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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2003-02, Vol.784 (2), p.351-365
• Main Authors: Tsikas, Dimitrios, Sawa, Masayuki, Brunner, Gorig, Gutzki, Frank-Mathias, Meyer, Hartmut H, Frölich, Jürgen C
• Format: Article
• Language: English
• Subjects: Animals; Chromatography, High Pressure Liquid - methods; cis-9,10-Epoxyoctadecanoic acid; Cytochrome P-450 Enzyme Inhibitors; Epoxy Compounds - metabolism; Fatty Acids - biosynthesis; Fatty Acids - metabolism; Gas Chromatography-Mass Spectrometry - methods; Hepatocytes - metabolism; Kinetics; Microsomes, Liver - metabolism; Oleic acid; Oleic Acid - metabolism; Oxidation-Reduction; Rats
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/S1570-0232(02)00821-8

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.