LC-IMS-HRMS for identification of biomarkers in untargeted metabolomics: The effects of pterostilbene and resveratrol consumption in liver steatosis, animal model

Untargeted metabolomics with the combination of ion mobility separation coupled to high resolution mass spectrometry (IMS-HRMS) was applied to investigate the impact of resveratrol and pterostilbene supplementation on the metabolic fingerprint of the Wistar rats liver with induced liver steatosis. RP-LC and HILIC in both ionisation modes were employed to analyse the liver samples (n = 40) from Wistar rats fed with a high-fat and high-fructose diet, supplemented or not with resveratrol and pterostilbene. After univariate and multivariate statistical analysis, 34 metabolites were highlighted in the different diets and elucidated. Despite the structural similarity, different alterations in liver metabolism were observed by the supplementations. Resveratrol treatment was characterised by the alteration in metabolism of 17 lysophospholipids, while pterostilbene affected some vitamins and derivatives, among others. IMS has demonstrated great potential in the elucidation process thanks to the additional structural descriptor the CCS (Å2), providing more confidence in the identification

Untargeted metabolomics approach for unraveling robust biomarkers of nutritional status in fasted gilthead sea bream (Sparus aurata)

A metabolomic study has been performed to identify sensitive and robust biomarkers of malnutrition in farmed fish, using gilthead sea bream (Sparus aurata) as a model. The metabolomic fingerprinting of serum from fasted fish was assessed by means of ultra-high performance liquid chromatography coupled to quadrupole time-offlight mass spectrometry. More than 15,000 different m/z ions were detected and Partial Least Squares–Discriminant analysis allowed a clear differentiation between the two experimental groups (fed and 10-day fasted fish) with more than 90% of total variance explained by the two first components. The most significant metabolites (up to 45) were elucidated on the basis of their tandem mass spectra with a broad representation of amino acids, oligopeptides, urea cycle metabolites, L-carnitinerelated metabolites, glutathione-related metabolites, fatty acids, lysophosphatidic acids, phosphatidylcholines as well as biotin- and noradrenaline-related metabolites. This untargeted approach highlighted important adaptive responses in energy and oxidative metabolism, contributing to identify robust and nutritionally-regulated biomarkers of health and metabolic condition that will serve to assess the welfare status of farmed fish.This work was supported by the Spanish MINECO (MI2-Fish, AGL2013-48560) and Plà de promoció de la investigació de la Universitat Jaume I (P1•1B2015-59) and the Generalitat Valenciana as research group of excellence (PROMETEO II/2014/023, PROMETEO II/2014/085, Collaborative Research on Environment and Food-Safety, ISIC/2012/016)

Aplicaciones de la metabolómica

III Congreso de Alimentación, Nutrición y Dietética. Combinar la nutrición comunitaria y personalizada: nuevos retos

Novel sampling strategy for alive animal volatolome extraction combined with GC-MS based untargeted metabolomics: Identifying mouse pup pheromones

In this study, we identify 11 mouse pup volatiles putatively involved in maternal care induction in adult females. For this purpose, we have adapted the dynamic headspace methodology to extract the volatolome of whole alive animals. Untargeted metabolomic methodology was used to compare the volatolome of neonatal (4–6 days) with elder pups until the age of weaning (21–23 days old). Pup volatolome was analyzed by gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) using automated thermal desorption for sample introduction. After data processing and multivariate statistical analysis, comparison with NIST spectral library allowed identifying compounds secreted preferentially by neonatal pups: di(propylen glycol) methyl ether, 4-nonenal, di(ethylene glycol) monobutyl ether, 2-phenoxyethanol, isomethyl ionone, tridecanal, 1,3-diethylbenzene, 1,2,4,5-tetramethylbenzene, 2-ethyl-p-xylene and tri(propylene glycol) methyl ether. Palmitic acid was enriched in the volatolome of fourth week youngsters compared to neonatal pups. The results demonstrated the great potential of the new sampling procedure combined with GC-MS based untargeted volatolomics to identify volatile pheromones in mammals.Funding for open access charge: CRUE-Universitat Jaume

Contributions of MS metabolomics to gilthead sea bream (Sparus aurata) nutrition. Serum fingerprinting of fish fed low fish meal and fish oil diets

The aim of this study was to evaluate the impact of fish meal (FM) and fish oil (FO) replacement by plant proteins and oils in the serum metabolome of two-year old gilthead sea bream (Sparus aurata) fed from early life stages with control and experimental diets. Randomly selected fish were overnight sampled and clotted serum was used for metabolomics fingerprinting by means of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. >12,500 different m/z ions were detected, and Partial Least Squares-Discriminant analysis separated fish fed control and plant-based diets, with a 71% of variance explained and 44% of variance predicted by the two first components. After variable importance in projection (VIP) and Benjamini-Hochberg test correction filtering, 50 endogenous compounds were elucidated as highly discriminant features of dietary treatment. Most of them were lipid-related compounds and reflected the different fatty acid composition of dietary oils, whereas changes in N-acyl taurines, cytidine and nucleoside related compounds would indicate changes in tissue repair and DNA degradation processes. Untargeted analysis also identified some exogenous compounds as markers of marine and vegetable raw materials. In the case of hercynine (antioxidant fungi and mycobacteria product), this was exemplified by a close lineal association between circulating and feed levels. Targeted approaches were focused on vitamins and a clear reduction of B12, indirectly assessed via methylmalonic acid levels, was found in fish fed vegetable diets. Conversely, serum riboflavin (B2) and pantothenic acid (B5) levels were consistently increased, which highlighted the close link between nutrition and gut microbiota

