Therapeutic paracetamol treatment in older persons induces dietary and metabolic modifications related to sulfur amino acids

Sulfur amino acids are determinant for the detoxification of paracetamol (N-acetyl-p-aminophenol) through sulfate and glutathione conjugations. Long-term paracetamol treatment is common in the elderly, despite a potential cysteine/glutathione deficiency. Detoxification could occur at the expense of anti-oxidative defenses and whole body protein stores in elderly. We tested how older persons satisfy the extra demand in sulfur amino acids induced by long-term paracetamol treatment, focusing on metabolic and nutritional aspects. Effects of 3 g/day paracetamol for 14 days on fasting blood glutathione, plasma amino acids and sulfate, urinary paracetamol metabolites, and urinary metabolomic were studied in independently living older persons (five women, five men, mean (+/- SEM) age 74 +/- 1 years). Dietary intakes were recorded before and at the end of the treatment and ingested sulfur amino acids were evaluated. Fasting blood glutathione, plasma amino acids, and sulfate were unchanged. Urinary nitrogen excretion supported a preservation of whole body proteins, but large-scale urinary metabolomic analysis revealed an oxidation of some sulfur-containing compounds. Dietary protein intake was 13% higher at the end than before paracetamol treatment. Final sulfur amino acid intake reached 37 mg/kg/day. The increase in sulfur amino acid intake corresponded to half of the sulfur excreted in urinary paracetamol conjugates. In conclusion, older persons accommodated to long-term paracetamol treatment by increasing dietary protein intake without any mobilization of body proteins, but with decreased anti-oxidative defenses. The extra demand in sulfur amino acids led to a consumption far above the corresponding population-safe recommendation

Identification of Pre-frailty Sub-Phenotypes in Elderly Using Metabolomics

Aging is a dynamic process depending on intrinsic and extrinsic factors and its evolution is a continuum of transitions, involving multifaceted processes at multiple levels. It is recognized that frailty and sarcopenia are shared by the major age-related diseases thus contributing to elderly morbidity and mortality. Pre-frailty is still not well understood but it has been associated with global imbalance in several physiological systems, including inflammation, and in nutrition. Due to the complex phenotypes and underlying pathophysiology, the need for robust and multidimensional biomarkers is essential to move toward more personalized care. The objective of the present study was to better characterize the complexity of pre-frailty phenotype using untargeted metabolomics, in order to identify specific biomarkers, and study their stability over time. The approach was based on the NU-AGE project (clinicaltrials.gov, NCT01754012) that regrouped 1,250 free-living elderly people (65–79 y.o., men and women), free of major diseases, recruited within five European centers. Half of the volunteers were randomly assigned to an intervention group (1-year Mediterranean type diet). Presence of frailty was assessed by the criteria proposed by Fried et al. (2001). In this study, a sub-cohort consisting in 212 subjects (pre-frail and non-frail) from the Italian and Polish centers were selected for untargeted serum metabolomics at T0 (baseline) and T1 (follow-up). Univariate statistical analyses were performed to identify discriminant metabolites regarding pre-frailty status. Predictive models were then built using linear logistic regression and ROC curve analyses were used to evaluate multivariate models. Metabolomics enabled to discriminate sub-phenotypes of pre-frailty both at the gender level and depending on the pre-frailty progression and reversibility. The best resulting models included four different metabolites for each gender. They showed very good prediction capacity with AUCs of 0.93 (95% CI = 0.87–1) and 0.94 (95% CI = 0.87–1) for men and women, respectively. Additionally, early and/or predictive markers of pre-frailty were identified for both genders and the gender specific models showed also good performance (three metabolites; AUC = 0.82; 95% CI = 0.72–0.93) for men and very good for women (three metabolites; AUC = 0.92; 95% CI = 0.86–0.99). These results open the door, through multivariate strategies, to a possibility of monitoring the disease progression over time at a very early stage

Use of high resolution mass spectrometry for identification of specific biomarkers of coffee consumption

