The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a potent chemoattractant for neutrophils and eosinophils, and its actions are mediated by the oxoeicosanoid (OXE) receptor, a member of the G proteincoupled receptor family. To define the requirements for activation of the OXE receptor, we have synthesized a series of 5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and 20 carbons, as well as a series of 20-carbon 5-oxo fatty acids, either fully saturated or containing between one and five double bonds. The effects of these compounds on neutrophils (calcium mobilization, CD11b expression, and cell migration) and eosinophils (actin polymerization) were compared with those of 5-oxo-ETE. The C 12 and C 14 analogs were without appreciable activity, whereas the C 16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C 18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C 20 analogs, the fully saturated compound had virtually no activity, whereas 5-oxo-6E-eicosenoic acid had only weak agonist activity. In contrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) and its 8-trans isomer were approximately equipotent with 5-oxo-ETE in activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fatty acid deficiency, by neutrophils. The main Mead acid metabolite identified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by 5-oxo-20:3 and two 6-trans isomers of leukotriene B 3 . We conclude that optimal activation of the OXE receptor is achieved with 5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter compound could potentially be formed under conditions of essential fatty acid deficiency.Metabolism of arachidonic acid by the 5-lipoxygenase (5-LO) pathway leads to the formation of leukotriene (LT) B 4 , LTC 4 , LTD 4 , and 5-HETE (Funk, 2001). LTB 4 , acting through the BLT 1 receptor, is a potent activator of neutrophils and lymphocytes. LTD 4 interacts with the cysteinyl-LT 1 and cysteinyl-LT 2 receptors to stimulate smooth muscle contraction, cytokine release from leukocytes, and various other responses. Although
Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP ؉ , the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B 4 , indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.Human sebaceous glands and the sebum they produce have a unique fatty acid profile in that they contain high levels of the ⌬ 6 C 16 fatty acid sapienic acid along with its ⌬ 5,8 elongation product sebaleic acid (1, 2). Sapienic acid is formed by the action of fatty acid desaturase-2 on palmitic acid (3). Although this enzyme is widely distributed, it does not normally act on palmitic acid but rather on linoleic acid or ␣-linolenic acid, which are much better substrates. However, only very small amounts of these polyunsaturated fatty acids (PUFA) 3 are found in sebaceous glands, which instead accumulate high levels of palmitic acid. This is possibly due to the preferential removal of linoleic acid by -oxidation in this tissue (4), leaving palmitic acid as the only substrate for fatty acid desaturase-2. Elongation of sapienic acid and insertion of a 5,6-double bond by ⌬ 5 -desaturase (fatty acid desaturase-1) results in...
5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of the 5-lipoxygenase (5-LO) product 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), formed by the microsomal enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH). 5-oxo-ETE is a chemoattractant for neutrophils and eosinophils, both in vitro and in vivo. To examine the substrate selectivity of 5-HEDH and to search for potential inhibitors, we prepared a series of 5S-hydroxy fatty acids (C 12 to C 20 containing zero to four double bonds) by total chemical synthesis and examined their metabolism by microsomes from monocytic U937 cells. Although most of these fatty acids were oxidized to their 5-oxo metabolites by 5-HEDH, 5-HETE seemed to be the best substrate. However, substrates containing less than 16 carbons, a methylated ␣-carboxyl group, or a hydroxyl group at the -end of the molecule were not substantially metabolized. Some of the fatty acids tested were fairly potent inhibitors of the formation of 5-oxo-ETE by 5-HEDH, in particular 5-hydroxy-6-octadecenoic acid and 5-hydroxy-6-eicosenoic acid. Both substances selectively inhibited 5-oxo-ETE formation by human peripheral blood mononuclear cells incubated with arachidonic acid and calcium ionophore without affecting the formation of leukotriene B 4 , 12-HETE, or 12-hydroxy-5,8,10-heptadecatrienoic acid. We conclude that the requirements for appreciable metabolism by 5-HEDH include a chain length of at least 16 carbons, a free ␣-carboxyl group, and a hydrophobic group at the -end of the molecule. 5-Hydroxy-⌬ 6 C 18 and C 20 fatty acids selectively inhibit 5-HEDH without inhibiting 5-LO, leukotriene A 4 hydrolase, 12-lipoxygenase, or cyclooxygenase. Such compounds may be useful in defining the role of 5-oxo-ETE and its mechanism of synthesis.The 5-lipoxygenase (5-LO) pathway results in the formation of a series of products with potent proinflammatory effects, including leukotrienes (LTs) B 4 , C 4 , and D 4 , and 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE). LTB 4 acts principally via the BLT 1 receptor and has potent stimulatory effects on neutrophils, monocytes, and lymphocytes, whereas LTD 4 acts through the cysLT 1 and cysLT 2 receptors and is an important mediator in asthma (Funk, 2001). 5-oxo-ETE is a potent activator of eosinophils, neutrophils, and monocytes, inducing cell migration, calcium mobilization, and actin polymerization (Norgauer et al., 1996;Sozzani et al., 1996;Powell and Rokach, 2005). It also stimulates degranulation and activates the respiratory burst, especially after priming with cytokines such as granulocyte macrophage-colony-stimulating factor (O'Flaherty et al., 1996a,b). It induces transendothelial migration of eosino-
The first total synthesis of 6(E),8(Z),11(Z),13(E) 5-oxo-15-HETE 4 was accomplished. The synthetic material was evaluated with calcium mobilization assay and compared with 5-oxo-ETE the natural ligand for the OXE receptor.
Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5oxo-ETE.
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