Dihydroxyeicosatetraenoic Acids Research Articles

The roles of eicosanoids in myocardial diseases.

Myocardial disease, the abnormalities of the cardiac muscle, is the leading cause of death in humans. Eicosanoids represent a large spectrum of lipid mediators with critical roles in physiological and pathophysiological conditions. Arachidonic acid (AA) is the major resource of eicosanoids and is metabolized via cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP) enzymes producing a diverse family of lipid mediators called eicosanoids, including prostanoids, leukotrienes (LTs), epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid (diHETEs), eicosatetraenoic acids (ETEs), and lipoxins (LXs). Beyond the well-established roles of eicosanoids in inflammation and vascular biology, a growing body of evidence showed that eicosanoids, especially CYP450 derived eicosanoids EETs, are preventive and therapeutic targets for many of the myocardial diseases. EETs not only ameliorate the cardiac injury and remodeling in different pathological models, but also attenuate subsequent hemodynamic disturbances and cardiac dysfunction. EETs have direct and indirect protective properties in the myocardium, and thus relieve dietetic cardiomyopathy and inflammatory cardiomyopathy. Moreover, EETs are capable to attenuate the ischemic cardiomyopathy, including the myocardial infarction and cardiac ischemic reperfusion injury. Multiple biological events and signaling networks are targeted during the myocardial protection of EETs, these are including mitochondria hemostasis, angiogenesis, oxidative stress, inflammatory response, metabolic regulation, endoplasmic reticulum (ER) stress and cell death. Additionally, eicosanoids from COX and LOX also have important roles in some of the myocardial diseases, such as cardiac hypertrophy and ischemic heart disease. This chapter summarizes the physiological and pathophysiological significance, and the signal mechanisms of the eicosanoids, especially the EETs, in myocardial diseases.

Multiassay nutritional metabolomics profiling of low vitamin A status versus adequacy is characterized by reduced plasma lipid mediators among lactating women in the Philippines: A pilot study

Low vitamin A (VA) status is common among lactating women in low-income countries. Lactation has substantial effects on mother's metabolism and VA is required in multiple biological processes, including growth, vision, immunity, and reproduction. The objective of this pilot study was to use metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus VA adequacy in lactating women. Plasma samples from lactating women who participated in a survey in Samar, Philippines, were selected from a cross-sectional study based on plasma retinol concentrations indicating low (VA–; n = 5) or adequate (VA+; n = 5) VA status (plasma retinol <0.8 or >1.05 µmol/L). The plasma results collected from 6 metabolomics assays (oxylipins, endocannabinoids, bile acids, primary metabolomics, biogenic amines, and lipidomics) were compared by group using liquid chromatography mass spectrometry. Twenty-eight metabolites were altered in the VA– versus VA+ status groups, with 24 being lipid mediators (P < .05). These lipid mediators included lower concentrations of arachidonic acid- and eicosapentaenoic acid-derived oxylipins, as well as lysophospholipids and sphingolipids, in the VA– group (P < .05). Chemical similarity enrichment analysis identified hydroxy-eicosatetraenoic acids, hydroxy-eicosapentaenoic acids, and dihydroxy-eicosatetraenoic acids as significantly altered oxylipin clusters (P < .0001, false discovery rate [FDR] P < .0001), as well as sphingomyelins, saturated lysophosphatidylcholines, phosphatidylcholines, and phosphatidylethanolamines (P < .001, FDR P < .01). The multiassay nutritional metabolomics profiling of low VA status compared with adequacy in lactating women was characterized by reduced lipid mediator concentrations. Future studies with stronger study designs and larger sample size are needed to confirm and validate these preliminary results.

