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 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.
Brekke
O L
OL
Department of Clinical Chemistry, Norwegian University of Science and Technology, Trondheim University Hospital, Norway.
Sagen
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Bjerve
K S
KS
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005227:Fatty Acids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D051379:Mice; D011721:Pyrenes; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D011721:Pyrenes; D014409:Tumor Necrosis Factor-alpha; C058827:1-pyrenyldiazomethane