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## Poster 15 – Associé FP6

# New insight in Metabolomics based study of Age Related Macular Degeneration (AMD): Lipoprotein profile and subclass analysis.

Matthieu Schoumacher<fnref n="2" />, Vincent Lambert, Justine Leenders <fnref n="1" />, Bernadette Govaerts <fnref n="2" />, Sylvain Hansen, Jean-Marie Rakic, Agnès Noël, Pascal De Tullio <fnref n="1" />

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly population in developed countries. 90% of all vision loss due to AMD result from the exudative form of this pathology, which is characterized by a choroidal neovascularization (CNV). Currently, diagnosis of AMD relies on ophthalmologic exams and treatments of the exudative form are based on the use of anti-angiogenic drug targeting vascular endothelial growth factors. Despite these advances, several clinical challenges have to be overcome. Among those, the identification of biomarkers that could allow to refine patient stratification, to follow disease progression and evaluate responses to treatment are mandatory. For this purpose, we decide to apply NMR-based metabolomics approach on both AMD patients and on a laser-induced murine choroidal neovascularization experimental model. In the clinical study, the metabolomics approach does not allow a complete differentiation between control and AMD patients. However, focusing only on AMD group, a clear-cut separation between active and non-active phases could be highlighted.

In the mouse model, discrimination between laser-induced and control mice occurs only when CNV is install ed.In both human and animal studies, lactate and lipoprotein profile were identified as the main biomarkers. Mechanistically, we demonstrated that lactate, plays a critical role in the onset of the inflammatory and angiogenic phases and could be correlated with the CNV development. Then, controlling lactate level appears as a new therapeutic approach of AMD.

On the other hand, lipoprotein profile is of particular interest for patient follow-up. Indeed, evaluation of lipoprotein profile change trough a simple methods allowed us to establish clear modification of profiles according to the active or non-active status of the patient and to the induced or non-induced status of the mice.

This work focuses on the lipoprotein profile and on the development of a methodology that could be used to characterize and compare the different profiles in the human and the mice studies.

Mots-Clés: NMR, metabolomics, AMD, Lipoprotein

<footnote n="1">¹ Centre Indisciplinaire de Recherche sur le Médicament (CIRM) – Institut de Pharmacie Quartier HOPITAL - Av. Hippocrate, 15 (Bât. B36) Sart-Tilman B-4000 Liège, Belgique</footnote>

<footnote n="2">² Institut de Statistique, Biostatistique et Sciences Actuarielles (ISBA) – Université Catholique de Louvain, Voie du Roman Pays 20, 1348 Louvain-La-Neuve, Belgique</footnote>
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## Poster 16 – Associé FP1

A combined metabolomics and lipidomics approach enables the stratification of acute-on-chronic liver failure patients according to their severity

**Noemie Butin**¹, **François Fenaille**¹, **Benoit Colsch**¹, **Christophe Junot**¹, **Joan Clària**²,³, **Richard Moreau**⁴,⁵, **Vicente Arroyo**⁵

Acute-on-Chronic Liver Failure (ACLF) is a recently recognized syndrome characterized by acute decompensation (AD) of cirrhosis, an organ/system failure(s) and extremely poor survival. ACLF can be triggered by a precipitating event (e.g. bacterial infection) and is invariably associated with exacerbated systemic inflammation. According to the European Foundation for the study of Chronic Liver Failure (EF-CLIF), patients with ACLF can be classified into three groups, essentially according to the number of impaired organs. In the present project, we investigated whether metabolomics and lipidomics can identify potential new diagnostic biomarkers of ACLF. In our study, a cohort of more of 800 serum samples from decompensated cirrhotic patients with and without ACLF were analyzed and compared to healthy subjects by LC-HRMS. Data mining procedures using multivariate and univariate analyses were then performed to highlight discriminant metabolites with a preliminary work on data processing to normalize data and to overcome analytical biases occurring in the analysis of a large biological cohort. Our data confirmed the metabolic and lipidomic cirrhosis signatures obtained in previous studies, especially regarding to the levels of glycerophosphatidylcholines, amino acids and energy metabolites. Furthermore, our approach enabled to discriminate between decompensated cirrhotic patients with ACLF and those without ACLF, and a specific metabolite signature associated with the ACLF grade was obtained. Moreover, we found that Kynurenine pathway is activated in patients with acute decompensation, culminate in patients with ACLF and may be involved in the pathogenesis of ACLF, clinical course and mortality.

