Blood glutathione disulfide: in vivo factor or in vitro artifact?
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.
Rossi
Ranieri
R
Department of Neuroscience, Pharmacology Unit, Via A. Moro 4, University of Siena, 53100 Siena, Italy.
Milzani
Aldo
A
Dalle-Donne
Isabella
I
Giustarini
Daniela
D
Lusini
Lorenzo
L
Colombo
Roberto
R
Di Simplicio
Paolo
P
D000818:Animals; D016477:Artifacts; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism; D005470:Fluorometry; D005978:Glutathione / Q000097:blood / Q000378:metabolism; D019803:Glutathione Disulfide / Q000097:blood* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000097:blood / Q000378:metabolism
D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D005978:Glutathione; D019803:Glutathione Disulfide