The incorporation and release of 12(S)-hydroxyeicosatetraenoic acid and its major metabolite, 8(S)-hydroxyhexadecatrienoic acid, from rabbit corneal lipids.

J S Hurst, H E Bazan, M Balazy

Index: Exp. Eye Res. 59(1) , 97-105, (1994)

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Abstract

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) is the predominant corneal lipoxygenase metabolite formed after injury. To investigate the metabolic fate of this eicosanoid in the tissue, [3H]12(S)-HETE was injected intracamerally into rabbits. Corneas were removed 1 to 18 hr after labeling. In some experiments, either entire corneas or the constituent tissues (epithelium, endothelium and stroma) were then incubated in oxygenated Ames' medium for different times. Eighteen hours after injection, the radioactivity was mainly incorporated into the membrane phospholipids, phosphatidyl choline (49%) and phosphatidyl ethanolamine (40%). Free 12(S)-HETE represented less than 1% of the total label. Analysis of the products after phospholipase A2 treatment indicated that the label was acylated in the sn-2 position. HPLC analysis of extracts from tissue and medium showed the presence of 12(S)-HETE and a more polar metabolite established as 8(S)-hydroxyhexadecatrienoic acid [8(S)-OH-16:3] by gas-chromatography-mass-spectrometry. Within 1 hr of injection, 27% of the tissue label was recovered as 8(S)-OH-16:3 and at 8 hr the ratio of incorporated 8(S)-OH-16:3 to 12(S)-HETE was 2:1. 8(S)-OH-16:3 was released into the medium faster than 12(S)-HETE. Metabolism was highest on the epithelial corneal surface. The mitochondrial beta-oxidation inhibitor, 4-pentenoic acid, did not inhibit the formation of 8(S)-OH-16:3 which suggested a peroxisomal beta oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)