Mass determination of the fatty acids released from tannin-stimulated rabbit alveolar macrophages

Previous studies with macrophages that had been prelabeled with [14C]arachidonic acid (20:4) have shown that condensed tannin is a potent agonist for the release of arachidonic acid. However, it has not been demonstrated that the percentage release of [14C]20:4 accurately reflects the metabolic activity of the endogenous 20:4 pool. In order to measure the 20:4 mass release relative to the total cellular 20:4 pool, the free fatty acids of freshly isolated alveolar macrophages were derivatized with a fluorescent reagent, and then separated and quantified by high-performance liquid chromatography. The amounts of esterified fatty acids were measured by gas chromatography of the methyl esters. Free fatty acid levels were compared to those of the total esterified plus unesterified fatty acids to determine the actual percentage release of each fatty acid. Tannin-stimulated release of 20:4 mass reflected that previously reported for the release of [14C]20:4 label but at a slower rate and at a much lower percentage indicating that [14C]20:4 had been incorporated into part of a more reactive pool. The specificity of the fatty acid release induced by tannin and beta-1,3-glucan, a known agonist for 20:4 release, was also examined. Both agonists promoted an increase in the levels of free 20:4 and of other fatty acids. A comparison of the absolute increases of each of the fatty acids indicated that tannin caused a preferential increase in the mass of free 20:4, whereas beta-1,3-glucan evoked a selective increase in the mass of 16:0.