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A subject's baseline FA composition may influence the ability of dietary highly unsaturated omega-3 FAs (n3-HUFA) to change circulating profiles of esterified FAs and their oxygenated metabolites. This study evaluates the influence of basal n3-HUFA and n3-oxylipin status on the magnitude of response to n3-HUFA consumption. Blood was collected from fasting subjects (n = 30) before and after treatment (4 weeks; 11 ± 2 mg/kg/day n3-HUFA ethyl esters). Esterified FAs were quantified in erythrocytes, platelets, and plasma by GC-MS. Esterified oxylipins were quantified in plasma by LC-MS/MS. Treatment with n3-HUFAs increased n3-HUFAs and decreased n6-HUFAs in all reservoirs and increased plasma n3-oxylipins without significantly changing n6-oxylipin concentrations. As subject basal n3-HUFAs increased, treatment-associated changes decreased, and this behavior was reflected in the percentage of 20:5n3 + 22:6n3 in red blood cell membrane FAs (i.e., the omega-3 index). To maintain an omega-3 index of 8% and thus reduce cardiovascular disease risk, our analyses suggest a maintenance dose of 7 mg/kg/day n3-HUFA ethyl esters for a 70-kg individual. These results suggest that the basal n3 index may have clinical utility to establish efficacious therapeutic experimental feeding regimens and to evaluate the USDA Dietary Guidelines recommendations for n3-HUFA consumption.

Arachidonate 5-lipoxygenase (ALOX5) gene variants that are common in people of African ancestry are associated with a differential cardiovascular disease (CVD) risk that may be ameliorated by intake of (n-3) PUFA, such as EPA or DHA. We conducted a double-masked, placebo (PL)-controlled trial of fish oil (FO) supplements to determine if changes in erythrocyte (n-3) PUFA composition, heart rate, blood pressure, and plasma lipid and lipoprotein concentrations are modified by genotype. Participants received 5 g/d FO (2 g EPA, 1 g DHA) or 5 g/d corn/soy oil (PL). A total of 116 healthy adults of African ancestry with selected genotypes (genotypes = "dd," "d5," and "55" with "d" representing the deletion of 1 or 2 Sp1 binding sites in the ALOX5 promoter and "5" indicating the common allele with 5 sites) were enrolled and 98 completed the study. FO caused significant increases (relative to PL) in erythrocyte EPA, DHA, and total (n-3) PUFA and a decrease in the (n-6) PUFA:(n-3) PUFA ratio in the low-CVD risk "d5" and "55" genotypes but not in the high-risk "dd" genotype. Similarly, HDL particle concentration decreased with FO relative to PL in the "d5" and "55" but not "dd" genotypes. The plasma TG concentration decreased significantly with FO relative to PL in the "d5" but not "dd" and "55" genotypes. No changes were seen in LDL particle or cholesterol concentrations, heart rate, or blood pressure. These findings indicate that the efficacy of FO supplements vary by ALOX5 genotype.

The efficacy of resveratrol as a preventive agent against the growth of t(4;11) acute lymphoblastic leukemia (ALL) was evaluated in NOD.CB17-Prkdcscid/J mice engrafted with the human t(4;11) ALL SEM cell line. SEM cells were injected into the tail vein and engraftment was monitored by flow cytometry. Once engraftment was observed, mice were injected intraperitoneally with resveratrol (10 mg/kg body weight) dissolved in dimethylsulfoxide (DMSO) or DMSO alone (control) every other day, or vincristine (0.5 mg/kg body weight) 3 times per week for 4 weeks (n=16 per group). Comparisons of the percent of human leukemia cells in blood and survival curves showed resveratrol did not inhibit progression of the disease. Liquid chromatography-tandem mass spectrometry analyses of mouse sera showed resveratrol was rapidly metabolized to glucuronidated and sulfated forms 1 h post-injection, with low to no resveratrol or metabolites observed in sera by 24-48 h. These data indicate that in contrast to findings in in vitro models, parenterally administered resveratrol does not have potential as a preventive agent against high risk t(4;11) ALL.

Background: Established clinical tests are commonly used in disease diagnosis, but tools that enhance identification of metabolic dysfunctions are needed. This study was conducted to identify typical and atypical metabolite temporal patterns in response to paired meal challenge tests., Design: Metabolic responses to high and low glycemic index (GI) meals were tested in 24 healthy pre-menopausal women, aged 20-50 y, with BMI of 25-30 kg/m2 using a cross-over design. On test days, blood glucose, insulin, leptin and non-esterified fatty acids were measured after an overnight fasting, and for 8 h following test meal consumption. The data were range scaled, and multivariate statistics were used to assess the presence of distinct response groups to the meal challenge tests., Results: As expected, participants showed higher circulating glucose and insulin in response to the high GI compared to the low GI meal challenge. However, using range-scaling and Principal Component Analysis, three distinct groups were identified based on differential responses to the paired challenges. Members of the most populated group (n = 18) displayed little deviation from the expected response to the two meal challenges. Two minor groups (n = 3/group) with distinct responses were observed, one suggestive of sub-clinical insulin resistance, and the other suggestive of hyperleptinemia., Conclusions: The differential responses of glucose, insulin and leptin to low and high glycemic test meals revealed three response groups. Dietary intervention studies traditionally evaluate group responses, and aim to identify the overall effect in the population studied. In contrast, our study analyzed the variance in the meal challenge responses, using an integrated physiological approach, rather than a reductionist approach. This phenotyping approach may be useful for detecting subclinical metabolic dysfunctions, and it could contribute to improved personalized nutrition management. This study is registered in ClinicalTrials.gov, record #200210295.

