Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: Derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography |
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| Authors: | Ken-ichi Kiyomiya Kohji Yamaki Noriyuki Nimura Toshio Kinoshita Sachiko Oh-ishi |
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| Affiliation: | 1. Department of Urology, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey;2. Program of Medical Laboratory Techniques, Vocational School of Health Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey;3. Department of Nutrition and Dietetics, Faculty of Health Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey;4. Department of Histology and Embryology, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey;5. Department of Medical Biochemistry, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey;6. Department of Urology, Faculty of Medicine, Akdeniz University, 07100 Antalya, Turkey;7. Department of Emergency Medicine, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey;1. Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algeria;2. Laboratoire de Valorisation et de Conservation des Ressources biologiques (VALCOR), Département de Biologie, Faculté des Sciences, Université de Boumerdes, Boumerdes, Algeria;3. LBN, Université de Montpellier, Montpellier, France;4. IRCM, Inserm, Université de Montpellier, ICM, Montpellier, France;5. Laboratoire de Biochimie, Biophysique, Biomathématique et Scientométrie (L3BS), Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia, Algeria;6. Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre, Université de Bouira, Bouira, Algeria |
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| Abstract: | 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 (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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