15-hydroxy-5-8-11-13-eicosatetraenoic-acid and 11-hydroxy-5-8-12-14-eicosatetraenoic-acid

Disease-specific treatment options for autosomal dominant polycystic kidney disease are limited. Clinical intervention early in life is likely to have the greatest effect. In a 3-year randomized double-blind placebo-controlled phase 3 clinical trial, the authors recently showed that pravastatin decreased height-corrected total kidney volume (HtTKV) progression of structural kidney disease over a 3-year period. However, the underlying mechanisms have not been elucidated.. Participants were recruited nationally from July 2007 through October 2009. Plasma and urine samples collected at baseline, 18 months, and 36 months from 91 pediatric patients enrolled in the above-mentioned clinical trial were subjected to mass spectrometry-based biomarker analysis. Changes in biomarkers over 3 years were compared between placebo and pravastatin-treated groups. Linear regression was used to evaluate the changes in biomarkers with the percent change in HtTKV over 3 years.. Changes in plasma concentrations of proinflammatory and oxidative stress markers (9- hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, and 15-hydroxyeicosatetraenoic acid [HETE]) over 3 years were significantly different between the placebo and pravastatin-treated groups, with the pravastatin group showing a lower rate of biomarker increase. Urinary 8-HETE, 9-HETE, and 11-HETE were positively associated with the changes in HtTKV in the pravastatin group.. Pravastatin therapy diminished the increase of cyclooxygenase- and lipoxygenase-derived plasma lipid mediators. The identified biomarkers and related molecular pathways of inflammation and endothelial dysfunction may present potential targets for monitoring of disease severity and therapeutic intervention of autosomal dominant polycystic kidney disease.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adolescent; Biomarkers; Child; Female; Humans; Hydroxyeicosatetraenoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Linoleic Acids; Male; Organ Size; Oxidative Stress; Polycystic Kidney, Autosomal Dominant; Pravastatin; Time Factors; Young Adult

The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Calibration; Chromatography, Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Molecular Structure; Postmenopause; Reference Standards; Reproducibility of Results; Solid Phase Extraction; Tandem Mass Spectrometry

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Topics: Animals; B-Lymphocytes; Cell Line; Cerebellum; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 1; Cyclooxygenase 2; Frataxin; Friedreich Ataxia; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Iron-Binding Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxylipins; Prostaglandins; Reactive Oxygen Species; Signal Transduction; Thromboxane B2; Transcription Factor AP-1

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

Topics: Animals; Aspirin; Celecoxib; Cell Line, Transformed; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Eicosanoids; Group IV Phospholipases A2; Hydroxyeicosatetraenoic Acids; Inflammasomes; Interleukin-1; Lipidoses; Macrophages; Male; Mice; Mice, Inbred C57BL; Pyrazoles; Receptors, Purinergic P2X7; Signal Transduction; Sulfonamides; Toll-Like Receptor 4

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acid Sequence; Amino Acid Substitution; Animals; Arachidonate 12-Lipoxygenase; Binding Sites; Humans; Hydroxyeicosatetraenoic Acids; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Rabbits; Recombinant Proteins; Sequence Homology, Amino Acid; Stereoisomerism; Substrate Specificity; Valine