Type 2 diabetes has profound effects on metabolism that can be detected in plasma. While increases in circulating non-esterified fatty acids (NEFA) are well-described in diabetes, effects on signaling lipids have received little attention. Oxylipins and endocannabinoids are classes of bioactive fatty acid metabolites with many structural members that influence insulin signaling, adipose function and inflammation through autocrine, paracrine and endocrine mechanisms. To link diabetes-associated changes in plasma NEFA and signaling lipids, we quantitatively targeted >150 plasma lipidome components in age- and body mass index-matched, overweight to obese, non-diabetic (n = 12) and type 2 diabetic (n = 43) African-American women. Diabetes related NEFA patterns indicated ∼60% increase in steroyl-CoA desaturase activity and ∼40% decrease in very long chain polyunsaturated fatty acid chain shortening, patterns previously associated with the development of nonalcoholic fatty liver disease. Further, epoxides and ketones of eighteen carbon polyunsaturated fatty acids were elevated >80% in diabetes and strongly correlated with changes in NEFA, consistent with their liberation during adipose lipolysis. Endocannabinoid behavior differed by class with diabetes increasing an array of N-acylethanolamides which were positively correlated with pro-inflammatory 5-lipooxygenase-derived metabolites, while monoacylglycerols were negatively correlated with body mass. These results clearly show that diabetes not only results in an increase in plasma NEFA, but shifts the plasma lipidomic profiles in ways that reflect the biochemical and physiological changes of this pathological state which are independent of obesity associated changes.

Metabolites of the cytochrome P450 (CYP) pathway may contribute to vasodilation of the vasculature. However, it is not known whether exercise affects their circulating concentrations. The authors determined effects of exercise intensity and duration on plasma concentrations of epoxy and dihydroxy metabolites of arachidonic acid. Their goal was to delineate the threshold workload, optimal workload, and duration required to produce increases in plasma concentrations of these vasoactive substances. Healthy volunteers (N = 14) performed maximal exercise testing on a bicycle ergometer during Visit 1. On separate days, subjects cycled for 20 min at 30%, 60%, and 80% of their maximal exercise intensity. The last day consisted of 40 min of exercise at 60% of maximal exercise intensity. Venous blood was obtained before, during, and after exercise for analysis. Compared with rest, increases were observed during the 80% workload at 20 min postexercise -14,15-DHET (0.77 ± 0.21 vs. 0.93 ± 0.27 nM) - and at 2 min postexercise: 11,12-DHET (0.64 ± 0.22 vs. 0.71 ± 0.24 nM; p < .05). Also compared with rest, 40-min values during the 60% workload were 14,15-DHET 0.79 ± 0.22 vs. 0.91 ± 0.31 nM and at 2 min post 14,15 EET 0.12 ± 0.06 vs. 0.21 ± 0.16 nM (p < .05). Results suggest the CYP metabolites (i.e., DHETs) are released during short-term high-intensity and long-term moderate-intensity exercise.

Consumption of industrially produced trans fatty acids (IP-TFA) has been positively associated with systemic markers of low-grade inflammation and endothelial dysfunction in cross-sectional studies, but results from intervention studies are inconclusive. Therefore, we conducted a 16 week double-blind parallel intervention study with the objective to examine the effect of IP-TFA intake on biomarkers of inflammation, oxidative stress, and endothelial dysfunction. Fifty-two healthy overweight postmenopausal women (49 completers) were randomly assigned to receive either partially hydrogenated soybean oil (15.7 g/day IP-TFA) or control oil without IP-TFA. After 16 weeks, IP-TFA intake increased baseline-adjusted serum tumor necrosis factor (TNF) α by 12% [95% confidence interval (CI): 5-20; P = 0.002] more in the IP-TFA group compared with controls. Plasma soluble TNF receptors 1 and 2 were also increased by IP-TFA [155 pg/ml (CI: 63-247); P < 0.001 and 480 pg/ml (CI: 72-887); P = 0.02, respectively]. Serum C-reactive protein, interleukin (IL) 6 and adiponectin and subcutaneous abdominal adipose tissue mRNA expression of IL6, IL8, TNFα, and adiponectin as well as ceramide content were not affected by IP-TFA, nor was urinary 8-iso-prostaglandin-F(2α). In conclusion, this dietary trial indicates that the mechanisms linking dietary IP-TFA to cardiovascular disease may involve activation of the TNFα system.

Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B(12)-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status. However, reported methods use between 100 and 1000 μL of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC-MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d(3)) were modified for use with 25 μL of human serum by scaling down sample processing volumes and analysis by UPLC-MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67 mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d(3) surrogate recovery averaged 93±14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25 μL of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5 μL of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected., (Published by Elsevier B.V.)

We evaluated the genetic contribution of the leukotriene (LT) pathway to risk of coronary artery disease (CAD) in 4,512 Caucasian and African American subjects ascertained through elective cardiac evaluation. Of the three previously associated variants, the shorter "3" and "4" alleles of a promoter repeat polymorphism in ALOX5 increased risk of CAD in African Americans (OR = 1.4, 95% CI 1.0-1.9; p = 0.04), whereas a haplotype of LTA4H (HapK) was associated with CAD in Caucasians (OR = 1.2, 95% CI 1.01-1.4; p = 0.03). In Caucasians, first-stage analysis of 254 haplotype-tagging SNPs in 15 LT pathway genes with follow-up of 19 variants in stage 2 revealed an LTA4H SNP (rs2540477) that increased risk of CAD (OR = 1.2, 95% CI 1.1-1.5; p = 0.003) and a PLA2G4A SNP (rs12746200) that decreased risk of CAD (OR = 0.7, 95% CI 0.6-0.9; p = 0.0007). The PLA2G4A rs12746200 variant also decreased risk of experiencing a major adverse cardiac event (MACE = myocardial infarction, stroke, or death) over 3 years of follow-up (HR = 0.7, 95% CI 0.5-0.9; p = 0.01), consistent with its cardioprotective effect. Functional experiments demonstrated that stimulated monocytes from carriers of LTA4H variants HapK or rs2540477 had 50% (p = 0.002) and 33% (p = 0.03) higher LTB(4) production, respectively, compared to non-carriers. These ex vivo results are consistent with LTB(4) being the direct product of the reaction catalyzed by LTA4H and its role in promoting monocyte chemotaxis to sites of inflammation, including the artery wall of atherosclerotic lesions. Taken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate atherogenic processes and the risk of CAD in humans.

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.