Mots-Clés: Metabolomics, ACLF, LC/MS

<footnote n="6">⁶ CEA Saclay, Laboratoire d'Etude du Métabolisme des Médicaments, MetaboHUB-Paris, DRF/IBITEC-S/SPI, 91191 Gif-sur-Yvette Cedex, France. – CEA, CNRS, Université Paris-Saclay, CEA Saclay 91191 Gif sur Yvette France – France</footnote>

<footnote n="7">⁷ European Foundation for the Study of Chronic Liver Failure (EF-CLIF), Barcelona, Spain – Travessera de Gràcia, 11, Planta 7ª, 08021 Barcelona, Spain, Espagne</footnote>

<footnote n="8">⁸ Department of Biochemistry and Molecular Genetics, Hospital Clinic, Institut d'Investigacions Biomèdiques August-Pi-Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain – Carrer del Rosselló, 149, 08036 Barcelona, Spain, Espagne</footnote>

<footnote n="9">⁹ INSERM and Université Paris Diderot-Paris 7, Centre de Recherche sur l'Inflammation (CRI), Paris and Clichy, France – Institut National de la Santé et de la Recherche Médicale - INSERM – France</footnote>

<footnote n="10">¹⁰European Foundation for the Study of Chronic Liver Failure (EF-CLIF), Barcelona, Spain – Espagne</footnote>
<figure><img src="image_127.png" /></figure>

## Poster 17 – Associé FP2

# Metabolic characterization of different phases of Parkinson's disease

David Mallet<fnref n="1" />, Thibault Duffourd<fnref n="1" />, Carole Carcenac<fnref n="1" />, Sébastien Carnicella<fnref n="1" />, Emmanuel Barbier<fnref n="1" />, Sabrina Boulet<fnref n="1" />, Florence Fauvelle<fnref n="1" />

**Context:** Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting almost 1% of the population beyond the age of 60. Currently, its diagnosis relies on the expression of the well-known motor symptoms (akinesia, rigidity, and tremor) which appear in the late stage of the disease. Detecting the disease earlier represents a key step to develop curative treatments which are so far only symptomatic. Long considered as a purely motor disease, PD is nevertheless also characterized by neuropsychiatric disorders (apathy, depression, anxiety...) that can develop during the early stages of the disease as well as later on.

**Objectives:** Our aim is to find specific molecular markers of early phases of PD, when only the neuropsychiatric symptoms are expressed, using 1HNMR-based metabolomics on serum samples and on brain tissues of a rodent model allowing investigation of different phases of PD.

**Methods:** The animal model is based on a specific, partial, bilateral 6-OHDA-induced lesion in dopaminergic neurons. For each rat, motor functions and apathetic-like behaviors were assessed using a stepping test and operant sucrose self-administration, respectively.

Both serum and intact tissue samples were analyzed using a CPMG pulse sequence by respectively liquid NMR at 950 MHz (IBS Grenoble) and HRMAS-NMR at 500 MHz (IRMaGE, CEA Grenoble). Spectra were submitted to multivariate statistics (SIMCA-v14) in order to investigate if metabolic profile is correlated to behavioral and histological data.

**Results/conclusion:** In our animal cohort we observed a gradation in the symptoms, well in line with PD progression, from only neuropsychiatric, to the expression of neuropsychiatric associated with motor symptoms. For more precise evaluation, a score based on behavioral performances and on the striatal dopaminergic denervation was built. Serum and tissue spectra showed a good correlation of this score with the gradation of symptoms at different phases of PD.

In both samples, the energetic pathways seems to be modified with the progression of the disease, while some amino acids, like alanine and serine, are also dysregulated prior to the appearance of motors symptoms. We will further use the same methodology in serum with *De Novo* patients.

