title: Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.
abstract: Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.
authors.affiliation: Center for Biomedical Engineering & Technology, and Department of Physiology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Legenzov
authors.affiliation: Center for Biomedical Engineering & Technology, and Department of Physiology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Sims
authors.affiliation: Center for Biomedical Engineering & Technology, and Department of Physiology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
authors.forename: Nathaniel D A
authors.initials: ND
authors.lastname: Dirda
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201, United States.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Rosen
authors.affiliation: Center for Biomedical Engineering & Technology, and Department of Physiology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
authors.forename: Joseph P Y
authors.initials: JP
authors.lastname: Kao
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005978:Glutathione / Q000032:analysis; D006801:Humans; D019169:Jurkat Cells; D009589:Nitrogen Oxides / Q000737:chemistry*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013601:T-Lymphocytes / Q000737:chemistry*
chemical_list: D004220:Disulfides; D009589:Nitrogen Oxides; D013438:Sulfhydryl Compounds; D005978:Glutathione; D004229:Dithiothreitol