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

O L Brekke,E Sagen,K S Bjerve

doi:10.1016/s0022-2275(20)37166-2

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.

Highlights

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
We have previously shown that tumor necrosis factor (TNF)-induced cytotoxicity in WEHI 164 fibrosarcoma cells is associated with enhanced release of 20:4n-6 [7],probably due to stimulation of PLA
We examined whether rTNF-a-induced release of endogenous fatty acids could be measured with the present method. rTNF-a (1 pg/l) enhances the release of ['Hlarachidonic acid after 1-2 h in WEHI cells, while cell death is only detectable after 4-5 h

Summary

Introduction

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. This paper reports an improved highly sensitive HPLC method using fluorescence detection of fatty acid PDAM derivatives designed to measure the release of fatty acids from a low number of cells. Some fatty acid PDAM derivativeswere isolated from the HPLC and fractions were reanalyzed on GLC.

Results

Conclusion