Dahinden C A, Clancy R M, Hugli T E
J Immunol. 1984 Sep;133(3):1477-82.
The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.
通过使用灵敏的博伊登小室分析法检测了白三烯B4（5S，12R-二羟基-6，14-顺式，8，10-反式-二十碳四烯酸）（LTB4）的趋化活性。将LTB4的活性与其他生物合成立体异构体进行了比较：5S，12R-二羟基-6，8，10-反式-14-顺式-二十碳四烯酸（6-反式LTB4）；5S，12S-二羟基-6，8，10-反式-14-顺式-二十碳四烯酸（12-表-6-反式LTB4），5S，12S-二羟二十碳四烯酸；代谢产物20-羟基-LTB4（20-OH LTB4）；甲基化LTB4（甲基-LTB4），以及相关的单羟二十碳四烯酸5-羟二十碳四烯酸（5-HETE）和12-羟二十碳四烯酸（12-HETE）。这些化合物通过几步反相和正相高效液相色谱法进行了纯化。LTB4在10⁻⁹M时表现出可测量的趋化活性，在10⁻⁷M时活性最大，半数有效剂量（ED50）为10⁻⁸M。LTB4异构体和单羟二十碳四烯酸的趋化性比先前报道的要低。单羟二十碳四烯酸（5-HETE和12-HETE）、异构体12-表-6-反式LTB4和5S，12S-二羟二十碳四烯酸在10⁻⁶至10⁻⁵M浓度范围内不能吸引中性粒细胞。如果这些化合物具有趋化性，那么其活性至少比LTB4低四个数量级。10⁻⁶至10⁻⁵M的异构体6-反式LTB4诱导趋化作用，外推的ED50值为10⁻⁵M，表明6位构型由顺式变为反式会使LTB4的趋化活性降低1000倍。相反，代谢产物20-OH LTB4至少与天然化合物LTB4活性相当。LTB4羧基的甲基化使其趋化活性降低两个数量级。这些结果表明LTB4受体具有高度的立体特异性，对羟基、三烯构型有严格依赖性，对羧基也有相当大的依赖性。LTB4分子脂肪族ω端的修饰对功能影响最小，这表明该分子这部分的疏水性对最佳活性并不重要。此外，我们提出LTB4的代谢产物作为体内生理性炎症介质可能比LTB4更重要。
