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Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay

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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 = H2N(HN)C–C(CH3)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.

Structures of spin traps, ESR spectra of their spin adducts of AAPH-derived free radical, and their Stern-Volmer’s plots for Trolox.

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Acknowledgments

This work was supported by a cooperative grant for innovative technology and advanced research in evolutional areas from the Ministry of Education, Science, Sports, and Culture of Japan. The authors wish to thank Professor Yoshinari Baba for his continuous guidance and encouragement through this work.

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Authors and Affiliations

  1. Section of Obstetrics and Gynecology, Department of Reproductive and Developmental Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki, 889-1692, Japan

    A. Nakajima, E. Matsuda, Y. Masuda, H. Sameshima & T. Ikenoue

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  1. A. Nakajima
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  2. E. Matsuda
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Correspondence to A. Nakajima.

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Nakajima, A., Matsuda, E., Masuda, Y. et al. Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay. Anal Bioanal Chem 403, 1961–1970 (2012). https://doi.org/10.1007/s00216-012-6021-8

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  • DOI: https://doi.org/10.1007/s00216-012-6021-8

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