Présentation poster : Y. FillâtreThe Second International Congress of Translational Research in Human Nutrition is organised by the Research Centre in Human Nutrition (CRNH) of Auvergne, of which INRA is a member, in collaboration with NuGO, European Association of universities and research institutes in the field of nutrigenomicsANR Phenomenep ALIA-2010-007 Conseil Regional Auvergne-FEDER post-doc grantAs part of the ANR PhenoMeNEp project, non-targeted profiling is used to identify potential biomarkers of plant food consumption. Using 24 hour dietary recall and food frequency questionnaire data, 144 high (median 974 grams/day) and 66 low (median 305 grams/day) consumers of fruit and vegetables were selected from the French SU.VI.MAX2 cohort. Morning spot urine samples from each subjec

Food metabolomics applied in cohorts to accelerate the discovery of nutritional biomarkers

communication orale : Claudine ManachThe Second International Congress of Translational Research in Human Nutrition is organised by the Research Centre in Human Nutrition (CRNH) of Auvergne, of which INRA is a member, in collaboration with NuGO, European Association of universities and research institutes in the field of nutrigenomics.The purpose of dietary assessment is to estimate usual and recent intake of foods, nutrients, bioactive compounds and food contaminants for exploration of associations with health outcomes and monitoring of population nutritional status. These data are still extremely difficult to obtain. Methods currently used are based on dietary questionnaires which have inherent limitations linked to self-reporting. A complementary approach to questionnaires is the use of biomarkers. However, only a few biomarkers have been properly validated, which do not cover the wide range offoods consumed. Metabolomics has emerged as a promising approach to discover nutritional biomarkers. Typically, plasma or urine samples collected before and after acute intake of a specific food are profiled using NMR or high resolution Mass Spectrometry (MS) and compared usingmultivariate statistics to pinpoint the signals reflecting the consumption of the target food. In a proof-of-concept study on citrus, we showed that urine profiling of cohort subjects stratified by consumption could be a more effective strategy for discovery of sensitive biomarkers of intake

Développement de techniques de préconcentration pour faciliter l’identification de biomarqueurs lors d’analyses métabolomiques.

National audienc

Développement d’une stratégie d’identification de métabolites inconnus à l’aide d’outil de prédiction de fragmentations in silico

National audienceCes dernières années, les approches métabolomiques ont ouvert de grandes perspectives dans le domaine de la nutrition. Elles permettent d’aborder les problèmes de bio complexité en décrivant de façon globale les métabolites générés ou modifiés dans certaines conditions nutritionnelles. La caractérisation et l’identification de ces métabolites est devenue l’étape limitante dans la découverte de potentiels biomarqueurs et la production de connaissances. Cependant l’apparition de nouveaux appareils alliant puissance résolutive, précision de mesure de masses, et capacité de fragmentation, accompagné de logiciels permettant de simuler les fragmentations in-silico est devenue une aide précieuse dans la recherche et l’identification de métabolites. Dans ce travail nous présentons le développement d’une stratégie d’identification d’inconnus lors d’une étude métabolomique sur urines visant à étudier les processus métaboliques chez des ours durant l’hibernation. Afin d’effectuer une caractérisation structurale permettant l’identification des métabolites d’intérêt mis en évidence par les analyses statistiques, notre démarche a été d’acquérir les spectres des inconnus en dépendant scan à une résolution de 100 000 en scan et 30000 en MSMS, puis de déterminer leur formule développée. La formule développée a été reconstruite à partir des masses précises des fragments, puis vérifiée en faisant générer les fragmentations par Mass Frontier sur la formule développée reconstituée. L’analyse et la comparaison des masses précises obtenues avec Mass Frontier et l’ensemble des masses précises expérimentales nous ont aidé à confirmer de façon putative mais avec une certaine confiance les formules développées correspondantes. Cette étude démontre l’intérêt de l’utilisation d’outils de prédiction de fragmentation in-silico dans le développement de workflows de traitement des données métabolomiques issues de spectrométrie de masse

Presence of mycotoxins in sugar beet pulp silage collected in France

Sugar beet pulp, a major by-product of the sugar industry, is a common feed component in cattle diets that is preserved on-farm as silage. This study was designed to investigate if sugar beet pulp silage could be a vehicle of common mycotoxins found in silages and other regulated mycotoxins. Samples (n = 40) favouring mouldy spots, if present, on the front face of open silages were collected in 2011 from 5 regions representing the main French sugar beet producing areas. Mycotoxins were extracted by QuEChERS procedure without any further clean-up and analyzed by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). The mycotoxins monitored were: aflatoxin B1, deoxynivalenol, gliotoxin, ochratoxin A, mycophenolic acid, patulin, penicillic acid, roquefortine C and zearalenone. Matrix-matched calibrations were used, yielding acceptable levels of recovery ranging from 64 to 168%, except for gliotoxin and roquefortine C for which recovery was lower (21 and 34%, respectively). Eight samples out of 40 (20%) were found to be positive. Mycophenolic acid and zearalenone were the most predominant of the mycotoxins studied. Mycophenolic acid was found in five of 40 samples at levels ranging from traces up to 1436 μg/kg. Zearalenone was found in three samples at concentrations of 1023, 4862 and 6916 μg/kg. The last 2 samples were at concentrations above the recommended limit of 2000 μg/kg. Ochratoxin A was detected in one sample at 15 μg/kg, which is below the recommended EU limit of 250 μg/kg. Roquefortine C was also detected but at low levels. To our knowledge, this study is the first to report on the presence of mycotoxins in sugar beet pulp silage. Contamination for the tested mycotoxins was low and did not seem to present a health risk for animals or consumers for the tested mycotoxins

Evaluation of oxidized phospholipids analysis by LC-MS/MS

Phospholipids (PLs) represent a class of metabolites of interest for evaluating the relationship between diet and the development of several metabolic diseases. Given that PLs are rich in unsaturated fatty acids, they can be oxidized. Because of their structure and reactivity, oxidized phospholipids (PLs-Ox) are increasingly recognized as markers of oxidative stress and of various diseases associated with inflammation. Therefore, there is a growing interest in studying PLs-Ox in lipidomics. Because of their limited commercial availability, very little information is currently available in databases to identify these molecules. The aim of this study is to acquire new knowledge about PLs-Ox in order to propose an analytical strategy for their analyses. For this purpose, a synthesis method of PLs-Ox, in auto-oxidation, has been developed and applied on phosphatidylcholine and phosphatidylethanolamine molecular species with various chain lengths, degree, and position of unsaturations. An analysis method based on mass (MS) and tandem mass spectrometry coupled to electrospray ionization was then developed and enabled the identification of a great diversity of long- and short-chain oxidation products. Formation kinetics of oxidation products was evaluated. Results showed that the formation of oxidized compounds was largely influenced by the degree of unsaturation on fatty acid chains. Oxidation time promotes the formation of some biologically important oxidation products. Coupling the MS method with liquid chromatography in flow injection analysis mode enabled the development of a full analytical strategy. Structural analysis of PLs-Ox allowed the enrichment of databases with important information to identify these molecules in biological matrices

Développement d’une méthode de préconcentration SPE permettant l’identification de métabolites inconnus dans le plasma par UHPLC-HRMS