Further exploration of the structure-activity relationship of dual soluble epoxide hydrolase/fatty acid amide hydrolase inhibitors

Fatty acid amide hydrolase (FAAH) is a membrane protein that hydrolyzes endocannabinoids, and its inhibition produces analgesic and anti-inflammatory effects. The soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids. EETs have anti-inflammatory and inflammation resolving properties, thus inhibition of sEH consequently reduces inflammation. Concurrent inhibition of both enzymes may represent a novel approach in the treatment of chronic pain. Drugs with multiple targets can provide a superior therapeutic effect and a decrease in side effects compared to ligands with single targets. Previously, microwave-assisted methodologies were employed to synthesize libraries of benzothiazole analogs from which high affinity dual inhibitors (e.g. 3, sEH IC50 = 9.6 nM; FAAH IC50 = 7 nM) were identified. Here, our structure-activity relationship studies revealed that the 4-phenylthiazole moiety is well tolerated by both enzymes, producing excellent inhibition potencies in the low nanomolar range (e.g. 6o, sEH IC50 = 2.5 nM; FAAH IC50 = 9.8 nM). Docking experiments show that the new class of dual inhibitors bind within the catalytic sites of both enzymes. Prediction of several pharmacokinetic/pharmacodynamic properties suggest that these new dual inhibitors are good candidates for further in vivo evaluation. Finally, dual inhibitor 3 was tested in the Formalin Test, a rat model of acute inflammatory pain. The data indicate that 3 produces antinociception against the inflammatory phase of the Formalin Test in vivo and is metabolically stable following intraperitoneal administration in male rats. Further, antinociception produced by 3 is comparable to that of ketoprofen, a traditional nonsteroidal anti-inflammatory drug. The results presented here will help toward the long-term goal of developing novel non-opioid therapeutics for pain management.

Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography–tandem mass spectrometry (LC–MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

Omega-3 polyunsaturated fatty acids protect against inflammation through production of LOX and CYP450 lipid mediators: relevance for major depression and for human hippocampal neurogenesis

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can exert antidepressant, anti-inflammatory and neuroprotective properties, but the exact molecular mechanism underlying their effects is still not fully understood. We conducted both in vitro and clinical investigations to test which EPA or DHA metabolites are involved in these anti-inflammatory, neuroprotective and antidepressant effects. In vitro, we used the human hippocampal progenitor cell line HPC0A07/03C, and pre-treated cells with either EPA or DHA, followed by interleukin 1beta (IL1β), IL6 and interferon-alpha (IFN-α). Both EPA and DHA prevented the reduction in neurogenesis and the increase in apoptosis induced by these cytokines; moreover, these effects were mediated by the lipoxygenase (LOX) and cytochrome P450 (CYP450) EPA/DHA metabolites, 5-hydroxyeicosapentaenoic acid (HEPE), 4-hydroxydocosahexaenoic acid (HDHA), 18-HEPE, 20-HDHA, 17(18)-epoxyeicosatetraenoic acid (EpETE) and 19(20)-epoxydocosapentaenoic acid (EpDPA), detected here for the first time in human hippocampal neurones using mass spectrometry lipidomics of the supernatant. In fact, like EPA/DHA, co-treatment with these metabolites prevented cytokines-induced reduction in neurogenesis and apoptosis. Moreover, co-treatment with 17(18)-EpETE and 19(20)-EpDPA and the soluble epoxide hydroxylase (sEH) inhibitor, TPPU (which prevents their conversion into dihydroxyeicosatetraenoic acid (DiHETE)/ dihydroxydocosapentaenoic acid (DiHDPA) metabolites) further enhanced their neurogenic and anti-apoptotic effects. Interestingly, these findings were replicated in a sample of n = 22 patients with a DSM-IV Major Depressive Disorder, randomly assigned to treatment with either EPA (3.0 g/day) or DHA (1.4 g/day) for 12 weeks, with exactly the same LOX and CYP450 lipid metabolites increased in the plasma of these patients following treatment with their precursor, EPA or DHA, and some evidence that higher levels of these metabolites were correlated with less severe depressive symptoms. Overall, our study provides the first evidence for the relevance of LOX- and CYP450-derived EPA/DHA bioactive lipid metabolites as neuroprotective molecular targets for human hippocampal neurogenesis and depression, and highlights the importance of sEH inhibitors as potential therapeutic strategy for patients suffering from depressive symptoms.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

