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;

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-γ-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 μg/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. © 2009 Elsevier B.V. All rights reserved

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-¿-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

Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples

LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its integration with software tools for data preprocessing and multivariate statistical analysis. The 2D LC-MS method was optimized in order to minimize peptide loss prior to sample injection and during the collection step after the first LC dimension, thus minimizing errors from off-column sample handling. The second dimension was run in fully automated mode, injecting onto a nanoscale LC-MS system a series of more than 100 samples, representing fractions collected in the first dimension (8 fractions/sample). As a model study, the method was applied to finding biomarkers for the antiinflammatory properties of zilpaterol, which are coupled to the β2-adrenergic receptor. Secreted proteomes from U937 macrophages exposed to lipopolysaccharide in the presence or absence of propanolol or zilpaterol were analysed. Multivariate statistical analysis of 2D LC-MS data, based on principal component analysis, and subsequent targeted LC-MS/MS identification of peptides of interest demonstrated the applicability of the approach. © 2006 American Chemical Society

Protein transport across the small intestine in food hypersensitivity

In view of the imminent deficiency of protein sources for human consumption in the near future, new protein sources need to be identified. However, safety issues such as the risk of allergenicity are often a bottleneck, due to the absence of predictive, validated and accepted methods for risk assessment. The current strategy to assess the allergenic potential of proteins focuses mainly on homology, stability and cross-reactivity, although other factors such as intestinal transport might be of added value too. In this review, we present an overview of the knowledge of protein transport across the intestinal wall and the methods currently being used to measure this. A literature study reveals that protein transport in sensitised persons occurs para-cellularly with the involvement of mast cells, and trans-cellularly via enterocytes, while in non-sensitised persons micro-fold cells and enterocytes are considered most important. However, there is a lack of comparable systematic studies on transport of allergenic proteins. Knowledge of the multiple protein transport pathways and which model system can be useful to study these processes may be of added value in the risk assessment of food allergenicity

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

Docosahexaenoic acid and eicosapentaenoic acid are converted by 3T3-L1 adipocytes to N-acyl ethanolamines with anti-inflammatory properties

n-3 PUFAs have beneficial health effects which are believed to be partly related to their anti-inflammatory properties, however the exact mechanisms behind this are unknown. One possible explanation could be via their conversion to N-acyl ethanolamines (NAEs), which are known to possess anti-inflammatory properties. Using fatty acid precursors we showed that 3T3-L1 adipocytes are indeed able to convert docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to their NAE derivatives docosahexaenoyl ethanolamine (DHEA) and eicosapentaenoyl ethanolamine (EPEA), respectively. This synthesis took place on top of an apparent background formation of these NAEs in standard culture medium. In addition we were able to demonstrate the presence of DHEA, but not of EPEA, in human plasma. DHEA and EPEA were found to decrease LPS induced adipocyte IL-6 and MCP-1 levels. Results of combined incubations with PPAR-¿ and CB2 antagonists suggest a role of these receptors in mediating the reduction of IL-6 by DHEA. Our results are in line with the hypothesis that in addition to other pathways, formation of N-acyl ethanolamines may contribute to the biological activity of n-3 PUFAs. Different targets, including the endocannabinoid system, may be involved in the immune-modulating activity of these “fish-oil-derived NAEs.

Inhibitory effects of the β2-adrenergic receptor agonist zilpaterol on the LPS-induced production of TNF-α in vitro and in vivo

In this study the anti-inflammatory properties of zilpaterol, a β2-adrenergic receptor (AR) agonist specifically developed as a growth promoter in cattle were investigated. Although zilpaterol has a different structure compared with the β2-AR agonists known to date, it was noted that it was able to bind to both the β2-AR (K i = 1.1 × 10-6) and the β1-AR (Ki = 1.0 × 10-5). Using lipopolysaccharide (LPS)-exposed U937 macrophages, the production of cyclic adenosine-3′, 5′-cyclic monophosphate (cAMP) and tumor necrosis factor alpha (TNF-α) were investigated. Zilpaterol inhibited TNF-α release and induced intracellular cAMP levels in a dose-dependent manner. The inhibition of TNF-α release and induction of cAMP production was mainly mediated via the β2-AR, as indicated by addition of β1- and β2-specific antagonists. The effects of zilpaterol were investigated in LPS-treated male Wistar rats after pretreatment with zilpaterol. Zilpaterol dosed at 500 μg/kg body weight reduced the TNF-α plasma levels. In conclusion, zilpaterol is a β2-adrenergic agonist and an inhibitor of TNF-α production induced by LPS both in vivo and in vitro. © 2005 Blackwell Publishing Ltd

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 propertie