title: Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). I. Direct evidence for cis-EODA formation from oleic acid oxidation by liver microsomes and isolated hepatocytes.
abstract: Oleic acid, cis-9-octadecenoic acid, is the major fatty acid in mammals. Its oxide, cis-9,10-epoxyoctadecanoic acid (cis-EODA), has been identified in blood and urine of humans, its origin is, however, still unknown. Lipid peroxidation and enzyme-catalyzed epoxidation of oleic acid are two possible sources. In the present article, we investigated by HPLC and GC-MS whether cis-EODA is formed enzymatically from oleic acid by the cytochrome P450 (CYP) system. Oleic acid, cis-EODA and its hydratation product threo-9,10-dihydroxyoctadecanoic acid (threo-DiHODA) were quantitated by HPLC as their p-bromophenacyl esters. For structure elucidation by GC-MS, the pentafluorobenzyl (PFB) esters of these compounds were isolated by HPLC and converted to their trimethylsilyl ether derivatives. Liver microsomes of rats, rabbits and humans oxidized oleic acid into cis-EODA. This is the first direct evidence for the enzymatic formation of cis-EODA from oleic acid. The epoxidation of oleic acid was found to depend on CYP, NADPH+H(+), and O(2). cis-EODA was measurable in incubates of liver microsomes for up to 30 min of incubation. Maximum cis-EODA concentrations were reached after 5-7 min of incubation and found to depend upon oleic acid concentration. Isolated rat hepatocytes hydratated cis-EODA into threo-DiHODA which was further converted to unknown metabolites. However, from incubation of oleic acid with these cells we could not detect threo-DiHODA or cis-EODA. Our study suggests that circulating and excretory cis-EODA may originate, at least in part, from CYP-catalyzed epoxidation of oleic acid. GC-MS of intact cis-EODA as its PFB ester in the negative-ion chemical ionization mode should be useful in investigating the physiological role of cis-EODA in man.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623, Hannover, Germany. tsikas.dimitros@mh-hannover.de
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sawa
authors.forename: Gorig
authors.initials: G
authors.lastname: Brunner
authors.forename: Frank Mathias
authors.initials: FM
authors.lastname: Gutzki
authors.forename: Hartmut H
authors.initials: HH
authors.lastname: Meyer
authors.forename: Jürgen C
authors.initials: JC
authors.lastname: Frölich
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D065607:Cytochrome P-450 Enzyme Inhibitors; D004852:Epoxy Compounds / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004852:Epoxy Compounds; D005227:Fatty Acids; D019301:Oleic Acid; C029355:dihydrosterculic acid