arachidonic-acid-omega-9-hydroperoxide and 8-11-12-trihydroxy-5-9-14-eicosatrienoic-acid

Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of (14)C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a (14)C-metabolite that migrated at 17.6 min. This (14)C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the (14)C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A(3)) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C(3)). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1+/-7.6%). Further testing with authentic trioxilin A(3) and C(3) revealed that trioxilin C(3) was the active metabolite (maximal relaxation: 16.6+/-1.3%). In conclusion, trioxilin C(3) acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Leukotrienes; Male; Rabbits; Vasodilation; Vasodilator Agents

The 30-50% ammonium sulfate fraction of the high speed supernatant (100,000 xg) of a rat lung homogenate is capable of catalysing the conversion of arachidonic acid into 8,11,12- and 10,11, 12-trihydroxyeicosatrienoic acids. This enzyme preparation was resolved through DEAE cellulose chromatography into three stages which were assayed with precursors specific for each stage. Thus in the first stage arachidonic acid is converted by 12-lipoxygenase into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) detected as the corresponding 12-hydroxy product (12-HETE). 12-HPETE in turn is converted into 8-hydroxy-11,12-epoxy-5,9,14-eicosatrienoic acid and 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid. These epoxides are in turn selectively converted through an epoxide hydrase into the respective triols. While the first and third stages were carried out by distinct fractions from the DEAE columns, the second i.e. conversion of 12-HPETE into epoxides, was detected in all fractions as was the reduction of 12-HPETE into 12-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Biotransformation; Chromatography, DEAE-Cellulose; Epoxide Hydrolases; Fatty Acids, Unsaturated; Leukotrienes; Lipoxygenase; Lung; Rats