title: A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells.
abstract: We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects. Cell incubation with AA (0.3-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry. Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA. Two epoxyeicosatrienoic acids derived from AA were also identified. Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects. Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets. Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions. The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: M J
authors.initials: MJ
authors.lastname: Holtzman
authors.forename: J
authors.initials: J
authors.lastname: Turk
authors.forename: A
authors.initials: A
authors.lastname: Pentland
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004817:Epidermis / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D013058:Mass Spectrometry; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; D009249:NADP; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D003577:Cytochrome P-450 Enzyme System