Enantioselective ultra-high performance liquid chromatography-tandem mass spectrometry method based on sub-2µm particle polysaccharide column for chiral separation of oxylipins and its application for the analysis of autoxidized fatty acids and platelet releasates

Oxylipins, the oxidation products of polyunsaturated fatty acids, are important signaling molecules in living organisms. Some of them have pro-inflammatory properties, while others act as pro-resolving agents. Oxylipins also play a major role in platelet biology and the progression of thrombo-inflammation. Depending on their structure, they may be pro-thrombotic or anti-thrombotic. For an unbiased biological interpretation, a detailed analysis of a broad spectrum of oxylipins including their stereoisomers is necessary. In our work, we developed for the first time an enantioselective UHPLC-ESI-MS/MS assay which allows quantifying individual oxylipin enantiomers. The assay made use of a sub-2µm particle-based amylose-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak IA-U) under MS-compatible reversed-phase conditions. It covered 19 enantiomeric pairs of oxylipins and one diasteromeric pair of a lipid mediator: 2 pairs of hydroxyoctadecadienoic acids (HODE), 6 pairs of hydroxyeicosatetraenoic acids (HETE), 5 pairs of hydroxyeicosapentaenoic acids (HEPE), 3 pairs of hydroxydocosahexaenoic acids (HDoHE) and one pair of each: resolvins D1, hydroxyeicosatrienoic acid (HETrE), hydroxyoctadecatrienoic acid (HOTrE) and dihydroxyeicosatetraenoic acid (DiHETE). The new method is fast and showed outstanding peak resolution for most of the isomeric pairs. Excellent method sensitivity (average LOD was equal to 2.7 pg on column) was obtained by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. The applicability of the method was verified by preliminary validation. It was then applied to analyze oxylipins produced by autoxidation of polyunsaturated fatty acids (PUFA) in air. Multiple oxylipins were found in each of the samples as racemic mixtures and served as reference substances for identification. Finally, the new enantioselective UHPLC method was applied to analyze releasates from platelets in resting state, and following activation with thrombin. The highest abundant oxylipin in the platelet releasate was 12(S)-HETE, but many other oxylipins were found in the thrombin activated samples, usually as single enantiomers (e.g. 12(S)-HEPE, 11(R)-HETE, 9(R)-HODE, 13-(S)-HODE, 14(S)-HDoHE). The latter was detected at about similar concentration in resting platelet releasates as well. 15-HETE showed elevated levels for both R-and S-enantiomers in releasates of thrombin-activated platelets. 12-HETrE was found presumably as both enantiomers, however, retention time inconsistencies indicate that the R-enantiomer is actually a different compound, maybe another constitutional isomer with different double-bond configuration.

Maximal exercise and plasma cytochrome P450 and lipoxygenase mediators: a lipidomics study

Epoxides derived from arachidonic acid (AA) are released during exercise and may contribute to vasodilation. However, exercise may also affect circulating levels of other epoxides derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX) pathways, many of whose exhibit cardiovascular activity in vitro. The effects of exercise on their levels have not been documented. We tested the hypothesis that acute, maximal exercise would influence the plasma concentrations of these vasoactive substances. We measured plasma CYP and LOX mediators derived from both the n − 3 and n − 6 fatty acid (FA) classes in healthy volunteers before, during and after short‐term exhaustive exercise. Lipid mediators were profiled by means of LC–MS/MS tandem mass spectrometry. A maximal Bruce treadmill test was performed to voluntary exhaustion. Exhaustive exercise increased the circulating levels of epoxyoctadecenoic (12,13‐EpOME), dihydroxyeicosatrienoic (5,6‐DHET), dihydroxyeicosatetraenoic acids (5,6‐DiHETE, 17,18‐DiHETE), but had no effect on the majority of CYP and LOX metabolites. Although our calculations of diol/epoxide ratios revealed preferred hydrolysis of epoxyeicosatrienoic acids (EEQs) into their diols (DiHETEs), this hydrolysis was resistant to maximal exercise. Our study is the first documentation that bioactive endogenous n − 3 and n − 6 CYP lipid mediators are released by short‐term exhaustive exercise in humans. In particular, the CYP epoxy‐metabolite status, 12,13‐EpOME/DiHOME, 5,6‐EET/DHET, 5,6‐EEQ/DiHETE and 17,18‐EEQ/DiHETE may contribute to the cardiovascular response during maximal exercise.

