HYDRA-VULGARIS OMEGA-10-LIPOXYGENASE IS USED IN-VIVO TO SYNTHESIZE NEW ALPHA-LINOLENIC ACID METABOLITES

Previous studies conducted in cytosolic extracts of the freshwater hyd rozoan Hydra vulgaris led to the finding of an abundant 11(R)-lipoxyge nase catalyzing the peroxidation of polyunsaturated fatty acid (PUFAs) on the tenth carbon atom from the aliphatic end (omega 10 peroxidatio n). Here we describe experiments aimed at identifying the actual metab olites generated in vivo by such enzymic activity, Homogenates of H. v ulgaris polyps were analyzed by HPLC. This showed the presence of thre e major components chromatographically identical to three metabolites obtained when incubating the homogenates with exogenous alpha-linoleni c acid (alpha-LA). The presence, in extracts of polyps prelabelled wit h [C-14]-alpha-linolenic acid, of radioactive metabolites displaying t he same chromatographic properties, substantiated the hypothesis that the natural products isolated in vivo are derived from alpha-LA. Gas c hromatographic analyses revealed that this was the most abundant PUFA in both free and phosphoglyceride-bound fatty acid pools. [H-1]-NMR an alysis of the endogenous substances, carried out in comparison with pr oducts obtained from exogenously incubated alpha-LA, indicated that th eir structures were those of 9-hydroxy-, 9-hydroperoxy- and 9-keto-oct adeca-10E-12Z-15Z-trienoic acids (9-alpha-HOTrE, -HPOTrE and -KOTrE). Hydra homogenates transformed 9-alpha-HPOTrE partly into 9-alpha-HOTrE and partly into 9-alpha-KOTrE. Chiral phase HPLC conducted on 9-alpha -HOTrE established that this metabolite was composed mostly of the R e nantiomer. These observations, and the finding that the presence of ex ogenous arachidonic acid in incubated homogenates significantly reduce s the production of alpha-LA metabolites, provide strong evidence that these compounds are produced by an enzymic activity identical to the previously-described H. vulgaris (R)-omega 10-lipoxygenase. Further ex periments suggested that alpha-LA, acting as the native substrate for this enzyme, is mainly esterified on the 2 position of Hydra phosphogl ycerides, and that the production of the alpha-LA metabolites describe d here for the first time from natural sources, can be potentially enh anced in vivo by stimuli activating phospholipase A(2).