**Mots-Clés:** Parkinson, Biomarkers, liquid 1HNMR, Tissu, HRMAS NMR (High resolution magic angle spinning NMR)

<footnote n="1">¹ Grenoble Institut des Neurosciences – CEA, CHU Grenoble, Inserm : U1216, Université Grenoble Alpes – France</footnote>
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**Communication Posters**

**Session Pharmacologie - Toxicologie**

<figure><img src="image_129.png" /></figure>

RÉSEAU FRANCOPHONE DE MÉTABOLOMIQUE ET FLUXOMIQUE
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Poster 18 – P18

# Metabolomic profiling of polarized human lung macrophages with liquid- and gas-chromatography coupled to mass spectrometry

Fanta Fall<fnref n="1" />, Natacha Lenuzza<fnref n="2" />, Elodie Lamy<fnref n="1" />, Marion Brollo<fnref n="3" />, Emmanuel Naline<fnref n="3" />,<fnref n="4" />, Philippe Devillier<fnref n="3" />,<fnref n="4" />, Etienne Thevenot<fnref n="2" />, Stanislas Grassin Delyle<fnref n="1" />,<fnref n="3" />

**Rationale:** Lung macrophages (LM) are in the first line of defense against inhaled pathogens. These cells can undergo phenotypic polarization to the proinflammatory M1 or to the immunomodulatory M2 phenotype. The objective of the present work was to characterize the metabolic alterations occurring during the experimental M1 or M2 LM polarization.

**Methods:** Human LM were obtained from resected lungs and cultured for 24h in medium alone or with 10 ng/mL LPS (M1 polarization) or 10 ng/mL IL-4 (M2 polarization). Cells and culture supernatants were subjected to extraction for metabolomic analysis with high-resolution LC-MS (HILIC and reverse phase -RP- chromatography in both negative and positive ionization modes) and GC-MS. The data were analyzed with the R software and the Workflow4Metabolomics online infrastructure.

**Results:** For the intracellular content, 7,527 features were detected in HILIC-MS, 1021 in RPMS and 193 in GC-MS. For supernatants, 1,894 features were detected in HILIC-MS, 2,187 in RP-MS and 274 in GC-MS. 52 features had expression levels significantly altered between experimental conditions, mainly with LPS. Annotation with HMDB suggested a regulation in the expression of metabolites from the eicosanoid (prostaglandin, leukotriene, lipoxin), retinoid, kynunerin and Krebs cycle pathways. Identification with reference standards was successful for several compounds and quantitative methods were developed for culture supernatants. In the presence of LPS, malic acid increased from 568±58 ng/mL to 902±111 ng/mL, quinolinic acid from 40±10 ng/mL to 108±20 ng/mL and kynurenin from 92±13 ng/mL to 280±45 ng/mL whereas tryptophan decreased from 173±22 mg/L to 51±10 mg/L.

**Conclusions:** Macrophages polarization is accompanied by changes in the cell metabolome. Metabolites differentially expressed are mainly involved in the promotion and regulation of inflammation. In depth analysis will allow a better understanding of the molecular mechanisms underlying LM polarization.

**Mots-Clés:** Lung macrophages, Proinflammatory, immunomodulators, Metabolomic, HILIC, Reverse phase

<footnote n="1">¹ Plateforme de spectrométrie de masse MasSpecLab, INSERM U1173, UFR Sciences de la Santé Simone Veil – Université Versailles - Saint Quentin en Yvelines – France</footnote>

<footnote n="2">² CEA, LIST, Laboratory for Data Sciences and Decision, MetaboHUB – CEA – France</footnote>

<footnote n="3">³ Département des maladies des voies respiratoires, Hôpital Foch, Suresnes, France – Hôpital Foch – France</footnote>

<footnote n="4">⁴ UPRES EA220, UFR Simone Veil - Santé, Université Versailles - Saint Quentin en Yvelines, Université Paris Saclay, Montigny le Bretonneux, France – UPRES EA220 – France</footnote>
<figure><img src="image_131.png" /></figure>

## Poster 19 – P19

# Identification des modulations du lipidome des cellules hépatiques HepaRG exposées à du tétrachloro-bisphénol A (TCBPA) par RMN du proton et du phosphore-31

Cécile Canlet<sup>1,2</sup>, Marie Tremblay-Franco<sup>2,1</sup>, Justine Bertrand Michel<sup>3</sup>, Roselyne Gautier<sup>1,2</sup>, Aurélie Batut<sup>3</sup>, Laurent Debrauwer<sup>2,1</sup>, Nicolas Cabaton<sup>1</sup>

