Lipoxygenase Metabolites Research Articles

Eicosanoids and Docosanoids in Plasma and Aorta of Healthy and Atherosclerotic Rabbits

Eicosanoids and docosanoids have been shown to be involved in atherosclerosis. Polyunsaturated fatty acids (PUFAs) are important nutrients that are metabolized by lipoxygenases and cyclooxygenases to various mono-hydroxy metabolites which can be further metabolized by specific enzymes to more complex eicosanoids and docosanoids. In this study a high-performance liquid chromatography methodology was established and rabbits were fed with a control or a high-cholesterol diet to induce atherosclerotic lesions to determine pro- or anti-inflammatory lipid mediators in atherosclerotic vessels. In aortic samples from atherosclerotic rabbits we determined for the first time various eicosanoids/docosanoids and observed an increased concentration of 12-lipoxygenase metabolites. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) in high-cholesterol versus control animals as well as increased ratios of 12-HETE/arachidonic acid ratios indicate that 12-lipoxygenase metabolites may have importance in atherosclerosis. In addition, decreased concentrations of the 5-lipoxygenase metabolite leukotriene B4 levels were detected in high-cholesterol animals. A positive correlation of total plaque area with plasma levels of 12-HETE and a negative correlation with aortic levels of endogenous PPARγ-ligand 13-oxo-octadecadienoic acid were found. This study let us conclude that the cholesterol content in the diet might influence atherosclerosis via increased 12-lipoxygenase- and cyclooxygenase-mediated pathways and reduced 5-lipoxygenase pathways.

Separation of 5-Lipoxygenase Metabolites Using Cyclodextrin-Modified Microemulsion Electrokinetic Chromatography and Head Column Field-Amplified Sample Stacking

A cyclodextrin-modified microemulsion electrokinetic chromatography method employing head column field-amplified sample stacking was developed for the analysis of arachidonic acid metabolites of the lipoxygenase pathways. The influence of the concentration of boric acid, the surfactant sodium dodecyl sulfate, the co-surfactant 1-butanol and the oil phase octane as well as the pH of the background electrolyte, the separation voltage and the separation temperature was studied. The optimized microemulsion consisting of 20 mM boric acid buffer, pH 9.0, 3.0 % (m/v) sodium dodecyl sulfate, 0.5 % (v/v) octane, 5.0 % (v/v) 1-butanol and 15 mM α-cyclodextrin enabled the separation of 20-hydroxy-leukotriene B4, leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid, 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid as well as the internal standard prostaglandin B1 in <10 min employing a separation voltage of 17.5 kV at a temperature of 23 °C. A matrix peak from solid-phase extraction sample workup co-migrated with 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid affecting peak integration. The addition of 5 % (v/v) 2-propanol to the microemulsion resulted in the separation of this eicosatetraenoic acid and the matrix components at the expense of analysis time and peak resolution between the diastereomers 6-trans-leukotriene B4 and 6-trans-12-epi-leukotriene B4. In summary, the MEEKC method appeared to be especially suitable for the more polar arachidonic acid metabolites.

Cell Survival Signalling through PPARδ and Arachidonic Acid Metabolites in Neuroblastoma

Retinoic acid (RA) has paradoxical effects on cancer cells: promoting cell death, differentiation and cell cycle arrest, or cell survival and proliferation. Arachidonic acid (AA) release occurs in response to RA treatment and, therefore, AA and its downstream metabolites may be involved in cell survival signalling. To test this, we inhibited phospholipase A2-mediated AA release, cyclooxygenases and lipoxygenases with small-molecule inhibitors to determine if this would sensitise cells to cell death after RA treatment. The data suggest that, in response to RA, phospholipase A2-mediated release of AA and subsequent metabolism by lipoxygenases is important for cell survival. Evidence from gene expression reporter assays and PPARδ knockdown suggests that lipoxygenase metabolites activate PPARδ. The involvement of PPARδ in cell survival is supported by results of experiments with the PPARδ inhibitor GSK0660 and siRNA-mediated knockdown. Quantitative reverse transcriptase PCR studies demonstrated that inhibition of 5-lipoxygenase after RA treatment resulted in a strong up-regulation of mRNA for PPARδ2, a putative inhibitory PPARδ isoform. Over-expression of PPARδ2 using a tetracycline-inducible system in neuroblastoma cells reduced proliferation and induced cell death. These data provide evidence linking lipoxygenases and PPARδ in a cell survival-signalling mechanism and suggest new drug-development targets for malignant and hyper-proliferative diseases.