Background: Intake of industrially produced trans fatty acids (TFAs) is, according to observational studies, associated with an increased risk of cardiovascular disease, but the causal mechanisms have not been fully elucidated. Besides inducing dyslipidemia, TFA intake is suspected to promote abdominal and liver fat deposition., Objective: We examined the effect of a high intake of TFA as part of an isocaloric diet on whole-body, abdominal and hepatic fat deposition, and blood lipids in postmenopausal women., Methods: In a 16-week double-blind parallel intervention study, 52 healthy overweight postmenopausal women were randomized to receive either partially hydrogenated soybean oil providing 15.7 g day(-1) of TFA or a control oil with mainly oleic and palmitic acid. Before and after the intervention, body composition was assessed by dual-energy X-ray absorptiometry, abdominal fat by magnetic resonance (MR) imaging, and liver fat by (1)H MR spectroscopy., Results: Compared with the control fat, TFA intake decreased plasma high-density lipoprotein (HDL)-cholesterol by 10%, increased low-density lipoprotein (LDL)-cholesterol by 18% and resulted in an increased LDL/HDL-cholesterol ratio (baseline adjusted mean (95% CI) difference between diet groups 0.41 (0.22; 0.60); P<0.001). TFA tended to increase the body fat (0.46 (-0.20; 1.17) kg; P=0.16) and waist circumference (1.1 (-0.1; 2.4) cm; P=0.08) more than the control fat, whereas neither abdominal nor liver fat deposition was affected by TFA., Conclusion: The adverse effect of dietary TFA on cardiovascular disease risk involves induction of dyslipidemia, and perhaps body fat, whereas weight gain-independent accumulation of ectopic fat could not be identified as a contributory factor during short-term intake.

Insulin resistance progressing to type 2 diabetes mellitus (T2DM) is marked by a broad perturbation of macronutrient intermediary metabolism. Understanding the biochemical networks that underlie metabolic homeostasis and how they associate with insulin action will help unravel diabetes etiology and should foster discovery of new biomarkers of disease risk and severity. We examined differences in plasma concentrations of >350 metabolites in fasted obese T2DM vs. obese non-diabetic African-American women, and utilized principal components analysis to identify 158 metabolite components that strongly correlated with fasting HbA1c over a broad range of the latter (r = -0.631; p<0.0001). In addition to many unidentified small molecules, specific metabolites that were increased significantly in T2DM subjects included certain amino acids and their derivatives (i.e., leucine, 2-ketoisocaproate, valine, cystine, histidine), 2-hydroxybutanoate, long-chain fatty acids, and carbohydrate derivatives. Leucine and valine concentrations rose with increasing HbA1c, and significantly correlated with plasma acetylcarnitine concentrations. It is hypothesized that this reflects a close link between abnormalities in glucose homeostasis, amino acid catabolism, and efficiency of fuel combustion in the tricarboxylic acid (TCA) cycle. It is speculated that a mechanism for potential TCA cycle inefficiency concurrent with insulin resistance is "anaplerotic stress" emanating from reduced amino acid-derived carbon flux to TCA cycle intermediates, which if coupled to perturbation in cataplerosis would lead to net reduction in TCA cycle capacity relative to fuel delivery.

The long-chain omega-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) have beneficial health effects, but the molecular mediators of these effects are not well characterized. Oxygenated n-3 FAs (oxylipins) may be an important class of mediators. Members of this chemical class include epoxides, alcohols, diols, and ketones, many of which have bioactivity in vitro. Neither the presence of n-3 oxylipins in human plasma nor the effect of n-3 FA ingestion on their levels has been documented. We measured plasma oxylipins derived from both the n-3 and n-6 FA classes in healthy volunteers (n = 10) before and after 4 weeks of treatment with prescription n-3 FA ethyl esters (4 g/day). At baseline, EPA and DHA oxylipins were detected in low (1-50 nM) range, with alcohols > epoxides >or= diols. Treatment increased n-3 oxylipin levels 2- to 5-fold and reduced selected n-6 oxylipins by approximately 20%. This is the first documentation that endogenous n-3 oxylipin levels can be modulated by n-3 FA treatment in humans. The extent to which the beneficial cardiovascular effects of n-3 FAs are mediated by increased n-3 and/or reduced n-6 oxylipin levels remains to be explored.

Background: Chronic systemic low-grade inflammation in obese subjects is associated with health complications including cardiovascular diseases, insulin resistance and diabetes. Reducing inflammatory responses may reduce these risks. However, available markers of inflammatory status inadequately describe the complexity of metabolic responses to mild anti-inflammatory therapy., Methods: To address this limitation, we used an integrative omics approach to characterize modulation of inflammation in overweight men during an intervention with the non-steroidal anti-inflammatory drug diclofenac. Measured parameters included 80 plasma proteins, >300 plasma metabolites (lipids, free fatty acids, oxylipids and polar compounds) and an array of peripheral blood mononuclear cells (PBMC) gene expression products. These measures were submitted to multivariate and correlation analysis and were used for construction of biological response networks., Results: A panel of genes, proteins and metabolites, including PGE2 and TNF-alpha, were identified that describe a diclofenac-response network (68 genes in PBMC, 1 plasma protein and 4 plasma metabolites). Novel candidate markers of inflammatory modulation included PBMC expression of annexin A1 and caspase 8, and the arachidonic acid metabolite 5,6-DHET., Conclusion: In this study the integrated analysis of a wide range of parameters allowed the development of a network of markers responding to inflammatory modulation, thereby providing insight into the complex process of inflammation and ways to assess changes in inflammatory status associated with obesity., Trial Registration: The study is registered as NCT00221052 in clinicaltrials.gov database.

Eicosanoids, including epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and other oxylipins derived from polyunsaturated fatty acids, have emerging roles in endothelial inflammation and subsequent atherosclerosis. Unlike eicosanoids in the prostanoid series, they are known to be esterified in cell lipids such as phospholipids and triglycerides; however, our understanding of these reservoirs is in its infancy. This review focuses on recent work identifying circulating oxylipins, primarily esterified with lipoprotein lipids, and their effects on markers of endothelial dysfunction. These oxylipins are known to be released by at least one lipase (lipoprotein lipase) and to mediate increased expression of inflammatory markers in endothelial cells, which coincides with the known roles of lipoproteins in endothelial dysfunction. The implications of the lipolytic release of lipoprotein-bound oxylipins for the inflammatory response, challenges to analysis of this oxylipin compartment, and the potential importance of non-arachidonate-derived oxylipins are discussed.

