12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Corneal-Injuries

Previous work has shown that, shortly after rabbit corneas are injured, arachidonic acid metabolism is activated, and 12-hydroxyeicosatetraenoic acid (12-HETE) is one of the main products formed (Bazan, H. E. P., Birkle, D. L., Beuerman, R., and Bazan, N. G. (1985) Invest. Ophthalmol. & Visual Sci. 26, 474-480; Bazan, H. E. P. (1987) Invest. Ophthalmol. Visual Sci. 28, 314-319). In order to determine whether this metabolite is a lipoxygenase product, anesthetized rabbit corneas injured in vivo, either cryogenically or by 1 M NaOH, were subsequently incubated in vitro with [14C] arachidonic acid in the presence of indomethacin. 12-HETE was the main metabolite produced, as established by gas chromatography-mass spectrometry. The (R)- and (S)-enantiomers of novel naphthoyl-pentafluorobenzoyl derivatives of 12-HETE were resolved by chiral-phase high performance liquid chromatography. The radiolabeled 12-HETE from whole cornea and from isolated epithelium, endothelium, or stroma eluted as a single peak co-chromatographing with the (S)-enantiomer and was detected both by UV absorbance at 234 nm and by radioactivity. In noninjured corneas a smaller peak of radiolabeled (12S)-HETE was also eluted from the chiral column. The stereochemistry was additionally confirmed by liquid chromatography-mass spectrometry. These studies suggest that (12S)-lipoxygenase is activated in the injured rabbit cornea.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Chromatography, High Pressure Liquid; Cornea; Corneal Injuries; Endothelium, Corneal; Epithelium; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Isomerism; Lipoxygenase; Mass Spectrometry; Rabbits