Michiel G.J. Balvers

Metabolic Effects of n-3 PUFA as Phospholipids Are Superior to Triglycerides in Mice Fed a High-Fat Diet: Possible Role of Endocannabinoids

Background n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides. Methodology/Principal Findings In a ‘prevention study’, C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ∼35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine. Conclusions/Significance Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT.

Development and validation of a quantitative method for the determination of 12 endocannabinoids and related compounds in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and specific LC-MS/MS method for the quantification of the endocannabinoids and related structures anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glycerol ether, O-arachidonoyl ethanolamide, dihomo-gamma-linolenoyl ethanolamide, docosatetraenoyl ethanolamide, N-arachidonoyl dopamine, N-arachidonyl glycine, N-oleoyl dopamine, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in human plasma was developed and validated. Compounds were extracted using acetonitrile followed by solid-phase extraction. Separation was performed on a Xterra C8 column using gradient elution coupled to a triple-quadrupole MS. LLOQ levels ranged from 0.02 to 1.75 microg/mL, LODs ranged from 0.0002 to 0.1266 ng/mL, and accuracies were >80% (except stearoyl ethanolamide at lowest spike level) at all spike levels.

Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators, in the intestinal tract

Following the discovery of the endocannabinoid arachidonoyl ethanolamide (anandamide) and other N-acyl-ethanolamines, several other compounds have been found in which amino acids or neurotransmitters rather than ethanolamide are linked to fatty acids. Studies have shown that the local availability of fatty acid precursors, which in turn is modulated by dietary intake of lipids, determines the pattern of conjugates formed. Less information is available whether the same might be true for the amines or neurotransmitters involved. We hypothesized that N-arachidonoyl-serotonin (AA-5-HT) and its analogs could be endogenously present in those tissues that have high contents of serotonin. We investigated the endogenous presence of N-acyl serotonins in different parts of the gastro-intestinal tract of pigs and mice. We discovered that AA-5-HT, oleoyl-serotonin, palmitoyl-serotonin, and stearoyl-serotonin were endogenously present, particularly in the jejunum and ileum. Their formation in vitro was stimulated by the addition of serotonin to intestinal tissue incubations. Furthermore, in a mouse study we showed that the pattern of formation is dependent on the relative amount of fatty acids in the diet. The formation of docosahexaenoyl-serotonin and eicosapentaenoyl-serotonin was elevated in mice fed with a diet containing fish oil. Preliminary data showed that several of the serotonin conjugates are able to inhibit glucagon-like peptide-1 secretion and FAAH activity in vitro. Taken together, our data suggest that N-acyl serotonins are a novel class of lipid mediators present in the gut with highly promising biological properties.

Plasma anandamide and other N-acylethanolamines are correlated with their corresponding free fatty acid levels under both fasting and non-fasting conditions in women

N-acylethanolamines (NAEs), such as anandamide (AEA), are a group of endogenous lipids derived from a fatty acid linked to ethanolamine and have a wide range of biological activities, including regulation of metabolism and food intake. We hypothesized that i) NAE plasma levels are associated with levels of total free fatty acids (FFAs) and their precursor fatty acid in fasting and non-fasting conditions and ii) moderate alcohol consumption alters non-fasting NAE levels. In a fasting and non-fasting study we sampled blood for measurements of specific NAEs and FFAs. In the fasting study blood was drawn after an overnight fast in 22 postmenopausal women. In the non-fasting study blood was sampled before and frequently after a standardized lunch with beer or alcohol-free beer in 19 premenopausal women. Fasting AEA levels correlated with total FFAs (r = 0.84; p < 0.001) and arachidonic acid levels (r = 0.42; p < 0.05). Similar results were observed for other NAEs with both total FFAs and their corresponding fatty acid precursors. In addition, AEA (r = 0.66; p < 0.01) and OEA levels (r = 0.49; p <0.02) positively related with BMI. Changes over time in non-fasting AEA levels were correlated with changes in total FFA levels, both after a lunch with beer (r = 0.80; 95% confidence interval: 0.54-0.92) and alcohol-free beer (r = 0.73; 0.41-0.89). Comparable correlations were found for other NAEs, without differences in correlations of each NAE between beer and alcohol free beer with lunch. In conclusion, i) in fasting and non-fasting states circulating anandamide and other N-acylethanolamines were associated with free fatty acid levels and ii) moderate alcohol consumption does not affect non-fasting NAE levels. This suggests that similar physiological stimuli cause the release of plasma N-acylethanolamines and free fatty acids in blood. The trials are registered at ClinicalTrials.gov numbers: NCT00524550 and NCT00652405.

