13-hydroxy-9-11-octadecadienoic-acid and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects.. In a randomized, crossover clinical trial, 14 healthy young volunteers followed a 14-day low-cholesterol/low-fat diet (LChF) and a 14-day isocaloric high-cholesterol/high-fat diet (HChF) in a random order. After each diet, we measured HDL concentrations of hydroxyeicosatetraenoic acids (HETE), hydroxyoctadecadienoic acids (HODE), and haptoglobin, as well as serum amyloid A (SAA) and paroxonase-1 activity (PON-1). HDL concentrations of 15-HETE (+254%, p = 0.002), 5-HETE (+116%, p = 0.004), 13-HODE (+102%, p = 0.049), and SAA levels (+75%, p = 0.007) were significantly higher after the HChF than after the LChF. Furthermore, haptoglobin was marginally increased (+32%, p = 0.091) while PON-1 activity was unaffected (-16%, p = 0.366) by the HChF.. In healthy subjects, a short-term elevation in dietary cholesterol and fat intake increases HDL lipid hydroperoxide content (15-HETE, 5-HETE, 13-HODE) and SAA levels, which are key features of dysfunctional HDL. This is the first study showing that a physiologic manipulation of dietary cholesterol and fat intake affects HDL lipidome and proteome in healthy subjects independently of weight changes.. NCT02549144.

Topics: Adult; Biomarkers; Cholesterol, Dietary; Cross-Over Studies; Diet, High-Fat; Female; Healthy Volunteers; Humans; Hydroxyeicosatetraenoic Acids; Italy; Linoleic Acids; Lipid Peroxides; Lipoproteins, HDL; Male; Postprandial Period; Prospective Studies; Serum Amyloid A Protein; Time Factors; Young Adult

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Eicosanoids; Ganglia, Spinal; Hydroxyeicosatetraenoic Acids; Hyperalgesia; Linoleic Acid; Linoleic Acids; Lysophospholipids; Mice; Rats; TRPV Cation Channels; Ultraviolet Rays

The 5-lipoxygenase (5-LO) enzymatic pathway is widely distributed within the central nervous system, and is upregulated in Alzheimer's disease. However, the mechanism whereby it may influence the disease pathogenesis remains elusive.. We evaluated the molecular mechanism by which 5-LO regulates amyloid β (Aβ) formation in vitro and in vivo by pharmacological and genetic approaches.. Here we show that 5-LO regulates the formation of Aβ by activating the cAMP-response element binding protein (CREB), which in turn increases transcription of the γ-secretase complex. Preventing CREB activation by pharmacologic inhibition or dominant negative mutants blocks the 5-LO-dependent elevation of Aβ formation and the increase of γ-secretase mRNA and protein levels. Moreover, 5-LO targeted gene disruption or its in vivo selective pharmacological inhibition results in a significant reduction of Aβ, CREB and γ-secretase levels.. These data establish a novel functional role for 5-LO in regulating endogenous formation of Aβ levels in the central nervous system. Thus, 5-LO pharmacological inhibition may be beneficial in the treatment and prevention of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Arachidonate 5-Lipoxygenase; Central Nervous System; Chemotactic Factors; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Female; Humans; Hydroxyeicosatetraenoic Acids; Immunoblotting; Leukotrienes; Linoleic Acids; Lipoxygenase Inhibitors; Mice; Mutation; Neuroblastoma; Transcription Factors; Transfection; Tumor Cells, Cultured; Up-Regulation

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Anti-Inflammatory Agents; Apolipoproteins E; Atherosclerosis; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Fatty Acids; Female; Hydroxyeicosatetraenoic Acids; Injections, Subcutaneous; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxides; Lipoproteins, HDL; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Oxidation-Reduction; Peptides; Species Specificity; Tandem Mass Spectrometry; Time Factors; Up-Regulation

The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) induces transcriptional repression of pro-inflammatory factors. Activation of PPARgamma is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARgamma was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARgamma. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARgamma in CECs.. LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of (14)C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARgamma.. Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85 ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARgamma agonist troglitazone and higher as with the known PPARgamma ligands 13-HODE and 15-HETE. Binding assays with (14)C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction.. High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARgamma and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Topics: Animals; Arachidonic Acid; Blotting, Western; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Colon; Epithelial Cells; HT29 Cells; Humans; Hydroxyeicosatetraenoic Acids; Immunoprecipitation; Interleukin-8; Ligands; Linoleic Acid; Linoleic Acids; Linolenic Acids; Molecular Structure; PPAR gamma; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Electrospray Ionization; Transfection

Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1alpha, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5alpha-androstane-3alpha,17beta-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1alpha and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1alpha induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1alpha, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Androstane-3,17-diol; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glucuronides; Glucuronosyltransferase; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Linoleic Acids; Lipid Metabolism; Male; Metribolone; Minor Histocompatibility Antigens; Prostate; Receptors, Androgen; RNA, Messenger

Conjugated linoleic acid (CLA) consists of a group of linoleic acid geometric isomers that have been shown to reduce tumor growth and metastasis in animal models of breast, prostate and colon cancer. To delineate a possible mechanism of action for CLA, we have recently shown that the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), could play a role in CLA alteration of mammary tumorigenesis. In this study, we determined how CLA could modulate 5-lipoxygenase activity. The t10, c12-CLA isomer reduced production of 5-HETE but not 12- and 15-HETE in MDA-MB-231 human breast tumor cells. That isomer and the c9, t11-CLA isomer decreased 5-HETE production by competition with the lipoxygenase substrate, arachidonic acid (AA). Interestingly, t10, c12-CLA reduced the expression of five-lipoxygenase activating protein (FLAP) but not the 5-lipoxygenase enzyme. Over-expression of FLAP abrogated t10, c12-CLA-reduced viability of MDA-MB-231 cells. These data suggest that the reduction of 5-HETE by t10, c12-CLA was due to competition with AA and the reduction of FLAP expression.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acids; Linoleic Acids, Conjugated; Membrane Proteins; Transfection

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cell Division; Cell Line; Cricetinae; DNA; DNA Replication; Embryo, Mammalian; Epidermal Growth Factor; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Mesocricetus; Oxidation-Reduction; Thymidine

An assay for the quantitative determination of the hydroxylation profile of long-chain fatty acids is described for gas chromatography negative-ion chemical ionization mass spectrometry and stable isotope dilution using [carboxyl-18O2]-labeled internal standards. The assay has been applied to the study of fatty acids isolated from body fluids, tissue, and cultured cells. Examples for the analyses of biological systems expressing 5-, 8-, 12-, or 15-lipoxygenase activity are given and the most important sources of analytical errors are addressed. Increased specificity compared to analysis by negative-ion chemical ionization, at the cost of sensitivity, can be achieved by the use of positive-ion electron impact ionization for the investigation of hydrogenated pentafluorobenzylester/trimethylsilylether derivatives. The method described provides complete, specific, and quantitative profiles of hydroxylated fatty acids originally present in biological samples or generated in vitro by incubation with polyunsaturated fatty acid substrates such as linoleic or arachidonic acid.

Topics: Animals; Cell Line; Female; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Mice

Lipoxygenase products were quantified in human mixed saliva and in saliva fractions obtained from a parotid or submandibular gland using gas chromatography-mass spectrometry and stable isotope dilution. In glandular saliva, only linoleic acid was detected at levels of 20-30 ng/ml. In contrast, mixed saliva showed a linoleic acid concentration of around 300 ng/ml, arachidonic acid levels of around 30 ng/ml, hydroxyoctadecadienoic acid (HODE) levels between 5 and 10 ng/ml, and hydroxyeicosatetraenoic acid (HETE) levels up to 25 ng/ml. By far the most abundant HETE was 12-HETE, and incubation experiments with arachidonic acid showed the presence of a substantial 12-lipoxygenase activity in human mixed saliva, but not in saliva fractions. This activity was identified as 12(S)-lipoxygenase activity by chiral analysis of the reaction product. Investigating mixed saliva and glandular saliva of patients with squamous cell carcinoma in the upper aerodigestive tract and of controls, most patients showed elevated levels of free arachidonic acid and elevated HETE levels. Besides a moderate increase in 12-HETE levels, markedly elevated concentrations of 5-HETE and 15-HETE were observed for the carcinoma patients. The level of free arachidonic acid and the quantitative HETE profile appear to be good markers for the inflammatory processes occurring in the oral mucosa and in saliva in response to the development of squamous cell carcinoma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Mouth Neoplasms; Parotid Gland; Saliva; Submandibular Gland

