MetaboLights

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.ac.uk/pub/databases/metabolights/studies/public/MTBLS561/FILES/151005s38.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS561/FILES/151005s56.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS561/FILES/151203s032.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS561/FILES/151005s72.rawprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS561For discovery of candidate biomarkers, the urinary fingerprints of the control group were compared with those of the TU treated group (excl. washout). To this end, urine samples from both treatment groups were extracted and analyzed in a random order, keeping the batches of the cows and calves separately. During the discovery phase, data was interpreted in a truly untargeted fashion. Following this, the classification performance of the selected candidate markers was further evaluated by considering the samples from the rapeseed-enriched diet and washout group. These samples were analyzed in a random order, whereby a targeted processing (XCalibur 3.0 software) of the acquired full-scan data was performed. During mass spectrometric analysis of urine extracts, quality control measures were taken by considering external and internal quality control (QC) samples. QC samples were prepared starting from pooled urine (equal volume contributions from at least 60 samples), which was extracted according to the standard protocol. External QC samples were used for instrument stabilization, whereas internal QC samples were included to monitor and correct for instrumental drift. Acquired full-scan MS data of study samples were imported into the SieveTM 2.2 software package (Thermo Fisher Scientific, San Jose, USA) to compose metabolic fingerprints. Metabolite features from total ion current chromatograms were extracted, applying peak alignment and integration. As primary parameters, a frame m/z width of 6 ppm, a frame time width of 0.75 min, and an intensity threshold of 1,000,000 were set. Each of the detected features was characterized through its retention time (tR) and m/z-value. For the discovery phase, the normalized multivariate data matrices from the TU treated and control group were analyzed by SIMCA 14.1 (Umetrics, Malmö, Sweden), whereby it was aimed to discover metabolome discrepancies for the two study groups. Data were log-transformed and pareto-scaled to induce normality and standardize the peak intensity ranges, respectively. In first instance, unsupervised segregation was checked by principal component analysis (PCA), allowing to evaluate clustering of QC samples and identify potential outliers. Subsequently, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed to model variation and establish separation between investigated treatment groups.MetaboLightsPublicUPLC-LTQ-MSThe UPLC system consisted of a Dionex Ultimate 3000 XRS pump, coupled to a Dionex Ultimate 3000 RS column compartment and autosampler (Dionex, Amsterdam, The Netherlands). Chromatographic separation was achieved on an Acquity HSS T3 column (1.8 µm, 2.1 x 100 mm) (Waters, Zellik, Belgium), whereby a gradient program using 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) was applied. Following proportions of solvent A were used: 0-1 min at 90%, 1-3 min from 90 to 79%, 3-5 min from 79 to 20%, 5-9 min from 20 to 0%, 9-12 min at 0%, 12-12.1 from 0 to 90%, followed by 3 min of re-equilibration. A constant flow rate of 300 µl/min and a column oven temperature of 25 °C were set.Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle. PMID:29649241UGhentBeata Pomianurinemass spectrometryFor extraction, a previously published methodology was adopted with some minor modifications[1]. In brief, 3 ml of urine was enriched with 50 ng PTU-d5 internal standard and supplemented with 1 mL phosphate buffer, containing 1% of the reducing agent DDT. Hereby, denaturing conditions (65 °C, 30 min) were imposed to impede protein-thyreostat interactions and avoid protein-based interference. Next, a 2-fold liquid-liquid extraction with 5 ml ethyl acetate was performed, after which the pooled supernatants were evaporated to dryness at 60 °C under a gentle stream of nitrogen. The remaining residue was dissolved in 200 µl ultrapure water (0.1% formic acid) and methanol (0.1% formic acid) (90/10, v/v). The injection volume was 10 µl. Ref: [1] Vanden Bussche J, Vanhaecke L, Deceuninck Y, Verheyden K, Wille K, Bekaert K, Le Bizec B, De Brabander HF. Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation. J Chromatogr A. 2010 Jun 25;1217(26):4285-93. doi: 10.1016/j.chroma.2010.04.030. PMID:20471019Bos taurushttps://www.ebi.ac.uk/metabolights/MTBLS561Beata Pomian. Faculty of Veterinary Medicine, Laboratory of Chemical Analysis. Salisburylaan 133, 9820 Merelbeke, Belgium. beata.pomian@ugent.be.Lieven Van Meulebroek. Faculty of Veterinary Medicine, Laboratory of Chemical Analysis. Salisburylaan 133, 9820 Merelbeke, Belgium. lieven.vanmeulenbroek@ugent.be.Targeted data was processed with Xcalibur Software 2.1 (Thermo Fisher Scientific). In particular, this involved the quantification of thiouracil.Treatmentbeata.pomian@ugent.beThe in vivo trial concerned a parallel-like design with various treatment groups. Within a first treatment group, test animals received a diet that was enriched with rapeseed cake, which intended to promote the semi-endogenous formation of TU. A second group of animals were fed a commercial diet during the entire course of the trial and thus served as control group. Finally, a group of animals was treated with TU on a daily basis (in the morning) for one week, which was followed by a washout period. Acclimation periods were incorporated for adaptation to the imposed dietary regimen. Each of the treatments was preceded by a 2-week acclimation phase during which all test animals were fed a commercial diet of concentrate (27% crude protein content) with ad libitum access to water and hay. Whereas this dietary regimen was maintained for the control and TU treated group, one fraction of the test animals received a diet in which 30% of the concentrate was replaced by rapeseed cake (37% crude protein content). With respect to the TU treated group, animals were orally administered a daily dose of 0.2 g TU per 100 kg body weight (TU analytical powder, Sigma Aldrich, St. Louis, MO, USA) by means of a filled capsule. Following this TU treatment, a short washout period was appended to assess the excretion profiles of potential markers in urine. Test animals were housed under controlled experimental conditions at the animal facilities of Centre d’Economie Rurale (CER, Marloie, Belgium), whereby animals were kept in separate half-covered pens. It was hereby opted to include both cows and calves since an age-dependent metabolic response towards TU treatment could be expected. All calves (female, 101.6 ± 14.7 kg, 3.3 ± 0.3 months) were of a mixed breed, whereas cows (female, 587.1 ± 98.9 kg, 4.0 ± 1.5 years) were either of a milk or meat breed. This in vivo study was approved by CER’s Ethical Committee (CE/Santé/ET004). Urine was collected in 5-ml aliquots, whereby samples were treated with EDTA (final concentration of 0.1 M) and 0.1 M hydrogen chloride (final pH 1) to inhibit thyreostat degradation during urine storage. Urine samples were stored at -20 °C and thawed prior to extraction.Metabolomicsultra-performance liquid chromatography-mass spectrometrythiouracilCattlebiomarkertargeted metabolitesultra-performance liquid chromatography-mass spectrometrythiouracilCattlebiomarkertargeted metabolitesMass spectrometric analysis was carried out using a high-resolution hybrid quadrupole Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA), which was equipped with a heated electrospray ionization source (HESI-II), operating in polarity switching mode. Instrumental settings for full-scan MS events involved a sheath gas flow rate of 2 arbitrary units (au), an auxiliary gas flow rate of 10 au, a sweep gas flow rate of 2 au, a capillary temperature of 250 °C, a heater temperature of 275 °C, a spray voltage of (-)3 kV, an S-lens RF level of 50 au, a mass