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Rationale: A high-fat diet accompanied by hypertriglyceridemia increases an individual’s risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. Objective: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. Methods and Results: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-α alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. Conclusions: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

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.

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease. Figure LC-MS/MS chromatogram of 104 oxylipins on a Triple Quadrupole MS system employing dynamic MRM in the Negative Ion Electrospray mode Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6226-x) contains supplementary material, which is available to authorized users.

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 LTB4 production, respectively, compared to non-carriers. These ex vivo results are consistent with LTB4 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. Electronic supplementary material The online version of this article (doi:10.1007/s00439-011-0963-3) contains supplementary material, which is available to authorized users.

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.

Food intake, physical activity (PA), and genetic makeup each affect health and each factor influences the impact of the other 2 factors. Nutrigenomics describes interactions between genes and environment. Knowledge about the interplay between environment and genetics would be improved if experimental designs included measures of nutrient intake and PA. Lack of familiarity about how to analyze environmental variables and ease of access to tools and measurement instruments are 2 deterrents to these combined studies. This article describes the state of the art for measuring food intake and PA to encourage researchers to make their tools better known and more available to workers in other fields. Information presented was discussed during a workshop on this topic sponsored by the USDA, NIH, and FDA in the spring of 2009.

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.

Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA beta-oxidation in the latter (i.e. summed C10- to C14-carnitine concentrations were approximately 300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c (r = 0.544, P < 0.0001; and r = -0.308, P < 0.05, respectively) and with increasing total plasma acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 acylcarnitines significantly stimulated nuclear factor kappa-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA beta-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance.

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.

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.

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 F1α 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 den- sity 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 pro- tein, indicating that the oxidized lipid load per particle was increased. Epoxides and diols increased ?2-fold in HDL and ?5-fold in VLDL, whereas LDL showed ?2-fold de- creases. The hydroxyeicosatetraenoic acids and hydroxy- octadecadienoic acids (HODEs) increased .4-fold in HDL and .20-fold in VLDL, whereas LDL showed ?2-fold de- creases 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.—Newman, J. W., G. A. Kaysen, B. D. Hammock, and G. C. Shearer. Proteinuria increases oxylipid concentrations in VLDL and HDL but not LDL particles in the rat. J. Lipid Res. 2007. 48: 1792-1800.

Mice deficient for zinc transporter 7 protein (ZnT7) are mildly zinc deficient with low body weight gain and body fat accumulation. To investigate the underlying mechanism of ZnT7 deficiency in body adiposity, we examined fatty acid composition and insulin sensitivity in visceral (epididymal) and subcutaneous fat pads from Znt7 knockout and control mice. We showed that ZnT7 deficiency had adverse effects on fatty acid metabolism and insulin action in subcutaneous fat but not in epididymal fat in mice, consistent with the ZnT7 protein expression pattern in adipose tissues. Importantly, we found that the expression of ZnT7 protein was induced by lipogenic differentiation and reached a peak when the adipocyte was fully differentiated in mouse 3T3-L1 adipocytes. We demonstrated, using Znt7 knockdown (Znt7KD) 3T3-L1 adipocytes, that reduction in Znt7 expression blunted activations of the signal transduction pathways that regulated both basal and insulin-stimulated glucose uptake in adipocytes, resulting in low glucose uptake and lipid accumulation. The expression of the signaling mediators critical for the initiation of pre-adipocyte differentiation, including Pparγ and C/Ebpα, appeared not to be affected by Znt7KD in 3T3-L1 adipocytes. These findings strongly suggest a role for ZnT7 in adipocyte lipogenesis.

