TUMOR NECROSIS FACTOR-INDUCED RELEASE OF ENDOGENOUS FATTY-ACIDS ANALYZED BY A HIGHLY SENSITIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY METHOD

A highly sensitive method to determine agonist-induced release of endo genous fatty acids from cells in culture was developed using high-perf ormance 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 comple te method using oleic acid was 93-98%. Tumor necrosis factor alpha (TN F-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(5) WEH I fibrosarcoma cells. In cells preincubated with 50 mu M 20:4n-6, the corresponding figures were 463 and 3379 pmol 20:4n-6/well. Simultaneou sly, nearly equimolar amounts of 22:4n-6 were released together with s lightly 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 maj or source of the released fatty acids. TNF-alpha increased the intrace llular concentration of unesterified 20:4n-6 and 22:4n-6 by 368% and 4 51%, respectively. This suggests that released 20:4n-6 is rapidly chai n elongated to 22:4n-6. The results indicate that the present method f acilitates studies on agonist-induced release of endogenous fatty acid s, and that TNF-induced fatty acid release seems to be less selective for 20:4n-6 than previously reported.