranieri, Milzani, Aldo, Dalle-Donne, Isabella, Giustarini, Daniela, Lusini, Lorenzo, Colombo, Roberto, Di Simplicio, Paolo
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Chromatography, High Pressure Liquid
Erythrocytes - metabolism
Fluorometry
Glutathione - blood
Glutathione - metabolism
Glutathione Disulfide - blood
Glutathione Disulfide - metabolism
Hematology
Hemoglobins - metabolism
Humans
Hydrogen-Ion Concentration
In Vitro Techniques
Investigative techniques, diagnostic techniques (general aspects)
Kinetics
Male
Medical sciences
Oxidation-Reduction
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Rats
Rats, Wistar
Sulfhydryl Compounds - blood
Sulfhydryl Compounds - metabolism
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Summary: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. 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. 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. 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.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/48.5.742