Omega-3 PUFAs prevent bone impairment and bone marrow adiposity in mouse model of obesity.

Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.

Triacylglycerols containing branched palmitic acid ester of hydroxystearic acid (PAHSA) are present in the breast milk and hydrolyzed by carboxyl ester lipase.

Breast milk is a complex mixture containing underexplored bioactive lipids. We performed an observational case-control study to compare the impact of delivery mode: caesarean section (CS) and vaginal birth (VB); and term (preterm and term delivery) on the levels of lipokines in human milk at different stages of lactation. Metabolomic analysis of the milk identified triacylglycerol estolides as a metabolic reservoir of the anti-inflammatory lipid mediator 5-palmitic acid ester of hydroxystearic acid (5-PAHSA). We found that triacylglycerol estolides were substrates of carboxyl ester lipase and 5-PAHSA-containing lipids were the least preferred substrates among tested triacylglycerol estolide isomers. This explained exceptionally high colostrum levels of 5-PAHSA in the VB group. CS and preterm birth negatively affected colostrum lipidome, including 5-PAHSA levels, but the lipidomic profiles normalized in mature milk. Mothers delivering term babies vaginally produce colostrum rich in 5-PAHSA, which could contribute to the prevention of intestinal inflammation in newborns.

Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity.

Preclinical evidence suggests that n-3 fatty acids EPA and DHA (Omega-3) supplemented as phospholipids (PLs) may be more effective than triacylglycerols (TAGs) in reducing hepatic steatosis. To further test the ability of Omega-3 PLs to alleviate liver steatosis, we used a model of exacerbated non-alcoholic fatty liver disease based on high-fat feeding at thermoneutral temperature. Male C57BL/6N mice were fed for 24 weeks a lard-based diet given either alone (LHF) or supplemented with Omega-3 (30 mg/g diet) as PLs (krill oil; ω3PL) or TAGs (Epax 3000TG concentrate; ω3TG), which had a similar total content of EPA and DHA and their ratio. Substantial levels of TAG accumulation (~250 mg/g) but relatively low inflammation/fibrosis levels were achieved in the livers of control LHF mice. Liver steatosis was reduced by >40% in the ω3PL but not ω3TG group, and plasma ALT levels were markedly reduced (by 68%) in ω3PL mice as well. Krill oil administration also improved hepatic insulin sensitivity, and its effects were associated with high plasma adiponectin levels (150% of LHF mice) along with superior bioavailability of EPA, increased content of alkaloids stachydrine and trigonelline, suppression of lipogenic gene expression, and decreased diacylglycerol levels in the liver. This study reveals that in addition to Omega-3 PLs, other constituents of krill oil, such as alkaloids, may contribute to its strong antisteatotic effects in the liver.

Additive Effects of Omega-3 Fatty Acids and Thiazolidinediones in Mice Fed a High-Fat Diet: Triacylglycerol/Fatty Acid Cycling in Adipose Tissue.

Long-chain n-3 polyunsaturated fatty acids (Omega-3) and anti-diabetic drugs thiazolidinediones (TZDs) exhibit additive effects in counteraction of dietary obesity and associated metabolic dysfunctions in mice. The underlying mechanisms need to be clarified. Here, we aimed to learn whether the futile cycle based on the hydrolysis of triacylglycerol and re-esterification of fatty acids (TAG/FA cycling) in white adipose tissue (WAT) could be involved. We compared Omega-3 (30 mg/g diet) and two different TZDs-pioglitazone (50 mg/g diet) and a second-generation TZD, MSDC-0602K (330 mg/g diet)-regarding their effects in C57BL/6N mice fed an obesogenic high-fat (HF) diet for 8 weeks. The diet was supplemented or not by the tested compound alone or with the two TZDs combined individually with Omega-3. Activity of TAG/FA cycle in WAT was suppressed by the obesogenic HF diet. Additive effects in partial rescue of TAG/FA cycling in WAT were observed with both combined interventions, with a stronger effect of Omega-3 and MSDC-0602K. Our results (i) supported the role of TAG/FA cycling in WAT in the beneficial additive effects of Omega-3 and TZDs on metabolism of diet-induced obese mice, and (ii) showed differential modulation of WAT gene expression and metabolism by the two TZDs, depending also on Omega-3.

Exercise training induces insulin-sensitizing PAHSAs in adipose tissue of elderly women.

