The extraction of leukotrienes (LTC4, LTD4, and LTE4) from tissue fluids: the metabolism of these mediators during IgE-dependent hypersensitivity reactions in lung.
The extraction of leukotrienes (LTC4, LTD4, and LTE4) from tissue fluids: the metabolism of these mediators during IgE-dependent hypersensitivity reactions in lung.
复制标题
从组织液中提取白三烯（LTC4、LTD4 和 LTE4）：这些介质在肺部 IgE 依赖性超敏反应期间的代谢。
DOI：
10.1016/0003-2697(83)90218-x
复制
发表时间：
1983
影响因子：
2.9
通讯作者：
Hugli,TE
中科院分区：
文献类型：
--
作者：
Clancy,RM;Hugli,TE
花生四烯酸的代谢产物称为白三烯，是一类在肺组织中具有强大而多样的生物学效应的脂质介质。白三烯C、D和E(LTC4、LTD4和LTE4)是免疫球蛋白E(IgE)介导的肺组织超敏反应的主要介质。因此，开发可靠和定量的分离技术来估计组织中这些介质的水平是很重要的。本研究采用有机萃取法从花生四烯酸代谢产物中分离出LTC4、LTD4和LTE4。5-羟基二十碳四烯酸和白三烯B4在水：乙醚或水：氯仿萃取物中能有效地萃取到有机层中，而含有结合肽的花生四烯酸代谢物(如LTC4、LTD4和LTE4)不能在这些有机溶剂中萃取。因此开发了一种萃取步骤，可以将LTC4、LTD4和LTE4定量地萃取到异丙醇：乙醚：水混合物的有机相中。这一步骤是两步提取法分离组胺、LTC4、LTD4和LTE4的关键步骤，回收率分别为100、85、75和57%。这种通过有机萃取获得这些介体的分离程序的一个优点是能够方便地处理许多样品。此外，提取样品的白三烯含量可以用豚鼠回肠生物测定法进行分析，而不受血管胺或血小板激活因子的干扰。通过这个提取过程，从富含白三烯的馏分中除去这些后一种物质。将组胺和LTC4加入到从分离的肺组织中回收的上清液中，用这种提取方法定量回收。组胺和LTC4也与对照肺组织孵育，孵育10min后，50%的组胺和40%的白三烯被回收。LTC4广泛分解为LTD4和LTE4。这些结果表明，肺组织含有有效的分解代谢途径，可以迅速代谢白三烯。致敏的豚鼠肺组织上清液仅含6.5nmol组胺/克湿重，可引起IgE介导的变态反应。抗原攻击组织的洗澡液中也含有LTD4，这是对照组织上清液中不存在的一种介质。从抗原攻击的组织中回收的LTD4量小于5pmoL/g湿组织。这里报道的提取组胺和白三烯的水平明显低于以前报道的使用生物测定分析未提取样本的水平。
The metabolites of arachidonic acid known as the leukotrienes are a class of lipid mediators which have potent and diverse biological effects in pulmonary tissue. Leukotrienes C, D, and E (LTC4, LTD4, and LTE4) are known to be principal mediators of immunoglobulin E (IgE)-mediated hypersensitivity reactions in lung tissue. It is therefore important to develop reliable and quantitative isolation techniques for estimating levels of these mediators in tissue. In this study, LTC4, LTD4, and LTE4were separated from other arachidonate metabolites by organic extraction procedures. 5-Hydroxyeicosatetraeonic acid and leukotriene B4extract efficiently into the organic layer of aqueous:ether or aqueous:chloroform extractions, whereas arachidonate metabolites containing conjugated peptides (e.g., LTC4, LTD4, and LTE4) failed to extract into these organic solvents. An extraction step was therefore developed that affords quantitative extraction of LTC4, LTD4, and LTE4into the organic phase of an isopropanol:ether:H2O mixture. This step is the key for a two-step extraction method that isolates histamine, LTC4, LTD4, and LTE4with a recovery of 100, 85, 75, and 57%, respectively. One advantage of this separation procedure for obtaining these mediators by organic extraction is an ability to expediently process many samples. Furthermore, the leukotriene content of extracted samples can be analyzed using the guinea pig ileum bioassay without interference from vasoamines or platelet-activating factor. These later substances are eliminated from leukotriene-enriched fractions by this extraction process. When histamine and LTC4were added to supernatant fluids recovered from isolated lung tissue, they were quantitatively recovered using this extraction method. Histamine and LTC4were also incubated with control lung tissue and, after 10 min of incubation, 50% of the histamine and 40% of the leukotriene were recovered. There was extensive catabolism of the LTC4to LTD4and LTE4. These results suggest that lung tissue contains potent catabolic pathways which rapidly metabolize the leukotrienes. Supernatant fluid from sensitized guinea pig lung tissue provoked by antigen to elicit an IgE-mediated hypersensitive reaction contained only 6.5 nmol histamine/g wet wt of tissue. Bathing fluid of the antigen-challenged tissue also contained LTD4, a mediator not present in supernatant fluid from control tissue. The quantity of LTD4recovered from antigen-challenged tissue was less than 5 pmol/g wet of tissue. Levels reported here for extracted histamine and leukotriene were significantly lower than those previously reported using bioassays to analyze nonextracted samples.