Krämer H J, Stevens J, Grimminger F, Seeger W
Department of Internal Medicine, Justus-Liebig University, Giessen, Germany.
Biochem Pharmacol. 1996 Oct 25;52(8):1211-7. doi: 10.1016/0006-2952(96)00473-x.
Dietary enrichment of membrane phospholipids with n-3 (fish-oil-derived) fatty acids has attracted attention as a putative therapeutic regimen for suppression of inflammatory and coagulatory events. Use of n-3 fatty-acid-enriched lipid infusions for parenteral nutrition results in micromolar concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) in the plasma-free fatty acid fraction. We investigated the influence of free EPA and DCHA on platelet thromboxane (Tx) A2 and A3 formation by using a recently developed high performance liquid chromatography-ELISA technique for separate quantification of the stable hydrolysis products TxB2 and TxB3. Washed human thrombocytes were incubated with free arachidonic acid (AA; 1 microM), A23187 (0.1 microM) or thrombin (5 U/mL) for stimulation; all regimens provoked large quantities of TxA2 in the absence of TxA3. Simultaneous admixture of free EPA or free DCHA to the incubation medium (concentration range, 0.01-50 microM) largely suppressed platelet TxA2 generation in response to all stimuli used in a dose-dependent manner. The effective concentration with 50% influence of arachidonic acid was 4.2 microM, whereas the inhibitory concentration with 50% effect of EPA and DCHA were both in the same order of magnitude but differed with the nature of the agonist (0.2-7 microM). Platelet (co-)incubation with EPA, but not DCHA, provoked dose-dependent synthesis of n-3-lipid-derived thromboxane: kinetics of formation and absolute quantities of TxA3 approximated 20% of the respective TxA2 data upon stimulation with AA. Both EPA and DCHA dose-dependently suppressed U46619-provoked platelet aggregation. We conclude that EPA and DCHA are potent competitive inhibitors of TxA2 generation by intact platelets, with EPA acting as poor substrate and DCHA being no substrate for the cyclooxygenase/thromboxane synthase complex. Enrichment of the plasma-free fatty acid fraction with n-3 lipids may offer a therapeutic regimen to suppress the synthesis of the potent proaggregatory and vasoconstrictory agent TxA2.
用n-3（鱼油衍生的）脂肪酸对膜磷脂进行膳食强化，作为一种抑制炎症和凝血事件的假定治疗方案已引起关注。在肠外营养中使用富含n-3脂肪酸的脂质输注会导致血浆游离脂肪酸部分中二十碳五烯酸（EPA）和二十二碳六烯酸（DCHA）的微摩尔浓度。我们使用最近开发的高效液相色谱-酶联免疫吸附测定技术来分别定量稳定水解产物血栓素B2（TxB2）和血栓素B3（TxB3），研究了游离EPA和DCHA对血小板血栓素（Tx）A2和A3形成的影响。将洗涤过的人血小板与游离花生四烯酸（AA；1微摩尔）、A23187（0.1微摩尔）或凝血酶（5单位/毫升）一起孵育以进行刺激；在没有TxA3的情况下，所有方案都会引发大量的TxA2。将游离EPA或游离DCHA同时加入孵育培养基（浓度范围为0.01 - 50微摩尔）会以剂量依赖的方式在很大程度上抑制血小板对所有所用刺激产生的TxA2。花生四烯酸产生50%影响的有效浓度为4.2微摩尔，而EPA和DCHA产生50%效应的抑制浓度在同一数量级，但因激动剂的性质而异（0.2 - 7微摩尔）。血小板与EPA（而非DCHA）共同孵育会引发剂量依赖性的n-3脂质衍生血栓素的合成：在用AA刺激时，TxA3的形成动力学和绝对量约为各自TxA2数据的20%。EPA和DCHA均以剂量依赖的方式抑制U46619诱导的血小板聚集。我们得出结论，EPA和DCHA是完整血小板产生TxA2的有效竞争性抑制剂，EPA作为环氧化酶/血栓素合酶复合物的不良底物，而DCHA则不是底物。用n-3脂质富集血浆游离脂肪酸部分可能提供一种治疗方案来抑制强效促聚集和血管收缩剂TxA2的合成。
