Phosphoproteomic Analysis of Dopamine D2 Receptor Signaling Reveals Interplay of G Protein- and ß-Arrestin-Mediated Effects.

Leveraging biased signaling of G protein-coupled receptors has been proposed as a promising strategy for the development of drugs with higher specificity. However, the consequences of selectively targeting G protein- or ß-arrestin-mediated signaling on cellular functions are not comprehensively understood. In this study, we utilized phosphoproteomics to gain a systematic overview of signaling induced by the four biased and balanced dopamine D2 receptor (D2R) ligands MS308, BM138, quinpirole, and sulpiride in an in vitro D2R transfection model. Quantification of 14,160 phosphosites revealed a low impact of the partial G protein agonist MS308 on cellular protein phosphorylation, as well as surprising similarities between the balanced agonist quinpirole and the inverse agonist sulpiride. Analysis of the temporal profiles of ligand-induced phosphorylation events showed a transient impact of the G protein-selective agonist MS308, whereas the ß-arrestin-preferring agonist BM138 elicited a delayed, but more pronounced response. Functional enrichment analysis of ligand-impacted phosphoproteins and treatment-linked kinases confirmed multiple known functions of D2R signaling while also revealing novel effects, for example of MS308 on sterol regulatory element-binding protein-related gene expression. All raw data were deposited in MassIVE (MSV000089457).

The beer component hordenine inhibits alcohol addiction-associated behaviours in mice.

Alcohol consumption is a widespread behaviour that may eventually result in the development of alcohol use disorder (AUD). Alcohol, however, is rarely consumed in pure form but in fruit- or corn-derived preparations, like beer. These preparations add other compounds to the consumption, which may critically modify alcohol intake and AUD risk. We investigated the effects of hordenine, a barley-derived beer compound on alcohol use-related behaviours. We found that the dopamine D2 receptor agonist hordenine (50 mg/kg) limited ongoing alcohol consumption and prophylactically diminished relapse drinking after withdrawal in mice. Although not having reinforcing effects on its own, hordenine blocked the establishment of alcohol-induced conditioned place preference (CPP). However, it independently enhanced alcohol CPP retrieval. Hordenine had a dose-dependent inhibitory effect on locomotor activity. Chronic hordenine exposure enhanced monoamine tissue levels in many brain regions. Further characterization revealed monoaminergic binding sites of hordenine and found a strong binding on the serotonin and dopamine transporters, and dopamine D3 , and adrenergic α1A and α2A receptor activation but no effects on GABAA receptor or glycinergic signalling. These findings suggest that natural ingredients of beer, like hordenine, may work as an inhibitory and use-regulating factor by their modulation of monoaminergic signalling in the brain.

In Vitro Reactivity of the Glucose Degradation Product 3,4-Dideoxyglucosone-3-ene (3,4-DGE) towards Abundant Components of the Human Blood Circulatory System.

3,4-Dideoxyglucosone-3-ene (3,4-DGE) is a glucose degradation product present in processed foods and medicinal products. Additionally, its constant formation from 3-deoxyglucosone in plasma has been suggested. Due to its α,ß-unsaturated dicarbonyl moiety, 3,4-DGE is highly reactive and has shown harmful effects in vitro. Here, we investigated the impact of major components of the human blood circulatory system on 3,4-DGE in vitro. Under physiological conditions, plasma concentrations of human serum albumin (HSA) reacted efficiently with 3,4-DGE, resulting in only 8.5% of the initial 3,4-DGE concentration after seven hours (vs. 83.4% without HSA, p < 0.001). Thereby, accessible thiol groups were reduced from 0.121 to 0.064 mol/mol HSA, whereas ketoprofen binding and esterase-like activity of HSA were not affected. Plasma concentrations of glutathione (GSH) reacted immediately and completely with 3,4-DGE, leading to two stereoisomeric adducts. Plasma concentrations of immunoglobulin G (IgG) bound to 3,4-DGE to a lower extent, resulting in 62.6% 3,4-DGE after seven hours (vs. 82.2% in the control, p < 0.01). Immobilized human collagen type IV did not alter 3,4-DGE concentrations. The results indicated that particularly HSA, GSH, and IgG readily scavenge 3,4-DGE after its appearance in the blood stream, which may be associated with a reduced antioxidative and cytoprotective activity for the living cells and, thus, the human organism by blocking free thiol groups.

Reactive dicarbonyl compounds cause Calcitonin Gene-Related Peptide release and synergize with inflammatory conditions in mouse skin and peritoneum.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene-Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Metal cations promote α-dicarbonyl formation in glucose-containing peritoneal dialysis fluids.

