Measuring the poise of thiol/disulfide couples in vivo.
The reduction potentials (E(h)) for the redox couples GSH/GSSG and cysteine/cystine (Cys/CySS) in plasma are useful indicators of systemic oxidative stress and other medically relevant physiological states. This article describes a sensitive method for determining plasma levels of GSH, GSSG, Cys, and CySS used to calculate the in vivo E(h) values. The method uses iodoacetate to alkylate free thiols, derivatization with dansyl chloride to fluorescently tag amino groups, and HPLC and fluorescence to separate, detect, and quantify the molecules. Benefits of the method, such as sensitivity and dynamic range, are described, as are caveats, such as the importance of preventing red blood cell hemolysis and limitations in quantification of GSSG. General principles of redox chemistry and previous studies showing that the compounds are more oxidized than predicted from their standard reduction potentials are reviewed. The calculated in vivo E(h) is a convenient and informative way of summarizing the redox environment of plasma and is also useful for studies of cerebrospinal fluid, lymph, bronchoalveolar lavage fluid, human biopsies, and a broad range of in vitro cell culture conditions.
Jones
Dean P
DP
Clinical Biomarkers Laboratory, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA. dpjones@emory.edu
Liang
Yongliang
Y
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000097:blood*; D003553:Cystine / Q000097:blood*; D005978:Glutathione / Q000097:blood*; D019803:Glutathione Disulfide / Q000097:blood*; D006801:Humans; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000097:blood*
D013438:Sulfhydryl Compounds; D003553:Cystine; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide