Qualitative and quantitative analysis of lipoxygenase products in bovine corneal epithelium by liquid chromatography-mass spectrometry with an ion trap.

Abstract

Electrospray ionization ion trap mass spectra of 5-, 12-, and 15-hydroperoxyeicosatetraenoic (HPETE), hydroxyeicosatetraenoic (HETE), and ketoeicosatetraenoic (KETE) acids were recorded. The HPETE were partly dehydrated to the corresponding KETE in the heated capillary of the mass spectrometer. 12-HPETE and 15-HPETE were also converted to KETE by collision-induced dissociation (CID) in the ion trap, whereas CID of 5-HPETE yielded little formation of 5-KETE. Subcellular fractions of bovine corneal epithelium were incubated with arachidonic acid (AA) and the metabolites were analyzed. 15-HETE and 12-HETE were consistently formed, whereas significant accumulation of HPETE and KETE was not detected. Biosynthesis of 12- and 15-HETE was quantified with octadeuterated 12-HETE and 15-HETE as internal standards. The average biosynthesis of 15-HETE and 12-HETE from 30 microM AA by the cytosol was 38 +/- 8 and below 3 ng/mg protein/30 min, respectively, which increased to 78 +/- 21 and 10 +/- 4 ng/mg protein/30 min in the presence of 1 mM free Ca2+. The microsomal biosynthesis was unaffected by Ca2+. The microsomes metabolized AA to 15-HETE as the main metabolite at a low protein concentration (0.3 mg/mL), whereas 12-HETE and 15-HETE were formed in a 2:1 ratio at a combined rate of 0.7 +/- 0.2 microg/mg protein/30 min at a high protein concentration (1.8 mg/mL). The level of 12-HETE in corneal epithelial cells was 50 +/- 13 pg/mg tissue, whereas the endogenous amount of 15-HETE was low or undetectable (<3 pg/mg tissue). Incubation of corneas for 20 min at 37 degrees C before processing selectively increased the amounts of 12-HETE in the epithelium fourfold to approximately 0.2 ng/mg tissue. We conclude that 12-HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.