Les bisphénols halogénés suscitent aujourd'hui un questionnement sociétal en toxicologie environnementale et alimentaire. Ces composés présentent une forte affinité pour le récepteur nucléaire PPARg et le tétrabromo-bisphenol A (TBBPA) a un effet avéré sur les phospholipides et la fluidité membranaire. Des études ont montré des altérations des concentrations en lipides plasmatiques et une inflammation au niveau hépatique

L'objectif de ce projet est d'approfondir l'exploration des voies lipidiques modulées par le TCBPA par deux approches complémentaires : la RMN et la spectrométrie de masse (MS), à partir des mêmes extraits de cellules HepaRG exposées au TCBPA, cellules métaboliquement compétentes et très proches d'hépatocytes humains. La RMN du proton permet de quantifier certaines familles de lipides et la RMN du phosphore-31 permet de quantifier de manière absolue les différentes classes de phospholipides (PL). Ces analyses ont été complétées par des approches classiques de lipidomique ciblée afin de comparer les résultats obtenus par les deux techniques et de montrer les avantages et les limites de chaque technique.

Des tests préliminaires ont été effectués afin d'optimiser la quantité de cellules nécessaire, le protocole d'extraction, le solvant de reprise des extraits lipidiques pour les analyses RMN. Les cellules HepaRG ont été ensemencées à un taux de 3 millions de cellules/puits et ont été exposées pendant 24h à différentes concentrations de TCBPA. Les extraits lipidiques ont été analysés par RMN du proton et du phosphore-31, et par MS. Pour les PL, les résultats obtenus en RMN sont similaires à ceux obtenus en MS. Bien que moins sensible que la MS, la RMN permet de quantifier de manière absolue les phospholipides. En revanche, seulement trois familles ont pu être quantifiées en RMN. Les analyses statistiques multivariées de toutes les espèces lipidiques quantifiées en RMN ont montré une perturbation du métabolisme lipidique dans les cellules exposées au TCBPA. Les analyses ciblées par MS des lipides neutres et acides gras totaux sont en cours pour préciser la nature des lipides modulés par l'exposition au TCBPA. Ces résultats préliminaires montrent d'ores et déjà la complémentarité des 2 approches.

Mots-Clés: lipidomique, RMN du proton, RMN du phosphore, bisphénols halogénés

<footnote n="1">1 Toxalim, Université de Toulouse, INRA, Toulouse</footnote>

<footnote n="2">2 Plateforme MetaToul-AXIOM, Toulouse – Institut National de la Recherche Agronomique - INRA - France</footnote>

<footnote n="3">3 Inserm U1048, Plateforme MetaToul-LIPIDOMIQUE, Toulouse – Institut National de la Santé et de la Recherche Médicale - INSERM – France</footnote>
<figure><img src="image_132.png" /></figure>

Poster 20 – P20

Untargeted profiling of toxicologically relevant metabolites: case
study of reactive aldehydes

**Loic Mervant**<sup>1,2</sup>, **Robin Costantino**<sup>1,2</sup>, **Jean-François Martin**<sup>1,2</sup>, **Laurent Debrauwer**<sup>1,2</sup>,
**Françoise Guéraud**<sup>2</sup>, **Emilien Jamin**<sup>1,2</sup>

Human exposure to toxic substances, particularly through diet, is a major societal concern, and the most
complete characterization of exposure is required to link food consumption and toxic effect. However, current
methods to assess exposure are mainly focused on some families of compounds. The concept of exposomics aims to
assess this exposure in a most comprehensive way by the use of untargeted methods.
Here we propose an untargeted UPLC-HRMS method to specifically profile electrophilic metabolites, which are
known to be toxic due to their reactivity towards biomolecules such as DNA, proteins... The main route to detoxify
these compounds is by conjugation to glutathione, and excretion in urine as mercapturic acids conjugates (MACs)
after enzymatic cleavage.

As proof of concept we applied this method in the toxicological context of colorectal cancer promotion linked to the
consumption of red meat/heme iron. Heme iron contained in red meat is known to catalyse lipid peroxidation in the
intestinal tract, leading to the formation of toxic alkenals. Urines of rats fed various oils with heme iron or free iron
supplementation were analysed by UPLC-MS, and untargeted MACs profiling was performed on QC samples by using
"all ion MS/MS mode" on a Waters Synapt G2-Si instrument since MACs display a characteristic neutral loss in
MS/MS. Then the corresponding MACs signals were measured in all urine samples.