Background: Lipid mediators are transported in plasma, inducing pro- or anti-inflammatory responses in target tissues. Each daily nut serving is associated with 30% reduction in risk for cardiovascular or ischemic heart disease. Walnuts are an abundant source of bioactives and of precursors to lipid mediator synthesis: linoleic (LA) and alpha-linolenic (aLA) acids. How walnuts impact plasma lipid mediators either by providing more substrate or more specifically via other bioactives is unknown. Objective: Measure lipid mediators from the cyclooxygenase (COX), lipoxygenase (LOX), cytochrome p450 (CYP), and auto-oxidative pathways in plasma from subjects consuming large and small amounts of walnuts. Design: 40 hypercholesterolemic, postmenopausal females were randomly assigned to a null dose (5g/d) or an active dose (40 g/d) of walnuts for 4 weeks. In a subset of 20 subjects, plasma lipid mediators synthesized from LA, aLA and other polyunsaturated fatty acids were measured by LC/MS/MS at baseline and final visits. Differences are reported as mean, 95% CIs. Results: Walnuts did not alter the compositional abundance of any plasma fatty acid except aLA, which was increased 1.46 fold [1.83, 1.16], p=0.003. Walnuts did modify plasma lipid mediator composition: specific pro-inflammatory COX metabolites, PGD2 and PGJ2/delta-12-PGJ2 were reduced by -72% [-43, -86] and -55% [-9, -77] respectively. LOX metabolites were almost uniformly depressed: the immediate mid-chain alcohols of LA (HODEs), dgLA (HETrEs), AA (HETEs), EPA (HEPEs) and DHA (HDoHEs) were reduced by 50-75% (p≤0.007), but aLA (HOTEs) were not. Metabolites of 5-LOX further downstream were also reduced: 5-KETE by 79% [-91, -50], and 6-trans-LTB4 by -89% [-76, -95]. Conversely, anti-inflammatory CYP-epoxides of LA (EpOME) and aLA (EpODE) were increased 82% [3, 223] and 143% [37, 331]. Epoxides of eicosanoids and docosanoids were unchanged (AA EpETrEs or EETs; EPA EpETEs; DHA EpDPEs). Conclusion: Walnut feeding reduces plasma pro-inflammatory COX and LOX metabolites while increasing some anti-inflammatory CYP metabolites. The effect occurs largely without changes in fatty acids, suggesting a molecular mechanism independent of simple changes in precursor abundance.

Obesity induces biochemical changes in lipid metabolism. The extent to which enzymatic and non-enzymatic lipid (per)oxidation products, oxylipins, are altered by obesity is of great interest. Confl...

Walnuts are a source of alpha-linolenic acid (ALA) and linoleic acid (LA). We examined the effects of short-term walnut intake on the reactive hyperemia index (RHI), a measure of microvascular function. We hypothesized that 4 weeks of 40g/day of walnuts would improve RHI compared to 5g/day of walnuts in hypercholesterolemic postmenopausal women. Microvascular function is influenced, in part, by locally produced vasodilators, such as nitric oxide and endothelial-derived hyperpolarizing factor (EDHF). Putative EDHF include cytochrome P450 epoxides of arachidonic acid (AA) known as the EpETrEs. Epoxides are also derived from ALA (EpODE) and LA (EpODE); however, their vascular influence is currently undefined. Four weeks of 40g/day of walnut intake increased RHI compared to 5g/day (2.61 ± 0.10 versus 2.24 ± 0.12, respectively, p = 0.027). Plasma EpODE and EpOME were significantly increased with 40g/day compared to 5g/day of walnut intake. The total change in plasma epoxides was strongly associated with the ch...

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 (KI = 15 nM) 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 μmol/L), the ATP-sensitive K + channel (K ATP ) inhibitor glibenclamide (1 μmol/L), the mitochondrial K ATP (mitoK ATP ) inhibitor 5-hydroxydecanoate (100 to 200 μmol/L), or the Ca 2+ -sensitive K + channel (K Ca ) inhibitor paxilline (10 μmol/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-3β (GSK-3β) 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.

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.

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.

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.