The vagal afferent pathway is important in short-term regulation of food intake, and decreased activation of this neural pathway with long-term ingestion of a high-fat diet may contribute to hyperphagic weight gain. We tested the hypothesis that expression of genes encoding receptors for orexigenic factors in vagal afferent neurons are increased by long-term ingestion of a high-fat diet, thus supporting orexigenic signals from the gut. Obesity-prone (DIO-P) rats fed a high-fat diet showed increased body weight and hyperleptinemia compared with low-fat diet-fed controls and high-fat diet-induced obesity-resistant (DIO-R) rats. Expression of the type I cannabinoid receptor and growth hormone secretagogue receptor 1a in the nodose ganglia was increased in DIO-P compared with low-fat diet-fed controls or DIO-R rats. Shifts in the balance between orexigenic and anorexigenic signals within the vagal afferent pathway may influence food intake and body weight gain induced by high fat diets.

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.

Hypertension and Type 2 diabetes are co-morbid diseases that lead to the development of nephropathy. sEH (soluble epoxide hydrolase) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor AUDA [12-(3-adamantan-1-yl-ureido)-dodecanoic acid] protects the kidney from the development of nephropathy associated with hypertension and Type 2 diabetes. Hypertension was induced in spontaneously diabetic GK (Goto-Kakizaki) rats using AngII (angiotensin II) and a high-salt diet. Hypertensive GK rats were treated for 2 weeks with either AUDA or its vehicle added to drinking water. MAP (mean arterial pressure) increased from 118+/-2 mmHg to 182+/-20 and 187+/-6 mmHg for vehicle and AUDA-treated hypertensive GK rats respectively. AUDA treatment did not alter blood glucose. Hypertension in GK rats resulted in a 17-fold increase in urinary albumin excretion, which was decreased with AUDA treatment. Renal histological evaluation determined that AUDA treatment decreased glomerular and tubular damage. In addition, AUDA treatment attenuated macrophage infiltration and inhibited urinary excretion of MCP-1 (monocyte chemoattractant protein-1) and kidney cortex MCP-1 gene expression. Taken together, these results provide evidence that sEH inhibition with AUDA attenuates the progression of renal damage associated with hypertension and Type 2 diabetes.

We previously demonstrated that defects in lipoprotein metabolism alter the distribution of oxygenated polyunsaturated fatty acids (PUFAs) in lipoprotein particles. If these oxidation products are released by lipoprotein lipase (LpL), then their delivery to peripheral tissues with bulk lipids could influence cellular function. Using 26-week-old normolipidemic and hyperlipidemic Zucker rats, we measured PUFA alcohols, epoxides, diols, ketones, and triols (i.e. oxylipins) in esterified and non-esterified fractions of whole plasma, VLDL, and LpL-generated VLDL-lipolysates. Whole plasma, VLDL, and lipolysate oxylipin profiles were distinct and altered by hyperlipidemia. While >90% of the whole plasma oxylipins were esterified, the fraction of each oxylipin class in the VLDL varied: 46% of alcohols, 30% of epoxides, 19% of diols, <10% of ketones, and <1% triols. Whole plasma was dominated by arachidonate alcohols, while the linoleate alcohols, epoxides, and ketones showed an increased prevalence in VLDL. LpL-mediated VLDL lipolysis of PUFA alcohols, diols and ketones was detected and the relative abundance of oxygenated linoleates was enhanced in the lipolysates, relative to their corresponding VLDL. In summary esterified oxylipins were seen to be LpL substrates with heterogeneous distributions among lipoprotein classes. Moreover, oxylipin distributions are changes within the context of obesity-associated dyslipidemia. These results support the notion that the VLDL-LpL axis may facilitate the delivery of plasma oxylipins to the periphery. The physiological implications of these findings are yet to be elucidated; however, these molecules are plausible indicators of systemic oxidative stress, and could report this status to the peripheral tissues.

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (-/-) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17beta-estradiol levels were reduced in female Cyp2j5 (-/-) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (-/-) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex-specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen-dependent mechanism.

Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid.

Neuronal expression of familial Alzheimer's disease-mutant human amyloid precursor protein (hAPP) and hAPP-derived amyloid-beta (Abeta) peptides causes synaptic dysfunction, inflammation and abnormal cerebrovascular tone in transgenic mice. Fatty acids may be involved in these processes, but their contribution to Alzheimer's disease pathogenesis is uncertain. We used a lipidomics approach to generate a broad profile of fatty acids in brain tissues of hAPP-expressing mice and found an increase in arachidonic acid and its metabolites, suggesting increased activity of the group IV isoform of phospholipase A(2) (GIVA-PLA(2)). The levels of activated GIVA-PLA(2) in the hippocampus were increased in individuals with Alzheimer's disease and in hAPP mice. Abeta caused a dose-dependent increase in GIVA-PLA(2) phosphorylation in neuronal cultures. Inhibition of GIVA-PLA(2) diminished Abeta-induced neurotoxicity. Genetic ablation or reduction of GIVA-PLA(2) protected hAPP mice against Abeta-dependent deficits in learning and memory, behavioral alterations and premature mortality. Inhibition of GIVA-PLA(2) may be beneficial in the treatment and prevention of Alzheimer's disease.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.

Pyridine is a prototypical inducer of cytochrome P450 (CYP) 2E1, an enzyme associated with cellular oxidative stress and membrane damage. To better understand the effect of this treatment on cellular lipids, the influence of pyridine exposure (100 mg/kg/day i.p. for 5 days) on fatty acids, fatty esters, and fatty alcohol ethers in brain, heart, liver, and adipose tissue from male Swiss Webster mice was investigated. Lipid levels in cholesterol esters, triglycerides, free fatty acids, cardiolipin, sphingomyelin, and glycerylphospholipids were quantified. Pyridine altered the level and composition of lipids involved in membrane structure (i.e., sphingomyelin, phosphatidylethanolamines, and plasmalogens), energy metabolism (i.e., free fatty acids), and long-chain fatty acid transport (i.e., cholesterol esters) in a tissue-specific manner. Subtle changes in cholesterol esters were observed in all tissues. Sphingomyelin in the brain and heart were depleted in monounsaturated fatty acids (1.4- and 1.5-fold, respectively), while the liver sphingomyelin concentrations increased (1.5-fold). Pyridine exposure also increased heart free fatty acids by 1.3-fold, enriched cardiac phosphatidylethanolamine in long-chain polyunsaturated fatty acids by 1.3-fold, and depleted cardiolipin-associated plasmalogens by 3.8-fold. Phosphatidylethanolamines in the brain were also enriched in both saturated fatty acids (1.2-fold) and polyunsaturated fatty acids (1.3-fold) but were depleted in plasmalogens (2.9-fold). In particular, the levels of phosphatidylethanolamine-associated arachidonic (AA) and docosahexaenoic acid (DHA) in both brain and cardiac tissues significantly decreased following pyridine exposure. Considering the hypothetical role of plasmalogens as membrane-bound reactive oxygen scavengers, the current findings suggest that the brain and heart should be the focus of future studies on the toxicity of pyridine, as well as other CYP 2E1 inducers.

