Measuring the Poise of Thiol/Disulfide Couples in vivo

• Type of Material: Article
• Authors: Dean P Jones, Emory University; Yongliang Liang, Emory University
• Language: English
• Date: 2009-11-15
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2009, Elsevier
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://ac.els-cdn.com/S0891584909004997/1-s2.0-S0891584909004997-main.pdf?_tid=e322c756-9a7a-11e3-ba8e-00000aacb360&acdnat=1392934013_76cdb28887bb0d4ceeafc5d718e506b8
• Title of Journal or Parent Work: Free Radical Biology and Medicine
• ISSN: 0891-5849
• Volume: 47
• Issue: 10
• Start Page: 1329
• End Page: 1338
• Grant/Funding Information: Support for research in the authors' laboratory was provided by NIEHS grants ES009047, ES011195 and ES016731.
• Keywords: redox signaling; oxidative stress; Glutathione; free radicals; human plasma; cysteine
• Research Categories: Biology, Physiology; Health Sciences, General; Biology, Cell

Abstract

The reduction potentials (Eh) 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 paper describes a sensitive method for determining plasma levels of GSH, GSSG, Cys and CySS used to calculate the in vivo Eh 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 reduction potential, Eh, 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.