Lipidomic Profiling of Influenza Infection Identifies Mediators that Induce and Resolve Inflammation

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

5-Hydroxyeicosatetraenoic acid and leukotriene D4 increase intestinal epithelial paracellular permeability

The loss of epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease, with levels of 5-lipoxygenase metabolites being increased in the mucosa of these patients. The objective of this study was to determine the effect of the eicosanoids produced by the 5-lipoxygenase pathway, leukotriene B4 and D4, and 5-hydroxyeicosatetraenoic acid on epithelial barrier function. Paracellular permeability was estimated from fluorescein isothiocyanate–dextran fluxes and transepithelial electrical resistance in differentiated Caco-2 cells. Our results suggest that leukotriene D4 and 5-hydroxyeicosatetraenoic acid altered both parameters. Identification of the receptors involved in these changes indicated that cysteinyl-leukotriene receptor 1 participates in the effects of leukotriene D4. For both eicosanoids, these effects were mediated by activation of the phospholipase C/Ca2+/protein kinase C pathway, in addition to cAMP-independent protein kinase A activation. Furthermore, we observed a correlation between increased paracellular permeability and the redistribution of occludin, and for leukotriene D4, the disorganization of the subapical actin ring and myosin light chain kinase activation. In conclusion, on the basis of our results, we propose that 5-lipoxygenase pathway metabolites participate in the disruption of epithelial barrier function that is characteristic of inflammatory bowel disease.

Cancer-Produced Metabolites of 5-Lipoxygenase Induce Tumor-Evoked Regulatory B Cells via Peroxisome Proliferator–Activated Receptor α

Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report in this study that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase pathway such as leukotriene B4 to activate the peroxisome proliferator-activated receptor α (PPARα) in B cells. Inactivation of leukotriene B4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells and/or tumor 5-lipoxygenase pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors

Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

Endogenous Modulators of TRP Channels

The transient receptor potential (TRP) superfamily consists of a large number of cation channels permeable to both monovalent and divalent cations. The 28 mammalian TRP channels can be divided into seven subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPN (no mechanopotential, NOMP) and the TRPA (ankyrin) groups. TRP channels are widely expressed in several cell types in every tissue and play a critical role in the regulation of various cell functions. Altogether these channels function as sensory transducers and detect chemical, thermal and mechanical stimuli. Endogenous substances acting on TRP channels can be released during the early stage of some pathological conditions. These substances can affect TRP channel functions and lead to the progression of diseases such as inflammation and chronic pain. For example, endogenous lipids, such as unsaturated fatty acids and their cyclooxygenase, lipoxygenase or epoxygenase related metabolites, were shown to modulate TRP channel activity by direct binding. Other lipidergic ligands include isoprene derivatives (e.g. diacylglycerol, lysophospholipids and resolvine) which play diverse activity on different TRP channels. This review focuses on lipidergic mediators which affect TRP channel activity. Opportunities to exploit TRP channels for novel therapeutic strategies will be discussed.

Involvement of PLA2, COX and LOX inRhinella arenarumoocyte maturation

In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.

Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

Leukocytes regulate retinal capillary degeneration in the diabetic mouse via generation of leukotrienes

Understanding the early pathogenesis of DR may uncover new therapeutic targets to prevent or slow the progression of this sight-threatening disorder. We investigated the role of leukocyte-mediated generation of LTs in regulation of retinal capillary degeneration and inflammation in the diabetic mouse. We generated (1) chimeric mice that lacked the ability to generate LTs by transplanting 5LO-/- bone marrow cells into ND.WT mice and into SD.WT mice and (2) "control" chimeric mice by transplanting WT bone marrow cells into 5LO-/- mice or into WT mice. Retinas from diabetic chimeric mice with WT marrow demonstrated capillary degeneration to the same extent as retinas from diabetic, nonchimeric WT mice. In contrast, retinas from diabetic chimeric mice with 5LO-/- marrow developed significantly less capillary degeneration and pericyte loss (P<0.05). In the retinas from chimeric mice with WT marrow, diabetes induced a rise in leukocyte adherence to the microvasculature, expression of the NF-κB p65 subunit, and ICAM1, superoxide generation, and retinal microvascular permeability, yet these characteristic responses were blunted by >50% in diabetic chimeras containing 5LO-/- leukocytes (P<0.05). Our data suggest the critical involvement of leukocytes and LTs in the regulation of inflammation and capillary degeneration in DR.

New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity

BackgroundThromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors.Results and discussionWe synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1.ConclusionsIn conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1.