Increased dietary linoleic acid has been associated with reduced blood pressure in clinical and animal studies possibly mediated by prostaglandins. Urinary linoleate and prostaglandin metabolite excretion were investigated in subjects exposed to a salt-loading/salt-depletion regimen. Twelve healthy subjects were recruited from the New Orleans population (before Hurricaine Katrina) and admitted to the Tulane-Louisiana State University-Charity Hospital General Clinical Research Center after a 5-day outpatient lead-in phase on a 160-mmol sodium diet. On inpatient day 1, the subjects were maintained on the 160-mmol sodium diet, and a 24-hour urine specimen was collected. On day 2, the subjects received 2 L of IV normal saline over 4 hours and continued on a 160-mmol Na(+) diet (total: 460 mmol of sodium). Two 12-hour urine collections were obtained. On day 3, the subjects received three 40-mg oral doses of furosemide, two 12-hour urine collections were obtained, and the subjects were given a 10-mmol sodium diet. Urinary oxidized lipids were measured by high-performance liquid chromatography-tandem quadrupole mass spectroscopy. The excretion of the urinary linoleate metabolites, dihydroxyoctadecamonoenoic acids, and trihydroxyoctadecamonoenoic acids increased significantly during intravenous salt loading as compared with day 1 and the salt-depleted periods. The urinary excretion of 6-keto- prostaglandin F1alpha was unaffected by salt loading but was dramatically increased 7- to 10-fold by salt depletion. Prostaglandin E2 excretion was positively correlated with sodium excretion. The salt-stimulated production of linoleic acid diols and triols may inhibit tubular sodium reabsorption, thereby assisting in the excretion of the sodium load.

We previously established that proteinuria alters the apolipoprotein content of lipoproteins. This study was conducted to establish whether proteinuria also alters the concentrations of oxidized lipids within lipoprotein density fractions. To this end, we induced passive Heymann nephritis in Sprague Dawley rats and measured an array of alkaline-stable oxylipids in VLDL, LDL, and HDL particles. Proteinuria increased the total oxylipid amounts in the HDL and VLDL fractions. More importantly, these levels were increased when expressed per unit lipoprotein protein, indicating that the oxidized lipid load per particle was increased. Epoxides and diols increased approximately 2-fold in HDL and approximately 5-fold in VLDL, whereas LDL showed approximately 2-fold decreases. The hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids (HODEs) increased >4-fold in HDL and >20-fold in VLDL, whereas LDL showed approximately 2-fold decreases in the HODEs. Therefore, nephrotic syndrome alters the lipoprotein oxylipid composition independently of an increase in total lipoprotein levels. These proteinuria-induced changes may be associated with the cardiovascular risk of lipoprotein oxidation.

Peroxisome proliferator activated receptor alpha (PPARα) agonists are anti-hyperlipidemic drugs that influence fatty acid combustion, phospholipid biosynthesis and lipoprotein metabolism. To evaluate impacts on other aspects of lipid metabolism, we applied targeted metabolomics to liver, heart, brain and white adipose tissue samples from male Swiss-Webster mice exposed to a 5 day, 500 mg/kg/day regimen of i.p. clofibrate. Tissue concentrations of free fatty acids and the fatty acid content of sphingomyelin, cardiolipin, cholesterol esters, triglycerides and phospholipids were quantified. Responses were tissue-specific, with changes observed in the liver > heart ≫ brain > adipose. These results indicate that liver saturated fatty acid-rich triglycerides feeds clofibrate-induced monounsaturated fatty acid (MUFA) synthesis, which were incorporated into hepatic phospholipids and sphingomyelin. In addition, selective enrichment of docosahexeneoic acid in the phosphatidylserine of liver (1.7-fold), heart (1.6-fold) and brain (1.5-fold) suggests a clofibrate-dependent systemic activation of phosphatidylserine synthetase 2. Furthermore, the observed ~20% decline in cardiac sphingomyelin is consistent with activation of a sphingomeylinase with a substrate preference for polyunsaturate-containing sphingomyelin. Finally, perturbations in the liver, brain, and adipose cholesterol esters were observed, with clofibrate exposure elevating brain cholesterol arachidonyl-esters ~20-fold. Thus, while supporting previous findings, this study has identified novel impacts of PPARα agonist exposure on lipid metabolism that should be further explored.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.

We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human soluble epoxide hydrolase, and obtained very potent compounds (K(I)=15nM) that are >10-fold more soluble than previously described sEH inhibitors. While our lead compound 2 showed low apparent bioavailability in dogs and rats, this series of compounds revealed that sEH inhibitor structures could accept large groups that could lead to better orally available drugs.

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.

Endothelial dysfunction contributes to the development of coronary heart disease (CHD). Soluble epoxide hydrolase metabolizes epoxyeicosatrienoic acids in the vasculature and regulates endothelial function. We sought to determine whether genetic variation in soluble epoxide hydrolase (EPHX2) was associated with the risk of CHD. We genotyped 2,065 Atherosclerosis Risk in Communities study participants (1,085 incident CHD cases, 980 non-cases) for 10 previously identified polymorphisms in EPHX2. Using a case-cohort design, associations between incident CHD risk and both non-synonymous EPHX2 polymorphisms and phase-reconstructed haplotypes were evaluated using proportional hazards regression. Individuals carrying the K55R polymorphism variant allele demonstrated higher apparent soluble epoxide hydrolase activity in vivo. Presence of the K55R variant allele was significantly more common among Caucasian CHD cases when compared with non-cases (20.8% versus 15.3%, respectively, P=0.012), and was associated with significantly higher risk of incident CHD (adjusted hazard rate ratio 1.45, 95% confidence interval 1.05-2.01, P=0.026). A significant association between the K55R variant allele and risk of CHD was not observed in African-Americans. The distribution of reconstructed haplotypes were significantly different in Caucasian cases when compared with non-cases (P=0.021). Significant differences in haplotype distribution were not observed in African-Americans (P=0.315). Genetic variation in EPHX2 was significantly associated with risk of incident CHD in Caucasians, implicating EPHX2 as a potential cardiovascular disease-susceptibility gene.