resolution of 70,000 full width at half maximum (FWHM), an automatic gain control (AGC) target of 3 x e^6 ions, and a m/z scan range from 100 to 800 Da. In addition to these full-scan MS events, separate MS/MS fragmentation experiments were performed for identification of revealed markers.6-(2-hydroxyethyl)-2-sulfanylidene-1H-pyrimidin-4-one3-[(2-amino-3-sulfanylpropanoyl)amino]-4-(carboxymethylamino)-4-oxobutanoic acidthiouracil2-sulfanyl-2,3-dihydro-1H-pyrimidin-4-one2H-tetrazol-2-ium-5-carboxamide3-thioureidopropionic acid3-(1,1-dioxo-3-thiolanyl)-5-methyl-2-thioxo-4-imidazolidinoneN-({2-[(ethylsulfonyl)amino]ethyl}sulfonyl)-L-alanineIn the European Union, the use of thyreostats for animal fattening purposes has been banned and monitoring plans have been established to detect potential abuse. However, this is not always straightforward as thyreostats such as thiouracil may also have a semi-endogenous origin. Therefore, this study aimed at defining urinary metabolites, which may aid in defining the origin of detected thiouracil. Hereto, a parallel-like randomized in vivo study was conducted in which calves (n = 8) and cows (n = 8) were subjected to either a control treatment, rapeseed-enriched diet to induce semi-endogenous formation, or thiouracil treatment. Urine samples (n = 330) were assessed through metabolic fingerprinting, employing liquid-chromatography and Q-ExactiveTM Orbitrap mass spectrometry. Urinary fingerprints comprised up to 40,000 features whereby multivariate discriminant analysis was able to point out significant metabolome differences between treatments (Q2(Y) ≥ 0.873). Using the validated models, a total of twelve metabolites (including thiouracil) were assigned marker potential. Combining these markers into age-dependent biomarker panels rendered a tool by which sample classification could be improved in comparison with thiouracil-based thresholds, and this during on-going thiouracil treatment (specificities ≥ 95.2% and sensitivities ≥ 85.7%), post-treatment (sensitivities ≥ 80% for ≥ 24 h after last administration), and simulated low-dose thiouracil treatment (exogenous thiouracil below 30 ng μL-1). Moreover, the metabolic relevance of revealed markers was supported by the suggested identities, for which a structural link with thiouracil could be determined in most cases. The proposed biomarker panels may contribute to a more justified decision-making in monitoring thiouracil abuse.Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design.Van Meulebroek Lieven L, Wauters Jella J, Pomian Beata B, Vanden Bussche Julie J, Delahaut Philippe P, Fichant Eric E, Vanhaecke Lynn LTaurine, dairy cow, Biological Markers, Viral Marker, Beef Cows, Viral, Surrogate Endpoints, Surrogate Endpoint, cow, Clinical Markers, Laboratory, Taurus Cattles, Bos indicus Cattles, Clinical Marker, Holstein Cow, Serum Markers, Taurus Cattle, Biochemical, End Point, Endpoint, Cattle, Taurine Cattles, Biochemical Markers, 3-dihydro-2-thioxo-, Serum, Indicine, Bos indicus, Dairy Cows, Biologic Marker, Immune Marker, Domestic Cows., Surrogate End Points, Surrogate Markers, 4(1H)-Pyrimidinone, Laboratory Markers, Bos grunniens, cattle, Marker, Biological, Surrogate End Point, Bos taurus, Bos primigenius taurus, 2, domestic cattle, Indicine Cattles, Yaks, Holstein, Cattles, Biologic Markers, Biomarker, Yak, Serum Marker, Clinical, Cows, End Points, Surrogate, Domestic Cow, Biological Marker, Endpoints, Dairy Cow, Bos bovis, Immunologic, Domestic, Dairy, Laboratory Marker, Taurine Cattle, Beef, Surrogate Marker, Bos Tauurus, Immunologic Markers, Immune, Beef Cow, oxen, Markers, ox, Biochemical Marker, Cow, Viral Markers, Taurus, Indicine Cattle, Zebu, domestic cow, Zebus, Bos indicus Cattle, bovine, Immunologic Marker, Biologic, Immune MarkersEuropean Economic Community, Common Markets, DUbc9, Plantar Prints, Food Patterns, Plantar Print, determination, hindlimb zeudopodium, leg, Print, posterior region of leg, atado, prevention, Relative, Tier, primary metabolites, Lwr, sura, European Coal and Steel Community, 2, Analysis, prevention