Thus, dozens of MACs could be detected without a priori with this approach, including expected ones (e.g. DHN-MA)
as well as unexpected MACs derived from aldehydes or other chemical classes. Interestingly, multivariate statistical
analyses carried out only on the MACs yielded a much better segregation of groups compared to results obtained
from a classic untargeted metabolomic approach. Therefore, our approach not only allowed to highlight metabolites
of lipid peroxidation, but also opens the way to the untargeted detection of toxicologically relevant compounds.

Mots-Clés: toxicology, reactive metabolites, mass spectrometry

<footnote n="1">¹ Axiom platform, MetaToul-Metabo-HUB, National Infrastructure for Metabolomics and Fluxomics, Toulouse – Institut national de la recherche agronomique (INRA) – France</footnote>

<footnote n="2">² Toxalim, UMR1331, Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse – Institut national de la recherche agronomique (INRA) – France</footnote>
<figure><img src="image_133.png" /></figure>

## Poster 21 – P21

# Variation du phénotype métabolique des mousses forestières liée à l'exposition de dépôts métalliques

Alain Paris<fnref n="1" />, Truong Anh Nguyen, Arul Marie <fnref n="2" />, Caroline Meyer, Sébastien Leblond <fnref n="3" />

La pollution atmosphérique par des éléments métalliques représente une menace pour la santé publique dans les zones urbaines et une cause majeure de contamination des écosystèmes terrestres. Ces dernières décennies, les émissions métalliques d'origine anthropique (industries, transports routiers, etc.) ont dominé les émissions naturelles. Les métaux émis dans l'atmosphère et transportés sur de longues distances se déposent au niveau des sols et des écosystèmes. Cependant, les effets chroniques toxiques sur la biosphère restent peu documentés.

L'évaluation métabolomique de l'impact des dépôts en métaux d'origine atmosphérique sur le métabolisme des mousses forestières a consisté en une analyse des corrélations canoniques pouvant exister entre les concentrations en métaux et métalloïdes mesurées par ICP-MS, d'une part, et les variations d'analytes détectés dans le métabolome d'extraits hydrosolubles de pièces végétales, brutes ou réduites en poudre au préalable, qui est établi par FIA-ESI-Q-ToF-MS en mode positif ou négatif, d'autre part. Trois espèces de mousse (Pseudoscleropodium purum, Hypnum cupressiforme, Thuidium tamariscinum) ont été prélevées au printemps 2016 sur 550 forestiers en France dans le cadre du dispositif de Biosurveillance des Retombées Atmosphériques des Métaux par les Mousses (BRAMM), inclus dans le volet européen de l'International Cooperative Programme – Vegetation. Dans cette pré-étude, 32 sites ont été choisis de façon à représenter les variations les plus contrastées en dépôts en métaux lourds (Cd, Pb, Hg) mesurées sur ces espèces sur tout le territoire.

L'approche métabolomique utilisée a permis de montrer qu'à partir des variables métaboliques détectées (entre 50 et 200 selon le mode d'ionisation et la matrice utilisés), une corrélation globale entre la variation des teneurs en Cd, Zn et Hg et celle en certains métabolites était établie significativement. Les trois espèces de mousse adoptent un comportement homéostatique similaire en réponse à la contamination chronique par ces métaux. L'identification de quelques marqueurs métaboliques a été tentée grâce à la base de données métaboliques Metlin.

Mots-Clés: pollution atmosphérique, métaux, bryophyte, FIA, ESI, Q, ToF, MS, analyse canonique

<footnote n="1">¹ UMR 7245 - Molécules de Communication et Adaptation des Micro-organismes (MCAM) – Museum National d'Histoire Naturelle, Centre National de la Recherche Scientifique – France</footnote>

<footnote n="2">² Molécules de Communication et Adaptation des Micro-Organismes (MCAM) – CNRS : UMR7245 – 57 rue Cuvier 75005 Paris, France</footnote>

<footnote n="3">³ MNHN – Museum National d'Histoire Naturelle – Rue Buffon, 75005 Paris, France</footnote>
<figure><img src="image_134.png" /></figure>