Conjugated linoleic acid (CLA) isomers have unique effects on tissue lipids. Here we investigated the influence of individual CLA isomers on the lipid weight and fatty acid composition of lipid metabolizing (i.e. liver and retroperitoneal adipose) and lipid sensitive (i.e. spleen and heart) tissues. Female mice (8 week old; n=6/group) were fed either a control or one of the two CLA isomer supplemented (0.5%) diets for 8 weeks. The cis-9, trans-11-CLA diet reduced the 18:1n-9 wt% by 20-50% in liver, adipose tissue, and spleen, reduced the spleen n-3 polyunsaturated fatty acid (PUFA) by 90%, and increased the n-6 PUFA wt% by 20-50% in all tissues except heart. The trans-10, cis-12-CLA reduced both the n-6 and n-3 PUFA wt% in liver (>50%), reduced the heart n-3 PUFA wt% by 25%, and increased the wt% of spleen n-3 PUFA by 700%. The functional consequences of such changes in tissue fatty acid composition need to be investigated.

Epoxyeicosatrienoic acid(s) (EET) have variable hemodynamic, anti-inflammatory, and growth regulatory effects, and inhibitors of their regulatory enzyme, soluble epoxide hydrolase (sEH), can mimic these effects. For this reason, sEH inhibitors are being studied as potential pharmaceuticals for the treatment of hypertension, atherosclerosis, and inflammatory diseases. We now show that a highly selective urea-based sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) attenuates human aortic vascular smooth muscle (HVSM) cell proliferation independently of any effect on sEH. CDU also inhibits endothelial cells when stimulated with basic fibroblast growth factor or serum. In addition, we demonstrate that EET, as well as several newer generation sEH inhibitors and a urea-based weak sEH inhibitor, do not affect proliferation in HVSM cells. Structure-activity relationships demonstrate that the addition of an acid group to the dodecyl carbon chain, changing the cyclohexyl group to an adamantyl group, and shortening the carbon chain to two carbons all abolish the antiproliferative effect. Our finding that a highly selective urea-based inhibitor of sEH can alter biology independently of its putative target enzyme suggests that there may be other useful properties of this class of compounds unrelated to their influence on epoxyeicosanoids. In addition, our results show that caution should be used when attempting to infer conclusions of EET biology based solely on the effects these inhibitors in tissue culture models, especially when used at micromolar concentrations.

Soluble epoxide hydrolase (sEH) inhibitors have been demonstrated to have cardiovascular protective actions. This hydrolase enzyme converts fatty acid epoxides to their corresponding diols, and this conversion can alter the biologic activity of these metabolites. We hypothesized that 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a sEH inhibitor, would protect stroke-prone spontaneously hypertensive rats from cerebral ischemia. AUDA was administered to 6-week-old male rats for 6 weeks, during which blood pressure was measured by telemetry. Cerebral ischemia was induced by middle cerebral artery occlusion, the size of the cerebral infarct was assessed after 6 hours of ischemia, and the results were expressed as a percentage of the hemisphere infarcted (%HI). Vascular structure and function were assessed using a pressurized arteriograph. Plasma levels of AUDA at the end of the treatment period averaged 5.0 +/- 0.4 ng/mL, and the urinary excretion rate was 99 +/- 21 ng/d. AUDA-treated rats had significantly smaller cerebral infarcts than control rats (36 +/- 4% vs 53 +/- 4% HI, treated versus control, P < 0.05, n = 6). This difference occurred independently of changes in blood pressure. AUDA treatment increased the passive compliance of the cerebral vessels but had no effect on vascular structure. The results of this study provide novel evidence suggesting that the sEH inhibitor AUDA is a possible therapeutic agent for ischemic stroke.

The present study tested the hypothesis that increasing epoxyeicosatrienoic acids by inhibition of soluble epoxide hydrolase (sEH) would lower blood pressure and ameliorate renal damage in salt-sensitive hypertension. Rats were infused with angiotensin and fed a normal-salt diet or an 8% NaCl diet for 14 days. The sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), was given orally to angiotensin-infused animals during the 14-day period. Plasma AUDA metabolite levels were measured, and they averaged 10+/-2 ng/mL in normal-salt angiotensin hypertension and 19+/-3 ng/mL in high-salt angiotensin hypertension on day 14 in the animals administered the sEH inhibitor. Mean arterial blood pressure averaged 161+/-4 mm Hg in normal-salt and 172+/-5 mm Hg in the high-salt angiotensin hypertension groups on day 14. EH inhibitor treatment significantly lowered blood pressure to 140+/-5 mm Hg in the normal-salt angiotensin hypertension group and to 151+/-6 mm Hg in the high-salt angiotensin hypertension group on day 14. The lower arterial blood pressures in the AUDA-treated groups were associated with increased urinary epoxide-to-diol ratios. Urinary microalbumin levels were measured, and ED-1 staining was used to determine renal damage and macrophage infiltration in the groups. Two weeks of AUDA treatment decreased urinary microalbumin excretion in the normal-salt and high-salt angiotensin hypertension groups and macrophage number in the high-salt angiotensin hypertension group. These data demonstrate that sEH inhibition lowers blood pressure and ameliorates renal damage in angiotensin-dependent, salt-sensitive hypertension.

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.

As of 2004, >73 million people were prescribed antiinflammatory medication. Despite the extensive number of current products, many people still suffer from their diseases or the pharmacological properties (side effects) of the medications. Therefore, developing therapeutic strategies to treat inflammation remains an important endeavor. Here, we demonstrate that the soluble epoxide hydrolase (sEH) is a key pharmacologic target for treating acute systemic inflammation. Lipopolysaccharide-induced mortality, systemic hypotension, and histologically evaluated tissue injury were substantially diminished by administration of urea-based, small-molecule inhibitors of sEH to C57BL/6 mice. Moreover, sEH inhibitors decreased plasma levels of proinflammatory cytokines and nitric oxide metabolites while promoting the formation of lipoxins, thus supporting inflammatory resolution. These data suggest that sEH inhibitors have therapeutic efficacy in the treatment and management of acute inflammatory diseases.

