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MOTIVATION: Plasma ionization is rapidly gaining popularity for mass spectrometry (MS)-based studies of volatiles and aerosols. However, data from plasma ionization are delicate to interpret as competing ionization pathways in the plasma create numerous ion species. There is no tool for detection of adducts and in-source fragments from plasma ionization data yet, which makes data evaluation ambiguous. SUMMARY: We developed DBDIpy, a Python library for processing and formal analysis of untargeted, time-sensitive plasma ionization MS datasets. Its core functionality lies in the identification of in-source fragments and identification of rivaling ionization pathways of the same analytes in time-sensitive datasets. It further contains elementary functions for processing of untargeted metabolomics data and interfaces to an established ecosystem for analysis of MS data in Python. AVAILABILITY AND IMPLEMENTATION: DBDIpy is implemented in Python (Version ≥ 3.7) and can be downloaded from PyPI the Python package repository (https://pypi.org/project/DBDIpy) or from GitHub (https://github.com/leopold-weidner/DBDIpy). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Ecosistema , Programas Informáticos , Espectrometría de Masas , Metabolómica , Biblioteca de GenesRESUMEN
A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded.
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Mass spectrometry is a popular and powerful analytical tool to study the effects of food processing. Industrial sampling, real-life sampling, or challenging academic research on process-related volatile and aerosol research often demand flexible, time-sensitive data acquisition by state-of-the-art mass analyzers. Here, we show a laboratory-scaled, miniaturized, and highly controllable setup for the online monitoring of aerosols and volatiles from thermal food processing based on dielectric barrier discharge ionization (DBDI) mass spectrometry (MS). We demonstrate the opportunities offered by the setup from a foodomics perspective to study emissions from the thermal processing of wheat bread rolls at 210 °C by Fourier transformation ion cyclotron resonance MS. As DBDI is an emerging technology, we compared its ionization selectivity to established atmospheric pressure ionization tools: we found DBDI preferably ionizes saturated, nitrogenous compounds. We likewise identified a sustainable overlap in the selectivity of detected analytes with APCI and electrospray ionization (ESI). Further, we dynamically recorded chemical fingerprints throughout the thermal process. Unsupervised classification of temporal response patterns was used to describe the dynamic nature of the reaction system. Compared to established tools for real-time MS, our setup permits one to monitor chemical changes during thermal food processing at ultrahigh resolution, establishing an advanced perspective for real-time mass spectrometric analysis of food processing.
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Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas/métodosRESUMEN
Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations.
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Espectrometría de Masas en Tándem , Vitamina B 12 , Vitamina B 12/análisis , Cromatografía Liquida , Reproducibilidad de los Resultados , IsótoposRESUMEN
Identification of chemically modified peptides in mass spectrometry (MS)-based glycation studies is a crucial yet challenging task. There is a need to establish a mode for matching tandem mass spectrometry (MS/MS) data, allowing for both known and unknown peptide glycation modifications. We present an open search approach that uses classic and modified peptide fragment ions. The latter are shifted by the mass delta of the modification. Both provide key structural information that can be used to assess the peptide core structure of the glycation product. We also leverage redundant neutral losses from the modification side chain, introducing a third ion class for matching referred to as characteristic fragment ions. We demonstrate that peptide glycation product MS/MS spectra contain multidimensional information and that most often, more than half of the spectral information is ignored if no attempt is made to use a multi-step matching algorithm. Compared to regular and/or modified peptide ion matching, our triple-ion strategy significantly increased the median interpretable fraction of the glycation product MS/MS spectra. For reference, we apply our approach for Amadori product characterization and identify all established diagnostic ions automatically. We further show how this method effectively applies the open search concept and allows for optimized elucidation of unknown structures by presenting two hitherto undescribed peptide glycation modifications with a delta mass of 102.0311 and 268.1768 Da. We characterize their fragmentation signature by integration with isotopically labeled glycation products, which provides high validity for non-targeted structure identification.
