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Qualitative and quantitative analysis of lipoxygenase products in bovine corneal epithelium by liquid chromatography‐mass spectrometry with an ion trap
Author(s) -
Liminga Maria,
Oliw Ernst
Publication year - 2000
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02664773
Subject(s) - chemistry , chromatography , mass spectrometry , arachidonic acid , hydroxyeicosatetraenoic acid , microsome , electrospray ionization , biochemistry , analytical chemistry (journal) , enzyme
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 30μM 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 Ca 2+ . The microsomal biosynthesis was unaffected by Ca 2+ . 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 μg/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°C before processing selectively increased the amounts of 12‐HETE in the epithelium fourfold to ∼0.2 ng/mg tissue. We conclude that 12‐HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.