Heat sterilization of peritoneal dialysis fluids (PDFs) leads to the formation of glucose degradation products (GDPs), which impair long-term peritoneal dialysis. The current study investigated the effects of metal ions, which occur as trace impurities in the fluids, on the formation of six major α-dicarbonyl GDPs, namely glucosone, glyoxal, methylglyoxal, 3-deoxyglucosone, 3-deoxygalactosone, and 3,4-dideoxyglucosone-3-ene. The chelation of metal ions by 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) during sterilization significantly decreased the total GDP content (585 µM vs. 672 µM), mainly due to the decrease of the glucose-oxidation products glucosone (14 µM vs. 61 µM) and glyoxal (3 µM vs. 11 µM), but also of methylglyoxal (14 µM vs. 31 µM). The glucose-dehydration products 3-deoxyglucosone, 3-deoxygalactosone, and 3,4-dideoxyglucosone-3-ene were not significantly affected by chelation of metal ions. Additionally, PDFs were spiked with eleven different metal ions, which were detected as traces in commercial PDFs, to investigate their influence on GDP formation during heat sterilization. Iron(II), manganese(II), and chromium(III) had the highest impact increasing the formation of glucosone (1.2-1.5 fold increase) and glyoxal (1.3-1.5 fold increase). Nickel(II) and vanadium(III) further promoted the formation of glyoxal (1.3 fold increase). The increase of the pH value of the PDFs from pH 5.5 to a physiological pH of 7.5 resulted in a decreased formation of total GDPs (672 µM vs 637 µM). These results indicate that the adjustment of metal ions and the pH value may be a strategy to further decrease the content of GDPs in PDFs.

Untargeted Proteomics-Based Profiling for the Identification of Novel Processing-Induced Protein Modifications in Milk.

Nonenzymatic post-translational protein modifications (nePTMs) affect the nutritional, physiological, and technological properties of proteins in food and in vivo. In contrast to the usual targeted analyses, the present study determined nePTMs in processed milk in a truly untargeted proteomic approach. Thus, it was possible to determine to which extent known nePTM structures explain protein modifications in processed milk and to detect and identify novel products. The method combined ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with bioinformatic data analysis by the software XCMS. The nePTMs detected by untargeted profiling of a ß-lactoglobulin-lactose model were incorporated in a sensitive scheduled multiple reaction monitoring method to analyze these modifications in milk samples and to monitor their reaction kinetics during thermal treatment. Additionally, we identified the structures of unknown modifications. Lactosylation, carboxymethylation, formylation of lysine and N-terminus, glycation of arginine, oxidation of methionine, tryptophan, and cysteine, oxidative deamination of N-terminus, and deamidation of asparagine and glutamine were the most important reactions of ß-lactoglobulin during milk processing. The isomerization of aspartic acid was observed for the first time in milk products, and N-terminal 4-imidazolidinone was identified as a novel nePTM.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

Design of a Prediction Model for the Differentiation of Pasteurized Milk from Heated ESL Milk by Peptide Profiling.

This study designs a prediction model to differentiate pasteurized milk from heated extended shelf life (ESL) milk based on milk peptides. For this purpose, quantitative peptide profiles of a training set of commercial samples including pasteurized (n = 20), pasteurized-ESL (n = 13), and heated-ESL (n = 16) milk are recorded by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Seven peptides are selected as putative markers, and cutoff levels and performance measures of each marker are defined by receiver operating characteristic (ROC) analysis. The accuracy of these peptides in the training set range between 71% and 90%. A prediction model is established based on the combined cutoff levels and evaluated by an independent blind test set. The processing method of 19 out of 20 unknown milk samples is predicted correctly achieving 95% accuracy. Five peptides of the prediction model are identified as αS1 -casein182-199 (m/z 2014.0), αS1 -casein180-199 (m/z 2216.1), αS1 -casein1-24 (m/z 2910.6), ß-casein108-125 (m/z 2126.0), and ß-casein106-125 (m/z 2391.2) indicating thermal release and the action of plasmin and cathepsins. Thus, the present study demonstrates that the milk peptide profile reflects even minor differences in production parameters.

Quantification of 24 circulating endocannabinoids, endocannabinoid-related compounds, and their phospholipid precursors in human plasma by UHPLC-MS/MS.