Nephrotic syndrome is characterized by increased triglycerides resulting from decreased clearance of VLDL and chylomicrons. These triglyceride-rich lipoproteins are structurally altered by interaction with HDL derived from animals with proteinuria and not as a consequence of hypoalbuminemia. HDL isolated from rats with massive proteinuria is depleted in apolipoprotein E (apoE). It is unknown at what threshold of urinary albumin loss HDL structure is altered, and it is unknown what effects proteinuria has on apolipoproteins other than apoE. Two models of albuminuria were used in Sprague-Dawley rats: Adriamycin and passive Heymann nephritis (HN). The adriamycin group was divided into minimal albumin excretion (MAE) and intermediate albumin excretion (MAE, 1 to 40; intermediate albumin excretion, 60 to 210 mg/d per 100 g body wt). Urinary albumin excretion exceeded 300 mg/d per 100 g body wt in the HN rats. HDL apolipoprotein composition was analyzed with SDS-PAGE densitometry and liquid chromatography-time of flight mass spectrometer mass spectrometry. HDL apoA-IV content relative to apoA-I was reduced at all levels of albuminuria (P < 0.0001). ApoE was not reduced in MAE but was significantly reduced in IAE (72%; P < 0.001). By contrast, apoA-II and apoC-III were each significantly increased with increasing UAE. ApoA-IV and apoE were decreased to approximately 10% of control in HDL isolated from rats with HN, whereas apoA-II, apoC-II, and apoC-III were each significantly increased relative to apoA-I. HDL is structurally altered by levels of albuminuria that are insufficient to change serum albumin levels and is progressively altered as albuminuria increases.

The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentrations of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass spectrometry. Arachidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids (DHETs) were also assessed in thecal and granulosa tissue fractions, and the expression of CYP epoxygenases was evaluated by immunoblots using available antisera. To evaluate soluble epoxide hydrolase (sEH) expression, the porcine sEH was cloned from ovarian tissue, expressed and purified for antibody generation. The follicular fluid oxylipin concentrations ranged from 1-150 nm depending on the compound and estrous stage. The follicular fluid concentrations of CYP-dependent oxylipins increased at estrus, as did sEH expression; however, significant changes in epoxides were not observed, and the 11,12-DHET peak was delayed. The ratio of 14,15-DHET:11,12-DHET across all samples correlated with the log of follicular fluid estradiol concentrations (P < 0.01). Epoxygenase activities were similar in theca and granulosa, varying little with follicular development, whereas the decline of a single CYP2J isoform at ovulation was observed by immunoblots. The sEH activity was higher in granulosa than in theca. Finally, the dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation, which may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies.

Objective: To determine cytochrome P450 (CYP450) and cyclooxygenase (COX) expression and metabolite regulation and renal damage in the early stages of obesity-related hypertension and diabetes., Research Methods and Procedures: Obese and lean Zucker rats at 10 to 12 weeks of age were studied. Blood pressure was measured in the conscious state using radiotelemetry. Blood glucose levels and body weight were measured periodically. Protein expression of CYP450 and COX enzymes in the kidney cortex, renal microvessels, and glomeruli was studied. The levels of CYP450 and COX metabolites in urine were measured, and urinary albumin excretion, an indicator of kidney damage, was measured., Results: Body weight and blood glucose averaged 432 +/- 20 grams and 105 +/- 5 mg/dl, respectively, in obese Zucker rats as compared with 320 +/- 8 grams and 91 +/- 5 mg/dl, respectively, in age-matched 10- to 12-week-old lean Zucker rats. Renal microvascular CYP4A and COX-2 protein levels were increased 2.3- and 17.0-fold, respectively, in obese Zucker rats. The protein expression of CYP2C11 and CYP2C23 was decreased 2.0-fold in renal microvessels isolated from obese Zucker rats when compared with lean Zucker rats. The urinary excretion rate of thromboxane B(2) was increased significantly in obese Zucker as compared with lean Zucker rats (22.0 +/- 1.8 vs. 13.4 +/- 1.0 ng/d). Urinary albumin excretion, an index of kidney damage, was increased in the obese Zucker rat at this early age., Discussion: These results suggest that increased CYP4A and COX-2 protein levels and decreased CYP2C11 and CYP2C23 protein levels occur in association with microalbuminuria during the onset of obesity-related hypertension and type 2 diabetes.

Epoxyeicosatrienoic acids (EET) have antihypertensive and anti-inflammatory properties and play a role in the maintenance of renal vascular function. A novel approach to increase EET levels is to inhibit epoxide hydrolase enzymes that are responsible for conversion of biologically active EET to dihydroxyeicosatrienoic acids (DHET). We hypothesized that soluble epoxide hydrolase (SEH) inhibition would improve renal vascular function and ameliorate hypertension induced renal damage. Chronic administration of the specific SEH inhibitor 1-cyclohexyl-3-dodecylurea (CDU, 3 mg/d) for 10 d lowered BP in angiotensin hypertensive rats. The contribution of renal vascular SEH to afferent arteriolar function in angiotensin hypertension was also assessed. SEH protein expression was increased in renal microvessels from hypertensive rats. Although CDU did not change afferent arteriolar responsiveness to angiotensin in normotensive animals, CDU treatment significantly attenuated afferent arteriolar diameter responses to angiotensin in hypertensive kidneys from 51% +/- 8% to 28% +/- 7%. Protection of the renal vasculature and glomerulus during chronic CDU administration was demonstrated by histology. Urinary albumin excretion, an index of renal damage, was also lower in CDU-treated hypertensive rats. These data demonstrate that SEH inhibition has antihypertensive and renal vascular protective effects in angiotensin hypertension and suggests that SEH inhibitors may be a useful therapeutic intervention for cardiovascular diseases.