Because of its oxidative modification during the acute-phase response to an aggression, low density lipoprotein (LDL) can be regarded as a source of lipid mediators that can act both to promote and inhibit inflammation. This can be exemplified by the production of anti-inflammatory oxidized fatty acids and proinflammatory lysophosphatidylcholine (LPC) during LDL oxidation. We have shown previously that oxidized LDL (oxLDL) plays an active role at the interface between innate and adaptive immunity by delivering instructive molecules such as LPC, which promotes mature dendritic cell (DC) generation from differentiating monocytes. It is shown in this study that LPC affects the signaling pathway of peroxisome proliferator-activated receptors (PPARs). LPC-induced DC maturation is associated with complete inhibition of PPARgamma activity and up-regulation of the activity of an uncharacterized nuclear receptor that bind peroxisome proliferator response element. Oxidized fatty acids generated during LDL oxidation are natural ligands for PPARgamma and inhibit oxLDL- and LPC-induced maturation. Inhibition experiments with synthetic PPARgamma ligands suggested a PPARgamma-dependent and independent effect of LPC on DC maturation. Therefore, the relative amount of oxidized fatty acids and LPC influences the immunological functions of oxLDL on DC, in part by regulating the PPAR pathway. By sensing the biochemical composition of lipoprotein particles, the innate immune system may thus identify various endogenous signals that influence the immune response during the acute-phase reaction. The therapeutic emulsion intralipid also blocks LPC action on PPAR activity and DC maturation. Intralipid may thus be an alternative therapeutic strategy for some chronic inflammatory diseases.

Topics: Acute-Phase Proteins; Cell Differentiation; Dendritic Cells; DNA-Binding Proteins; Fat Emulsions, Intravenous; Growth Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Ligands; Linoleic Acids; Linoleic Acids, Conjugated; Lipoproteins, LDL; Lysophosphatidylcholines; Oxidation-Reduction; Receptors, Cytoplasmic and Nuclear; Signal Transduction; T-Lymphocytes; Thiazolidinediones; Transcription Factors

Topics: Animals; Arachidonic Acid; Chlorocebus aethiops; COS Cells; Cyclooxygenase 1; Cyclooxygenase 2; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Kinetics; Male; Membrane Proteins; Microsomes; Prostaglandin-Endoperoxide Synthases; Prostaglandins G; Recombinant Proteins; Seminal Vesicles; Stereoisomerism; Substrate Specificity; Transfection

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.

Topics: Animals; Cyclooxygenase 1; Cyclooxygenase 2; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Membrane Proteins; Mice; Molecular Conformation; Mutagenesis, Site-Directed; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Serine

Arachidonic acid is converted to prostaglandin G(2) (PGG(2)) by the cyclooxygenase activities of prostaglandin endoperoxide H synthases (PGHSs) 1 and 2. The initial, rate-limiting step is abstraction of the 13-proS hydrogen from arachidonate which, for PGG(2) formation, is followed by insertion of O(2) at C-11, cyclization, and a second O( 2) insertion at C-15. As an accompaniment to ongoing structural studies designed to determine the orientation of arachidonate in the cyclooxygenase site, we analyzed the products formed from arachidonate by (a) solubilized, partially purified ovine (o) PGHS-1; (b) membrane-associated, recombinant oPGHS-1; and (c) a membrane-associated, recombinant active site mutant (V349L oPGHS-1) and determined kinetic values for formation of each product. Native forms of oPGHS-1 produced primarily PGG(2) but also several monohydroxy acids, which, in order of abundance, were 11R-hydroxy-5Z, 8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid (15S-HETE), and 15R-HETE. V349L oPGHS-1 formed primarily PGG(2), 15S-HETE, and 15R-HETE but only trace amounts of 11R-HETE. With native enzyme, the K(m) values for PGG(2), 11-HETE, and 15-HETE formation were each different (5.5, 12.1, and 19.4 microM, respectively); similarly, the K(m) values for PGG(2) and 15-HETE formation by V349L oPGHS-1 were different (11 and 5 microM, respectively). These results establish that arachidonate can assume at least three catalytically productive arrangements within the cyclooxygenase site of oPGHS-1 leading to PGG(2), 11R-HETE, and 15S-HETE and/or 15R-HETE, respectively. IC(50) values for inhibition of formation of the individual products by the competitive inhibitor, ibuprofen, were determined and found to be the same for a given enzyme form (i.e. 175 microM for oPGHS-1 and 15 microM for V349L oPGHS-1). These latter results are most simply rationalized by a kinetic model in which arachidonate forms various catalytically competent arrangements only after entering the cyclooxygenase active site.