Apart from the generation of potent inflammatory mediators, the effects of fatty acid oxygenase activation, per se, on the host cell have not been well-delineated. Fatty acid oxygenases were activated in rat basophilic leukemia cells (RBL-1) by incubating them for 2-4 hr with 33-300 microM of arachidonic acid (AA) or linoleic acid (LA). As a control, the cells were incubated with one of two analogs of these fatty acids which are not oxygenase substrates: eicosatetraynoic acid or linoelaidic acid. Effects of oxygenase activation on cell viability were monitored by an assay for mitochondrial function. Cytotoxicity occurred in incubations with exogenous AA or LA in direct proportion to the substrate concentration but was not found in the control incubations or in incubations with the principal monohydroxylated AA products, 5-, 15-, and 12-HETE. Nordihydroguaiaretic acid (80 microM) and alpha-tocopherol (100 microM) significantly decreased the cell death observed during incubations with AA or LA. It is concluded that extensive oxygenase activation can result in cell death from intermediates produced proximal to the stable monohydroxylated derivatives.

Topics: Animals; Arachidonic Acid; Cell Survival; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Linoleic Acid; Linoleic Acids; Lipoxygenase; Masoprocol; Rats; Tumor Cells, Cultured; Vitamin E

Fatty acid-derived inflammatory mediators are considered to play an important role in airway hyperresponsiveness of asthmatic patients. The pulmonary macrophage may be an important source for these mediators in airway tissue. We investigated the metabolism of arachidonic acid and linoleic acid by human bronchoalveolar lavage cells, mainly comprising pulmonary macrophages. Arachidonic was mainly metabolized by 5-lipoxygenase, giving rise to the formation of leukotriene B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). Linoleic acid was converted to 5 major metabolites, including the 9-hydroxy and 13-hydroxy derivatives, 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE). The formation of HODEs could be inhibited by cyclooxygenase inhibitors as well as lipoxygenase inhibitors, indicating that both enzymic species play a role in the generation of HODEs.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase Inhibitors; Macrophages

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

Topics: Animals; Antithrombins; Arachidonic Acid; Arachidonic Acids; Carbon Radioisotopes; Epithelial Cells; Epithelium; Fatty Acids; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Male; Perfusion; Trachea

There is convincing evidence that normal cultured human keratinocytes possess a 15-lipoxygenase activity which, however, does not appear to manifest itself without cell membrane damage. When "activated", this enzyme transforms arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE), and linoleic acid into 13-hydroxyoctadecadienoic acid, presumably by peroxidase action on their respective hydroperoxy intermediates. Normal but not membrane-damaged keratinocytes metabolize exogenous 5-HETE, principally by esterifying the eicosanoid intact, primarily in the triacylglycerol fraction. In the present study, membrane-damaged keratinocytes were found to transform 5-HETE to 5,15-diHETE and also to a lipoxin-like group of tetraenes. Similar, if not identical, tetraenes were produced by action of the keratinocyte enzyme on 5(S),15(S)-diHETE, which points to the role of the latter as an intermediate between 5-HETE and the tetraenes. A direction for further study of the mechanism of the "activation" step is presented.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Enzyme Activation; Esterification; Humans; Hydroxyeicosatetraenoic Acids; Keratinocytes; Kinetics; Linoleic Acids

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Cell Adhesion; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Neoplasms; Neutrophils; Platelet Adhesiveness; Tumor Cells, Cultured

Episodes of fever, serositis, and arthritis in familial Mediterranean fever (FMF) suggested circulating mediators of acute inflammation (e.g., neutrophil activation). The mean serum neutrophil-aggregating activity of 51 FMF patients was 2.5 +/- 0.2 cm2/min, compared to 1.0 +/- 0.1 cm2/min in 20 normal controls (P less than 0.0002). Lipid extracts of FMF sera retained neutrophil-aggregating activity and had UV absorbance peaks at 269 and 279 nm, indicating the presence of lipids with a conjugated triene structure. Chromatography of extracts yielded peaks that were coeluted with reference dihydroxyicosatetraenoic acids, had UV absorbance peaks at 259, 269, and 279 nm, and possessed neutrophil-aggregating activity. The presence of leukotriene B4 was excluded by chromatography following methyl-esterification. Monohydroxy compounds identified in FMF extracts by gas chromatography/mass spectrometry included 5-hydroxyicosatetraenoic acid, and 9- and 13-hydroxyoctadecadienoic acids. Hydroxy acids were present in 19 of 31 FMF sera and absent in extracts of sera from 8 patients with active systemic lupus erythematosus, 7 with fever from infection, and 12 normal controls. The finding of circulating mono- and dihydroxy fatty acids in FMF suggests that defects in the formation or elimination of these compounds might play a role in the pathogenesis of FMF.

Topics: Cell Aggregation; Familial Mediterranean Fever; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Neutrophils