DEY, APARAJITA, ROGER S. WILLIAMS, DAVID M. POLLOCK, DAVID W. STEPP, JOHN W. NEWMAN, BRUCE D. HAMMOCK, AND JOHN D. IMIG. Altered Kidney CYP2C and cyclooxygenase-2 levels are associated with obesity-related albuminuria. Obes Res. 2004;12: 1278 –1289. 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 B2 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.

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slowrelease nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC‐ MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems. q 2003 Elsevier Ltd. All rights reserved.

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 -9/10-phosphonoxy-hydroxy-octadecanoic acid) with K m = 21 ± 0.3 μM, V Max = 338 ± 12 nmol⋅min −1 ⋅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.

Background: Oxylipins mediate inflammation, vascular tension, and more. Their presence in lipoproteins could explain why lipoproteins mediate nearly identical activities. Methods: To determine how oxylipins are distributed in the lipoproteins of hypertriglyceridemic subjects, and whether omega-3 fatty acids alter them in a manner consistent with improved cardiovascular health, we recruited 15 dyslipidemic subjects whose levels of low density lipoprotein cholesterol (LDL-C) were at goal but who remained hypertriglyceridemic (200–499 mg/dL). They were treated them with the indicated dose of 4 g/d omega-3 acid ethyl esters (P-OM3) for 8 weeks. Measured oxylipins included mid-chain alcohols (HETEs, HEPEs and HDoHEs), ketones (KETEs), epoxides (as EpETrEs, EpETEs, and EpDPEs). Results: At baseline, arachidonate-oxylipins (HETEs, KETEs, and EpETrEs) were most abundant in plasma with the greatest fraction of total abundance (mean |95% CI|) being carried in high density lipoproteins (HDL); 42% |31, 57| followed by very low density lipoproteins (VLDL); 27% |20, 36|; and LDL 21% |16, 28|. EPA- and DHA-derived oxylipins constituted less than 11% of total. HDL carried alcohols and epoxides but VLDL was also rich in ketones. Treatment decreased AA-derived oxylipins across lipoprotein classes (223% |233, 212|, p=0.0003), and expanded EPA2(322% |241, 422|, p,0.0001) and DHA-derived oxylipins (123% |80, 176|, p,0.0001). Conclusions: Each lipoprotein class carries a unique oxylipin complement. P-OM3 treatment alters the oxylipin content of all classes, reducing pro-inflammatory and increasing anti-inflammatory species, consistent with the improved inflammatory and vascular status associated with the treatment. Trial Registration: ClinicalTrials.gov NCT00959842

Improved vascular function after the incorporation of walnuts into controlled or high-fat diets has been reported; however, the mechanism(s) underlying this effect of walnuts is(are) poorly defined. The objective of the current study was to evaluate the acute and short-term effects of walnut intake on changes in microvascular function and the relationship of these effects to plasma epoxides, the cytochrome-P450-derived metabolites of fatty acids. Thirty-eight hypercholesterolemic postmenopausal women were randomized to 4 weeks of 5 g or 40 g of daily walnut intake. All outcomes were measured after an overnight fast and 4 h after walnut intake. Microvascular function, assessed as the reactive hyperemia index (RHI), was the primary outcome measure, with serum lipids and plasma epoxides as secondary measures. Compared to 5 g of daily walnut intake, consuming 40 g/d of walnuts for 4 weeks increased the RHI and Framingham RHI. Total cholesterol and low- and high-density cholesterol did not significantly change after walnut intake. The change in RHI after 4 weeks of walnut intake was associated with the change in the sum of plasma epoxides (r=0.65, P=.002) but not with the change in the sum of plasma hydroxyeicosatetraenoic acids. Of the individual plasma epoxides, arachidonic-acid-derived 14(15)-epoxyeicosatrienoic acid was most strongly associated with the change in microvascular function (r=0.72, P