and control, sural region, multicellular organismal biosynthetic process, European Common Market, European Economic, Animalia, Mass Spectrum Analysis, lower extremity zeugopod, average, treatment, single-organism biosynthetic process, Man (Taxonomy), crus of hindlimb, reference sample, Analyses, Union, Economic Community, CG12352, Food, DmelCG4063, preventive measures, lower extremity middle limb segment, urinary aspects, scientific observation, disease management, Therapies, Plantar, Tbl1, TBL1, calf, DmAAF34715, middle limb segment of hind limb, posterior curral region, Posterior part of leg, Therapy, Communities, preventive therapy, animalia, Modern, Normalcy, Spectrum Analysis, hind epipodium, Spectroscopy, European, span., Market, Relative Risks, Common, regio cruris posterior, hbl, Individual, END, l(2)02858, posterior part of leg, l(2)05487, Posterior curral region, l(2)05486, European Economic Communities, whole organism, CG4063, Risk, Calf of leg, i56, European Atomic Energy Community, Spectrometry, Treatments, E-2f, human, i105, E-2g, Health, Dietary Patterns, label, metabolites, Koerper, hindlimb zeugopod, Community, Policies, hindlimb zeugopodium, human being, Tb11, Common Market, secondary metabolites, 3-dihydro-2-thioxo-, Spectrum Analyses, Normalcies, hind limb middle limb segment, Patterns, Human, FBXW4, regio surae, 4(1H)-Pyrimidinone, dip4, Homo sapiens, zeugopod of hind limb, Diets, Mass, zeugopod of hindlimb, Ubc 9, Animal, hindlimb epipodium, Man, hind limb zeudopodium, Mass Spectroscopy, Normalities, study, Fingerprints, urinary levels, Food Pattern, DmelCG3018, Ebi, EBI, metazoa, Ubc-9, lower leg, semi, European Communities, l(2)01519, Pattern, crus, DmelCG12352, liquid, Dietary Pattern, Chromatographies, Economic Communities, HHT1, Prints, Controlled, hind limb zeugopod, span, Individual Health, measuring, UBC9, Controlling, adequate, Edg, European Common Markets, Ubc9, body, intermediate segment of free lower limb, whole body, Sural region, SMAP55, EEC, Fingerprint, MS, FBgn0010602, middle limb segment of hindlimb, Metazoa, chemical analysis, dUbc9, dUBC9, ubc9, calf of leg, Foods, Mass Spectrum Analyses, Relative Risk, Mass Spectrum, Normality, prophylaxis, Dietary, Risks, metabolite, Posterior leg region, Euratom, l(2)k16213, European Common, European Community, Therapeutic, posterior leg region, concentration, control, Dmubc9, shank, Modern Man, Treatment, hindlimb middle limb segment, assay, ORW1, CG3018, DmUbc9, Posterior region of legEuropean Economic Community, Common Markets, Plantar Prints, Food Patterns, Plantar Print, Biological Markers, Viral Marker, Surrogate Endpoints, Laboratory, PNT-P1, DmelCG17077, Print, Biochemical, Endpoint, EY3-1, Pnt, Profiles, Serum, prevention, Classifications, Donor Artificial Insemination, Laboratory Markers, Tier, hierarchies, hierarchy, png, Biological, systematics, CDA2, European Coal and Steel Community, 2, Analysis, D-ets-2, 3520, prevention and control, Donor Artificial, multicellular organismal biosynthetic process, European Common Market, Pnt-P1, Metabolic Profiles, European Economic, Animalia, Mass Spectrum Analysis, treatment, single-organism biosynthetic process, me75, reference sample, Analyses, Union, Economic Community, D17Mit170, T1, Food, preventive measures, Immune, Markers, urinary aspects, Viral Markers, sample, disease management, Therapies, Plantar, 3-dihydro-2-thioxo-., Therapy, Communities, preventive therapy, Viral, Surrogate Endpoint, animalia, Systematics, D-Ets-2, ets94F, Biochemical Markers, 0123/09, Tl3, Biologic Marker, Tl2, Spectrum Analysis, Spectroscopy, Taxonomies, Marker, European, Market, RENBP, Common, Heterologous Insemination, European Economic Communities, pntP2, whole organism, Pointed-P1, End Points, European Atomic Energy Community, Ets94F, Spectrometry, Immunologic, Laboratory Marker, Treatments, AGE, ptd, Artificial Insemination, PntP2, Dietary Patterns, Biochemical Marker, PntP1, E(E2F)3D, Koerper, PNTP2, PNTP1, Community, 0998/12, Clinical Markers, taxonomy, Common Market, Clinical