Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta.
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.
Pfister
Sandra L
SL
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Spitzbarth
Nancy
N
Nithipatikom
Kasem
K
Falck
John R
JR
Campbell
William B
WB
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D066298:In Vitro Techniques; D015289:Leukotrienes / Q000378:metabolism*; D008297:Male; D011817:Rabbits; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000378:metabolism*
D015289:Leukotrienes; D014665:Vasodilator Agents; C021201:12-HPETE; C019121:8,11,12-trihydroxy-5,9,14-eicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid