title: Simultaneous evaluation of one-electron reducing systems and radical reactions in cells by nitroxyl biradical as probe.
abstract: In the present study, a novel probe for the simultaneous evaluation of one-electron reducing systems (electron transport chain) and one-electron oxidizing systems (free radical reactions) in cells by electron chemical detection was developed. Six-membered cyclic nitroxyl radicals (2,2,6,6-tetramethylpiperidine-1-oxyl; TEMPO series) are sensitive to one-electron redox systems, generating the hydroxylamine form [TEMPO(H)] via one-electron reduction, and the secondary amine form [TEMPO(N)] via one-electron oxidation in the presence of thiols. In contrast, the sensitivities of five-membered cyclic nitroxyl radicals (2,2,5,5-tetramethylpyrrolidine-1-oxyl; PROXYL series) to the one-electron redox systems are comparatively low. The electron chemical detector can detect 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), TEMPO(H) and PROXYL but not TEMPO(N). Therefore, nitroxyl biradical, TEMPO-PROXYL, as a probe for the evaluation of one-electron redox systems was employed. TEMPO-PROXYL was synthesized by the conjunction of 4-amino-TEMPO with 3-carboxyl-PROXYL via the conventional dicyclohexyl carbodiimide reaction. TEMPO-PROXYL, TEMPO(H)-PROXYL and TEMPO(N)-PROXYL were simultaneously quantified by HPLC with Coularray detection. Calibration curves for the quantification of TEMPO-PROXYL, TEMPO(H)-PROXYL and TEMPO(N)-PROXYL were linear in the range from 80 nm to 80 μm, and the lowest quantification limit of each molecule was estimated to be <80 nm. The relative standard deviations at 0.8 and 80 μm were within 10% (n = 5). Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Araki
authors.affiliation: Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Koshiishi
mesh_terms: D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004583:Electrons; D005419:Flavonoids / Q000097:blood* / Q000493:pharmacokinetics; D057230:Limit of Detection; D051379:Mice; D011794:Quercetin / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005419:Flavonoids; C519462:guaijaverin; C021304:hyperoside; D011794:Quercetin
keywords: caco-2 cells; coularray; electron transport chain; hplc; nitroxyl radicals; one-electron redox