Topics: Animals; Arachidonic Acid; Catalytic Domain; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Hydroxyeicosatetraenoic Acids; Ibuprofen; Isoenzymes; Male; Microsomes; Models, Chemical; Mutagenesis, Site-Directed; Mutation; Prostaglandin-Endoperoxide Synthases; Prostaglandins G; Seminal Vesicles; Sheep; Stereoisomerism

25-hydroxycholesterol (25-OHC) is an oxidized derivative of cholesterol that has been implicated in the early development of arteriosclerosis. Changes in arterial smooth muscle cell (SMC) migration and proliferation have also been linked to the pathophysiology of arteriosclerosis. SMCs undergo "activation" in response to vascular injury by changing phenotypically and by increasing prostaglandin G/H synthase-2 (PGHS-2) protein levels and eicosanoid release. Activation is thought to be important in atheroma formation and arteriosclerosis progression. 25-OHC induces SMCs to change morphologically, increase PGHS-2, and increase eicosanoid release. Confluent monolayers were treated with 25-OHC (10 microg/mL) or the PGHS-2 inducer interleukin-1beta (1 ng/mL) for 18 hours at 37 degrees C. The 18-hour treatment resulted in morphological changes. After uptake of [(14)C]arachidonic acid, released radiolabeled arachidonic acid products were extracted and chromatographed by both normal and reverse-phase high-performance liquid chromatography systems. 25-OHC-treated cells increased their prostaglandin production, with the major component comigrating with a prostaglandin-E(2) standard. HETEs and epoxyeicosatrienoic acids were not affected. Immunoprecipitation analysis of treated and control cell lysates using anti-PGHS-1 and -2 and anti-alpha-actin primary antibodies indicated PGHS-2 induction over control and no change in contractile proteins. These changes are consistent with SMC activation, which occurs in vascular injury models. The notion that oxysterols can activate vascular SMCs may be important in ultimately understanding the pathophysiology of atheroma formation.

Topics: Actins; Animals; Arachidonic Acid; Carbon Radioisotopes; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Endothelium, Vascular; Hydroxycholesterols; Hydroxyeicosatetraenoic Acids; Isoenzymes; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Pulmonary Artery; Rabbits; Subcellular Fractions

To investigate the cooperation of chondrocytes and polymorphonuclear cells (PMN) in the biosynthesis of leukotrienes (LT).. PMN, resting and interleukin-1beta-stimulated cultured human chondrocytes, and mixtures of both cell types were incubated with A23187 and/or 14C-arachidonic acid (14C-AA). To explore the presence of LTC4 synthase and LTA4 hydrolase, the chondrocytes were incubated with authentic LTA4. Eicosanoids were analyzed using high performance liquid chromatography techniques.. Chondrocytes formed only prostaglandin E2 and minor amounts of 15-HETE and 11-HETE, the production of all of which was inhibited by 1 microM indomethacin. Incubation of PMN and chondrocytes produced more LTC4 from endogenous and exogenous AA, and more LTB4 from endogenous AA, than incubation of PMN alone, which was consistent with the presence of LTC4 synthase and LTA4 hydrolase activities in chondrocytes. Chondrocytes also slightly increased the level of PMN production of all 5-lipoxygenase (5-LO)-derived products from endogenous AA.. Human chondrocytes form eicosanoids from AA only by the cyclooxygenase pathway. Chondrocytes cooperate in the transcellular biosynthesis of LT since they possess LTA4 hydrolase and LTC4 synthase activities and increase metabolism by the 5-LO pathway in PMN.