N-acyl amines of docosahexaenoic acid and other n–3 polyunsatured fatty acids – from fishy endocannabinoids to potential leads

N–3 long‐chain polyunsaturated fatty acids (n–3 LC‐PUFAs), in particular α‐linolenic acid (18:3n‐3), eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3) are receiving much attention because of their presumed beneficial health effects. To explain these, a variety of mechanisms have been proposed, but their interactions with the endocannabinoid system have received relatively little attention so far. However, it has already been shown some time ago that consumption of n–3 LC‐PUFAs not only affects the synthesis of prototypic endocannabinoids like anandamide but also stimulates the formation of specific n–3 LC‐PUFA‐derived conjugates with ethanolamine, dopamine, serotonin or other amines. Some of these fatty amides show overlapping biological activities with those of typical endocannabinoids, whereas others possess distinct and sometimes largely unknown receptor affinities and other properties. The ethanolamine and dopamine conjugates of DHA have been the most investigated thus far. These mediators may provide promising new leads to the field of inflammatory and neurological disorders and for other pharmacological applications, including their use as carrier molecules for neurotransmitters to target the brain. Furthermore, combinations of n–3 LC‐PUFA‐derived fatty acid amides, their precursors and FAAH inhibitors offer possibilities to optimise their effects in health and disease.

Time-dependent effect of in vivo inflammation on eicosanoid and endocannabinoid levels in plasma, liver, ileum and adipose tissue in C57BL/6 mice fed a fish-oil diet.

Eicosanoids and endocannabinoids/N-acylethanolamines (NAEs) are fatty acid derived compounds with a regulatory role in inflammation. Considering their complex metabolism, it is likely that inflammation affects multiple compounds at the same time, but how lipid profiles change in plasma and other tissues after an inflammatory stimulus has not been described in detail. In addition, dietary fish oil increases levels of several n-3 fatty acid derived eicosanoids and endocannabinoids, and this may lead to a broader change in the profiles of bioactive lipids. In the present study mice were fed a diet containing 3% w/w fish oil for 6 weeks before receiving i.p. saline or 3 mg/kg lipopolysaccharide (LPS) to induce an inflammatory response. Eicosanoid and endocannabinoid/NAE levels (in total 61 metabolites) in plasma, liver, ileum, and adipose tissue were quantified using targeted lipidomics after 2, 4, 8, and 24 h, respectively. Tissue- and time-dependent effects of LPS on bioactive lipid profiles were observed. For example, levels of CYP derived eicosanoids in the ileum were markedly affected by LPS, whereas this was less pronounced in the plasma and adipose tissue. For some compounds, such as 9,10-DiHOME, opposing effects of LPS were seen in the plasma compared to the other tissues, suggesting differential regulation of bioactive lipid levels after an inflammatory stimulus. Taken together, our results show that plasma levels do not always correlate with the effects found in the tissues, which underlines the need to measure profiles and pathways of mediators involved in inflammation, including endocannabinoid-like structures, in both plasma and tissues.

Recommended intakes of vitamin D to optimise health, associated circulating 25-hydroxyvitamin D concentrations, and dosing regimens to treat deficiency: workshop report and overview of current literature.