Endocannabinoids and endocannabinoid-related compounds (ERCs) are involved in many physiological processes. They are released on demand from phosphoinositide and N-acylphosphatidyl ethanolamine (NAPE) precursors and comprise 2-monoacylglycerols (2-MGs) and FA ethanolamides (FEAs). Despite the abundance of advanced quantitative methods, however, their determined concentrations in blood plasma are inconsistent because 2-MGs and FEAs undergo artifactual de novo formation, chemical isomerization, and degradation during sample collection and storage. For a comprehensive survey of these compounds in blood and plasma, we have developed and validated an ultra-HPLC-MS/MS method to quantify 24 endocannabinoids, ERCs, and their phospholipid precursors. Immediate acidification of EDTA-blood to pH 5.8 blocked artifactual FEA formation for at least 4 h on ice. The 2-MGs were stabilized after plasma harvest with 0.5 M potassium thiocyanate at pH 4.7. FEA and MG plasma concentrations in six healthy volunteers ranged between 0.04-3.48 and 0.63-6.18 ng/ml, respectively. Interestingly, only 1-5% of circulating FEAs were present in their free form, while the majority was bound to NAPEs. Similarly, 97% of 2-arachidonoylglycerol (2-AG) was bound to a potential phosphoinositide pool. The herein-described stabilization and extraction methods may now be used to reliably and comprehensively quantify endocannabinoids, ERCs, and their phospholipid precursors in clinical studies.

Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4-Dideoxyglucosone-3-ene (3,4-DGE).

Glucose degradation products (GDPs) are formed from glucose and other reducing sugars during heat treatment, for example, in heat-sterilized peritoneal dialysis fluids or foods. Because of their reactive mono- and dicarbonyl structure, they react readily with proteins, resulting in the formation of advanced glycation end products (AGEs), loss of protein functionality, and cytotoxicity. Among the GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) exerts the strongest effects despite its relatively low concentration levels. The goal of the present study was therefore to identify the structure of specific protein modifications deriving from 3,4-DGE. A nonapeptide containing the reactive amino acids lysine, arginine, and cysteine was incubated with 3,4-DGE and the dominant GDPs 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in concentrations as present in peritoneal dialysis fluids (235 µM 3-DG, 100 µM 3-Gal, and 11 µM 3,4-DGE). Glycation rate and product formation were determined by ultra-HPLC-MS/MS (UHPLC-MS/MS). 3,4-DGE showed the strongest glycation activity. After 2 h of incubation, 3,4-DGE had modified 57% of the nonapeptide, whereas 3-DG had modified only 2% and 3-DGal had modified 29% of the peptide. A stable 3,4-DGE-derived cysteine modification was isolated. Its structure was determined by comprehensive NMR and MS experiments to be [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC), which represents a novel cysteine-AGE derived from 3,4-DGE. The results indicate that 3,4-DGE might contribute to a severe loss of protein functionality by forming cysteine-specific AGEs, such as HHPC.

Targeted mass spectrometry to monitor nuclear accumulation of endogenous Nrf2 and its application to SH-SY5Y cells stimulated with food components.

The Nrf2 signaling pathway is highly significant for redox homeostasis. Hence, nutrients and drugs activating Nrf2 can prevent oxidative stress-mediated medical conditions. After activation, Nrf2 accumulates in the cell nucleus; therefore, stimulation of Nrf2 by food components and drugs is usually monitored by measuring nuclear Nrf2 levels. The present study developed a targeted mass spectrometry method for the highly reliable quantification of nuclear Nrf2 levels. Three Nrf2-specific peptides were detected after enzymatic digestion of the nuclear fraction by the developed protocol for micro-liquid chromatography-tandem mass spectrometry in scheduled multiple reaction monitoring mode (microLC-MS/MS-sMRM). The method also identified nuclear Nrf2 unequivocally and specifically in the SDS-PAGE fraction of 100-150 kDa. Moreover, highly precise and linear relative quantification was achieved (mean relative standard deviation 8.3%; coefficient of determination 0.998). Incubation experiments in SH-SY5Y neuroblastoma cells revealed significantly up to 6-fold elevated nuclear Nrf2 levels after stimulation with 10 µM carnosol (rosemary), 10 µM sulforaphane (broccoli), or 20 µM cinnamaldehyde (cinnamon). Our results were in very good accordance with conventional Nrf2 western blotting and were highly correlated with the food components' effect on the expression levels of NAD(P)H dehydrogenase [quinone] 1 and thioredoxin reductase 1, two major Nrf2-regulated cytoprotective enzymes. The newly developed microLC-MS/MS-sMRM method shows broad applicability and can serve as a highly selective and reliable method to analyze Nrf2 activation. Graphical abstract ᅟ.

Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1).

Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct Nɛ-(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed advanced glycation end products (AGEs) are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using methylglyoxal-derived hydroimidazolone 1 (MG-H1) as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.

Snack food as a modulator of human resting-state functional connectivity.

OBJECTIVE: To elucidate the mechanisms of how snack foods may induce non-homeostatic food intake, we used resting state functional magnetic resonance imaging (fMRI), as resting state networks can individually adapt to experience after short time exposures. In addition, we used graph theoretical analysis together with machine learning techniques (support vector machine) to identifying biomarkers that can categorize between high-caloric (potato chips) vs. low-caloric (zucchini) food stimulation. METHODS: Seventeen healthy human subjects with body mass index (BMI) 19 to 27 underwent 2 different fMRI sessions where an initial resting state scan was acquired, followed by visual presentation of different images of potato chips and zucchini. There was then a 5-minute pause to ingest food (day 1=potato chips, day 3=zucchini), followed by a second resting state scan. fMRI data were further analyzed using graph theory analysis and support vector machine techniques. RESULTS: Potato chips vs. zucchini stimulation led to significant connectivity changes. The support vector machine was able to accurately categorize the 2 types of food stimuli with 100% accuracy. Visual, auditory, and somatosensory structures, as well as thalamus, insula, and basal ganglia were found to be important for food classification. After potato chips consumption, the BMI was associated with the path length and degree in nucleus accumbens, middle temporal gyrus, and thalamus. CONCLUSION: The results suggest that high vs. low caloric food stimulation in healthy individuals can induce significant changes in resting state networks. These changes can be detected using graph theory measures in conjunction with support vector machine. Additionally, we found that the BMI affects the response of the nucleus accumbens when high caloric food is consumed.

Regioisomeric distribution of 9- and 13-hydroperoxy linoleic acid in vegetable oils during storage and heating.

BACKGROUND: The oxidative deterioration of vegetable oils is commonly measured by the peroxide value, thereby not considering the contribution of individual lipid hydroperoxide isomers, which might have different bioactive effects. Thus, the formation of 9- and 13-hydroperoxy octadecadienoic acid (9-HpODE and 13- HpODE), was quantified after short-term heating and conditions representative of long-term domestic storage in samples of linoleic acid, canola, sunflower and soybean oil, by means of stable isotope dilution analysis-liquid chromatography-mass spectroscopy. RESULTS: Although heating of pure linoleic acid at 180 °C for 30 min led to an almost complete loss of 9-HpODE and 13-HpODE, heating of canola, sunflower and soybean oil resulted in the formation of 5.74 ± 3.32, 2.00 ± 1.09, 16.0 ± 2.44 mmol L-1 13-HpODE and 13.8 ± 8.21, 10.0 ± 6.74 and 45.2 ± 6.23 mmol L-1 9-HpODE. An almost equimolar distribution of the 9- and 13-HpODE was obtained during household-representative storage conditions after 56 days, whereas, under heating conditions, an approximately 2.4-, 2.8- and 5.0-fold (P ≤ 0.001) higher concentration of 9-HpODE than 13-HpODE was detected in canola, soybean and sunflower oil, respectively. CONCLUSION: A temperature-dependent distribution of HpODE regioisomers could be shown in vegetable oils, suggesting their application as markers of lipid oxidation in oils used for short-term heating. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Global Food-Related Challenges: What Chemistry Has Achieved and What Remains to Be Done.

"… Food chemistry has the important task to improve food security by the development of applicable strategies to give perishable products longer shelf lives. The discovery of unknown food contaminants and adulterants that are not being searched for is crucial in order to avoid scandals and to provide safe and pure food for the consumer …" Read more in the Guest Editorial by Monika Pischetsrieder.

Targeted label-free quantification of interleukin-8 in PMA-activated U937 cell secretome by nanoLC-ESI-MS/MS-sSRM.

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12-myristate 13-acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin-8 (IL-8). Next, a label-free nanoLC-ESI-MS/MS-sSRM method for quantification of IL-8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM-MS using one proteotypic peptide as precursor ion and four mass transitions. Label-free quantification was performed by external calibration using IL-8 standard. Validation results indicated that the method was suited for the quantification of IL-8 in the secretome. The maximal IL-8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL-8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation.

