title: A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes.
abstract: 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-(14)C]arachidonic acid. The majority of the (14)C 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 (14)C 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.
authors.affiliation: Department of Medicine/Dermatology and Biochemistry, Vanderbilt University, 607 Light Hall (0146), Nashville, TN 37232-0146, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Du
authors.forename: Valery
authors.initials: V
authors.lastname: Yermalitsky
authors.forename: David L
authors.initials: DL
authors.lastname: Hachey
authors.forename: Setti G
authors.initials: SG
authors.lastname: Jagadeesh
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Diane S
authors.initials: DS
authors.lastname: Keeney
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002853:Chromatography, Liquid; D000072467:Cytochrome P450 Family 2; D008084:Lipoxygenase / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008861:Microsomes / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D012867:Skin / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: C067514:12-hydroxy-5,8,14-eicosatrienoic acid; D016718:Arachidonic Acid; D009249:NADP; D006899:Mixed Function Oxygenases; D008084:Lipoxygenase; D001189:Aryl Hydrocarbon Hydroxylases; C033914:Cyp2b19 protein, mouse; D000072467:Cytochrome P450 Family 2; D015126:8,11,14-Eicosatrienoic Acid