La métabolomique est un outil de phénotypage puissant en nutrition permettant de mieux comprendre les mécanismes biologiques impliqués dans les processus physiopathologiques et d’identifier des biomarqueurs de déviations métaboliques. Cependant cette étape d’identification est devenue un challenge important en métabolomique et constitue un verrou analytique majeur, de par leurs diversités chimiques des métabolites présents dans les biofluides et leurs faibles concentrations. Dans ce contexte, le développement de méthodes de préconcentration est une nécessité pour pouvoir donner une signification physiologique aux profils différenciés que fournit l’analyse des données métabolomiques. Une étude préliminaire avait permis de montrer que parmi plusieurs techniques de préparation testées (SPME, SBSE et SLE), l’extraction SLE donnait les meilleurs résultats. Ce travail, qui constitue la suite du projet, a été consacré au développement de l’extraction SPE et sera par la suite comparé avec la méthode SLE préalablement testée.Pour pouvoir répondre à ces objectifs, 19 molécules de propriétés physico-chimiques diverses (pKa, logP, …) et mimant un échantillon biologique ont été sélectionnées parmi des acides aminés, des acides organiques, des hormones... Une injection de ce mélange en chromatographie semi-préparative a été réalisée afin de séparer et de collecter nos composés en plusieurs fractions. Ensuite, une extraction sur phase solide SPE a été développée pour pré concentrer ces fractions à l’aide d’un robot Freedom Evo 200 (Técan). Enfin, une analyse en mode scan sur une chaîne UHPLC Ultimate 3000 couplée à un spectromètre de masse haute résolution LTQ Orbitrap Vélos (Thermo Scientific) est réalisée afin d’évaluer les performances de la pré concentration mise en jeu.Lors de l’étape d’optimisation, la colonne semi-préparative, les cartouches SPE ainsi que les différents paramètres d’extraction (volume d’échantillon, volume et solvants de lavage, volume et solvants d’élution et le temps de séchage) ont été optimisés de sorte à obtenir les intensités les plus importantes pour un maximum de composés. Les premiers résultats obtenus montrent clairement que la cartouche SPE Oasis MCX 30mg 1 mL (Waters) est la plus performante. En effet, elle permet d’extraire, à partir de fractions chromatographiques collectées, à la fois les composés polaires et basiques mais également les composés neutres. Celle-ci est donc la cartouche de choix pour la suite de l’optimisation.Pour conclure, cette technique sera ensuite utilisée sur ces extraits concentrés en vue de réaliser des analyses structurales de potentiels biomarqueurs, pour aider à la caractérisation et à l’identification de nouveaux métabolites dans des échantillons biologique

Comparison of lycopene and tomato effects on biomarkers of oxidative stress in vitamin E deficient rats

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceBACKGROUND: Cohort studies suggested that individuals with higher intake of tomatoes and tomato products have a lower risk of degenerative diseases. Lycopene, an antioxidant and antiproliferative carotenoid, has been hypothesized to be responsible for the health benefits of tomatoes. However, studies demonstrated a higher potential of tomatoes compared to lycopene to reduce oxidative stress or carcinogenesis. AIM OF THE STUDY: Our study aimed at distinguishing lycopene effect from that of tomato on oxidative stress, by using yellow tomato, a tomato variety devoid of lycopene. METHODS: Effects of feeding with none (control), 16% freeze-dried yellow tomato (YT), 16% freeze-dried red tomato (RT) or 0.05% lycopene beadlets (LB) were compared in a rat model with mild oxidative stress induced by low vitamin E diet (LVED). Four groups of 10 rats were fed ad libitum for 6 weeks. Physiological parameters such as ingesta, body, spleen and liver weights, cholesterol and triglycerides (TG) levels were assessed. Lycopene and vitamin E concentrations and oxidative stress biomarkers were measured in the plasma and/or liver and/or heart tissue of the rats. RESULTS: RT, YT, and LB had no effect on rats' ingesta, body and spleen weights. RT, YT and LB had no effect on plasma cholesterol concentration. RT decreased TG level compared to control, YT and LB (P < 0.05). Rats fed RT or LB accumulated lycopene in plasma in contrast with rats fed YT. Heart level of thiobarbituric reactive species (TBARS) in rats fed RT or YT was lower than that in the control and the LB fed rats (P < 0.05). Despite similar concentrations of lycopene in plasma and liver, rats fed LB showed a significantly higher heart level of TBARS than rats fed tomatoes. RT increased erythrocyte superoxide dismutase (eSOD) activity compared with LB and nitric oxide (NO) level compared with control and LB. LB decreased ferric reducing ability of plasma (FRAP) level compared with control, RT and LB (P < 0.05). CONCLUSION: Our study showed for the first time that tomatoes, containing or not containing lycopene, have a higher potential than lycopene to attenuate and or to reverse oxidative stress-related parameters in a mild oxidative stress context