Standarization of assay methods of endogenous prostaglandins and leukotrienes

本项目组由1984年京都前列腺（PG）会议测定研讨会的发言人组成，根据研讨会的结论，重点研究了以下项目。清除方法的建立：用本课题组成员自己的方法（RIA、EIA、GC-MS，有或没有清除）测定已知量（1 ng）的人血浆供试品中的6-Keto- <PGF_（1a）>，作为盲样，以比较清除方法。结果表明：（1）大多数成员使用的分离PGs的Sep-Pak C18被有机溶剂释放，干扰了RIA和EIA。(2)因此，Sep-Pak C18应与HPLC或TLC结合使用，以消除不纯物质。(3)回收率测定用放射性标记化合物的纯度以及标准品校准曲线之间的差异也会引起值的变化. 11-乙酰-（DH-）酶免疫分析法的建立：建立了人工合成的11-乙酰<TXB_2>-（<TXB_2>DH-）代谢产物DH-的酶免疫分析法<TXB_2><TXB_2><TXB_2>。用本试剂盒测定，<TXB_2>未观察到采血过程中DH-值升高，DH-血药浓度持续时间<TXB_2>长于<TXB_2>。原发<TXB_2>性高血压患者尿DH-值与大鼠尿DH-值呈良好的相关性<TXB_2>，而TXA合成酶抑制剂<TXB_2>仅轻微降低大鼠尿DH-值.白三烯（LT）测定方法：在使用商业RIA试剂盒之前必须进行清洁;在pH 3.5下酸化样品，然后通过Sep-Pak C18分离LT，使我们能够<LTB_4>毫无困难地测定免疫反应性LT。值得注意的是，HPLC上的12-HETE与来自乙醇的不纯物质共迁移，表明检测12-HETE时应谨慎。

This project team, composed of speakers of assay workshop in Kyoto Prostaglandin (PG) Conference, 1984, has focused the research on the following items, according to the conclusion of the workshop.1. Establishment of clean-up method: 6-Keto- <PGF_(la)> in a test sample of human plasma containing a known amount (1 ng) was measured as a blind sample by own method of the team members (RIA, EIA, GC-MS with or without cleaning-up) to compare cleaning method. Variation of the values was rather small, but the following criticisms were made: (1) Sep-Pak C18, which was used by most members for separation of PGs, releases by organic solvent materials, which disturb RIA and EIA. (2) Thus, Sep-Pak C18 should be used in combination with HPLC or TLC for elimination of impure substances. (3) Purity of radio-labelled compounds for testing recovery as well as a difference between calibration curves of the standard also caused variation of the values.2. Development of enzyme-immunoassay of 11-dehydro- <TXB_2> (DH- <TXB_2> ): Enzyme-immuno-assay of DH- <TXB_2> , a metabolite of <TXB_2> , has been developed, as <TXB_2> is generated artificially. Determination with the present kit revealed that an increase in DH- <TXB_2> values during blood collection was not observed and duration of blood concentration of DH- <TXB_2> was longer than that of <TXB_2> . DH- <TXB_2> value in urine from patients of essential hypertension was well correlated to that of <TXB_2> , but TXA synthetase inhibitor attenuated DH- <TXB_2> value in rat urine only slightly.3. Leukotriene (LT) assay method: Cleaning-up must be made before using commercial RIA kits; acidification of samples at pH 3.5, followed by separation of LTs by Sep-Pak C18, enabled us to determine <LTB_4> and immunoreactive LTs without difficulty. It should be noticeable that 12-HETE on HPLC co-migrates with that of impure materials from ethanol, indicating that caution should be taken when 12-HETE is detected.