Kiyomiya K, Yamaki K, Nimura N, Kinoshita T, Oh-ishi S
Prostaglandins. 1986 Jan;31(1):71-82. doi: 10.1016/0090-6980(86)90226-1.
White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.
大鼠放血后，从其胸腔冲洗液中收集白细胞。将胸腔细胞与1微摩尔佛波醇肉豆蔻酸酯乙酸酯（PMA）的孵育混合物用酸化乙醇提取，并用Sep-pak C18纯化。所得含有前列腺素（PG）和血栓素（TX）的馏分与9-蒽重氮甲烷（ADAM）反应。通过硅胶Sep-pak去除污染物和降解的试剂后，将样品应用于十八烷基硅烷硅胶的反相高效液相色谱，并通过荧光检测器进行监测。 authentic PGD2、PGE2、PGF2α、6-酮-PGF1α、6-酮-PGE1、TXB2、15-酮-PGE2、13,14-二氢-15-酮-PGF2α和13,14-二氢-15-酮-PGE2的ADAM衍生物在0.5-25纳克范围内显示出峰高的线性回归线。通过使用该方法，在大鼠胸腔细胞与PMA的孵育混合物中检测到了PGD2、6-酮-PGF1α和TXB2。该结果阐明了在PMA诱导的大鼠胸膜炎渗出物中发现的这些PG和TX的来源。借助Sep-pak进行净化的ADAM-HPLC方法可能是检测生物材料中一系列花生四烯酸代谢物的有用工具。