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Péptidos , Espectrometría de Masas en Tándem , Glicosilación , Iones , Fragmentos de Péptidos , Péptidos/química , Espectrometría de Masas en Tándem/métodosRESUMEN
After ingestion of food commodities, the gastrointestinal tract (GIT) poses the first barrier against xenobiotics and pathogens. Therefore, it is regularly confronted with external stressors potentially affecting the inflammatory response and the epithelial barrier. Alternaria mycotoxins such as alternariol (AOH) and altertoxin II (ATX-II) are frequently occurring food and feed contaminants that are described for their immunomodulatory capacities. Hence, this study aimed at exploring the effect of AOH and ATX-II as single compounds or binary mixtures on the immune response and epithelial homeostasis in noncancerous colon epithelial cells HCEC-1CT. Both toxins suppressed mRNA levels of proinflammatory mediators interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and secretion of IL-8, as well as mRNA levels of the matrix metallopeptidase 2 (MMP-2). Binary combinations of AOH and ATX-II reduced the response of the single toxins. Additionally, AOH and ATX-II modified immunolocalization of transmembrane proteins such as integrin ß1, zona occludens 1 (ZO-1), claudin 4 (Cldn 4), and occludin (Ocln), which support colonic tissue homeostasis and intestinal barrier function. Moreover, the cellular distribution of ZO-1 was affected by ATX-II. Mechanistically, these effects could be traced back to the involvement of several transcription factors. AOH activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2), governing cell metabolic competence and structural integrity. This was accompanied by altered distribution of the NF-κB p65 protein, an important regulator of inflammatory response. ATX-II also induced AhR and Nrf2 translocation, albeit failing to substantiate the effect of AOH on the colonic epithelium. Hence, both toxins coherently repress the intestinal immune response on the cytokine transcriptional and protein levels. Furthermore, both mycotoxins affected the colonic epithelial integrity by altering the cell architecture.
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Alternaria , Micotoxinas , Alternaria/química , Alternaria/metabolismo , Colon , Células Epiteliales/metabolismo , Inmunidad , Interleucina-8/metabolismo , Lactonas/metabolismo , Micotoxinas/toxicidad , Factor 2 Relacionado con NF-E2/metabolismo , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Nonalcoholic beers are becoming increasingly popular, in part due to consumers' awareness of a healthier lifestyle. Additionally, consumers are demanding diversification in the product range, which can be offered by producing nonalcoholic beers using non-Saccharomyces yeasts for fermentation to create a wide variety of flavors. So far, little is known about the nutritionally relevant byproducts that these yeasts release during wort fermentation and whether these yeasts can be considered safe for food fermentations. To gain insights into this, the B vitamins of four different nonalcoholic beers fermented with the yeast species Saccharomycodes ludwigii, Cyberlindnera saturnus (two strains), and Kluyveromyces marxianus were analyzed. Furthermore, a total of 16 beers fermented with different non-Saccharomyces yeast strains were analyzed for biogenic amines. Additionally, stress tolerance tests were performed at 37°C and in synthetic human gastric juice in vitro. B vitamins were found in the four nonalcoholic beers in nutritionally relevant amounts so they could serve as a supplement for a balanced diet. Biogenic amines remained below the limit of determination in all 16 beers, and thus likely had no influence, while the stress tolerance tests gave a first indication that seven yeast strains could possibly tolerate the human gastric juice milieu.
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Cerveza , Complejo Vitamínico B , Humanos , Cerveza/análisis , Complejo Vitamínico B/metabolismo , Levaduras/metabolismo , FermentaciónRESUMEN
OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Aminofenoles , Aminopiridinas , Benzodioxoles , Cromatografía Liquida , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Indoles , Isótopos , Mutación , Pirazoles , Piridinas , Pirrolidinas , Quinolonas , Espectrometría de Masas en TándemRESUMEN
1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.