Vitamin D is a fat-soluble hormone that traditionally has been linked to bone health. Recently, its involvement has been extended to other (extra-skeletal) disease areas, such as cancer, CVD, energy metabolism and autoimmune diseases. Vitamin D deficiency is a worldwide problem, and several recommendation-setting bodies have published guidelines for adequate vitamin D intake and status. However, recommendations from, for example, the Health Council of the Netherlands do not provide advice on how to treat vitamin D deficiency, a condition that is often encountered in the clinic. In addition, these recommendations provide guidelines for the maintenance of ‘minimum levels’, and do not advise on ‘optimum levels’ of vitamin D intake/status to further improve health. The NutriProfiel project, a collaboration between the Gelderse Vallei Hospital (Ede, the Netherlands) and the Division of Human Nutrition of Wageningen University (Wageningen, the Netherlands), was initiated to formulate a protocol for the treatment of vitamin deficiency and for the maintenance of optimal vitamin D status. To discuss the controversies around treatment of deficiency and optimal vitamin D status and intakes, a workshop meeting was organised with clinicians, scientists and dietitians. In addition, a literature review was conducted to collect recent information on optimal intake of vitamins, their optimal circulating concentrations, and effective dosing regimens to treat deficiency. This information has been translated into the NutriProfiel advice, which is outlined in this article.

Measurement of palmitoylethanolamide and other N-acylethanolamines during physiological and pathological conditions

Palmitoylethanolamide (PEA) belongs to the N-acyl ethanolamines (NAEs), a group of endogenous compounds involved in a variety of physiological processes, including energy homeostasis and inflammation. This review focuses on the analysis of PEA in plasma and tissues and discusses effects of diet and some pathological processes on PEA levels. Originally isolated from egg yolk, PEA has been detected in a variety of tissues and plasma of different species. The compound is present at relatively high levels compared to other NAEs and now mostly analysed using liquid chromatography coupled to mass spectrometry. PEA plasma concentrations show marked fluctuations during the day. However, concentrations in tissues are likely to be more relevant than those in plasma. Most studies suggest that compared to other NAEs, tissue PEA tissue levels are not influenced by changes in dietary fatty acid composition. Effects of inflammation and disease on PEA tissue levels show differences between different models and studies. Therefore, more research is needed on the endogenous role and tissue kinetics of PEA during disease. The rediscovery of the therapeutic potential of PEA has fuelled research and the development of new pharmaceutical formulations. With regard to this there is a need for better kinetic data and models, preferably also on its tissue disposition. Moreover, it is important to learn more about effects of exogenous PEA on the kinetics of other NAEs (and endocannabinoids) and effects of inhibiting its breakdown using inhibitors of the degrading enzymes fatty acid amide hydrolase or N-acylethanolamine-hydrolyzing acid amidase.

Liquid chromatography-tandem mass spectrometry analysis of free and esterified fatty acid N-acyl ethanolamines in plasma and blood cells

The origin of N-acyl ethanolamides (NAEs) in plasma is not well understood, and it is possible that NAEs are present in plasma in esterified form. To test this hypothesis, a new and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of arachidonoyl ethanolamide, 2-arachidonoyl glycerol, docosahexaenoyl ethanolamide, dihomo-γ-linolenoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in 100 μl of human plasma using a simple acetonitrile extraction step. Using this method, we determined (i) free and esterified NAE levels in human plasma, (ii) free and esterified NAE levels in plasma of mice fed with diets with different amounts of n-3 fatty acids, and (iii) esterified NAE levels in blood cells. Murine and human plasma extracts contained 20- to 60-fold higher levels of esterified NAEs than free NAEs. Moreover, the effect of dietary n-3 fatty acids on murine free plasma NAE profiles was similar for esterified NAEs. Finally, esterified NAEs were also present in murine blood cells, and their pattern followed the same diet effect as observed for free and esterified NAEs in plasma. Together, these data point to the presence of previously ignored pools of esterified NAEs in plasma and blood cells that correlated well with free NAE levels in plasma.

Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages.

N‐docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long‐chain polyunsaturated n‐3 fatty acid docosahexaenoic (DHA; 22: 6n‐3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti‐inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune‐modulating effects of DHEA.