The histamine receptors (HRs) represent a subclass of G protein-coupled receptors (GPCRs) and comprise four subtypes. Due to their numerous physiological and pathological effects, HRs are popular drug targets for the treatment of allergic reactions or the regulation of gastric acid secretion. Hence, an understanding of the functional selectivity of HR ligands has gained importance. These ligands can bind to specific GPCRs and selectively activate defined pathways. Supporting the activation of a therapeutically necessary pathway without the activation of other signaling cascades can result in drugs with more specific activity and fewer side effects. To evaluate the cellular consequences resulting from receptor binding, comprehensive analyses of cellular protein alterations upon incubation with ligands are required. For this purpose, endothelial cells are treated with histamine, as the endogenous ligand of HRs, to obtain a global overview of its cellular effects. Quantitative proteomics and pathway analyses of histamine-treated and untreated cells reveal enrichment of the nuclear factor-κB and tumor necrosis factor signaling pathways, cytokine-cytokine receptor interactions, complement and coagulation cascades, and acute inflammatory processes upon histamine treatment. This strategy offers the opportunity to monitor HR-mediated signaling in a multidimensional manner.

Identification of the oleic acid ethanolamide (OEA) isomer cis-vaccenic acid ethanolamide (VEA) as a highly abundant 18:1 fatty acid ethanolamide in blood plasma from rats and humans.

The endocannabinoid system is important in various physiological pathways, especially the regulation of food intake. It consists of endocannabinoids like 2-arachidonoyl-glycerol (2-AG) or the fatty acid ethanolamide archachidonoyl-ethanolamide (AEA) with binding affinity to cannabinoid receptors. Further, fatty acid ethanolamides (FAEAs) influence the endocannabinoid system without affecting cannabinoid receptors by using independent physiological pathways. Among FAEAs, oleic acid ethanolamide (OEA) gained importance because of its promising ability to reduce food intake. By ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS), we detected a chromatographically separated molecule in plasma samples from rats and humans with identical mass and fragmentation patterns as those of OEA. Via synthesis and extensive analysis of ethanolamides of different cis/trans- and position isomers of oleic acid (cis9-18:1), we could identify the unknown molecule as vaccenic acid (cis11-18:1) ethanolamide (VEA). In this study we identified VEA as the most abundant 18:1 FAEA in rat plasma and the second most abundant 18:1 FAEA in human plasma.

Three-Step Test System for the Identification of Novel GABAA Receptor Modulating Food Plants.

Potentiation of γ-amino butyric acid (GABA)-induced GABAA receptor (GABAAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1ß2-GABAAR expressed in Xenopus laevis oocytes. Positively tested food plants were then subjected to quantification of GABA content by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to exclude test foods, which evoke a TEVC-response by endogenous GABA. In the third step, specificity of GABAA-modulating activity was assessed by TEVC analysis of Xenopus laevis oocytes expressing the homologous glycine receptor (GlyR). The three-component test was then applied to screen 10 aqueous extracts of food plants for their GABAAR activity. Thus, hop cones (Humulus lupulus) and Sideritis sipylea were identified as the most potent specific GABAAR modulators eliciting significant potentiation of the current by 182 ± 27 and 172 ± 19 %, respectively, at the lowest concentration of 0.5 µg/mL. The extracts can now be further evaluated by in vivo studies and by structural evaluation of the active components.

Qualitative Profiling of Polyglucose Degradation Products in Peritoneal Dialysis Fluids.

Heat sterilization of peritoneal dialysis (PD) fluids leads to partial degradation of the osmotic agent to form reactive carbonyl structures, which significantly reduce the biocompatibility of PD fluids and impair long-term PD therapy. Hence, it is important to know the exact composition of the degradation products to improve biocompatibility of PD fluids. Our study conducted targeted screening for degradation products in polyglucose (icodextrin)-containing PD fluids (pGDPs) by applying o-phenylenediamine (OPD) to form stable derivatives, which were analyzed by ultrahigh-performance liquid chromatography with hyphenated diode array tandem mass spectrometry (UHPLC-DAD-MS/MS). For the first time, specific degradation products of polyglucose, namely, 4-deoxyglucosone (4-DG) and 3,4-dideoxypentosone (3,4-DDPS), could be identified in PD fluids. Further, a reaction product of 5-hydroxymethylfurfural (5-HMF) and OPD could be characterized to be (5-(1H-benzo[d]imidazol-2-yl)furan-2-yl)methanol. Additionally, 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal), both known to be present in glucose-based PD fluids, were also detected in polyglucose-containing fluids. Trapping a hitherto unknown degradation product with OPD yielded 1,4-bis(1H-benzo[d]imidazol-2-yl)-3,4-dihydroxybutan-1-one, which was present in heat- as well as filter-sterilized PD fluids.