An Alternative Pathway to Leukotriene B4 Enantiomers Involving a 1,8-Diol-Forming Reaction of an Algal Oxylipin.

Oxidized lipids function as tissue hormones in mammals. An important class of these oxylipins are the immunomodulatory leukotrienes (LTs). Besides mammals, marine algae produce bioactive oxylipins, including LTs. The novel acid-labile oxylipin, (5 R,8 S)-dihydroxy eicosatetraenoic acid, from the edible alga Gracilaria vermiculophylla rearranges via a 1,8-diol-forming mechanism to inflammatory LTB4 enantiomers that exhibit an enantio-specific potency toward LTB4 receptor 1. This alternative pathway to a well-known leukotriene may explain food poisoning after Gracilaria consumption.

Human Metabolome Changes after a Single Dose of 3,4-Methylenedioxymethamphetamine (MDMA) with Special Focus on Steroid Metabolism and Inflammation Processes.

The intake of 3,4-methylenedioxymethamphetamine (MDMA) is known to increase several endogenous substances involved in steroid and inflammation pathways. Untargeted metabolomics screening approaches can determine biochemical changes after drug exposure and can reveal new pathways, which might be involved in the pharmacology and toxicology of a drug of abuse. We analyzed plasma samples from a placebo-controlled crossover study of a single intake of MDMA. Plasma samples from a time point before and three time points after the intake of a single dose of 125 mg MDMA were screened for changes of endogenous metabolites. An untargeted metabolomics approach on a high-resolution quadrupole time-of-flight mass spectrometer coupled to liquid chromatography with two different chromatographic systems (reversed-phase and hydrophobic interaction liquid chromatography) was applied. Over 10 000 features of the human metabolome were detected. Hence, 28 metabolites were identified, which showed significant changes after administration of MDMA compared with placebo. The analysis revealed an upregulation of cortisol and pregnenolone sulfate 4 h after MDMA intake, suggesting increased stress and serotonergic activity. Furthermore, calcitriol levels were decreased after the intake of MDMA. Calcitriol is involved in the upregulation of trophic factors, which have protective effects on brain dopamine neurons. The inflammation mediators hydroxyeicosatetraenoic acid, dihydroxyeicosatetraenoic acid, and octadecadienoic acid were found to be upregulated after the intake of MDMA compared with placebo, which suggested a stimulation of inflammation pathways.

Production of lipid mediators across different disease stages of dextran sodium sulfate-induced colitis in mice

Although several studies have revealed the role of different lipid mediators in colitis, the comprehensive analysis of their production across different phases of colitis remained unclear. Here, we performed the following analysis in the dextran sodium sulfate (DSS)-induced colitis model using LC-MS/MS. Oral administration of 2% DSS in mice for 4 days resulted in severe intestinal inflammation by day 7, which gradually subsided by day 18. Based on the disease scoring index (assigned on the basis of fecal condition and weight loss), we defined the phases of colitis as induction (days 0-4), acute inflammation (days 4-7), recovery (days 7-9), and late recovery (days 9-18). Across all phases, 58 lipid mediators were detected in the inflamed colon tissue. In the induction phase, the production of n-6 fatty acid-derived prostaglandin E2 and thromboxane B2 increased by ∼2-fold. In the acute inflammation phase, the production of n-6 fatty acid-derived leukotrienes increased by >10-fold, while that of n-3 fatty acid-derived hydroxyeicosapentaenoic acids and dihydroxyeicosatetraenoic acids decreased. In the recovery phase, a precursor of protectin D1 (17-hydroxydocosahexaenoic acid) increased over 3-fold. These observations suggested dynamic changes in the production of lipid mediators across different phases of the disease and their potential regulation in healing colitis.

Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology.

Cytochrome P450s enzymes catalyze the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acid and hydroxyeicosatetraeonic acid (HETEs). 20-HETE is a vasoconstrictor that depolarizes vascular smooth muscle cells by blocking K+ channels. EETs serve as endothelial derived hyperpolarizing factors. Inhibition of the formation of 20-HETE impairs the myogenic response and autoregulation of renal and cerebral blood flow. Changes in the formation of EETs and 20-HETE have been reported in hypertension and drugs that target these pathways alter blood pressure in animal models. Sequence variants in CYP4A11 and CYP4F2 that produce 20-HETE, UDP-glucuronosyl transferase involved in the biotransformation of 20-HETE and soluble epoxide hydrolase that inactivates EETs are associated with hypertension in human studies. 20-HETE contributes to the regulation of vascular hypertrophy, restenosis, angiogenesis and inflammation. It also promotes endothelial dysfunction and contributes to cerebral vasospasm and ischemia-reperfusion injury in the brain, kidney and heart. This review will focus on the role of 20-HETE in vascular dysfunction, inflammation, ischemic and hemorrhagic stroke and cardiac and renal ischemia reperfusion injury.

Urinary CYP eicosanoid excretion correlates with glomerular filtration in African-Americans with chronic kidney disease

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid (AA), i.e., 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), play an important role in the regulation of renal tubular and vascular function. The present study for the first time profiled HETEs and epoxygenase derived dihydroxyeicosatetraenoic acid diHETEs levels in spot urines and plasma in 262 African American patients from the University of Mississippi Chronic Kidney Disease Clinic and 31 African American controls. Significant correlations in eGFR and urinary 20-HETE/creatinine and 19-HETE/creatinine levels were observed. The eGFR increased by 17.47 [p=0.001] and 60.68 [(p=0.005]ml/min/for each ng/mg increase in 20-HETE and 19-HETE levels, respectively. Similar significant positive associations were found between the other urinary eicosanoids and eGFR and also with 19-HETE/urine creatinine concentration and proteinuria. We found that approximately 80% of plasma HETEs and 30% diHETEs were glucuronidated and the fractional excretion of 20-HETE was less than 1%. These results suggest that there is a significant hepatic source of urinary 20-HETE glucuronide and EETs with extensive renal biotransformation to metabolites which may play a role in the pathogenesis of CKD.

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.

Cytochrome P450 metabolites of arachidonic acid are elevated in stroke patients compared with healthy controls

CYP450AAM [arachidonic acid metabolites of the CYP450 (cytochrome P450) enzyme system] have a range of biological functions. CYP450AAM are involved in the pathogenesis of hypertension, renal function and vascular function, yet their role in stroke has not been clarified. We aimed at determining the levels of circulating CYP450 metabolites in patients with acute ischaemic stroke (<96h) compared with healthy age- and gender-matched controls. This was a retrospective case-controlled study of 44 acute ischaemic stroke patients and 44 matched controls. A subset of acute ischaemic stroke patients was available for follow-up. Acute ischaemic stroke patients had elevated plasma CYP450AAM, including 20-HETE (20-hydroxyeicosatetraenoic acid) (1921±170 compared with 1108±170pmol/l, P<0.001), EETs (epoxyeicosatrienoic acids) (77.88±3.34 compared with 35.35±3.34 nmol/l, P<0.0001) and DiHETEs (dihydroxyeicosatetraenoic acids) (92.87±4.61 compared with 68.17±4.61 nmol/l, P<0.0001), as well as increased plasma F2-isoprostane levels (3754±538 compared with 1947±538pmol/l, P<0.02), the latter a marker of oxidative stress, compared with controls. In a subset analysis of the stroke patients, plasma 20-HETE, EETs and F2-isoprostanes were attenuated 30days after the stroke. Baseline 20-HETE levels were also associated with lesion size and functional indices within the stroke patients. The present study highlights the elevation in CYP450AAM and oxidative stress in acute ischaemic stroke patients. Further investigation of the effect this has on long-term clinical outcome or whether this can be modified by treatment is warranted.