Topics: Arachidonic Acid; Calcimycin; Cartilage, Articular; Cells, Cultured; Chondrocytes; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Dinoprostone; Epoxide Hydrolases; Glutathione Transferase; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Knee Joint; Leukotriene B4; Leukotriene C4; Neutrophils

Inhibition of the formation of pro-inflammatory eicosanoids such as leukotrienes and 12-hydroxyeicosatetraenoic acid by 15-hydroxyeicosatetraenoic acid (15-HETE) has been reported. Psoriatic dermis synthesizes reduced levels of 15-HETE and it has been postulated to play a role in the pathophysiology of this disease. Interleukin-1 stimulates the production of prostaglandin E2 in fibroblasts, but its effect on the synthesis of 15-HETE is at present unknown. The aim of this study was to investigate the modulation of 15-HETE formation by interleukin-1 in dermal fibroblasts. Cells were treated with recombinant interleukin-1 alpha or beta prior to incubation with exogenous 14C-arachidonic acid, and eicosanoids were analyzed by HPLC. Interleukin-1 significantly increased the production of 15-HETE, but also 12-hydroxy-heptadecatrienoic acid, 11-hydroxyeicosatetraenoic acid, and prostaglandins, in a concentration- and time-dependent fashion. No significant differences between the two types of interleukin-1 were found. Dexamethasone (10 nM), and the protein synthesis inhibitors actinomycin D (1 microM) and cycloheximide (3 micrograms/ml) completely abolished the effect of interleukin-1 on 15-HETE formation. Whereas indomethacin (0.5-25 microM) strongly inhibited the synthesis of 15-HETE, aspirin (100-1000 microM) was unable to significantly inhibit its formation in both untreated and interleukin-treated fibroblasts. Aspirin inhibited the 15-HETE produced by cyclooxygenase from ram seminal vesicles, although to a lesser extent than indomethacin. In cell-free extracts, the activity concerning the synthesis of 15-HETE was associated with the microsomal fraction (100,000 x g pellet). Overall, these results strongly suggest that interleukin-1 increases 15-HETE formation mainly through the expression of new cyclooxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Cells, Cultured; Chromatography, High Pressure Liquid; Cycloheximide; Dactinomycin; Dexamethasone; Dose-Response Relationship, Drug; Eicosanoids; Epidermal Cells; Epidermis; Female; Fibroblasts; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Time Factors

Arachidonic acid metabolism via cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase was investigated in thoracic aortic tissue obtained from rabbits fed either standard rabbit chow or chow containing 2% cholesterol. Aortic strips were incubated with [14C]arachidonic acid and A23187. Metabolites from extracted media were resolved by high-pressure liquid chromatography (HPLC). Normal and cholesterol-fed rabbit aortas synthesized prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs). The major cyclooxygenase products were 6-keto-PGF1 alpha and PGE2. Basal aortic 6-keto-PGF1 alpha production was slightly reduced in cholesterol-fed compared with normal rabbits. 12(S)- and 15(S)-HETE were the major aortic lipoxygenase products from both normal and cholesterol-fed rabbits. The structures were confirmed by gas chromatography-mass spectrometry (GC-MS). Only cholesterol-fed rabbit aortas metabolized arachidonic acid via cytochrome P-450 epoxygenase to the epoxyeicosatrienoic acids (EETs). 14,15-, 11,12-, 8,9-, and 5,6-EET were identified based on comigration on HPLC with known 14C-labeled standards and typical mass spectra. Incubation of normal aorta with 14,15-EET decreased the basal synthesis of 6-keto-PGF1 alpha. The other EETs were without effect. The four EET regioisomers relaxed the norepinephrine-precontracted normal and cholesterol-fed rabbit aorta. The relaxation response to 14,15-EET was greater in aortas from cholesterol-fed rabbits. These studies demonstrate that hypercholesterolemia, before the development of atherosclerosis, alters arachidonic acid metabolism via both the cyclooxygenase and epoxygenase pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Arachidonic Acids; Carbon Radioisotopes; Cholesterol, Dietary; Clotrimazole; Diet, Atherogenic; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Kinetics; Masoprocol; Metyrapone; Muscle, Smooth, Vascular; Rabbits; Reference Values; Stereoisomerism

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.