## Poster 22 – P22

# Central and peripheral metabolomics in a model of depression: biosignatures of drug responder and non-responder mice

Sylvie Mavel<fnref n="1" />, Antoine Lefèvre<fnref n="2" />, Marc Legrand<fnref n="1" />, Laurent Galineau<fnref n="1" />, Lydie Nadal-Desbarats<fnref n="1" />,<fnref n="2" />, Catherine Belzung<fnref n="1" />, Patrick Emond<fnref n="1" />,<fnref n="2" />

Major depressive disorder is a heterogeneous disorder with a wide spectrum of symptoms leading to disability, suicide and physical disorders. Selective serotonin reuptake inhibitors (SSRIs) are ones of the most commonly used drugs of treatment of depression. For unknown reasons, a great number of patients do not show any improvement during drug treatment (30-40%). The underlying molecular mechanisms of depression remain unknown. To improve knowledge of pathophysiology and to search biomarkers to help prognostic of antidepressant treatment response in patient, we employed a LC-HRMS metabolomics approach in a BALB/c induced mouse model of depression to investigate metabolic changes in 6 specific brain regions, after 5 weeks with a fluoxetine treatment. From 770 cumulated targeted metabolites, 68 discriminant Variable Importance in Projection (VIP) were shown to discriminate vehicle-treated group from drug responder and non-responder mice, after OPLS-DA. This central metabolomics approach is completed by a peripheral analysis through plasma analysis. A less robust OPS-DA model was obtained, but 27 discriminant metabolites (over 197 robustly analyzed) were altered in plasma. Comparison of central VIP with peripheral VIP leads to 7 common metabolites. This study shows the complementary of these two compartments, i.e. cerebral and plasma metabolome.

From this, specific brain regions shown particular metabolism which could explain some aspects of the depressive pathophysiology and some biomarkers could be proposed to anticipate antidepressant treatment response.

<footnote n="1">¹ UMR 1253, iBrain, Université de Tours, Inserm, Tours, France</footnote>

<footnote n="2">² Université François-Rabelais, PPF Analyse des Systèmes Biologiques (ASB), 10 Bv Tonnellé, 37044 Tours, France</footnote>
<figure><img src="image_135.png" /></figure>

**Communication Posters**

**Session Alimentation**

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RESEAU FRANCOPHONE DE METABOLOMIQUE ET FLUXOMIQUE
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Poster 23 – P23

# De bonnes habitudes alimentaires: impact sur le profil métabolique d'échantillons de sérum analysés par RMN du proton

Tony Palama<fnref n="1" />, Marinos Fysekidis <fnref n="2" />,<fnref n="3" />, Nadia Bouchemal <fnref n="1" />, Mohamed Triba <fnref n="1" />, Philippe Savarin <fnref n="1" />

Les mécanismes métaboliques en lien avec la nutrition-santé restent peu explorés jusqu'ici. Leur élucidation est cruciale pour mieux comprendre la survenue des maladies et améliorer les stratégies de prévention. Le but de l'étude Nutrivasc réalisée par l'équipe de recherche en épidémiologie nutritionnelle (EREN) était d'étudier les relations entre la qualité nutritionnelle de l'alimentation évaluée par les recommandations du Programme National Nutrition Santé (PNNS) et des facteurs de risques cardiovasculaires. Nous avons utilisé des échantillons de sérum collectés lors de cette étude pour en faire l'analyse du métabolome. L'utilisation de la métabolomique dans le domaine de l'épidémiologie est une approche récente et alternative permettant d'explorer ces relations afin de découvrir de nouveaux biomarqueurs. Objectifs: 1/ Comparer les profils métaboliques RMN des sérum des individus ayant un mauvais comportement alimentaire (score bas PNNS) de ceux qui respectent les recommandations nutritionnelles (score PNNS élevé). 2/ Identifier les biomarqueurs durant la période postprandiale dans les deux groupes. Matériels et méthodes: 49 individus appartenant au 1er et 4ème quartile des scores PNNS ont été sélectionnés. Lors de l'étude, des prélèvements sanguins ont été réalisés à jeun (T0), puis 1, 2, et 3 heures (T+1h, T+2h et T+3h) après la prise d'un petit déjeuner standardisé. Les échantillons ont été analysés par résonance magnétique nucléaire (RMN) du proton 1H. Les spectres acquis sur un spectromètre RMN Bruker à 500 MHz en utilisant la séquence CPMG ont été soumis à des analyses statistiques multivariées (ACP, OPLS). Résultats: Les métabolites responsables de la principale source de variabilité ont été identifiés. En prenant en compte l'ensemble de la population aucune discrimination n'a été possible entre le 1er et le 4ème quartile par ACP et OPLS. Cependant une discrimination des échantillons, suivant le temps de prélèvement, a été observée. Les métabolites discriminants ont été identifiés par comparaison avec la base de donnée HMDB (www.hmdb.ca) et l'analyse des spectres RMN 2D (J-resolved, TOSCY, HSQC).