Epoxyeicosatrienoic acids induce growth inhibition and calpain/caspase-12 dependent apoptosis in PDGF cultured 3T6 fibroblast

Previous studies have demonstrated that arachidonic acid (AA) metabolites released by the cyclooxygenase pathway is involved in serum-induced 3T6 fibroblast cycle progression and proliferation. However, these results also suggest that other AA cascade pathways might be involved. Recently, we also described the role of hydroxyeicosatetraenoic acids, which are produced by cytochrome P450 monooxygenases (CYP), in 3T6 fibroblast growth. AA can be also metabolized by the epoxygenase activity of CYP-producing epoxyeicosatrienoic acids (EETs). Finally, the cytosolic epoxide hydrolases catalyze the hydration of the EETs, transforming them into dihydroxyeicosatetraenoic acids (DHETEs). In this work, we have studied the role of the EETs/DHETEs on 3T6 fibroblasts growth. Our results show that PDGF stimulates 3T6 fibroblast proliferation and [3H]thymidine incorporation, while the addition of 5,6-EET, 8,9-EET, 11,12-EET or 14,15-EET (0.1-1 microM) inhibit these processes. Furthermore, 5,6-DHETE and 11,12-DHETE (0.1-1 microM) also inhibit cell proliferation and DNA synthesis. Interestingly, this growth inhibition was correlated with an induction of apoptosis. Thus, we observed that in the presence of PDGF, EETs or DHETEs (0.1-1 microM) induce phosphatidylserine externalization (as measured by annexin V-binding) and DNA fragmentation (as quantified using a TUNEL assay). Our results show that calpain, as well as caspase-12 and caspase-3, are involved in these events. Therefore, EETs and DHETEs have anti-proliferative and pro-apoptotic effects on PDGF-stimulated 3T6 fibroblasts.

Role of endogenous CYP450 metabolites of arachidonic acid in maintaining the glomerular protein permeability barrier

This study examined the metabolism of arachidonic acid (AA) by cytochrome P-450 enzymes in isolated glomeruli and the effects of selective inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatetraenoic acids (EETs) on glomerular permeability to albumin (P(alb)). Glomeruli avidly produced 20-HETE, EETs, dihydroxyeicosatetraenoic acids (diHETEs), and HETEs when incubated with exogenous AA. N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 microM) selectively inhibited the formation of 20-HETE by 95% and increased P(alb) from 0.00 +/- 0.08 to 0.73 +/- 0.10 (n = 43 glomeruli, 4 rats). Addition of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (20-5,14-HEDE; 1 microM) opposed the effects of HET0016 (10 microM) to increase P(alb) (0.21 +/- 0.10, n = 36 glomeruli, 4 rats). Preincubation of glomeruli with exogenous AA to increase basal production of 20-HETE had a similar effect. We also examined the effect of an epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MSPPOH; 5 microM), on P(alb). MSPPOH (5 microM) significantly increased P(alb) but had no effect on the synthesis of EETs in glomeruli incubated with AA. However, MSPPOH (5 microM) selectively reduced epoxygenase activity by 50% in glomeruli incubated without added AA. Pretreatment with 8,9-EET (100 nM) attenuated the effects of MSPPOH (5 microM) on P(alb). These results indicate that glomeruli produce 20-HETE, EETs, diHETEs, and HETEs and that endogenously formed 20-HETE and EETs play an essential role in the maintenance of the glomerular permeability barrier to albumin.

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S),12(S)-dihydroxyeicosatetraenoic acid and 14(R),15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5- and 15-hydroxyeicosatetraenoic acids, and 5,15-dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)-dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R),15(S)-dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 μm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)-dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 × 105 for 15-HETE, 1.4 × 105 for 12-HETE, and 0.9 × 105 for 5-HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.—Dadaian, M., and P. Westlund. Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets. J. Lipid Res. 1999. 40: 940–947.