title: Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe.
abstract: Superoxide (O(2)(•-)) plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific O(2)(•-) probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, O(2)(•-) preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O(2)(•-), CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O(2)(•-). This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV-Vis absorbance (ε=15,900 M(-1) cm(-1)) at 540 nm, providing an additional O(2)(•-) detection method. More than five-fold higher reactivity of CT02-H for O(2)(•-) relative to CT-03 was demonstrated, with a second-order rate constant of 1.7×10(4) M(-1) s(-1) compared to 3.1×10(3) M(-1) s(-1) for CT-03. CT02-H exhibited high specificity for O(2)(•-) as evidenced by its inertness to other oxidoreductants. The O(2)(•-) generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low O(2)(•-) flux with a detection limit of 0.34 nM/min over 120 min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of O(2)(•-), with a sensitivity limit of ~2×10(-13) mol (10 nM with 20-μl injection volume). Based on the O(2)-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O(2) concentration and O(2)(•-) generation and was shown to provide sensitive detection of extracellular O(2)(•-) generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of O(2)(•-) by either EPR or HPLC methods.
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Yangping
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Song
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: De Pascali
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Liu
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.|Department of Pharmacology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Villamena
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013481:Superoxides / Q000737:chemistry*; D019820:Xanthine / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry
chemical_list: D005609:Free Radicals; D006575:Heterocyclic Compounds, 3-Ring; D013481:Superoxides; D019820:Xanthine; D014969:Xanthine Oxidase