Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: Derivatization of prostaglandins with 9-ANTHRYLDIAZOMETHANE (cas 10401-59-9) for fluorometric determination by high performance liquid chromatography

Add time:08/05/2019    Source:sciencedirect.com

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 μM 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 (cas 10401-59-9) (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, PGF2α, 6-keto-PGF1α, 6-keto-PGE1, TXB2, 15-keo-PGE2, 13, 14-dihydro-15-keto-PGF2α 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-PGF1α 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.

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