Objectives: Endocannabinoid system (ECS) overactivation is associated with increased adiposity and likely contributes to type 2 diabetes risk. Elevated tissue cannabinoid receptor 1 (CB1) and circulating endocannabinoids (ECs) derived from the n-6 polyunsaturated acid (PUFA) arachidonic acid (AA) occur in obese and diabetic patients. Here we investigate whether the n-3 PUFA docosahexaenoic acid (DHA) in the diet can reduce ECS overactivation (that is, action of ligands, receptors and enzymes of EC synthesis and degradation) to influence glycemic control. This study targets the ECS tonal regulation of circulating glucose uptake by skeletal muscle as its primary end point. Design: Male C57BL/6J mice were fed a semipurified diet containing DHA or the control lipid. Serum, skeletal muscle, epididymal fat pads and liver were collected after 62 and 118 days of feeding. Metabolites, genes and gene products associated with the ECS, glucose uptake and metabolism and inflammatory status were measured. Results: Dietary DHA enrichment reduced epididymal fat pad mass and increased ECS-related genes, whereas it reduced downstream ECS activation markers, indicating that ECS activation was diminished. The mRNA of glucose-related genes and proteins elevated in mice fed the DHA diet with increases in DHA-derived and reductions in AA-derived EC and EC-like compounds. In addition, DHA feeding reduced plasma levels of various inflammatory cytokines, 5-lipoxygenase-dependent inflammatory mediators and the vasoconstrictive 20-HETE. Conclusions: This study provides evidence that DHA feeding altered ECS gene expression to reduce CB1 activation and reduce fat accretion. Furthermore, the DHA diet led to higher expression of genes associated with glucose use by muscle in mice, and reduced those associated with systemic inflammatory status.

Background: Total weight loss induced by energy restriction is highly variable even under tightly controlled conditions. Identifying weight-loss discriminants would provide a valuable weight management tool and insights into body weight regulation. Objective: This study characterized responsiveness to energy restriction in adults from variables including the plasma metabolome, endocrine and inflammatory markers, clinical indices, body composition, diet, and physical activity. Methods: Data were derived from a controlled feeding trial investigating the effect of 3–4 dairy product servings in an energy-restricted diet (2092 kJ/d reduction) over 12 wk. Partial least squares regression was used to identify weight-loss discriminants in 67 overweight and obese adults. Linear mixed models were developed to identify discriminant variable differences in high- vs. low-weight–loss responders. Results: Both pre- and postintervention variables (n = 127) were identified as weight-loss discriminants (root mean squared error of prediction = 1.85 kg; Q2 = 0.43). Compared with low-responders (LR), high-responders (HR) had greater decreases in body weight (LR: 2.7 ± 1.6 kg; HR: 9.4 ± 1.8 kg, P < 0.01), BMI (in kg/m2; LR: 1.0 ± 0.6; HR: 3.3 ± 0.5, P < 0.01), and total fat (LR: 2.2 ± 1.1 kg; HR: 8.0 ± 2.1 kg, P < 0.01). Significant group effects unaffected by the intervention were determined for the respiratory exchange ratio (LR: 0.86 ± 0.05; HR: 0.82 ± 0.03, P < 0.01), moderate physical activity (LR: 127 ± 52 min; HR: 167 ± 68 min, P = 0.02), sedentary activity (LR: 1090 ± 99 min; HR: 1017 ± 110 min, P = 0.02), and plasma stearate [LR: 102,000 ± 21,000 quantifier ion peak height (QIPH); HR: 116,000 ± 24,000 QIPH, P = 0.01]. Conclusions: Overweight and obese individuals highly responsive to energy restriction had accelerated reductions in adiposity, likely supported in part by higher lipid mobilization and combustion. A novel observation was that person-to-person differences in habitual physical activity and magnitude of weight loss were accompanied by unique blood metabolite signatures. This trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT00858312","term_id":"NCT00858312"}}NCT00858312.

