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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

A Biosynthetic Pathway Generating 12-Hydroxy-5,8,14-eicosatrienoic Acid from Arachidonic Acid Is Active in Mouse Skin Microsomes

Departments of Biochemistry (L.D., V.Y., D.S.K.), Pharmacology (D.L.H.), and Medicine/Dermatology (D.S.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (S.G.J., J.R.F.); and Veterans Administration Tennessee Valley Healthcare System, Nashville, Tennessee (D.S.K.)

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-¹⁴C]arachidonic acid. The majority of the ¹⁴C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal ¹⁴C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Address correspondence to: Dr. Diane S. Keeney, Department of Medicine/Dermatology and Biochemistry, Vanderbilt University, 607 Light Hall (0146), Nashville, TN 37232-0146. E-mail: diane.keeney{at}vanderbilt.edu

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