Mots clefs : métabolomique clinique, RMN, analyse multivariée, serum

<footnote n="1">¹ Laboratoire Chimie, Structures, Propriétés de Biomatériaux et d'Agents Thérapeutiques (CSPBAT) – Université Paris 13, Sorbonne Paris Cité - CNRS (France) : UMR7244 – France</footnote>

<footnote n="2">² Service d'endocrinologie diabétologie, nutrition (Hôpital Jean Verdier) – Assistance publique Hôpitaux de Paris (AP-HP), université Paris 13, Hôpital Jean Verdier – France</footnote>

<footnote n="3">³ Equipe de Recherche en Epidémiologie Nutritionnelle (EREN) – Université Paris 13, Sorbonne Paris Cité,, Inserm (U1153), Inra (U1125), Centre d'Epidémiologie et Statistiques Paris Cité, CNAM, COMMUE Sorbonne-Paris-Cité, F-93017 Bobigny – France</footnote>
<figure><img src="image_138.png" /></figure>

Poster 24 – P24

Modelling hepatic metabolic changes during the onset of obesity using
NMR metabolomics arterio-venous blood exploration in minipigs

**Nathalie Poupin**<sup>1</sup>, **Marie Tremblay-Franco**<sup>1,2</sup>, **Aurélien Amiel**<sup>1,2</sup>, **Cécile Canlet**<sup>1,2</sup>, **Didier Rémond**<sup>3</sup>, **Laurent Debrauwer**<sup>1,2</sup>, **Dominique Dardevet**<sup>3</sup>, **Fabien Jourdan**<sup>1</sup>, **Isabelle Savary-Auzeloux**<sup>3</sup>, **Sergio Polakof**<sup>3</sup>

Phenotypic alterations associated with obesity are well known, but metabolic adaptations occurring during its onset are poorly characterized. This study aims at understanding the hepatic metabolic processes modulated during the onset of obesity by combining paired arterial and venous metabolome analyses with computational modeling. It relies on the principle that differences in the metabolite composition of blood flowing in and out of the liver reflect its metabolic functioning. Blood samples were collected in a mini-pig model of obesity [1], consisting in over nutrition with a high-fat-high-sucrose (HFHS diet) for 60 days. Blood was sampled at the fasting state in 5 catheterized mini-pigs from incoming (abdominal artery and portal vein) and outgoing hepatic vessels at days 0 and 60 of HFHS feeding. From 1H-NMR analyses, we identified metabolites with significantly changed levels between arterious and venous blood. Calculated 1H-NMR integration ratios between arterious and venous blood allowed us to identify the metabolites with significant positive (resp. negative) balance between inflow and outflow, reflecting a release (resp. uptake) by the liver. These data were analyzed within the frame of an hepatic genome-scale metabolic network model using constraint-based modeling approaches to predict the changes in intra-tissular metabolic fluxes [2]. Constraints were set on exchange reactions in the metabolic model to enforce uptake and release of metabolites in accordance to experimental data and *in silico* flux balance analysis methods were used to predict possible flux ranges for hepatic metabolic reactions. We predicted that HFHS was associated with changes in the glucose metabolism and the sources of gluconeogenesis, but also in the catabolism of tryptophan and lysine, for which further support was found through complementary biochemical and molecular analyses.

Polakof S, *et al*. Metabolic adaptations to HFHS overfeeding: how whole body and tissues postprandial metabolic flexibility adapt in Yucatan mini-pigs. *Eur J Nutr*. 2018;57(1):119-35.