Dichlorodiphenyltrichloroethane (DDT) has been used extensively to control malaria, typhus, body lice and bubonic plague worldwide, until countries began restricting its use in the 1970s. Its use in malaria control continues in some countries according to recommendation by the World Health Organization. Individuals exposed to elevated levels of DDT and its metabolite dichlorodiphenyldichloroethylene (DDE) have an increased prevalence of diabetes and insulin resistance. Here we hypothesize that perinatal exposure to DDT disrupts metabolic programming leading to impaired metabolism in adult offspring. To test this, we administered DDT to C57BL/6J mice from gestational day 11.5 to postnatal day 5 and studied their metabolic phenotype at several ages up to nine months. Perinatal DDT exposure reduced core body temperature, impaired cold tolerance, decreased energy expenditure, and produced a transient early-life increase in body fat in female offspring. When challenged with a high fat diet for 12 weeks in adulthood, female offspring perinatally exposed to DDT developed glucose intolerance, hyperinsulinemia, dyslipidemia, and altered bile acid metabolism. Perinatal DDT exposure combined with high fat feeding in adulthood further impaired thermogenesis as evidenced by reductions in core temperature and in the expression of numerous RNA that promote thermogenesis and substrate utilization in the brown adipose tissue of adult female mice. These observations suggest that perinatal DDT exposure impairs thermogenesis and the metabolism of carbohydrates and lipids which may increase susceptibility to the metabolic syndrome in adult female offspring.

Introduction of vegetable ingredients in fish feed has affected the fatty acid composition in farmed Atlantic salmon (Salmo salar L). Here we investigated how changes in fish feed affected the metabolism of mice fed diets containing fillets from such farmed salmon. We demonstrate that replacement of fish oil with rapeseed oil or soybean oil in fish feed had distinct spillover effects in mice fed western diets containing the salmon. A reduced ratio of n-3/n-6 polyunsaturated fatty acids in the fish feed, reflected in the salmon, and hence also in the mice diets, led to a selectively increased abundance of arachidonic acid in the phospholipid pool in the livers of the mice. This was accompanied by increased levels of hepatic ceramides and arachidonic acid-derived pro-inflammatory mediators and a reduced abundance of oxylipins derived from eicosapentaenoic acid and docosahexaenoic acid. These changes were associated with increased whole body insulin resistance and hepatic steatosis. Our data suggest that an increased ratio between n-6 and n-3-derived oxylipins may underlie the observed marked metabolic differences between mice fed the different types of farmed salmon. These findings underpin the need for carefully considering the type of oil used for feed production in relation to salmon farming.

Elevated levels of endocannabinoid system (ECS) ligands have been associated with increased adiposity and obesity, and are likely modulators of type II diabetes pathogenesis. High CB1 receptor expression and circulating arachidonic acid (AA)-derived endocannabinoids (EC) have been found in both obese and diabetic patients. We wanted to determine whether dietary docosahexaenoic acid (DHA) could influence endocannabinoid tone, which includes the action of ligands, receptors, and EC synthesis and degradation enzymes, to prevent an overactive ECS whilst improving aspects of euglycemia. To assess this, male C57bl/6 mice were fed a semi-purified diet containing DHA or control for 118 d. Serum, gastrocnemius, epididymal fat pads, and liver were collected after 62 and 118 d of feeding. DHA diet fed mice had lower epididymal fat pad masses compared to the control diet group. A compensatory response resulted in DHA fed mice with higher EC receptors and synthesis/degradation enzyme mRNA expression. However, AA-deriv...

Global metabolite analysis grouping various pathways represents a powerful and practical means of establishing links to health and disease status. Lipid-based metabolites, such as the arachidonate (AA)-derived ligands of the endocannabinoid system (ECS), appear to regulate whole body energy balance and shift anabolic pathways, with overactivation of the ECS associated with obesity and type 2 diabetes. Since ECS activation relies on lipid-derived ligands, namely AA, we conducted an investigation to determine how dietary PUFA influence the ECS to affect glucose clearance. C57BL/6 mice were fed a control or DHA containing semi-purified diet for 118 d. Serum, gastrocnemius, and liver were collected after 62 d and 118 d for metabolomics analyses. Epididymal fat mass was lower in mice given DHA but no difference in body weights or food intakes were observed compared to controls. Metabolite profiles portrayed a shift from glucose use to favoring fatty acid oxidation with DHA. Higher levels of DHA-containing glyc...

