Brekke O L, Sagen E, Bjerve K S
Department of Clinical Chemistry, Norwegian University of Science and Technology, Trondheim University Hospital, Norway.
J Lipid Res. 1997 Sep;38(9):1913-22.
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.
利用高效液相色谱和荧光检测技术，开发了一种用于测定培养细胞中激动剂诱导内源性脂肪酸释放的高灵敏度方法。脂肪酸用1-芘重氮甲烷进行选择性衍生化，然后在LC18反相柱上使用乙腈-水梯度进行分离。检测限约为20飞摩尔，使用油酸时整个方法的回收率为93%-98%。使用2.7×10⁶个WEHI纤维肉瘤细胞，肿瘤坏死因子α（TNF-α）使内源性花生四烯酸（20:4n-6）的细胞外释放量在每4小时内从21皮摩尔/孔增加到153皮摩尔/孔。在预先用50微摩尔20:4n-6孵育的细胞中，相应的数据为463皮摩尔和3379皮摩尔20:4n-6/孔。同时，几乎等摩尔量的22:4n-6与稍少量的24:4n-6、16:0、16:1n-9和18:1n-9一起释放。细胞脂质脂肪酸分析表明，磷脂酰胆碱是释放脂肪酸的主要来源。TNF-α使未酯化的20:4n-6和22:4n-6的细胞内浓度分别增加了368%和451%。这表明释放的20:4n-6迅速链延长为22:4n-6。结果表明，本方法有助于研究激动剂诱导的内源性脂肪酸释放，并且TNF诱导的脂肪酸释放对20:4n-6的选择性似乎比先前报道的要低。