Untargeted metabolomics based on ultra-high-performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry for biomarker discovery of orange intake in a cross-over trial

Pòster presentat al congrés "Understanding Human Diseases Through Metabolomics: Interactions Among the Genome, Proteome, Gut Microbiome and Nutrition", Metabolomics and Human Health, Gordon Research Conference (February 3 - 8, 2019 Ventura, CA, United States)Diet is one of the most important lifestyle factors associated with health status. Currently, one of the main limitations of nutritional epidemiology and nutritional genomics is the difficulty in the measurements of dietary intake. In observational studies carried out in a large number of participants, the most commonly applied tools for estimating dietary intake are based on self-reporting, including food frequency questionnaires (FFQ) for the assessment of regular consumption (usually 1-year), or 24-h recalls for 1-day assessment. However, such instruments for data collection may contain several recall bias and other systematic or random errors that may have a great effect in the subsequent associations found. Although in recent years, it has been an improvement in increasing the validity and precision of food questionnaires due to the use of the new information technologies (Figure 1), these instruments are still biased and additional information based on objective biomarkers of food intake is needed

Gas chromatography-mass spectrometry based untargeted volatolomics for smoked seafood classification

With the increase of the demand of low flavouring smoked seafood products, there is a need of methodologies able to distinguish between different seafood treatments, as not all of them are allowed in all markers. Following this objective, in the present work an untargeted volatolomics approach was applied to identify volatile markers that demonstrate that Cold smoked products can be distinguished from Tasteless smoke neither Carbon monoxide treated seafood, which are prohibited in the European Union. The use of dynamic headspace for the volatile extraction followed by thermal desorption in combination with Gas Chromatography (GC) coupled to single quadrupole Mass Spectrometry (MS) has been employed for the determination of volatile composition of smoked fish. Data processing consisted on the use of PARADISe software, applied for GC/MS data treatment, followed by the multivariate analysis with PLS_Toolbox (MATLAB), and finally the creation and validation of statistical classification model. All 107 variables obtained allowed the construction of a model reaching the correct classification of 97% of the blind samples, while a simplified model with only 11 variables correctly classified up to 93% of the blind samples. These 11 compounds were elucidated to develop subsequent target volatolomics approaches, if needed. Ordered according to the importance in the classification model, the elucidated compounds were: 3-methylcyclopentanone, ethylbenzene, 2-methyl-2-cyclopenten-1-one, 2-methyl-benzofuran, furfuryl alcohol, 2-acetylfuran, acetophenone, guaiacol, 1-hydroxy-2-butanone, 4-vinylguaicol and acetoin. The results demonstrated the great potential of untargeted volatolomics for smoked seafood treatments classification

Untargeted metabolomics approach using UHPLC-IMS-QTOF MS for surface body samples to identify low-volatility chemosignals related to maternal care in mice

The present study is focused on the determination of low-volatile chemosignals excreted or secreted by mouse pups in their early days of life involved in maternal care induction in mice adult females. Untargeted metabolomics was employed to differentiate between samples collected with swabs from facial and anogenital area from neonatal mouse pups receiving maternal care (first two weeks of life) and the elder mouse pups in the weaning period (4th week old). The sample extracts were analysed by ultra-high pressure liquid chromatography (UHPLC) coupled to ion mobility separation (IMS) in combination with high resolution mass spectrometry (HRMS). After data processing with Progenesis QI and multivariate statistical analysis, five markers present in the first two weeks of mouse pups life and putatively involved in materno-filial chemical communication were tentatively identified: arginine, urocanic acid, erythro-sphingosine (d17:1), sphingosine (d18:1) and sphinganine. The four-dimensional data and the tools associated to the additional structural descriptor obtained by IMS separation were of great help in the compound identification. The results demonstrated the great potential of UHPLC-IMS-HRMS based untargeted metabolomics to identity putative pheromones in mammals.Funding for open access charge: CRUE-Universitat Jaume

Ultra-Performance Liquid Chromatography-Ion Mobility Separation-Quadruple Time-of-Flight MS (UHPLC-IMS-QTOF MS) Metabolomics for Short-Term Biomarker Discovery of Orange Intake: A Randomized, Controlled Crossover Study

A major problem with dietary assessments is their subjective nature. Untargeted metabolomics and new technologies can shed light on this issue and provide a more complete picture of dietary intake by measuring the profile of metabolites in biological samples. Oranges are one of the most consumed fruits in the world, and therefore one of the most studied for their properties. The aim of this work was the application of untargeted metabolomics approach with the novel combination of ion mobility separation coupled to high resolution mass spectrometry (IMS-HRMS) and study the advantages that this technique can bring to the area of dietary biomarker discovery, with the specific case of biomarkers associated with orange consumption (Citrus reticulata) in plasma samples taken during an acute intervention study (consisting of a randomized, controlled crossover trial in healthy individuals). A total of six markers of acute orange consumption, including betonicines and conjugated flavonoids, were identified with the experimental data and previous literature, demonstrating the advantages of ion mobility in the identification of dietary biomarkers and the benefits that an additional structural descriptor, as the collision cross section value (CCS), can provide in this area