title: Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay.
abstract: The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.
authors.affiliation: Section of Obstetrics and Gynecology, Department of Reproductive and Developmental Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan. akanaka@med.miyazaki-u.ac.jp
authors.forename: A
authors.initials: A
authors.lastname: Nakajima
authors.forename: E
authors.initials: E
authors.lastname: Matsuda
authors.forename: Y
authors.initials: Y
authors.lastname: Masuda
authors.forename: H
authors.initials: H
authors.lastname: Sameshima
authors.forename: T
authors.initials: T
authors.lastname: Ikenoue
mesh_terms: D000578:Amidines / Q000737:chemistry*; D002138:Calibration; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals; D013113:Spin Labels; D014466:Ultraviolet Rays
chemical_list: D000578:Amidines; D005609:Free Radicals; D013113:Spin Labels; C046728:2,2'-azobis(2-amidinopropane)