15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and 11-12-15-trihydroxyeicosatrienoic-acid

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Cytochrome P-450 Enzyme Inhibitors; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Leukotrienes; Lipid Peroxides; Miconazole; Microsomes; Models, Biological; Rabbits; Vasodilator Agents

Incubation of 15-hydroperoxide of 5,8,11,14,17-eicosapentaenoic acid (15-HPEPE) with 5-lipoxygenase enzyme, partially purified from potato tubers, resulted in the generation of 6 isomers of lipoxin A5. These compounds were identified by GC/MS analysis of their methyl ester and trimethylsilyl ether derivatives with C-values: 23.3, 24.5, 24.6, 24.9, 25 and 25.2 respectively. The major products of the enzymatic reaction on 15-HPEPE are 5,15-DiHEPE and 13,14,15-trihydroxy eicosatetraenoic acid as identified by RP-HPLC and GC/MS analysis. There are also two new trihydroxyl compounds of eicosapentaenoic acid identified as 11,12,15-trihydroxy and 11,14,15-trihydroxy eicosatetraenoic acid respectively. The transformation of these two trihydroxyl compounds may be due to non-enzymatic rearrangement of 15-HPEPE.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonate 5-Lipoxygenase; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxins; Mass Spectrometry; Molecular Structure; Plants; Solanum tuberosum