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Alcaloides de Pirrolicidina , Animales , Cromatografía Liquida/métodos , Alcaloides de Pirrolicidina/análisis , Leche/química , Espectrometría de Masas en Tándem/métodos , Tropanos/análisis , Cromatografía Líquida de Alta PresiónRESUMEN
Folates are one of the essential micronutrients for all living organisms. Due to inadequate dietary intake, folate deficiency remains prevalent in humans. Genetically diverse germplasms can potentially be used as parents in breeding programs and also for understanding the folate regulatory network. Therefore, we investigated the natural genetic diversity of folates and their physiological regulation in pak choi (Brassica rapa subsp. Chinensis) germplasm. The total folate concentration ranged from 52.7 µg 100 gFW-1 to 166.9 µg 100 gFW-1, with 3.2-fold variation. The main folate vitamer was represented by 5-CH3-H4folate, with 4.5-fold variation. The activities of GTP cyclohydrolase I and aminodeoxy chorismate synthase, the first step of folate synthesis, were high in high folate accessions and low in low folate accessions. Analysis of the transcription levels of 11 genes associated with folate metabolism demonstrated that the difference in folate concentrations may be primarily controlled at the post-transcriptional level. A general correlation between total folate and their precursors was observed. Folate diversity and chlorophyll content were tightly regulated through the methyl cycle. The diverse genetic variation in pak choi germplasm indicated the great genetic potential to integrate breeding programs for folate biofortification and unravel the physiological basis of folate homeostasis in planta.
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Brassica rapa , Brassica rapa/genética , Ácido Fólico , FitomejoramientoRESUMEN
Prolonged exposure to mycotoxins, even in subtoxic concentrations, might contribute to modulate pro- or anti-inflammatory cascades and ultimately have long-term consequences on our health. In line, there is an increasing need to describe and comprehend the potential immunomodulatory effects of toxins that can be produced from fungi proliferating even in a domestic environment like, for instance, Alternaria alternata. Taking this as a starting point, we investigated the effects of one of the most potent genotoxic compounds produced by this fungi type, namely altertoxin II (ATXII) on THP-1 macrophages. In noncytotoxic concentrations (0.1-1 µM), ATXII inhibited the activation of the transcription factor NF-κB, and this event was accompanied by significant mitochondrial superoxide production (1 µM ATXII). Both responses seemed dependent on membrane structure and morphology since they were modulated by the coincubation with the cholesterol complexing agent methyl-ß-cyclodextrin (MßCD, 10-50 µM). Moreover, toxicity of ATXII was enhanced by cholesterol load (cholesterol-MßCD). The mycotoxin induced also lipid peroxidation (1-10 µM, ATXII) possibly streaming down at the mitochondrial level and suppressing NF-κB activation in THP-1 macrophages.
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Antiinflamatorios/farmacología , Benzo(a)Antracenos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Macrófagos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Antiinflamatorios/química , Benzo(a)Antracenos/química , Células Cultivadas , Humanos , Macrófagos/metabolismo , Mitocondrias/metabolismo , Estructura Molecular , FN-kappa B/metabolismo , Relación Estructura-Actividad , Células THP-1RESUMEN
Vitamin B6 comprises an important set of molecules tightly interwoven with the human amino acid, fatty acid, and carbohydrate metabolism. Analytical methods striving for the quantification of individual B6 vitamers so far mostly rely on methods based on HPLC in combination with fluorescence detection, but their application encounters multiple difficulties due to the chemical divergence of the single vitamers. The present study describes the development of a method based on LC-MS/MS and stable isotope dilution assay (SIDA) for the simultaneous quantification of five vitamers (PN, PL, PM, PMP, and PNG) of the B6 group in food samples. [13C3]-PN, [13C3]-PL, and [13C6]-PNG were applied as internal standards for the analysis of PN, PL, and PNG. PM and PMP were quantified via matrix-matched calibration referring to [13C3]-PN. The developed method was validated using starch matrix. The limits of detection and quantification ranged from 0.0028 to 0.02 mg/kg and from 0.0085 to 0.059 mg/kg, respectively, for all analytes. Calculated recoveries varied from 92 to 111%. Intra-injection precisions ranged from 0 to 9%, inter-day precisions from 4 to 10%, and intra-day precisions from 4 to 10%. A total of 14 plant-based food samples including fruits, vegetables, and cereals were examined for their content of vitamin B6 using the validated method. Furthermore, the first quantitation of PNG without enzymatic steps or divergent internal standards was undertaken utilizing LC-MS/MS and SIDA.
