Tumor necrosis factor-induced release of endogenous fatty acids analyzed by a highly sensitive high-performance liquid chromatography method

Abstract

A highly sensitive method to determine agonist-induced release of endogenous fatty acids from cells in culture was developed using high-performance liquid chromatography and fluorescence detection. Fatty acids were selectively derivatized with 1-pyrenyldiazomethane and separated on a LC18 reversed phase column using an acetonitrile-water gradient. The detection limit was approx. 20 fmol and the recovery of the complete method using oleic acid was 93-98%. Tumor necrosis factor alpha (TNF-alpha) increased the extracellular release of endogenous arachidonic acid (20:4n-6) from 21 to 153 pmol/well per 4 h using 2.7 x 10(6) WEHI fibrosarcoma cells. In cells preincubated with 50 microM 20:4n-6, the corresponding figures were 463 and 3379 pmol 20:4n-6/well. Simultaneously, nearly equimolar amounts of 22:4n-6 were released together with slightly lower amounts of 24:4n-6, 16:0, 16:1n-9, and 18:1n-9. Analysis of cell lipid fatty acids showed that phosphatidylcholine was the major source of the released fatty acids. TNF-alpha increased the intracellular concentration of unesterified 20:4n-6 and 22:4n-6 by 368% and 451%, respectively. This suggests that released 20:4n-6 is rapidly chain elongated to 22:4n-6. The results indicate that the present method facilitates studies on agonist-induced release of endogenous fatty acids, and that TNF-induced fatty acid release seems to be less selective for 20:4n-6 than previously reported.