Standarization of assay methods of endogenous prostaglandins and leukotrienes

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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.