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Cromatografía Liquida/métodos , Grano Comestible/química , Frutas/química , Técnicas de Dilución del Indicador , Isótopos/química , Espectrometría de Masas en Tándem/métodos , Verduras/química , Vitamina B 6/análisis , Calibración , Estándares de Referencia , Vitamina B 6/químicaRESUMEN
The photochemical transformation of Maillard reaction products (MRPs) under simulated sunlight into mostly unexplored photoproducts is reported herein. Non-enzymatic glycation of amino acids leads to a heterogeneous class of intermediates with extreme chemical diversity, which is of particular relevance in processed and stored food products as well as in diabetic and age-related protein damage. Here, three amino acids (lysine, arginine, and histidine) were reacted with ribose at 100 °C in water for ten hours. Exposing these model systems to simulated sunlight led to a fast decay of MRPs. The photodegradation of MRPs and the formation of new compounds have been studied by fluorescence spectroscopy and nontargeted (ultra)high-resolution mass spectrometry. Photoreactions showed strong selectivity towards the degradation of electron-rich aromatic heterocycles, such as pyrroles and pyrimidines. The data show that oxidative cleavage mechanisms dominate the formation of photoproducts. The photochemical transformations differed fundamentally from "traditional" thermal Maillard reactions and indicated a high amino acid specificity.
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Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a large group of secondary plant metabolites comprising more than 660 compounds. Exhibiting geno- and hepatotoxic properties, they are responsible for multiple cases of food and feed poisoning over the last 100 years. For food and feed safety reasons, relevant PA/PANO should be monitored extensively in the main sources of PA/PANO intake. In this study, a sensitive analytical method was developed for detecting a broad range of 44 commercially available PA/PANO compounds, and in-house validation procedures were performed for several (herbal) teas. Various extraction solvents and procedures, as well as solid phase extraction materials for sample clean-up and analyte concentration, were tested to establish the methods' efficiency and effectiveness. Chromatographic conditions were optimised to obtain the best possible separation of isomers for the 44 PA/PANO analytes. The final method was proven very sensitive and accurate, with detection limits ranging from 0.1 to 7.0 µg/kg and precisions between 0.7 and 16.1%. For 40 of the analytes, the recovery rates ranged from 60.7 to 128.8%. The applicability and trueness of the method were examined by analysing tea samples from a local supermarket and comparing them to a reference material. At least one PA/PANO analyte was detected in 17 of the 18 samples under investigation, and the sum contents of the samples ranged from 0.1 to 47.9 µg/kg. Knowledge of the PA/PANO composition in a sample can be used to indicate the botanical origin of the impurity and, thus, the geographical region of cultivation.