Adverse effects of aging can be delayed with life-style interventions. We examined how exercise training (ET) alone or combined with omega-3 polyunsaturated fatty acid (PUFA) affects serum and adipose tissue (AT) lipidome in older women. Fifty-five sedentary older women were included in the physical activity program and given either sunflower (Placebo) or wax esters-rich (Calanus) oil capsules for 4 months. Serum and subcutaneous abdominal AT samples were acquired while maximum rates of oxygen consumption (VO2 max), insulin sensitivity (hyperinsulinemic-euglycemic clamps) and comprehensive lipidome profiles were determined before and after the study. ET increased VO2 max in both groups. Lipidomics profiling revealed unusual serum triacylglycerols and phospholipids with ether-bound alkyls in the Calanus group, while ET generally induced shorter-chain triacylglycerols in AT, suggesting increased de novo lipogenesis. The latter was positively associated with whole-body insulin sensitivity. Unexpectedly, insulin-sensitizing lipokines from the family of branched palmitic acid esters of hydroxy stearic acid (PAHSAs) were elevated in both serum and AT after ET, while PAHSAs-containing triacylglycerols were detected in AT. ET stimulated beneficial changes in AT, including PAHSAs synthesis. Although the added value of omega-3 PUFA supplementation was not proven, our discovery can help understand the nature of the metabolic benefits of exercise.

Untargeted metabolomics identifies trimethyllysine, a TMAO-producing nutrient precursor, as a predictor of incident cardiovascular disease risk.

Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.

Development of Tetramethylenedisulfotetramine (TETS) Hapten Library: Synthesis, Electrophysiological Studies, and Immune Response in Rabbits.

There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABAA ) receptors. General synthetic approach toward two groups of analogues was developed. Screening of the resulting library of compounds by FLIPR or whole-cell voltage-clamp revealed that, despite the structural differences, some of the TETS analogues retained GABAA receptor inhibition; however, their potency was an order of magnitude lower. Antibodies raised in rabbits against some of the TETS analogues conjugated to protein recognized free TETS and will be used for the development of an immunoassay for TETS.

Evaluation of direct analysis in real time ionization-mass spectrometry (DART-MS) in fish metabolomics aimed to assess the response to dietary supplementation.

Ambient mass spectrometry employing a direct analysis in real time (DART) ion source coupled to a medium high-resolution/accurate mass time-of-flight mass spectrometer (TOFMS) was used as a rapid tool for metabolomic fingerprinting to study the effects of supplemental feeding with cereals (triticale) on the composition of muscle metabolites of common carp (Cyprinus carpio L.). First, the sample extraction and DART-TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in the extracts obtained from common carp muscle. To this end, a simultaneous (all-in-one) extraction procedure was developed employing water and cyclohexane mixture as the extraction solvents. Under these conditions both polar as well as non-polar metabolites were isolated within a single extraction step. Next, the metabolomic fingerprints (mass spectra) of a large set of common carp muscle extracts were acquired. Finally, the experimental data were statistically evaluated using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Using this approach, differentiation of common carp muscle in response to dietary supplementation (feeding with and without cereals) was feasible. Correct classification was obtained based on the assessment of polar and as well as non-polar extracts fingerprints. The current study showed that DART-TOFMS metabolomic fingerprinting represents a rapid and powerful analytical strategy enabling differentiation of common carp muscles according to feeding history by recording metabolomic fingerprints of ionizable components under the conditions of ambient MS.

Critical assessment of extraction methods for the simultaneous determination of pesticide residues and mycotoxins in fruits, cereals, spices and oil seeds employing ultra-high performance liquid chromatography-tandem mass spectrometry.