Hepoxilin A3 Facilitates Neutrophilic Breach of Lipoxygenase-Expressing Airway Epithelial Barriers

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.

Arachidonic acid metabolism in human prostate cancer

The arachidonic acid pathway is important in the development and progression of numerous malignant diseases, including prostate cancer. To more fully evaluate the role of individual cyclooxygenases (COXs), lipoxygenases (LOXs) and their metabolites in prostate cancer, we measured mRNA and protein levels of COXs and LOXs and their arachidonate metabolites in androgen-dependent (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cell lines, bone metastasis-derived MDA PCa 2a and MDA PCa 2b cell lines and their corresponding xenograft models, as well as core biopsy specimens of primary prostate cancer and nonneoplastic prostate tissue taken ex vivo after prostatectomy. Relatively high levels of COX-2 mRNA and its product PGE2 were observed only in PC-3 cells and their xenografts. By contrast, levels of the exogenous 12-LOX product 12-HETE were consistently higher in MDA PCa 2b and PC-3 cells and their corresponding xenograft tissues than were those in LNCaP cells. More strikingly, the mean endogenous level of 12-HETE was significantly higher in the primary prostate cancers than in the nonneoplastic prostate tissue (0.094 vs. 0.010 ng/mg protein, respectively; p=0.019). Our results suggest that LOX metabolites such as 12-HETE are critical in prostate cancer progression and that the LOX pathway may be a target for treating and preventing prostate cancer.

Chemometrics-guided development of a cyclodextrin-modified micellar electrokinetic chromatography method with head-column field amplified sample stacking for the analysis of 5-lipoxygenase metabolites

A new sensitive method using α-cyclodextrin-modified micellar electrokinetic chromatography has been developed to separate and quantify arachidonic acid metabolites of the lipoxygenase pathways in human polymorphonuclear leukocytes, i.e. leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, and 15(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid. The electrophoresis system was optimized with regard to the pH, boric acid, SDS and α-cyclodextrin concentration as well as separation voltage and temperature using a three level resolution IV fractional factorial design and a five level circumscribed central composite design. The resulting optimized conditions included 80mM sodium borate buffer, pH 10.07, containing 16.6mM sodium dodecyl sulfate, and 15mM α-cyclodextrin, using a separation voltage of 12.5kV at 23°C. Sensitivity was enhanced employing head-column field amplified sample stacking which resulted in limits of quantification between 30 and 50ng/mL and limits of detection between 10 and 17ng/mL after solid phase extraction of the lipoxygenase products. The method was validated according to the recommendations of the International Conference on Harmonization and applied to the determination of the lipoxygenase metabolites in polymorphonuclear leukocytes upon stimulation with Ca2+-ionophore A23187 and arachidonic acid. Robustness was confirmed using a three level resolution IV fractional factorial design. The novel method is suitable for the analysis of various arachidonic acid metabolites produced by cells and may be used for evaluation of lipoxygenase inhibitors.

Influence of cigarette smoking on synthesis of eicosanoids, isoprostanes and lipoxygenase metabolites in apical periodontitis

Arachidonic acid (AA) is metabolized to eicosanoids and isoprostanes (IPs) via different pathways. The presence of granuloma in apical periodontitis (AP) is linked with inflammation and the synthesis of metabolites of AA. ObjectiveWe investigated the conversion rate of 14C labelled arachidonic acid (14C-AA), the lipoxygenases (LOX) products and the endogenous synthesis of eicosanoids and IPs in extracted granuloma. Furthermore, we assessed if there are markers for bone destruction and the influence of cigarette smoking. Patients and methodsIn 46 patients with symptoms and corresponding radiological signs of AP, teeth were extracted including the periapical granuloma. The endogenous synthesis of eicosanoids and IPs, the conversion rate of 14C-AA and LOX products in extracted granuloma were analyzed. ResultsWe found that smoking increases significantly the synthesis of IPs and LOX-metabolites in granuloma. Furthermore, smoking may have contributed to significant differences in qualitative and quantitative profile of eicosanoids, IPs and the conversion rate of 14C-AA independent of the size of the granuloma. ConclusionsOur data demonstrate that in smokers with granuloma due to AP products of lipid peroxidation as 8-iso-PGF2α and products of the LOX-pathway are increased at the expense of cyclooxygenase products. The size of granuloma did not influence the amount of synthesized eicosanoids, IPs or LOX-metabolites out of 14C-AA whereas cigarette smoking was a significantly influencing and modifiable risk factor.