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Cromatografía Liquida/métodos , Alcaloides de Pirrolicidina/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Té/química , Tés de Hierbas/análisis , Contaminación de Alimentos/análisis , Límite de Detección , Reproducibilidad de los ResultadosRESUMEN
Milk fatty acids (MFA) are a proxy for the prediction of CH4 emission from cows, and prediction differs with diet. Our objectives were (1) to compare the effect of diets on the relation between MFA profile and measured CH4 production, (2) to predict CH4 production based on 6 data sets differing in the number and type of MFA, and (3) to test whether additional inclusion of energy-corrected milk (ECM) yield or dry matter intake (DMI) as explanatory variables improves predictions. Twenty dairy cows were used. Four diets were used based on corn silage (CS) or grass silage (GS) without (L0) or with linseed (LS) supplementation. Ten cows were fed CS-L0 and CS-LS and the other 10 cows were fed GS-L0 and GS-LS in random order. In feeding wk 5 of each diet, CH4 production (L/d) was measured in respiration chambers for 48 h and milk was analyzed for MFA concentrations by gas chromatography. Specific CH4 prediction equations were obtained for L0-, LS-, GS-, and CS-based diets and for all 4 diets collectively and validated by an internal cross-validation. Models were developed containing either 43 identified MFA or a reduced set of 7 groups of biochemically related MFA plus C16:0 and C18:0. The CS and LS diets reduced CH4 production compared with GS and L0 diets, respectively. Methane yield (L/kg of DMI) reduction by LS was higher with CS than GS diets. The concentrations of C18:1 trans and n-3 MFA differed among GS and CS diets. The LS diets resulted in a higher proportion of unsaturated MFA at the expense of saturated MFA. When using the data set of 43 individual MFA to predict CH4 production (L/d), the cross-validation coefficient of determination (R2CV) ranged from 0.47 to 0.92. When using groups of MFA variables, the R2CV ranged from 0.31 to 0.84. The fit parameters of the latter models were improved by inclusion of ECM or DMI, but not when added to the data set of 43 MFA for all diets pooled. Models based on GS diets always had a lower prediction potential (R2CV = 0.31 to 0.71) compared with data from CS diets (R2CV = 0.56 to 0.92). Models based on LS diets produced lower prediction with data sets with reduced MFA variables (R2CV = 0.62 to 0.68) compared with L0 diets (R2CV = 0.67 to 0.80). The MFA C18:1 cis-9 and C24:0 and the monounsaturated FA occurred most often in models. In conclusion, models with a reduced number of MFA variables and ECM or DMI are suitable for CH4 prediction, and CH4 prediction equations based on diets containing linseed resulted in lower prediction accuracy.
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Alimentación Animal/análisis , Bovinos/metabolismo , Ácidos Grasos/metabolismo , Metano/metabolismo , Leche/metabolismo , Animales , Dieta/veterinaria , Ácidos Grasos/análisis , Femenino , Lino/química , Lino/metabolismo , Lactancia , Leche/química , Poaceae/metabolismo , Ensilaje/análisis , Zea mays/metabolismoRESUMEN
Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed-safflower oil mix (EFA; rich in n-3 FA), Lutalin®, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.
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Adenosina Trifosfato/metabolismo , Industria Lechera , Dieta , Membrana Eritrocítica/metabolismo , Ácidos Grasos/farmacología , Animales , Bovinos , Conexinas/metabolismo , Suplementos Dietéticos , Membrana Eritrocítica/efectos de los fármacos , Femenino , Proteínas de la Membrana/metabolismoRESUMEN
Background: High-fat diets (HFDs) have been linked to low-grade inflammation and insulin resistance. Objective: The main purpose of the present study was to assess whether acute overfeeding with an HFD affects insulin sensitivity, gut barrier function, and fecal microbiota in humans. Methods: In a prospective intervention study, 24 healthy men [mean ± SD: age 23.0 ± 2.8 y, body mass index (in kg/m2) 23.0 ± 2.1] received an HFD (48% of energy from fat) with an additional 1000 kcal/d (as whipping cream) above their calculated energy expenditure for 7 d. Insulin sensitivity (hyperinsulinemic euglycemic clamp), gut permeability (sugar and polyethylene glycol absorption tests, plasma zonulin), and gut microbiota profiles (high-throughput 16S rRNA gene sequencing) were assessed before and after overfeeding, and 14 