Skeletal muscle uncoupling protein 3 (UCP3) is thought to facilitate fatty acid oxidation (FAO) and protect against excess mitochondrial (MITO) reactive oxygen species (ROS); however, this has yet ...

Weight loss (WL) induced by energy restriction is highly variable even in controlled clinical trials. An integrative analysis of the plasma metabolome coupled to traditional clinical variables may ...

Although the effects of fish oil supplements on airway inflammation in asthma have been studied with varying results, the independent effects of the fish oil components, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), administered separately, are untested. Here, we investigated airway inflammation and hyperresponsiveness using a mouse ovalbumin exposure model of asthma assessing the effects of consuming EPA (1.5% wt/wt), DHA (1.5% wt/wt), EPA plus DHA (0.75% each), or a control diet with no added omega-3 polyunsaturated fatty acids. Consuming these diets for 6 weeks resulted in erythrocyte membrane EPA contents (molar %) of 9.0 (± 0.6), 3.2 (± 0.2), 6.8 (± 0.5), and 0.01 (± 0.0)%; DHA contents were 6.8 (± 0.1), 15.6 (± 0.5), 12.3 (± 0.3), and 3.8 (± 0.2)%, respectively. The DHA group had the highest bronchoalveolar lavage (BAL) fluid eosinophil and IL-6 levels (P < 0.05). Similar trends were seen for macrophages, IL-4, and IL-13, whereas TNF-α was lower in omega-3 polyunsaturated fatty acid groups than the control (P < 0.05). The DHA group also had the highest airway resistance, which differed significantly from the EPA plus DHA group (P < 0.05), which had the lowest. Oxylipins were measured in plasma and BAL fluid, with DHA and EPA suppressing arachidonic acid-derived oxylipin production. DHA-derived oxylipins from the cytochrome P450 and 15-lipoxygenase pathways correlated significantly with BAL eosinophil levels. The proinflammatory effects of DHA suggest that the adverse effects of individual fatty acid formulations should be thoroughly considered before any use as therapeutic agents in asthma.

Novel plasma metabolite patterns reflective of improved metabolic health (insulin sensitivity, fitness, reduced body weight) were identified before and after a 14-17 wk weight loss and exercise intervention in sedentary, obese insulin-resistant women. To control for potential confounding effects of diet- or microbiome-derived molecules on the systemic metabolome, sampling was during a tightly-controlled feeding test week paradigm. Pairwise and multivariate analysis revealed intervention- and insulin-sensitivity associated: (1) Changes in plasma xeno-metabolites ("non-self" metabolites of dietary or gut microbial origin) following an oral glucose tolerance test (e.g. higher post-OGTT propane-1,2,3-tricarboxylate [tricarballylic acid]) or in the overnight-fasted state (e.g., lower γ-tocopherol); (2) Increased indices of saturated very long chain fatty acid elongation capacity; (3) Increased post-OGTT α-ketoglutaric acid (α-KG), fasting α-KG inversely correlated with Matsuda index, and altered patterns of malate, pyruvate and glutamine hypothesized to stem from improved mitochondrial efficiency and more robust oxidation of glucose. The results support a working model in which improved metabolic health modifies host metabolism in parallel with altering systemic exposure to xeno-metabolites. This highlights that interpretations regarding the origins of peripheral blood or urinary "signatures" of insulin resistance and metabolic health must consider the potentially important contribution of gut-derived metabolites toward the host's metabolome.