Serum oxylipin profiles in IgA nephropathy patients reflect kidney functional alterations

Immunoglobulin A nephropathy (IgAN) is a leading cause of chronic kidney disease, frequently associated with hypertension and renal inflammation. ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil (FO) improve kidney function in animal models, but have inconsistent metabolic effects in humans. Oxylipin profiles in serum from IgAN patients supplemented with either FO or corn oil (CO) placebo were analyzed by liquid chromatography coupled to tandem mass spectrometry. EPA cyclooxygenase and lipoxygenase metabolites, and EPA and DHA epoxides and diols were increased in response to FO supplementation, as were total epoxides and epoxide/diol ratios. Several of these metabolites were drivers of separation as assessed by multivariate analysis of FO patients pre- vs. post-supplementation, including 17,18-dihydroxyeicosatrienoic acid, prostaglandin D3, prostagalandin E3, Resolvin E1, 12-hydroxyeicosapentaenoic acid, and 10(11)-epoxydocosapentaenoic acid. In patients whose proteinuria improved, plasma total oxylipins as well as several hydroxyoctadecadienoic acids, hydroxyeicosatetraenoic acids, and leukotriene B4 metabolites were among the metabolites that were significantly lower than in patients whose proteinuria either did not improve or worsened. These data support the involvement of oxylipins in the inflammatory component of IgAN as well as the potential use of oxylipin profiles as biomarkers and for assessing responsiveness to ω-3 fatty acid supplementation in IgAN patients.

Effect of 15-lipoxygenase metabolites on angiogenesis: 15(S)-HPETE is angiostatic and 15(S)-HETE is angiogenic

15(S)-Hydroxyeicosatetraenoic acid [15(S)-HETE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] are the products of arachidonic acid formed in the 15-lipoxygenase pathway. They have opposing effects on the inflammatory process. The present study was designed to examine the role of these metabolites on angiogenesis, which is critically associated with inflammatory conditions. Chick chorio-allantoic membrane (CAM), rat aortic rings and human umbilical vein endothelial cells (HUVECs) in culture were used to study the effect of 15(S)-HETE and 15(S)-HPETE on angiogenesis. Biochemical markers of angiogenesis were analysed by ELISA. 15(S)-HETE increased vessel density in chick CAM, induced sprouting in rat aortic rings and increased endothelial cell-cell contact and formation of tubular network-like structures in HUVECs. Furthermore, it up-regulated the expression of CD31, E-selectin and vascular endothelial growth factor (VEGF) in HUVECs, indicating its pro-angiogenic effect. 15(S)-HPETE, on the other hand, decreased vessel density in chick CAM, down-regulated the expression of E-selectin (<35%), VEGF (<90%) and CD31 (<50%) and did not produce sprouting in aortic rings, suggesting an anti-angiogenic property. 15(S)-HETE-mediated up-regulation of CD 31 and VEGF was reversed by treatment with 15(S)-HPETE. These results indicate the divergent effects of hydroxy and hydroperoxy products of 15-LOX on angiogenesis, highlighting the role of these products in the co-dependence of inflammation and angiogenesis.

Cysteinyl leukotrienes mediate the response of submucosal ganglia from rat colon to bradykinin

The aim of the present study was to find out the mechanism by which the inflammatory mediator, bradykinin, induces an increase of the cytosolic Ca2+ concentration ([Ca2+]i) in enteric neurons. For this purpose, ganglia in the isolated submucosa from rat colon were loaded with the Ca2+-sensitive dye, fura-2, and were exposed to bradykinin (2·10−8mol/l). Under control conditions, the kinin evoked a transient increase in [Ca2+]i. Preincubation with quinacrine or arachidonyltrifluoromethylketone (AACOCF3), i.e. blockers of cytosolic phospholipase A2, prevented the raise of [Ca2+]i. This inhibition was mimicked by 5,8,11,14-eicosatetrayonic acid (ETYA), an inhibitor of cyclooxygenases as well as lipoxygenases, and by BWA4C, a selective inhibitor of lipoxygenases, whereas indomethacin was ineffective, suggesting the mediation of the kinin response by a lipoxygenase metabolite. Indeed, a leukotriene, leukotriene D4 (LTD4), mimicked the effect of bradykinin. The LTD4 receptor blocker, MK-571, inhibited the increase in [Ca2+]i evoked by LTD4 and by bradykinin. Consequently, bradykinin receptors in submucosal ganglia from rat colon are coupled to a stimulation of phospholipase A2, the release of arachidonic acid and the production of LTD4, which seems to be finally responsible for the change in the cytosolic Ca2+ concentration.