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J. Lipid Res.
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A more recent version of this article appeared on November 1, 2005

Papers In Press, published online ahead of print August 16, 2005
J. Lipid Res., doi:10.1194/jlr.D500006-JLR200
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Submitted on February 18, 2005
Revised on July 28, 2005
Accepted on August 1, 2005

Quantification of lipid alkyl radicals via the combination of nitroxyl radical-spin trapping method and HPLC with post-column thermal decomposition

Ichiro Koshiishi, Kazunori Tsuchida, Tokuko Takajo, and Makiko Komatsu

Faculty of Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama 362-0806

Corresponding Author: 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 quantification of lipid alkyl radicals via nitroxyl radical spin trapping technique. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts by using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (Cmdelta P) (PO/W = 3) as a spin trapping agent. The resulting Cmdelta P-lipid alkyl radical adducts were determined by HPLC with post column on-line thermal decomposition system, in which the adducts were degraded into nitroxyl radical by heating at 100°C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detector (ECD). Through the present method, we, for the first time, succeeded to determine the lipid alkyl radicals generated from linoleic acid, linolenic acid and arachidonic acid via soybean lipoxygenase-1 or radical initiator, 2,2’-azobis(2,4-dimethyl-valeronitrile) (AMVN).


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