Leukotriene B$ sb4$ (LTB$ sb4)$ has previously been reported to be metabolized by porcine polymorphonuclear leukocytes (PMNL) to 10,11-dihydro-LTB$ sb4$ and 10,11-dihydro-12-oxo-LTB$ sb4$ (Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369). The 10,11-dihydro-LTB$ sb4$ fraction, prepared as described above, was resolved into two components by normal-phase (NP) high pressure liquid chromatography (HPLC). These products were identified as 10,11-dihydro-LTB$ sb4$ and 10,11-dihydro-12-epi-LTB$ sb4$ by gas-chromatography mass-spectrometry (GC-MS) and by co-chromatography with authentic chemically synthesized compounds. In the presence of NAD$ sp+,$ a microsomal 12-hydroxyeicosanoid dehydrogenase converted LTB$ sb4$ to 12-oxo-LTB$ sb4,$ which was identified by GC-MS. This enzyme catalyzes the initial rate-limiting step in the formation of dihydro metabolites of LTB$ sb4.$ 12-Oxo-LTB$ sb4$ was rapidly metabolized to 10,11-dihydro-12-oxo-LTB$ sb4$ by a cytosolic 10,11-reductase in the presence of NADH or NADPH. LTB$ sb4$ was not converted to any products by this fraction. / 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) was metabolized by intact porcine PMNL to 12-oxo-5,8,14-eicosatrienoic acid, 12(R)-hydroxy-5,8,14-eicosatrienoic acid and 12(S)-hydroxy-5,8,14-eicosatrienoic acid, which were identified by GC-MS and nuclear magnetic resonance spectroscopy. The latter two compounds were separated by NP-HPLC following derivatization with methoxy-(trifluoromethyl)phenylacetic acid and were identified by co-chromatography with authentic compounds. 12(S)-HETE was metabolized by 12-hydroxyeicosanoid dehydrogenase, in the presence of NAD$ sp+,$ to 12-oxo-ETE, identified by its chromatographic and UV spectral properties. 13-Hydroxy-9,11-octadecadienoic acid (13-HODE) was metabolized by porcine PMNL to 13-hydroxy-9-octadecenoic acid and 13-oxo-9-octadecenoic acid which were identified by GC-MS. Specificity studies indicated that the porcine PMNL 12-hydroxyeicosanoid dehydrogenase preferentially oxidizes substrates with a 12-hydroxyl group preceded by at least two conjugated double bonds and followed by a 2-cis-octenyl group.
Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.39359 |
Date | January 1992 |
Creators | Wainwright, Sandra L. (Sandra Lee) |
Publisher | McGill University |
Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
Language | English |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Format | application/pdf |
Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
Relation | alephsysno: 001277315, proquestno: NN74936, Theses scanned by UMI/ProQuest. |
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