Topics: Animals; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Indomethacin; Linoleic Acids; Linoleic Acids, Conjugated; Stereoisomerism; Substrate Specificity

It has previously been reported that rat embryonic tissue produces various prostanoids. This report demonstrates that rat embryo homogenates synthesized various lipoxygenase metabolites, including 12-hydroxyeicosatetraenoic acid (12-HETE) as the major metabolite, 5-HETE, and 15-HETE. The cyclooxygenase product 11-HETE was also formed. Product identification was based on radioimmunoassay and comparison of reverse-phase- and straight-phase-high-pressure liquid chromatography retention times with authentic standards. Additional evidence was the observation that the lipoxygenase inhibitor nordihydroguaiaretic acid inhibited HETE formation. It appears that, under the same (though not necessarily optimal) experimental conditions, lipoxygenase metabolites predominate quantitatively over cyclooxygenase pathway products and that 11-day embryonic tissue produces more HETEs than either 12-day or 13-day embryo homogenates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Embryo, Mammalian; Gestational Age; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Masoprocol; Radioimmunoassay; Rats; Rats, Inbred Strains

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.

Topics: Adrenergic alpha-Agonists; Animals; Arachidonic Acid; Arachidonic Acids; Cell Division; Cell Line; Cyclic AMP; Cyclooxygenase Inhibitors; Dinoprostone; Hydroxyeicosatetraenoic Acids; Indomethacin; Norepinephrine; Prostaglandins E; Rats; Thymidine; Thyroid Gland; Thyrotropin; Time Factors

Current strict O2 management may be precipitating more severe retinopathy of prematurity than would occur with a more lenient approach. Hypoxemia in an animal model has also been found to worsen retinal neovascularization. It has recently been shown that the hydroxyeicosatetraenoic acids can modulate angiogenesis. 15-Hydroxyeicosatetraenoic acid is proangiogenic, whereas 12-hydroxyeicosatetraenoic acid is an antiangiogenic metabolite. In vitro exposure of paired human neonatal vessels (n = 7) to hypoxia enhanced the production of total vascular hydroxyacids (232 +/- 36 pmol/mg of protein [experimental group] nu 168 +/- 31 pmol [control group]; P less than .01). The increase in vascular 15-hydroxyeicosatetraenoic acid under hypoxic conditions was even more significant (P less than .001). However, platelet production of 12-hydroxyeicosatetraenoic acid was not significantly affected by hypoxia. These observations suggest a possible biochemical basis for the abnormal angiogenic process that occurs during the proliferation phase of the retinopathy of prematurity. The production of local hydroxyeicosatetraenoic acids in tissues manifesting abnormal neovascularization needs to be further evaluated.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neovascularization, Pathologic; Oxygen; Retinopathy of Prematurity; Umbilical Arteries; Vasoconstrictor Agents

Human umbilical arteries convert arachidonic acid into three hydroxy-eicosatetraenoic acids as well as 6-ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and D2 and thromboxane B2. Two of these hydroxy derivatives of arachidonic acid were purified by reverse-phase HPLC and identified by GC-MS as 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) while a third, presumed dihydroxy derivative has not yet been identified. Both the cyclooxygenase and HETE synthesizing activities were found to be localized mainly in the microsomal fraction (100 000 X g pellet) (51 and 61% of total, respectively), and approx. 25% of both activities was found in the 10 000 X g pellet. The formation of these HETEs was inhibited by the cyclooxygenase inhibitors indomethacin and aspirin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. Production of immunoreactive 15-HETE as well as 6-ketoprostaglandin F1 alpha were also decreased significantly when arterial segments were incubated in the presence of either indomethacin or aspirin. Indomethacin inhibited the formation of both prostanoids and HETEs by microsomes in a concentration-dependent and time-dependent manner. The ID50 values for indomethacin against HETE synthesizing activity and against cyclooxygenase were 4.5 and 3.8 microM, respectively. The inactivation constants were found to be 0.09 and 0.08 min-1 for HETE synthesizing activity and cyclooxygenase, respectively. These two microsomal activities were solubilized in parallel with Tween-20. Incubation with three distinct monoclonal antibodies against different epitopes on cyclooxygenase precipitated both cyclooxygenase and HETE synthesizing activity. Each of these activities was recovered in the immune pellets. These studies demonstrate that in human umbilical arteries 11-HETE, 15-HETE and a presumed di-HETE are the products of cyclooxygenase.