Marker, ETS2, Ets2, 3-dihydro-2-thioxo-, Spectrum Analyses, AID, Aid, Patterns, Discriminant, Surrogate End Points, 4(1H)-Pyrimidinone, Surrogate Markers, DMPOINT1A, pntegfr, 0608/07, Diets, Mass, Low, Animal, Metabolic Profile, aid, Mass Spectroscopy, Insemination, study, Biomarker, Fingerprints, urinary levels, Taxonomy, Clinical, Food Pattern, pointed-RC, Metabolomes, Profile, Biological Marker, metazoa, European Communities, EK3-2, Pattern, l(3)07825, Immunologic Markers, HEL-S-284, HIGM2, CG17077, liquid, Dietary Pattern, Chromatographies, Economic Communities, Immunologic Marker, l(3)j1B7, Heterologous, Prints, Biologic, Controlled, Controlling, Ets, cou, European Common Markets, body, RnBP, Serum Markers, GlcNAc 2-epimerase, End Point, whole body, Immune Marker, Human Donor, EEC, Fingerprint, N-acetyl-D-glucosamine 2-epimerase, MS, Lr, Surrogate End Point, Metazoa, Donor, pnt-P1, pnt-P2, Mass Spectrum Analyses, l(3)s118306, Biologic Markers, Artificial, Mass Spectrum, Serum Marker, Metabolic, Arp2, Surrogate, ARP2, prophylaxis, Dietary, Endpoints, Euratom, Surrogate Marker, sample population, European Common, Ets58AB, European Community, Therapeutic, control, Discriminant Analyses, Bra, renin-binding protein, Treatment, POINT, CG8705, Immune MarkersTaurine, dairy cow, Biological Markers, Viral Marker, Beef Cows, Viral, Surrogate Endpoints, Surrogate Endpoint, cow, Clinical Markers, Laboratory, Taurus Cattles, Bos indicus Cattles, Clinical Marker, Holstein Cow, Serum Markers, Taurus Cattle, Biochemical, End Point, Endpoint, Cattle, Taurine Cattles, Biochemical Markers, 3-dihydro-2-thioxo-, Serum, Indicine, Bos indicus, Dairy Cows, Biologic Marker, Immune Marker, Domestic Cows., Surrogate End Points, Surrogate Markers, 4(1H)-Pyrimidinone, Laboratory Markers, Bos grunniens, cattle, Marker, Biological, Surrogate End Point, Bos taurus, Bos primigenius taurus, 2, domestic cattle, Indicine Cattles, Yaks, Holstein, Cattles, Biologic Markers, Domestic Cows, Biomarker, Yak, Serum Marker, Clinical, Cows, End Points, Surrogate, Domestic Cow, Biological Marker, Endpoints, Dairy Cow, Bos bovis, Immunologic, Domestic, Dairy, Laboratory Marker, Taurine Cattle, Beef, Surrogate Marker, Bos Tauurus, Immunologic Markers, Immune, Beef Cow, oxen, Markers, ox, Biochemical Marker, Cow, Viral Markers, Taurus, Indicine Cattle, Zebu, domestic cow, Zebus, Bos indicus Cattle, bovine, Immunologic Marker, Biologic, Immune MarkersfalseDiscovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle (Cow samples)In the European Union, the use of thyreostats for animal fattening purposes has been banned because of their carcinogenic and teratogenic effects, whereby any residues in derived food products may hold a risk for human health. In this regard, potential abuse is monitored by targeted analysis of thyreostats with the application of a zero-tolerance policy. However, such a strategy is no longer adequate because of the semi-endogenous origin of some thyreostats, including thiouracil (TU). Additionally, the use of recommended TU concentration levels (10 or 30 µg/l) is also not fully conclusive. Therefore, this study aimed at defining urinary metabolite markers by which the true origin of detected TU could be ascertained. To this end, an in vivo study was carried out in which 11 calves were subjected to either a control treatment, a rapeseed-enriched diet (known to induce semi-endogenous formation) or a TU treatment. Urine samples (n = 165) were assessed through metabolic fingerprinting, employing ultra-high performance liquid-chromatography and Q-Exactive Orbitrap mass spectrometry. Urinary fingerprints were interpreted by multivariate data-analysis. The targeted Calf samples assay for this study can be found in the MetaboLights study MTBLS554. Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS554' target='_blank'>MTBLS554</a>2018-02-222017-10-24MTBLS561MTBLC140201MTBLC140071MTBLC140206MTBLC140209MTBLC140216MTBLC140230MTBLC140231MTBLC34853029649241CHEBI:140201CHEBI:140071CHEBI:140206CHEBI:140209CHEBI:140216CHEBI:140230CHEBI:140231CHEBI:348530