This study addresses a current trend in chemical food safety control represented by an effort to integrate analyses of various groups of food contaminants/toxicants into a single, high-throughput method. The choice of optimal sample preparation step is one of the key conditions to achieve good performance characteristics. In this context, we investigated the possibility to expand the scope of the three multi-analyte extraction procedures employed earlier in other studies for rapid isolation of either pesticides or mycotoxins from plant matrices. Following procedures were tested: A - aqueous acetonitrile extraction followed by partition (QuEChERS-like method), B - aqueous acetonitrile extraction, and C - pure acetonitrile extraction. On the list of target analytes, we had 288 pesticides (including 'troublesome' acidic, basic and base-sensitive compounds) together with 38 mycotoxins (including all EU regulated ones and many 'emerging' toxins on the European Food Safety Authority (EFSA) list). The matrices selected for the experiments, apple baby food, wheat flour, spices and sunflower seeds, represented various composition categories in terms of moisture, fat and extractable compounds (e.g. pigments and essential oils) content. In preliminary experiments, acceptable recoveries (70-120%) for most of analytes were obtained by the analysis of spiked matrices, regardless which extraction procedure was used. However, when analysing dry samples with incurred pesticide residues/mycotoxins, the method C did not enable efficient extraction of some common contaminants. Procedure A, thanks to a higher matrix equivalent compared to the method B and relatively less pronounced matrix effects, enabled lower quantification limits for all analyte/matrix combinations, with the exception of polar mycotoxins and/or pesticides. Higher recoveries for the latter group of analytes could be achieved by the method B; on the other hand, extraction efficiency of non-polar pesticides from fatty matrix was rather poor by this method.

Streamlining sample preparation and gas chromatography-tandem mass spectrometry analysis of multiple pesticide residues in tea.

In this work, a new rapid method for the determination of 135 pesticide residues in green and black dry tea leaves and stalks employing gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) with a triple quadrupole was developed and validated. A substantial simplification of sample processing prior to the quantification step was achieved: after addition of water to a homogenised sample, transfer of analytes into an acetonitrile layer was aided by the addition of inorganic salts. Bulk co-extracts, contained in the crude organic extract obtained by partition, were subsequently removed by liquid-liquid extraction using hexane with the assistance of added 20% (w/w) aqueous NaCl solution. The importance of matrix hydration prior to the extraction for achieving good recoveries was demonstrated on tea samples with incurred pesticide residues. For most of the analytes, recoveries in the acceptable range of 70-120% and repeatabilities (relative standard deviations, RSDs) ≤20% were achieved for both matrices at spiking levels of 0.01, 0.1 and 1 mg kg(-1). Under optimised GC-MS/MS conditions, most of the analytes gave lowest calibration level ≤0.01 mg kg(-1), permitting the control at the maximum residue levels (MRLs) laid down in Regulation (EC) No 396/2005. The developed method was successfully applied to the determination of pesticide residues in real tea samples.

Rapid analysis of caffeine in various coffee samples employing direct analysis in real-time ionization-high-resolution mass spectrometry.

The development and use of a fast method employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) for the quantitative analysis of caffeine in various coffee samples has been demonstrated in this study. A simple sample extraction procedure employing hot water was followed by direct, high-throughput (<1 min per run) examination of the extracts spread on a glass rod under optimized conditions of ambient mass spectrometry, without any prior chromatographic separation. For quantification of caffeine using DART-TOFMS, an external calibration was used. Isotopically labeled caffeine was used to compensate for the variations of the ion intensities of caffeine signal. Recoveries of the DART-TOFMS method were 97% for instant coffee at the spiking levels of 20 and 60 mg/g, respectively, while for roasted ground coffee, the obtained values were 106% and 107% at the spiking levels of 10 and 30 mg/g, respectively. The repeatability of the whole analytical procedure (expressed as relative standard deviation, RSD, %) was <5% for all tested spiking levels and matrices. Since the linearity range of the method was relatively narrow (two orders of magnitude), an optimization of sample dilution prior the DART-TOFMS measurement to avoid saturation of the detector was needed.

Ambient mass spectrometry employing direct analysis in real time (DART) ion source for olive oil quality and authenticity assessment.

A novel approach for the authentication of olive oil samples representing different quality grades has been developed. A new type of ion source, direct analysis in real time (DART), coupled to a high-resolution time-of-flight mass spectrometer (TOFMS) was employed for the comprehensive profiling of triacylglycerols (TAGs) and/or polar compounds extracted with a methanol-water mixture. The main parameters influencing the ionization efficiency of TAGs were the type of sample solvent, degree of sample dilution, ion beam temperature, and presence of a dopant (ammonia vapors). The ionization yield of polar compounds depended mainly on a content of water in the extract and ion beam temperature. Using DART-TOFMS, not only differentiation among extra virgin olive oil (EVOO), olive pomace oil (OPO) and olive oil (OO) could be easily achieved, but also EVOO adulteration with commonly used adulterant, hazelnut oil (HO), was feasible. Based on the linear discriminant analysis (LDA), the introduced method allowed detection of HO addition of 6 and 15% (v/v) when assessing DART-TOFMS mass profiles of polar compounds and TAGs, respectively.