The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury.
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.
Hurst
J S
JS
Department of Ophthalmology, Louisiana State University Eye Center, School of Medicine, New Orleans 70112.
Balazy
M
M
Bazan
H E
HE
Bazan
N G
NG
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003315:Cornea / Q000201:enzymology / Q000473:pathology; D065306:Corneal Injuries; D004728:Endothelium, Corneal / Q000201:enzymology; D004848:Epithelium / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis* / Q000302:isolation & purification; D007536:Isomerism; D008084:Lipoxygenase / Q000378:metabolism*; D013058:Mass Spectrometry; D011817:Rabbits
D006893:Hydroxyeicosatetraenoic Acids; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D008084:Lipoxygenase