8-11-12-trihydroxy-5-9-14-eicosatrienoic-acid and 8-hydroxy-11-12-epoxyeicosa-5-9-14-trienoic-acid

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Bacterial Proteins; Biotransformation; Epoxide Hydrolases; Fatty Acids, Unsaturated; Metabolic Networks and Pathways; Molecular Structure; Myxococcus xanthus

12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity.. Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A. All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A. Trioxilin A

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; HEK293 Cells; Humans; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Receptors, Thromboxane; Vasodilation; Vasodilator Agents

The barnacle life cycle has two key stages at which eicosanoids are believed to be involved in cellular communication pathways, namely the hatching of nauplii and the settlement of cypris larvae. Barnacle egg-hatching activity has previously been reported to reside in a variety of eicosanoids, including 8-hydroxyeicosapentaenoic acid and a number of tri-hydroxylated polyunsaturated fatty acid derivatives, the trioxilins. The production of the eicosapentaenoic acid metabolite trioxilin A4 (8,11,12-trihydroxy-5,9,14,17-eicosatetraenoic acid) by the barnacles Balanus amphitrite and Elminius modestus was confirmed using a combination of high-performance liquid chromatography and gas chromatography, both linked to mass spectrometry. In addition, both species also generated trioxilin A3 (8,11,12-trihydroxy-5,9,14-eicosatrienoic acid; an arachidonic acid-derived product), 8,11,12-trihydroxy-9,14,17-eicosatrienoic acid (a omega3 analogue of trioxilin A3; derived from omega3 arachidonic acid) and 10,13,14-trihydroxy-4,7,11,16,19-docosapentaenoic acid (a docosahexaenoic acid-derived product). In contrast to earlier reports, trioxilin A3 had no E. modestus egg-hatching activity at any of the concentrations tested (10(-9)-10(-6) mol l(-1)). The unstable epoxide precursor hepoxilin A3, however, caused significant levels of hatching at 10(-6) mol l(-1). Furthermore, the stable hepoxilin B3 analogue PBT-3 stimulated hatching at 10(-7) mol l(-1). Neither trioxilin A3, hepoxilin A3 or PBT-3 at 0.25-30 micromol l(-1) served as settlement cues for B. amphitrite cypris larvae.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Larva; North Carolina; Ovum; Reproduction; Signal Transduction; Thoracica; United Kingdom

We recently found that normal human epidermis produces relatively high amounts of hepoxilins and trioxilins in vitro. Therefore, the aim of this study was to demonstrate the presence of these compounds in psoriatic lesions. Extracts from scales of patients with chronic stable plaque psoriasis were analyzed by a combination of high performance liquid chromatography and gas chromatography-mass spectrometry techniques. We found that the levels of hepoxilin B3 were more than 16-fold higher in psoriatic scales than in normal epidermis (3.2+/-2.3 and < 0.2 ng per mg, respectively), whereas hepoxilin A3 was not detected in any sample. Trioxilins were semiquantitated and referred to 12-hydroxyeicosatetraenoic acid, ratios of trioxilins A3 and B3 12-hydroxyeicosatetraenoic acid in psoriatic lesions were 0.65+/-0.23 and 0.32+/-0.28, respectively, and they were not detected in normal epidermis. The presence of a great amount of trioxilin A3 strongly suggests that hepoxilin A3 was present in psoriatic lesions and it was totally degraded to trioxilin A3 during the analysis procedure. Our results demonstrate that hepoxilins and trioxilins are produced by human skin in vivo and that the levels of these compounds are increased in psoriasis. The reported biologic activities of hepoxilins indicate that they could amplify and maintain the inflammatory response. Our results reinforce the idea that these compounds could play a role as mediators in the inflammatory response in skin, particularly in psoriasis.

Topics: 8,11,14-Eicosatrienoic Acid; Chromatography, High Pressure Liquid; Epidermis; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Psoriasis; Reference Values

Hepoxilins (Hx) are biologically active metabolites of arachidonic acid (AA) formed regioselectively from 12(S)-HPETE by 'hepoxilin synthase'. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the course of our studies we investigated whether docosahexaenoic acid (DHA) was a substrate for hepoxilin formation. We used two tissues, the pineal gland and hippocampal slices. Tissues were incubated alone or with AA (20 microg/ml) or DHA (20 microg/ml). After 60 min at 37 degrees C, samples were acid-extracted to convert Hx into their stable trioxilin (TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to determine the structures of the DHA metabolites, and as PFB-TMSi derivatives by GC/MS in the NICI mode using SIM to simultaneously quantify TrX products of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593. Results show good conversion of both substrate fatty acids by the rat pineal gland and hippocampal slices, into the 3-series (21.3 +/- 5.8 and 12.5 +/- 2.2 ng/microg protein, respectively) and 5-series TrX (12.3 +/- 2.7 and 2.9 +/- 0.4 ng/microg protein, respectively). Surprisingly though, experiments with DHA, in both tissues, also showed formation of TrX derived from endogenous AA (3-series) (10.4 +/- 8.3 and 3.1 +/- 2.1 ng/microg protein, respectively). These experiments demonstrate previously unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated during DHA stimulation of the tissue, we carried out separate experiments with hippocampal slices in which the neutral lipids and phospholipids were labeled with [14C]AA. DHA caused a time-dependent appearance of free [14C]AA which was released mostly from the TG pool. Measurement of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results demonstrate that DHA competes with AA for acylation into the metabolically active TG fraction, and both fatty acids are converted into hepoxilins of the corresponding series.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Chromatography, Thin Layer; Docosahexaenoic Acids; Gas Chromatography-Mass Spectrometry; Hippocampus; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Male; Molecular Structure; Pineal Gland; Rats; Rats, Wistar

Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Anthracenes; Arachidonic Acid; Chromatography, High Pressure Liquid; Eicosanoids; Esters; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Pineal Gland; Rats; Spectrometry, Fluorescence

1. The vascular activity of two stereoisomers of hepoxilin A3 (HxA3) (8R and 8S) and of its glutathione conjugate, hepoxilin A3-C (HxA3-C) (8R and 8S), was investigated on rat helicoidal strips of thoracic aorta and longitudinal strips of portal vein. 2. Neither of the hepoxilins tested had a direct effect on the tone of the aortic strip or on the spontaneous contractions of the portal vein. However, the noradrenaline (NA)-induced response of these vessels, as expressed by the dose required for half maximal contraction, (EC50) was greater in HxA3 (8S)- and HxA3-C (8R)-treated aorta. Increased frequency and strength of spontaneous contractions of the portal vein were detected at lower concentrations of NA in the presence of hepoxilins. 3. The threshold dose for both hepoxilins was 10(-8) M and their effect was not dose-related beyond 10(-8) M. The effect of hepoxilin appeared after a 45 min incubation period and could be observed even if the compounds were washed out after 15 min. 4. Stereochemical specificity was observed. The 8S isomer of HxA3 was active in potentiating the NA-induced contraction of these vessels while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. In both tissues, HxA3 (8S) was more potent than its glutathione conjugate, HxA3-C (8R). 5. In calcium-free buffer or in the presence of a calcium channel blocker (nifedipine 1 microM), no potentiation of NA-induced contraction by hepoxilins could be observed, suggesting the involvement of extracellular calcium in the actions of hepoxilins.6. These experiments suggest that hepoxilins may be involved in the modulation of vascular tone and contractility.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Calcium; Drug Synergism; Glutathione; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nifedipine; Norepinephrine; Portal Vein; Rats; Rats, Inbred Strains; Stereoisomerism

In this paper we describe the release of hepoxilin A3 (HxA3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A3 (TrXA3), and a second pathway involves conjugation of HxA3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A3-C (HxA3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA3 and HxA3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA3 and to a lesser extent HxA3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Epoxide Hydrolases; gamma-Glutamyltransferase; Glutathione; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred Strains; Vasoconstriction

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Carbon Radioisotopes; Epoxide Hydrolases; Evoked Potentials; Fatty Acids, Unsaturated; Glutathione; Hippocampus; Indicators and Reagents; Male; Rats; Rats, Inbred Strains; Trichloroepoxypropane

Bolus intravenous injection of arachidonic acid (10 mg/kg) in the rat led to the appearance of hepoxilin A3 in the circulation. The product was assayed as the Me t-BDMSi derivative of its stable trihydroxy product trioxilin A3, by capillary gas chromatography-electron impact mass spectrometry using the stable deuterium isotope dilution technique. Hepoxilin A3, was undetected in blood samples taken prior to the injection of arachidonic acid, but rapidly appeared (4.62 +/- 1.3 ng/ml blood, n = 3) within 1 minute after injection of arachidonic acid. The plasma concentration of insulin increased by 36% over the same period after injection of arachidonic acid. These experiments demonstrate for the first time the formation of this new class of insulin secretagogues in vivo and their temporal correlation with plasma insulin concentrations in vivo.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Insulin; Insulin Secretion; Rats

Arachidonic acid and 12-hydroperoxyeicosa-5,8, 10, 14-tetraenoic acid are converted by a 0-30% ammonium sulfate fraction (Fraction A) of the high speed supernatant of rat lung into two hydroxy epoxides (EH-1 and EH-2) which have been purified by high performance liquid chromatography. These hydroxy epoxides are converted quantitatively into two triols (10,11,12- from EH-1 and 8,11,12- from EH-2) by a 30-50% ammonium sulfate fraction (Fraction B) of the high speed supernatant. We propose the structures, 8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid (EH-2) and 10-hydroxy-11,12-epoxyeicosa-5,8,14-trienoic acids (EH-1) for these intermediates on the basis of mass spectral interpretation of several derivatives including the lithium aluminum hydride reduction product of both natural and 18Oxygenated derivatives.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Epoxy Compounds; Ethers, Cyclic; Fatty Acids, Unsaturated; Isomerism; Lung; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Tritium