Pranav Patel

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F2-iPs, PGF2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F3-iPs, 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F3-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R2 median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F2- and F3-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs.

Structural Requirements for Activation of the 5-Oxo-6E,8Z, 11Z,14Z-eicosatetraenoic Acid (5-Oxo-ETE) Receptor : Identification of a Mead Acid Metabolite with Potent Agonist Activity

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 protein-coupled 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 C12 and C14 analogs were without appreciable activity, whereas the C16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C20 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 B3. 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.

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells.

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D3. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP+ over NAD+ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE-->5-HETE) is NADPH-dependent and is maximal at pH 6. Although the K(m) for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the V(max) is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP(+) (K(m) 139 nM), inhibited by NADPH (K(i) 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP+ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP+ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP+ to NADPH.

Human Neutrophils Convert the Sebum-derived Polyunsaturated Fatty Acid Sebaleic Acid to a Potent Granulocyte Chemoattractant

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 B4, 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.

5-Oxo-ETE Receptor Antagonists

5-Oxo-ETE is the most powerful eosinophil chemoattractant among lipid mediators. Eosinophil infiltration into the lungs of asthmatics may be responsible for the late phase of inflammatory asthma. We have designed and synthesized a 5-oxo-ETE receptor antagonist, the purpose of which is to prevent eosinophil migration to the lung during an asthma attack and thereby reduce asthma symptoms.

Inhibition of 5-oxo-6,8,11,14-eicosatetraenoic acid-induced activation of neutrophils and eosinophils by novel indole OXE receptor antagonists.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that is a potent granulocyte chemoattractant, which induces the infiltration of eosinophils into human skin when injected intradermally. It could therefore be an important proinflammatory mediator in eosinophilic diseases such as asthma and allergic rhinitis, and the OXE receptor, which mediates its actions, is therefore an attractive drug target. Using a structure-based approach in which substituents mimicking the essential polar (C1-C5) and hydrophobic (C15-C20) regions of 5-oxo-ETE were incorporated on an indole scaffold, we identified two potent selective OXE antagonists with IC50 values of about 30 nM. Neither compound displayed agonist activity and both inhibited 5-oxo-ETE-induced chemotaxis and actin polymerization and were relatively resistant to metabolism by rat liver homogenates. The active enantiomers of these racemic antagonists were even more potent, with IC50 values of <10 nM. These selective OXE antagonists could potentially be useful therapeutic agents in allergic diseases such as asthma.

Enhanced formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by cancer cells in response to oxidative stress, docosahexaenoic acid and neutrophil-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid

The 5-lipoxygenase (5-LO) product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent chemoattractant for myeloid cells, is known to promote the survival of prostate cancer cells. In the present study, we found that PC3 prostate cancer cells and cell lines derived from breast (MCF7) and lung (A-427) cancers contain 5-hydroxyeicosanoid dehydrogenase (5-HEDH) activity and have the ability to synthesize 5-oxo-ETE from its precursor 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when added as an exogenous substrate. H(2)O(2) strongly stimulated the synthesis of 5-oxo-ETE and induced dramatic increases in the levels of both glutathione disulfide and NADP(+). The effects of H(2)O(2) on 5-oxo-ETE and NADP(+) were blocked by N-ethylmaleimide (NEM), indicating that this effect was mediated by the glutathione reductase-dependent generation of NADP(+), the cofactor required by 5-HEDH. 5-Oxo-ETE synthesis was also stimulated by agents that have cytotoxic effects on tumor cells, including 4,7,10,13,16,19-docosahexaenoic acid, tamoxifen and MK-886. Because PC3 cells have only modest 5-LO activity compared with inflammatory cells, we investigated their ability to contribute to the transcellular biosynthesis of 5-oxo-ETE from neutrophil-derived 5-HETE. Stimulation of neutrophils with arachidonic acid and calcium ionophore in the presence of PC3 cells led to a large and selective increase in 5-oxo-ETE synthesis compared with controls in which PC3 cell 5-oxo-ETE synthesis was selectively blocked by pretreatment with NEM. The ability of prostate tumor cells to synthesize 5-oxo-ETE may contribute to tumor cell proliferation as well as the influx of inflammatory cells, which may further induce cell proliferation through the release of cytokines. 5-Oxo-ETE may be an attractive target in cancer therapy.

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes.

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.

Eicosapentaenoic-acid-derived isoprostanes: synthesis and discovery of two major isoprostanes.

The stereospecific synthesis of two all-syn-EPA-derived isoprostanes (iPs), 5-epi-8,12-iso-iPF(3alpha)-VI 17 and 8,12-iso-iPF(3alpha)-VI 18, has been accomplished. These two synthetic probes have been used to discover and identify their presence in human urine. The eventual quantitative measurement of these two iPs may be a valuable index of oxidative stress in people with eicosapentaenoic acid- (EPA) and docosahexaenoic acid- (DHA) enriched phospholipids.

Enantio- and stereospecific syntheses of 15(R)-Me-PGD2, a potent and selective DP2-receptor agonist.

The first total synthesis of 15(R)-Me-PGD2 3 is reported. The synthesis is based on the enantioselective and stereospecific syntheses of synthon 17 and its attachment to the five-membered ring by a olefin cross metathesis reaction. This approach permits the introduction of a side chain with a predetermined stereogenic center into the prostanoid ring, resulting in the synthesis of 15R-methyl prostaglandin D2 and allows rapid access to other prostanoids.