d after intervention. Additionally, inflammation markers such as high-sensitivity C-reactive protein, lipopolysaccharide-binding protein, leptin, high-molecular-weight adiponectin, calprotectin, regulated on activation normal, T cell expressed and secreted (RANTES), and monocyte chemoattractant protein-1 were measured in plasma by ELISA. Finally, lipid parameters were analyzed in serum by a laboratory service. Results: Although participants gained 0.9 ± 0.6 kg (P < 0.001) body weight, overnutrition was not associated with a significant change in insulin sensitivity (M value and glucose disposal). Overfeeding for 7 d resulted in elevated serum total (10.2%), LDL (14.6%) and HDL (14.8%) cholesterol concentrations (P < 0.01). In contrast, fasting plasma triglyceride significantly declined (29.3%) during overfeeding (P < 0.001). In addition, there were no significant changes in inflammatory markers. Urine excretion of 4 sugars and polyethylene glycol, used as a proxy for gut permeability, and plasma concentration of zonulin, a marker of paracellular gut permeability, were unchanged. Moreover, overfeeding was not associated with consistent changes in gut microbiota profiles, but marked alterations were observed in a subgroup of 6 individuals. Conclusions: Our findings suggest that short-term overfeeding with an HFD does not significantly impair insulin sensitivity and gut permeability in normal-weight healthy men, and that changes in dominant communities of fecal bacteria occur only in certain individuals. The study was registered in the German Clinical Trial Register as DRKS00006211.
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Productos Lácteos , Dieta Alta en Grasa , Heces/microbiología , Microbioma Gastrointestinal , Adulto , Biomarcadores/sangre , Índice de Masa Corporal , Metabolismo Energético , Estudios de Seguimiento , Humanos , Resistencia a la Insulina , Masculino , Permeabilidad , Estudios Prospectivos , ARN Ribosómico 16S/genética , Estadísticas no Paramétricas , Adulto JovenRESUMEN
RATIONALE: Atropine (Atr) and scopolamine (Scp) are toxic secondary plant metabolites of species within the Solanaceae genus that can accidentally or intentionally reach the food store chain by inaccurate harvesting of any plant material, e.g. for herbal tea infusions. Ingestion may cause severe anticholinergic poisoning thus requiring risk-oriented determination in food and beverages. The suitability of matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), should be characterized for simultaneous analysis. METHODS: We herein present the first MALDI-TOF MS(/MS) procedure for quantitative determination of both alkaloids in herbal tea infusions and Solanaceae plant material. A standard additions procedure using triply deuterated Atr as internal standard was developed and validated. RESULTS: Tropane alkaloids were detected without interferences and the standard additions procedure allowed reliable quantification. Intraday and interday precision were less than 17% and corresponding accuracies were between 77% and 112%. Limits of detection in the spotting solution were found at 5 ng/mL (Atr) and 0.5 ng/mL (Scp). The assay was applied to diverse tea infusions as well as to berries and leaves of deadly nightshade and angel's trumpet. CONCLUSIONS: The usefulness of MALDI-TOF MS(/MS) for investigations of plant-derived samples to prove contaminations by small basic compounds was demonstrated. The elaborate procedure is reliable but quite laborious to obtain quantitative results, but MALDI-TOF MS(/MS) was also shown to be a valuable tool for rapid qualitative screening for Atr and Scp in plant extracts.
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Atropina/análisis , Escopolamina/análisis , Solanaceae/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tés de Hierbas/análisis , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Límite de Detección , Extractos Vegetales/químicaRESUMEN
[13C3]-labelled vitamers (PN, PL and PM) of the B6 group were prepared starting from [13C3]-propionic acid. [13C3]-PN was synthesized in ten linear steps with an overall yield of 17%. Hereby, higher alkyl homologues of involved esters showed a positive impact on the reaction outcome of the intermediates in the chosen synthetic route. Oxidation of [13C3]-PN to [13C3]-PL was undertaken using potassium permanganate and methylamine followed by acid hydrolysis of the imine derivative. [13C3]-PM could be prepared from the oxime derivative of [13C3]-PN by hydrogenation with palladium.