Blood glutathione disulfide: in vivo factor or in vitro artifact?

Clin Chem. 2002 May;48(5):742-53.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Artifacts*
  • Chromatography, High Pressure Liquid
  • Erythrocytes / metabolism
  • Fluorometry
  • Glutathione / blood
  • Glutathione / metabolism
  • Glutathione Disulfide / blood*
  • Glutathione Disulfide / metabolism
  • Hemoglobins / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Kinetics
  • Male
  • Oxidation-Reduction
  • Rats
  • Rats, Wistar
  • Sulfhydryl Compounds / blood
  • Sulfhydryl Compounds / metabolism

Substances

  • Hemoglobins
  • Sulfhydryl Compounds
  • Glutathione
  • Glutathione Disulfide