Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 47, Issue 10, Pages 1329-1338Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2009.08.021
Keywords
Glutathione; Cysteine; Oxidative stress; Redox signaling; Human plasma; Free radicals
Funding
- NIEHS [ES009047, ES011195, ES016731]
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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. (C) 2009 Elsevier Inc. All rights reserved.
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