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J. Biol. Chem., Vol. 282, Issue 5, 2899-2910, February 2, 2007

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Arachidonate-derived Dihomoprostaglandin Production Observed in Endotoxin-stimulated Macrophage-like Cells^*

Richard Harkewicz, Eoin Fahy, Alexander Andreyev, and Edward A. Dennis¹

From the Departments of Pharmacology, Chemistry, and Biochemistry, University of California, San Diego, La Jolla, California 92093-0601 and the San Diego Supercomputing Center and University of California, San Diego, La Jolla, California 92093-0505

Eicosanoids, including the prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, epoxyeicosatetraenoic acids, and related compounds, are biosynthetic, bioactive mediators derived from arachidonic acid (AA), a 20:4(n-6) fatty acid. We have developed a comprehensive and sensitive mass spectral analysis to survey eicosanoid release from endotoxin-stimulated RAW 264.7 macrophage-like cells that is capable of detecting over 70 diverse eicosanoids and eicosanoid metabolites, should they be present. We now address the question: Are biologically significant eicosanoids being overlooked? Herein, we illustrate a general approach to diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometry (DIMPLES/MS), demonstrated for one substrate (AA) and its resultant products (eicosanoids). RAW cells were incubated in medium supplemented with deuterium-labeled AA. When the cells are stimulated, two sets of eicosanoids are produced, one from endogenous AA and the other from the supplemented (exogenous) deuterium-labeled form. This produces a signature mass spectral "doublet" pattern, allowing for a comprehensive and diverse eicosanoid search requiring no previous knowledge or assumptions as to what these species may be, in contrast to traditional methods. We report herein observing unexpected AA metabolites generated by the cells, some of which may constitute novel bioactive eicosanoids or eicosanoid inactivation metabolites, as well as demonstrating differing metabolic pathways for the generation of isomeric prostaglandins and potential peroxisome proliferator-activated receptor activators. Unexpectedly, we report observing a series of 1a, 1b-dihomologue prostaglandins, products of adrenic acid (22:4(n-6)), resulting from the two-carbon elongation of AA by the RAW cells.

Received for publication, October 27, 2006 , and in revised form, November 17, 2006.

^* This work was supported by the LIPID MAPS Large Scale Collaborative Grant GM069338 from the U.S. National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and structures.

¹ To whom correspondence should be addressed: Depts. of Pharmacology, Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601. Tel.: 858-534-3055; Fax: 858-534-7390; E-mail: edennis{at}ucsd.edu.

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