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Papers In Press, published online ahead of print December 1, 2008
J. Lipid Res., doi:10.1194/jlr.D800034-JLR200

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Journal of Lipid Research, Vol. 49, 2668-2677, December 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Preparation of pure lipid hydroperoxides

Daigo Ibusuki^*, Kiyotaka Nakagawa^*, Akira Asai, Shinichi Oikawa, Yuichi Masuda^*, Toshihide Suzuki and Teruo Miyazawa^1,*

^* Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Department of Medicine, Nippon Medical School, Tokyo 113-8602, Japan
Faculty of Pharmaceutical Science, Teikyo University, Kanagawa 199-0195, Japan

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of six figures.

Published, JLR Papers in Press, July 18, 2008.

This work was supported by KAKENHI (20228002) of JSPS, Japan.

¹ To whom correspondence should be addressed. e-mail: miyazawa{at}biochem.tohoku.ac.jp

Increasing evidence of lipid peroxidation in food deterioration and pathophysiology of diseases have revealed the need for a pure lipid hydroperoxide (LOOH) reference as an authentic standard for quantification and as a compound for biological studies in this field. Generally, LOOH is prepared from photo- or enzymatically oxidized lipids; however, separating LOOH from other oxidation products and preparing pure LOOH is difficult. Early studies showed the usability of reaction between hydroperoxide and vinyl ether for preparation of pure LOOH. Because the reactivity of vinyl ether with LOOHs other than fatty acid hydroperoxides has never been reported, here, we employed the reaction for preparation of a wide variety of pure LOOHs. Phospholipid, cholesteryl ester, triacylglycerol, or fatty acid was photo- or enzymatically oxidized; the resultant crude sample containing hydroperoxide was allowed to react with a vinyl ether [2-methoxypropene (MxP)]. Liquid chromatography (LC) and mass spectrometry confirmed that MxP selectively reacts with LOOH, yielding a stable MxP adduct (perketal). The lipophilic perketal was eluted at a position away from that of intact LOOH and identified and isolated by LC. Upon treatment with acid, perketal released the original LOOH, which was finally purified by LC. Using our optimized purification procedures, for instance, we produced 75 mg of pure phosphatidylcholine hydroperoxide (>99%) from 100 mg of phosphatidylcholine. Our developed method expands the concept of the perketal method, which provides pure LOOH references. The LOOHs prepared by the perketal method would be used as "gold standards" in LOOH methodology.

Supplementary key words oxidative stress • lipid peroxidation • lipid hydroperoxide standard • 2-methoxypropene

Abbreviations: ChL, cholesteryl linoleate; CL, chemiluminescence; ESI, electrospray ionization; LA, linoleic acid; LAMe, methyl linoleate; LC, liquid chromatography; LLL, trilinoleoylglycerol; LOOH, lipid hydroperoxide; LOX-1, lipoxygenase-1; MS, mass spectrometry; MxP, 2-methoxypropene; PLPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; PLPCOOH, 1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine; PLPCOOMxP, MxP adduct of PLPCOOH; PLPE, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine; PLPS, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoserine; PPTS, pyridinium p-toluenesulfonate; RB, rose bengal; TIC, total ion chromatogram; Tris, tris(hydroxymethyl)aminomethane; UV, ultraviolet; XIC, extracted ion chromatogram

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