Epoxide hydrolases play an important role in detoxifying epoxides that arise from the metabolism of xenobiotic and endogenous compounds. Both the soluble and microsomal forms of epoxide hydrolase (sEH and mEH, respectively) have been detected in the rat testis. Because of the important role the epididymis plays in sperm maturation and protection, the present study evaluated the presence and activity of these two epoxide hydrolases in the rat epididymis. Using Western blotting, protein bands consistent in size with both mEH and sEH were detected in the caput, corpus, and cauda of the epididymis. The mEH immunoreactive bands in the epididymis ( approximately 50 kDa) were consistent with mEH detected in the liver and kidney. The sEH immunoreactive bands in the epididymis ( approximately 65 kDa) were consistent with a recombinant sEH standard and sEH detected in the liver, kidney, and testis. The presence of mEH and sEH in the epididymis was supported by observations from substrate-based enzyme assays. Results indicated that epididymal mEH can hydrolyze [(3)H]-cis-stilbene oxide to the corresponding diol at levels approximately 9% of the kidney. Epididymal sEH hydrolyzed the substrate [(3)H]-trans-diphenylpropene oxide to the corresponding diol and this activity was inhibited by cyclohexyl-dodecyl urea. Arachidonic acid epoxygenase activity was detected in epididymal S9 fractions, suggesting that fatty acid metabolism by epididymal cytochrome P450s can form epoxides that subsequently become substrates for epididymal sEH. Results from the present study indicate that the epididymis contains at least two active forms of epoxide hydrolase. The role of these enzymes in the detoxification of xenobiotic epoxides is well known, although it is unclear what cellular role they may play in the formation of biologically active metabolites in the epididymis.

This paper compares two previously published methods, an Aroclor estimation method and a mixing model method, that relate Aroclor contamination to congener specific data in environmental samples. The Aroclor estimation method, which is consistent with U.S. EPA Method 8082, uses a limited set of congener specific data to estimate Aroclor contributions to the sample, while the mixing model method uses the full congener data to model sample compositions as linear combinations of Aroclors. The performance of these methods are compared, using 181 samples at a variety of trophic levels, in terms of (a) total PCB concentrations, (b) compositional modification levels from original Aroclors, and (c) determination of the Aroclor mixture or mixtures best describing the sample (Aroclor speciation). We find that the two methods agree in all three terms for samples of low trophic level, but disagree for samples of higher tropic levels. Most significantly, the comparison reveals systematic overestimation of total PCB content by the Aroclor estimation method for samples at high trophic levels. The implication is that Aroclor determinations using persistent congeners cannot reliably be used as surrogates for total PCB concentration. The strengths and weaknesses of each method are detailed.

Objective: Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD)., Methods: The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry., Results: Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations., Conclusion: Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.

The metabolism of the soluble epoxide hydrolase (sEH) inhibitor, 1-cyclohexyl-3-dodecyl-urea (CDU), was studied in rat and human hepatic microsomes. The microsomal metabolism of CDU enhanced sEH inhibition potency of the reaction mixture and resulted in the formation of several metabolites. During the course of this study, a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry analytical method was developed to investigate simultaneously the production of these metabolites. In both rat and human hepatic microsomes, CDU was ultimately transformed into the corresponding omega-carboxylate; however, the rodent tissue appeared to perform this transformation more rapidly. After a 60-min incubation in rat hepatic microsomes, the percentage of residual CDU, the omega-carboxylate, and the intermediary omega-hydroxyl were about 20%, 20%, and 50%, respectively. Carbon monoxide inhibited the metabolism of CDU by rat hepatic microsomes, suggesting that the initial step is catalyzed by cytochrome P450. Further metabolism was enhanced by the addition of NAD, suggesting that dehydrogenases are associated with intermediate metabolic steps. Regardless, the ultimate product of microsomal metabolism, 12-(3-cyclohexyl-ureido)-dodecanoic acid, is also an excellent sEH inhibitor with several hundred-fold higher solubility, supporting the hypothesis that CDU has prodrug characteristics. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties and improved metabolic stability.

Swine vesicular disease virus (SVDV) is an Enterovirus of the family Picornaviridae that causes symptoms indistinguishable from those of foot-and-mouth disease virus. Phylogenetic studies suggest that it is a recently evolved genetic sublineage of the important human pathogen coxsackievirus B5 (CBV5), and in agreement with this, it has been shown to utilize the coxsackie and adenovirus receptor (CAR) for cell entry. The 3.0-A crystal structure of strain UK/27/72 SVDV (highly virulent) reveals the expected similarity in core structure to those of other picornaviruses, showing most similarity to the closest available structure to CBV5, that of coxsackievirus B3 (CBV3). Features that help to cement together and rigidify the protein subunits are extended in this virus, perhaps explaining its extreme tolerance of environmental factors. Using the large number of capsid sequences available for both SVDV and CBV5, we have mapped the amino acid substitutions that may have occurred during the supposed adaptation of SVDV to a new host onto the structure of SVDV and a model of the SVDV/CAR complex generated by reference to the cryo-electron microscopy-visualized complex of CBV3 and CAR. The changes fall into three clusters as follows: one lines the fivefold pore, a second maps to the CAR-binding site and partially overlaps the site for decay accelerating factor (DAF) to bind to echovirus 7 (ECHO7), and the third lies close to the fivefold axis, where the low-density lipoprotein receptor binds to the minor group of rhinoviruses. Later changes in SVDV (post-1971) map to the first two clusters and may, by optimizing recognition of a pig CAR and/or DAF homologue, have improved the adaptation of the virus to pigs.

The gene EPXH2 encodes for the soluble epoxide hydrolase (sEH), an enzyme involved in the regulation of cardiovascular and renal physiology containing two distinct domains connected via a proline-rich linker. The C-terminal domain containing the EH catalytic activity has been well studied. In contrast, a function for the N-terminal domain, which has high homology to the haloacid dehalogenase family of phosphatases, has not been definitively reported. In this study we describe the N-terminal domain as a functional phosphatase unaffected by a number of classic phosphatase inhibitors. Assuming a functional association between these catalytic activities, dihydroxy lipid phosphates were rationalized as potential endogenous substrates. A series of phosphorylated hydroxy lipids were therefore synthesized and found to be excellent substrates for the human sEH. The best substrate tested was the monophosphate of dihydroxy stearic acid (threo-910-phosphonoxy-hydroxy-octadecanoic acid) with K(m) = 21 +/- 0.3 microM, V(Max) = 338 +/- 12 nmol x min(-1) x mg(-1), and k(cat) = 0.35 +/- 0.01 s(-1). Therefore dihydroxy lipid phosphates are possible candidates for the endogenous substrates of the sEH N-terminal domain, which would represent a novel branch of fatty acid metabolism with potential signaling functions.