International audience; The anti-atherogenic effects of omega 3 fatty acids, namely eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) are well recognized but the impact of dietary intake on bioactive lipid mediat or profiles remains unclear. Such a profiling effort may offer novel targets for future studies into the mechanism of action of omega 3 fatty acids. The present study aimed to determine the impact of DHA supplementation on the profiles of polyunsaturated fatty acids (PUFA) oxygenated metabolites and to investigate their contribution to therosclerosis prevention. A special emphasis was given to the non-enzymatic metabolites knowing the high susceptibility of DHA to free radical-mediated peroxidation and the increased oxidative stress associated with plaque formation. Atherosclerosis prone mice (LDLR -/-) received increasing doses of DHA (0, 0.1, 1 or 2% of energy) during 20 weeks leading to a dose -dependent reduction of atherosclerosis (R2=0.97, p=0.02), trig lyceridemia (R2=0.97, p=0.01) and cholesterolemia (R2=0.96, p

Objective: Alzheimer’s disease (AD) is a neurodegenerative disease of aging with unknown causative factors. Accumulating evidence suggests that inflammation and neurovascular dysfunction play important roles in AD. The postprandial period following a moderately high-fat meal is associated with vascular inflammation in young, healthy individuals; however, this relationship has not been investigated in Alzheimer’s patients despite their exaggerated inflammatory state. Methods: Patients with AD and age-matched control subjects were recruited through the UC Davis Alzheimer’s Disease Center. All subjects consumed a moderately high-fat breakfast meal. Fasting and postprandial blood samples were collected for lipid, lipoprotein, and oxylipin analyses, as well as assays for cytokine levels and monocyte activation. Results: The plasma lipid analyses revealed similar levels of triglycerides and esterified oxylipins between groups, but there was an interaction between postprandial non-esterified fatty acid (NEFA) levels and body mass index in the AD group compared to the control subjects. The AD group also had increased behenic acid and decreased linoleic and oleic acids in the postprandial period; however, these were not significantly different. Inflammatory assays revealed elevated fasting levels of interleukin (IL)-10 and IL-12 p70, but no change in monocyte activation in the AD group. Conclusion: The postprandial period following a moderately high-fat meal is not associated with an exaggerated inflammatory state in Alzheimer’s patients, and basal esterified oxylipin profiles do not indicate elevated oxidative stress. However, the baseline inflammatory state during fasting in AD patients includes elevated levels of plasma IL-10 and IL-12 p70, which may indicate a balance between immune responses mediated by these interleukins.

BackgroundDiets rich in dairy and/or calcium (Ca) have been associated with reductions in adiposity and inflammation, but the mechanisms underlying this remain to be fully elucidated. Oxylipins and endocannabinoids are bioactive lipids, which influence energy homeostasis, adipose function, insulin signaling, and inflammation. Our objective was to determine if these metabolites associate with metabolic and inflammatory phenotypes stemming from dietary Ca and dairy in diet induced obese mice.MethodsIn one study, C57BL6/J mice were fed high fat diets (45% energy) with varying dietary matrices for 12weeks: soy protein and Ca adequate (0.5%; CONTROL), soy protein and high Ca (1.5%; HighCa), or nonfat-dry-milk based high Ca (NFDM). In a second study, mice were pre-fattened for 12weeks on the CONTROL high fat diet, and then fed one of three high fat diets for an additional 8weeks: CONTROL, HighCa, or NFDM. In both studies, adiposity and associated metabolic and inflammatory outcomes were measured and a targeted lipidomics analysis was performed on plasma collected during the post-absorptive condition.ResultsAs reported previously, mice fed NFDM had less body fat and reduced mRNA markers of adipose inflammation (p

