Biotransformation of linoleic acid by porcine leukocytes

Abstract

Linoleic acid is an important essential fatty acids of leukocyte cell membrane phospholipids from some animals, e.g. from pigs and rabbits, and is a known substrate for lipoxygenase(s), especially in plant systems. Lipoxygenase activity has also been well documented in leukocytes using arachidonic acid as a substrate. These findings and our own interest in the fate of linoleic acid have prompted us to investigate the biotransformation of this essential fatty acids in leukocytes. Porcine leukocytes were isolated from whole blood by dextrane precipitation of the erythrocytes and by centrifugation. Broken cells were incubated with exogenous linoleic acid and four major biotransformation products, X 1, X 2, X 3 and X 4, were formed. Following isolation by silicagel column chromatography and thin layer chromatography, the products were derivatized and characterized by GC/MS. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation, and various types of derivatives were made in order to facilitate the structure elucidation. The major product X 1, which represented 60.5% of the total metabolites formed, was identified as 13-hydroxy-9,11-octadecadienoic acid. Product X 2 (16.2%) was shown to be 11-hydroxy-12,13-epoxy-9-octadecenoic acid. Products X 3 and X 4 (respectively 5.2 and 7.5%) resulted in identical thermore, each of the products X 3 and X 4 was shown to be a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic acid (70%) and 9,10,13-trihydroxy-12-octadecenoic acid (30%). With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butylboronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol position. The metabolism of linoleic acid in porcine leukocytes is analogous to that by cereal lipoxygenases. A major difference however is that porcine leukocyte lipoxygenase predominantly yields products, which arise through 13-lipoxygenation, whereas, in cereals, transformation products of 9-hydroperoxy-10,12-octadecadienoic acid are formed to the same extent as metabolites of 13-hydroperoxy-9,11-octadecadienoic acid.