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Journal of Lipid Research, Vol. 47, 2515-2524, November 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Oxidized derivatives of -3 fatty acids: identification of IPF₃-VI in human urine¹

John A. Lawson^*, Seongjin Kim, William S. Powell, Garret A. FitzGerald^* and Joshua Rokach^2,

^* Institute for Translational Medicine and Therapeutics and Department of Pharmacology, The University of Pennsylvania, Philadelphia, PA 19104
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901
Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2

¹ The iP nomenclature used throughout this manuscript was previously reported (Rokach, J., S. P. Khanapure, S. W. Hwang, M. Adiyaman, J. A. Lawson, and G. A. FitzGerald. 1997. Prostaglandins. 54: 853–873). Another nomenclature is also in use (Taber, D. F., J. D. Morrow, and L. J. Roberts II. 1997. Prostaglandins. 53: 63–67).

Published, JLR Papers in Press, August 30, 2006.

² To whom correspondence should be addressed. e-mail: jrokach{at}fit.edu

Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from 3-PUFAs have been found to be below detection limits of available assays. Because of the interest in 3-PUFA dietary supplementation, we developed specific methods to measure nPF₄-VI and iPF₃-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF₄-VI was below the detection limit of the assay, we conclusively identified iPF₃-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were 300 pg/mg creatinine. Our failure to detect nPF₄-VI may have been due to its rapid metabolism by ß-oxidation to iPF₃-VI, which we showed to occur in rat liver homogenates. In contrast, iPF₃-VI is highly resistant to ß-oxidation in vitro. Thus iPF₃-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) ß-oxidation of nPF₄-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA.

Supplementary key words isoprostanes • arachidonic acid • eicosapentaenoic acid • docosahexaenoic acid • neuroprostane F₄-VI • mass spectrometry • lipid peroxidation • 3-polyunsaturated fatty acids

Abbreviations: AA, arachidonic acid; AD, Alzheimer's disease; BDS, Base Deactivated Silica; DHA, 4,7,10,13,16,19-docosahexaenoic acid; ECNI, electron capture/negative ion; EPA, 5,8,11,14,17-eicosapentaenoic acid; iP, isoprostane; i.s., internal standard; LC, liquid chromatography; MS/MS, tandem mass spectrometry; nP, neuroprostane; PFB, pentafluorobenzyl; SPE, solid-phase extraction; TMS, trimethylsilyl

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