Topics: Arachidonic Acid; Arachidonic Acids; Aspirin; Catechols; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Infant, Newborn; Masoprocol; Prostaglandin-Endoperoxide Synthases; Subcellular Fractions; Umbilical Arteries

Our previous data suggested that lipoxygenation of endogenously released arachidonic acid (AA) is a critical step in stimulus-secretion coupling in the pancreatic beta cell. In the current study using monolayer cultures of neonatal rat islet cells, exogenous arachidonic acid (AA) (5 micrograms/ml) potently stimulated insulin release in the presence of a substimulatory glucose concentration, and potentiated release induced by glucose. Since the latter stimulatory effect of AA is prevented by inhibitors of the lipoxygenase pathway, we examined the effects of various lipoxygenase pathway products on glucose-induced insulin secretion. The mediator was not one of the stable end-products of either limb of the lipoxygenase pathway: 12- or 5-hydroxyeicosatetraenoic acid (HETE) (0.5-2000 ng/ml) did not alter insulin release, whereas 11-HETE, 15-HETE, leukotriene (LT)B4 and the delta 6 trans isomers of LTB4, LTC4 and 11-trans LTC4 all inhibited insulin release. Furthermore, diethylcarbamazine, a selective leukotriene synthesis inhibitor, did not prevent AA- or glucose-induced insulin release, arguing against a role for LTs as the mediator of AA's stimulatory effect. However, the unstable intermediate 12-hydroperoxyeicosatetraenoic acid (12-HPETE), and positional isomers of 12-HPETE, potentiated glucose-induced insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Diethylcarbamazine; Glucose; Hydroxyeicosatetraenoic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Leukotriene B4; Lipoxygenase; Rats

Phytohemagglutinin (PHA)-induced mitogenesis of mixed mouse spleen lymphocyte populations, measured by [3H]thymidine incorporation, was completely inhibited by micromolar concentrations of certain hydroxyeicosatetraenoic acids (HETE's). These are lipoxygenase metabolites of arachidonic acid which are synthesized in considerable concentrations by macrophages, lymphocytes, and other components of the immune system when appropriately stimulated. In the studies described here, the structural requirements for the maximum antimitogenic activities were examined. A series of monohydroxylated HETE's were prepared using a singlet oxygen photochemical procedure or by enzymatic synthesis from arachidonic acid substrate, and isolated by HPLC. Isomers containing different numbers of double bonds were synthesized using the appropriate unsaturated fatty acid as substrate, and the functional importance of the OH and carboxylic functions was tested using various acetoxy- and carbomethoxy derivatives. A serum-free mitogenesis assay system was used for testing, which minimized binding of the fatty acids by serum proteins and increased the inhibitory potency of the various HETE's several-fold. It was found that inhibition of cell proliferation was related to: hydroxyl proximity to the center of the eicosatetraenoic acid, decreasing in the order: 9 greater than 11 greater than 12 greater than 15 greater than 8 much greater than 5; the number of double bonds in the fatty acid chain, decreasing in the order: 15-OH, 20:4 greater than 15-OH 20:3 much greater than 15-OH, 20:2 much greater than 15-OH, 20:0; and the 15-position functional group as well as the 1-carboxylic group, decreasing in the order: 15-hydroxy, 1-carboxylic greater than 15-acetoxy, 1-carboxylic much greater than 15-hydroxy, 1-carbomethoxy greater than 15-acetoxy, 1-carbomethoxy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxygenase; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Phytohemagglutinins; Structure-Activity Relationship

