Structural Characterization of Monohydroxyeicosatetraenoic Acids and Dihydroxy- and Trihydroxyeicosatrienoic Acids by ESI-FTICR

The fragmentation characteristics of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids were investigated by electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD). The fragmentation patterns of these compounds were associated with the number and positions of the hydroxyl substituents. The fragmentation is more complicated with increasing number of the hydroxyl groups of the compounds. In general, the major carbon–carbon cleavage of [M − H]− ions occurred at the α-position to the hydroxyl group, and the carbon–carbon cleavage occurred when there was a double-bond at the β-position to the hydroxyl group. SORI-CID and IRMPD produced some common fragmentation patterns; however, each technique provided some unique patterns that are useful for structural identification of these compounds. This study demonstrated the application of FTICR via the identification of regioisomers of trihydroxyeicosatrienoic acids in rabbit aorta samples

Chronic hypoxia enhances 15-lipoxygenase-mediated vasorelaxation in rabbit arteries

15-Lipoxygenase (15-LO-1) metabolizes arachidonic acid (AA) to 11,12,15-trihydroxyeicosatrienoic acids (THETAs) and 15-hydroxy-11,12-epoxyeicosatrienoic acids (HEETA) that dilate rabbit arteries. Increased endothelial 15-LO-1 expression enhances arterial relaxations to agonists. We tested the effect of hypoxia on 15-LO-1 expression, THETA and HEETA synthesis, and relaxations in rabbit arteries. The incubation of rabbit aortic endothelial cells and isolated aortas in 0.7% O2 increased 15-LO-1 expression. Rabbits were housed in a hypoxic atmosphere of 12% O2 for 5 days. 15-LO-1 expression increased in the endothelium of the arteries of rabbits in 12% O2 compared with room air. THETA and HEETA synthesis was also enhanced in aortas and mesenteric arteries. AA hyperpolarized the smooth muscle cells in indomethacin- and phenylephrine-treated mesenteric arteries of hypoxic rabbits from −29.4 ± 1 to −50.1 ± 3 mV. The hyperpolarization to AA was less in arteries of normoxic rabbits (from −26.0 ± 2 to −37 ± 2 mV). This AA-induced hyperpolarization was inhibited by the 15-LO inhibitor BW-755C. Nitric oxide and prostaglandin-independent maximum relaxations to acetylcholine (79.7 ± 2%) and AA (38.3 ± 4%) were enhanced in mesenteric arteries from hypoxic rabbits compared with the normoxic rabbits (49.7 ± 6% and 19.9 ± 2%, respectively). These relaxations were inhibited by BW-755C and nordihydroguaiaretic acid. Therefore, hypoxia increased the relaxations to agonists in the rabbit mesenteric arteries by enhancing endothelial 15-LO-1 expression and synthesis of the hyperpolarizing factors THETA and HEETA