Abstract
Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to 10,11-dihydro and 10,11-dihydro-12-oxo products by porcine polymorphonuclear leukocytes (PMNL) (Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) Biochemistry 29, 10126-10135). These 10,11-dihydro metabolites could either have been formed by the direct reduction of the 10,11-double bond of the substrate, as previous evidence suggested, or via an initially formed 12-oxo intermediate. To gain some insight into the mechanism for the formation of dihydro products by this pathway, we investigated the metabolism of leukotriene B4 (LTB4), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(12(S)-HETE), and 12(R)-HETE by subcellular fractions from porcine PMNL. In the presence of NAD+ and a microsomal fraction from PMNL, each of the above 12-hydroxyeicosanoids was converted to a single product with a lambda max approximately 40 nm higher than that of the substrate, indicating that the conjugated diene or triene chromophore had been extended by one double bond, presumably by oxidation of the 12-hydroxyl group to an oxo group. In the case of LTB4, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB4. LTB4 was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 by the 1500 x g supernatant in the presence of NAD+. Negligible amounts of dihydro products were formed in the presence of NADH or NADPH, suggesting that initial oxidation of the 12-hydroxyl group is a requirement for reduction of the 10,11-double bond. Consistent with this hypothesis, 12-oxo-LTB4 was rapidly metabolized to 10,11-dihydro-12-oxo-LTB4 by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB4, were formed by the microsomal fraction. These results indicate that the initial step in the formation of 10,11-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic delta 10-reductase in the presence of NADH.
Highlights
From the Meakins-Christie Laboratories, Respiratory Health Network of Centers of Excellence, Dept. of Medicine, McGill University, Montreal, Quebec, Canada, H2X 2P2, and the Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada H3A l A 1
We have previously reported that the major pathway for the metabolism of LTB, [22] and 12(S)-HETE [20] by porcine PMNL is the formation of lO,ll-dihydro, 10,ll-dihydro12-epi,and 10,11-dihydro-12-oxoproducts
The present study has clearly shown that the initial step in the formation of dihydro products by this pathway is oxidation of the 12hydroxyl group of the substrate by a microsomal 12-hydroxyeicosanoiddehydrogenase to give a 12-oxointermediate (Fig. 8).We are unable to conclude from our results whether this enzyme is presentin the plasma membrane or the endoplasmic reticulum of PMNL, since experiments in which we measured the activities of enzymes which are markers for the endo
Summary
Unlabeled LTB, was prepared by incubation of arachidonic acid and A23187 (10 p ~ w)ith porcine PMNL which were preincubated with ETYA [32, 33]. Unlabeled 12(S)-HETE was prepared under identical conditions except that ETYA was not present. Unlabeled 12(R)-HETEwas prepared by incubation of arachidonic acid with a homogenate of sea urchin eggs (Strongylocentrotus purpurutus) as described by Brash et al [34]. [1-"C]LTB4 was prepared by incubation of [l-'4C]arachidonic acid with purified human PMNL and A23187 (10 pM) for 5 min at 37"C [35]. 12(S)-[1-'4C]HETEwas prepared by incubation of [l-'4C]arachidonic acid with human platelets [36]. [9@-3H]PGF2w,as prepared by reducing PGE, with tritiated NaBH4,followed bypurification by RP-HPLC on aSpherisorb ODS2 column (Jones Chromatography Ltd.) with a gradient between acetonitrile/water/acetic acid (30:700.02) and acetonitrile/water/ acetic acid (4060:0.02) over 30 min at a flow rate of 1.5 ml/min Unlabeled 12(R)-HETEwas prepared by incubation of arachidonic acid with a homogenate of sea urchin eggs (Strongylocentrotus purpurutus) as described by Brash et al [34]. [1-"C]LTB4 was prepared by incubation of [l-'4C]arachidonic acid with purified human PMNL and A23187 (10 pM) for 5 min at 37"C [35]. 12(S)-[1-'4C]HETEwas prepared by incubation of [l-'4C]arachidonic acid with human platelets [36]. [9@-3H]PGF2w,as prepared by reducing PGE, with tritiated NaBH4,followed bypurification by RP-HPLC on aSpherisorb ODS2 column (Jones Chromatography Ltd.) with a gradient between acetonitrile/water/acetic acid (30:700.02) and acetonitrile/water/ acetic acid (4060:0.02) over 30 min at a flow rate of 1.5 ml/min
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