The effects of 5-, 8-, 9-, 11-, 12-, and 15-monohydroxyeicosatetraenoic acid (HETE) (0.1-100 nM) on mucous glycoprotein release from cultured human airways were determined. Each of the HETE was an active secretagogue of mucus at concentrations greater than 1-10 nM with 12- and 15-HETE, the most active. Both 5- and 9-hydroperoxyeicosatetraenoic acid (HPETE) were also active as secretagogues at 100 nM, although of somewhat lower potency. As cultured airways were capable of responding to HETE with mucous glycoprotein release, it was of interest to identify and quantitate airway HETE formation. Accordingly, airways were incubated with tracer quantities of [14C]arachidonate for 16-48 h, and the spontaneous formation of 5-, 12- and 11- and/or 15-HETE was measured by high-pressure liquid chromatography. Indeed, sizeable quantities of 11- and/or 15- greater than 5- greater than 12-HETE were generated. This HETE generation was increased by the addition of 25 micrograms/ml of arachidonate and was reduced somewhat after 18-21 d in continuous tissue culture. Reversed anaphylaxis of human airways using anti-human IgE markedly increased the HETE formation, resulting in the production of micromolar concentrations of 5- and 11- and/or 15-HETE. Thus, human airways not only are capable of responding to the presence of HETE with mucous glycoprotein release, but also generate (both spontaneously and in response to anaphylaxis) at least three species of HETE, and do so in quantities capable of acting as mucus secretagogues.

Topics: Anaphylaxis; Arachidonic Acids; Culture Techniques; Humans; Hydroxyeicosatetraenoic Acids; Immunoglobulin E; Leukotriene B4; Lung; Mucus

A procedure using high performance liquid chromatography (HPLC) is described for the separation of major primary cyclooxygenase metabolites (prostacyclin metabolite-6ketoPGF1 alpha, thromboxane B2, and prostaglandins F2 alpha, E2, and D2), leukotrienes (C4, B4, and D4), monohydroxyeicosatetraenoic acids (15-, 11-, 12-, and 5HETEs), and free arachidonic acid. It is therefore possible to quantitate major arachidonic acid metabolites by a single chromatographic procedure. Using this technique we have determined that a major arachidonic acid metabolite of human lung macrophages co-elutes with leukotriene B4.

Topics: Arachidonic Acids; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung; Macrophages; Prostaglandins; Prostaglandins F; SRS-A; Thromboxane B2

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Mice; SRS-A

Arachidonic acid is the precursor of several potent derivatives that regulate physiological functions in the cardiovascular system. Thromboxane (TXA2) and prostacyclin (PGI2) are synthesized by the cyclooxygenase enzyme. The proaggregatory and vasoconstrictive TXA2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of PGI2 synthesized by blood vessels. Arachidonic acid is also converted via a 5-lipoxygenase to leukotrienes, the vasoconstrictive components of SRSA. We have shown that this latter pathway is regulated by 15-HETE, a product of the 15-lipoxygenase present in lymphocytes. Confluent cultures of rat aorta smooth muscle cells (RSM) were superfused briefly with [14C]arachidonic acid. The products were isolated and analyzed by thin-layer chromatography-radioautography, high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. Prostacyclin (PGI2) was identified as the major product both by its biological properties in a platelet aggregation assay and by the mass spectrum of its tetra-trimethylsilylether-methyl ester derivative. Minor quantities of PGE2, PGD2, and PGF2 alpha were also synthesized. Three other compounds with chromatographic properties of mono-hydroxy eicosanoic acids were also formed in major amounts. These were shown to be cyclooxygenase products since their synthesis, together with that of prostacyclin, was blocked by the cyclooxygenase inhibitors aspirin (0.2 mM) and indomethacin (10 microM). Quantities of the hydroxy-eicosanoids were isolated from large scale incubations by silicic acid chromatography. Following methylation and reduction with platinum oxide/H2, the compounds were converted to their trimethylsilylether derivatives and analyzed by gas-liquid chromatography-mass spectrometry. The compounds were identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and hydroxy-5,8,10-heptadeca-trienoic acid (HHT) by simultaneous ion monitoring of characteristic ions at M/e ratios of 287, 258, 229 for 11-HETE and 343, 314, 173 for 15-HETE, and by comparison with the mass spectra of authentic samples. Rat smooth muscle cells, prelabeled by 24-hour incubation with [14C]arachidonic acid, released large amounts of prostacyclin together with enhanced amounts of 11- and 15-HETE in response to physiological levels of thrombin (0.5-5 units/ml). These experiments demonstrate that, in addition to the thromboxan

