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Journal of Lipid Research, Vol. 46, 2506-2513, November 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Ichiro Koshiishi^1,*, Kazunori Tsuchida^*, Tokuko Takajo^* and Makiko Komatsu

^* Nihon Pharmaceutical University, Ina-machi, Kita-Adachi-gun, Saitama 362-0806, Japan
Applied Biosystems Japan, Ltd., Chuo-ku, Tokyo 104-0032, Japan

Published, JLR Papers in Press, August 16, 2005. DOI 10.1194/jlr.D500006-JLR200

¹ To whom correspondence should be addressed. e-mail: ikoshi{at}nichiyaku.ac.jp

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100°C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection.

With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).

Abbreviations: AMVN, 2,2'-azobis(2,4-dimethyl-valeronitrile); CmP, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl; ECD, electrochemical detection; epoxy-C_18:2, linoleate epoxyallyl radical; LC/MS/MS, liquid chromatography-tandem mass spectrometry

Supplementary key words spin-trapping • high-performance liquid chromatography • lipoxygenases

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