Genomics and bioinformatics have the vast potential to identify genes that cause disease by investigating whole-genome databases. Comparison of an individual's geno-type with a genomic database will allow the prescription of drugs to be tailored to an individual's genotype. This same bioinformatic approach, applied to the study of human metabolites, has the potential to identify and validate targets to improve personalized nutritional health and thus serve to define the added value for the next generation of foods and crops. Advances in high-throughput analytic chemistry and computing technologies make the creation of a vast database of metabolites possible for several subsets of metabolites, including lipids and organic acids. In creating integrative databases of metabolites for bioinformatic investigation, the current concept of measuring single biomarkers must be expanded to 3 dimensions to 1) include a highly comprehensive set of metabolite measurements (a profile) by multiparallel analyses, 2) measure the metabolic profile of individuals over time rather than simply in the fasted state, and 3) integrate these metabolic profiles with genomic, expression, and proteomic databases. Application of the knowledge of individual metabolism will revolutionize the ability of nutrition to deliver health benefits through food in the same way that knowledge of genomics will revolutionize individual treatment of dis-ease with pharmaceuticals.

Sediment reference sites were used to establish toxicity standards against which to compare results from sites investigated in San Francisco Bay (California, USA) monitoring programs. The reference sites were selected on the basis of low concentrations of anthropogenic chemicals, distance from active contaminant sources, location in representative hydrographic areas of the Bay, and physical features characteristic of depositional areas (e.g., fine grain size and medium total organic carbon [TOC]). Five field-replicated sites in San Francisco Bay were evaluated over three seasons. Samples from each site were tested with nine toxicity test protocols and were analyzed for sediment grain size and concentrations of trace metals, trace organics, ammonia, hydrogen sulfide, and TOC. The candidate sites were found to have relatively low concentrations of measured chemicals and generally exhibited low toxicity. Toxicity data from the reference sites were then used to calculate numerical tolerance limits to be used as threshold values to determine which test sites had significantly higher toxicity than reference sites. Tolerance limits are presented for four standard test protocols, including solid-phase sediment tests with the amphipods Ampelisca abdita and Eohaustorius estuarius and sea urchin Strongylocentrotus purpuratus embryo/larval development tests in pore water and at the sediment-water interface (SWI). Tolerance limits delineating the lowest 10th percentile (0.10 quantile) of the reference site data distribution were 71% of the control response for Ampelisca, 70% for Eohaustorius, 94% for sea urchin embryos in pore water, and 87% for sea urchins embryos exposed at the SWI. The tolerance limits are discussed in terms of the critical values governing their calculation and the management implications arising from their use in determining elevated toxicity relative to reference conditions.

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron < desmethyl diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects.

Soluble epoxide hydrolase (sEH) is suggested to alter the mode of action and increase the toxic potency of fatty acid epoxides. To characterize the structural features necessary for sEH-dependent epoxy fatty acid toxicity, 75 aliphatic compounds were assayed for cytotoxicity in the presence and absence of sEH. Three groups of aliphatic epoxide-diol pairs were described by their observed differential toxicity. Group I compounds were typified by terminal epoxides whose toxicity was reduced in the presence of sEH. Group II compounds were toxic in either their epoxide or diol form, but toxicity was unaffected by sEH. Group III compounds exhibited sEH-dependent toxicity and were therefore used to investigate the structural elements required for cytotoxicity in this study. The optimal structure for group III compounds appeared to be a fatty acid 18-20 atoms long (e.g., a carbon backbone plus a terminal heteroatom) with an epoxide positioned between C-7 and C-12. In the absence of sEH, replacement of epoxides with a vicinal diol was required for toxicity. While diol stereochemistry was unimportant, vicinal diol-induced toxicity exhibited fewer positional constraints to toxicity than sEH-dependent epoxide toxicity. Tested fatty acids and esters with neither an epoxide nor a vicinal diol were not toxic. These data support the hypothesis that long-chain epoxy fatty acid methyl esters are potential pro-toxins metabolized by sEH to more toxic diols. Furthermore, our results suggest that the endogenous compounds, leukotoxin methyl ester, 9,10(Z)-epoxyoctadec-12(Z)-enoic acid methyl ester, and isoleukotoxin methyl ester, 12, 13(Z)-epoxyoctadec-9(Z)-enoic acid methyl ester, are structurally optimized to elicit the observed effect.