Qualitative and quantitative analysis of lipoxygenase products in bovine corneal epithelium by liquid chromatography-mass spectrometry with an ion trap.
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.
Liminga
M
M
Department of Pharmaceutical Biosciences, Uppsala University, Uppsala Biomedical Center, Sweden. Maria.Liminga@farmbio.uu.se
Oliw
E
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D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D019573:Epithelium, Corneal / Q000378:metabolism*; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis / Q000378:metabolism; D015289:Leukotrienes / Q000032:analysis* / Q000378:metabolism; D008054:Lipid Peroxides / Q000032:analysis / Q000378:metabolism; D008084:Lipoxygenase / Q000032:analysis / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D013347:Subcellular Fractions
D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D008054:Lipid Peroxides; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C021201:12-HPETE; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C025348:arachidonic acid 5-hydroperoxide; D008084:Lipoxygenase