Mass spectrometric analysis of leukotriene A₄ and other chemically reactive metabolites of arachidonic acid

Abstract

The biosynthesis of prostaglandins and leukotrienes proceeds through the formation of chemically reactive intermediates leukotriene A 4 (LTA 4 ) and prostaglandin H 2 (PGH 2 ) which in aqueous solutions have chemical half-lives of 3 s and 3 min, respectively. Prostacyclin (PGI 2 ) is another chemically reactive prostanoid that has a chemical half-life of 3-4 min. The recent development of reversed phase HPLC stationary phases that are stable to elevated pH (pH 10 -12) without significant column damage has permitted direct analysis of these acid-sensitive eicosanoids. Using electrospray ionization, molecular anions [M Ϫ H] Ϫ of these compounds were observed at m/z 317 for LTA 4 and m/z 351 for both PGH 2 and PGI 2 . The mechanism of formation of ions derived from collisional activation of LTA 4 was studied using stable isotope labeled and chemical analogs of LTA 4 and found to involve formation of highly conjugated anions at m/z 261 and 163. The collisional activation of the molecular anion of PGH 2 yielded a product ion spectrum identical to that observed for the isomeric prostaglandins PGE 2 and PGD 2 . However, it was possible to baseline separate PGE 2 , PDG 2 , and PGH 2 by reversed phase HPLC using basic HPLC mobile phases. The collisional activation of PGI 2 led to a family of abundant ions including highly conjugated carbon-centered and oxygen-centered radical species (m/z 245 and 205) likely derived from the attack of the carboxylate anion on the cyclic enolether of PGI 2 as well as the most abundant product ion (m/z 215) which formed following loss of neutral hexanal and water. The structures of these product ions were consistent with high resolution measurements measured in a quadrupole time-of-flight mass

Stabilization of Leukotriene A4 by Epithelial Fatty Acid-binding Protein in the Rat Basophilic Leukemia Cell

“…Aliquots (50 l) were removed at various time points between 2 and 30 min and added to ethanol (100 l) containing 100 ng of internal standard, which was either 15-oxo-ETE or 20-trifluoro LTA 4 . These samples were analyzed using either an in-line photo diode array or a triple quadrupole mass spectrometer (API-3000; PE-Sciex, Thornhill, Canada) as previously described (26). In either case, the log ratio (peak area of LTA 4 )/(peak area of its internal standard) versus time was plotted, and the half-life was calculated using the slope of the resulting line.…”

COX-mediated endothelium-dependent contractions: from the past to recent discoveries

“…All prostanoids are able to bind with TP receptors, albeit with different affinities [82] . Thromboxane A 2 is the most potent agonist at TP receptors.…”

Endothelium-Dependent Contractions in Hypertension

“…46,55,93,94 It is unknown whether or not this lack of responsiveness of IP receptors initiates a positive feedback on the endothelial cells, leading to the abundant overexpression of PGI 2 synthase and the predominant release of PGI 2 by endothelial cells stimulated by acetylcholine or A23187. However, the large amounts of PGI 2 become important enough to bind with TP receptors 55,95 ( Figure 2). The contraction of the latter on TP receptor activation is attributable to the combination of an increased entry of Ca 2ϩ resulting from the opening of both receptoroperated and voltage-gated Ca 2ϩ channels and Rho kinasemediated sensitization of the myofilaments.…”