A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes

Liping Du, Valery Yermalitsky, David L. Hachey, Setti G. Jagadeesh, J R Falck, Diane S. Keeney

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 [1-14C]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.

Original languageEnglish (US)
Pages (from-to)371-379
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume316
Issue number1
DOIs
StatePublished - Jan 2006

Fingerprint

Biosynthetic Pathways
Microsomes
Arachidonic Acid
Epidermis
NADP
Skin
Prostaglandin-Endoperoxide Synthases
High Pressure Liquid Chromatography
Lipoxygenases
Arachidonic Acids
Keto Acids
Acids
Lipoxygenase
Electrospray Ionization Mass Spectrometry
Reverse-Phase Chromatography
Keratinocytes
Skin Diseases
Liquid Chromatography
Cytochrome P-450 Enzyme System
Hydrolysis

ASJC Scopus subject areas

  • Pharmacology

Cite this

A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes. / Du, Liping; Yermalitsky, Valery; Hachey, David L.; Jagadeesh, Setti G.; Falck, J R; Keeney, Diane S.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 316, No. 1, 01.2006, p. 371-379.

Research output: Contribution to journalArticle

Du, Liping ; Yermalitsky, Valery ; Hachey, David L. ; Jagadeesh, Setti G. ; Falck, J R ; Keeney, Diane S. / A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes. In: Journal of Pharmacology and Experimental Therapeutics. 2006 ; Vol. 316, No. 1. pp. 371-379.
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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 [1-14C]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.",
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