Topics: Animals; Aorta; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Epoprostenol; Hydroxyeicosatetraenoic Acids; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Rats; Thrombin

Particulate fractions and slices from fetal calf aorta convert arachidonic acid to 6-oxoprostaglandin F1 alpha (6-oxoPGF1 alpha), 6,15-dioxoPGF1 alpha, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11h-20:4), and 15-hydroxy-5,8,11,13-icosatetraenoic acid (15h-20:4). In some cases, small amounts of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12h-20:4) were also detected. The products were all identified by gas chromatography-mass spectrometry after purification by normal phase and argentation high pressure liquid chromatography. Both 11h-20:4 and 15h-20:4 appeared to be formed by prostaglandin endoperoxide synthetase rather than by lipoxygenases, since their formation was inhibited by indomethacin but not by nordihydroguaiaretic acid. The formation of 12h-20:4, on the other hand, was stimulated by indomethacin, probably due to increased substrate availability. The formation of hydroxyicosatetraenoic acids was markedly stimulated by adrenaline. Substantial amounts of 6,15-dioxoPGF1 alpha were formed from arachidonic acid by particulate fractions from fetal calf blood vessels, especially in the presence of relatively high substrate concentrations. The formation of this product was stimulated by methemoglobin and inhibited by adrenaline, glutathione, and tryptophan. It would appear that particulate fractions from fetal calf aorta convert arachidonic acid to 15-hydroperoxyPGI2, which can either be reduced in the presence of various cofactors to form PGI2 or dehydrated to give 15-oxoPGI2. The formation of hydroperoxides from arachidonic acid could be an important factor in regulating PGI2 synthesis in aorta, since PGI2 synthetase is strongly inhibited by such intermediates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acids; Cattle; Ductus Arteriosus; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Prostaglandins F, Synthetic

During allergic and inflammatory reactions, arachidonic acid is oxidized by lipoxygenases to a variety of biologically active products, including leukotrienes. The mechanisms for regulation of the different lipoxygenase activities are not well defined. We report here that [14C]arachidonic acid metabolism by the 5- and 15-lipoxygenase activities in rabbit leukocytes and the 12-lipoxygenase in rabbit platelets is inhibited by various hydroxyeicosatetraenoic acids (HETEs). 15-HETE was the most effective inhibitor of the 5- and 12-lipoxygenases, whereas similar inhibitory potencies were observed for 5-HETE and 12-HETE acting on the 15-lipoxygenase. These three enzyme pathways were all least sensitive to their own products HETEs. To determine which structural characteristics of 15-HETE are essential for inhibition of the 5-lipoxygenase, various derivatives were prepared and purified by high pressure liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The inhibitory potencies of 15-HETE analogs with different degrees of unsaturation were in the order of three double bonds greater than 4 greater than 2 greater than 0. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was four times more potent than 15-HETE. The 15-acetoxy, 15-keto and methyl ester derivatives were of comparable activity to 15-HETE, and the 15-acetoxy methyl ester derivative was less potent. Based upon the observed patterns of inhibition, we postulate that complex interregulatory relationships exist between the various lipoxygenases, and that cells containing these lipoxygenases may interact with each other via their lipoxygenase metabolites.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors; Neutrophils; Rabbits; Species Specificity; Structure-Activity Relationship