Mots-Clés: arterio venous differences, metabolic networks, constraint, based modelling, obesity, mini, pig

<footnote n="1">¹ UMR 1331 Toxalim - INRA-ENVT-INP-EIP-UPS – Institut national de la recherche agronomique [Toulouse] – France</footnote>

<footnote n="2">² Metatoul Axiom platform - Metabohub national Infrastructure INRA [Toulouse] - France</footnote>

<footnote n="3">³ INRA UMR1019 Unité de Nutrition Humaine – Université Clermont Auvergne, INRA, UNH – France</footnote>
<figure><img src="image_139.png" /></figure>

## Poster 25 – P25

# Metabolomic and lipidomic analysis of serum and liver samples following Curcuma longa extract supplementation in high-fructose and saturated fat fed rats

Fabrice Tranchida<fnref n="1" />, Laetitia Shintu<fnref n="1" />, Zo Rakotoniaina<fnref n="1" />, Léopold Tchiakpe<fnref n="2" />, Mehdi Yemloul<fnref n="1" />, Valérie Deyris<fnref n="1" />, Pierre Stocker<fnref n="3" />, Nicolas Vidal<fnref n="3" />, Abel Hiol⁴, Stefano Caldarelli<fnref n="1" />

Fructose consumption from corn syrup, a common sweetener used in the food industry, has increased dramatically over the past few decades in industrialized countries, and its impact on health has been recently reviewed. Similarly, the intake of saturated fats has risen during, the same time period. It has been reported that the current high dietary intake of fructose and saturated fats (HFS) contributes to the epidemic of insulin resistance (IR). Native to Southeast Asia, Curcuma longa (CL) also known as turmeric is one of the most studied natural products. A metabolomics approach based on NMR spectroscopy and GC-MS was performed to highlight metabolic variations of serum and liver tissues of rats fed either a standard diet, a HFS diet, or a HFS diet with the addition of a Curcuma longa extract at a dose nutritionally relevant with common human use (1% in curcuminoids) for ten weeks. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) on the NMR profiles and fatty acid composition (determined by GC/MS) showed a clear discrimination between HFS groups and controls involving metabolites such as glucose, glycogen, amino acids, acetate, choline, pyruvate, creatine, phosphocholine/glycerophosphocholine, lysophosphatidylcholine, phosphatidylethanolamine, and β-hydroxybutyrate, VLDL as well as an increase of MUFAs and a decrease of n-6 and n-3 PUFAs. Although the administration of CL did not counteract the IR induced by the HFS diet, discriminating metabolites were observed between groups fed HFS alone or with addition of a Curcuma longa extract, namely some n-6 PUFA, n-3 PUFA, betaine and glycoproteins, glutamine, methanol respectively in liver and serum samples. CL extract addition in rats appears to increase some of the natural defences preventing the development of fatty liver by acting on the choline metabolism (betaine was found to rise by 35%) to increase fat export from the liver. Whereas in serum, a depression of the alcohol oxidation, detected through an enhanced concentration of methanol, and a possible activation of the hexosamine biosynthesis pathway, consistent with a reduction of glutamine and glycoprotein levels were observed. All of these points to possible specific and local, rather than systemic, protective effects of Curcuma longa extract.

Mots-Clés: Fructose, Saturated fatty acid, Curcuma longa, Metabolomics, NMR, Serum, Liver

<footnote n="1">¹ Institut Sciences Moléculaires de Marseille UMR 7313 – Aix-Marseille Université - AMU - France</footnote>

<footnote n="2">² Laboratoire de Nutrition-Dietétique, Faculté de Pharmacie, Marseille – Aix-Marseille Université AMU – France</footnote>

<footnote n="3">³ Institut de Chimie Radicalaire UMR 7273 – Aix-Marseille Université - AMU - France ⁴ CIRAD UMR QualiSud – Université de la Réunion – France</footnote>
<figure><img src="image_140.png" /></figure>

## Poster 26 – P26

# Evaluation de l'approche non-ciblée pour l'analyse des biotoxines marines dans les coquillages via la plateforme Galaxy w4m

Sophie Mompelat¹, Inès Dom, Vincent Hort, Gwenaëlle Lavison-Bompard, Marina Nicolas, Thierry Guerin, Dominique Hurtaud-Pessel, Ronel Bire

¹Laboratoire de Fougères, Laboratoire National et Européen de Référence pour les Résidus de Médicaments Vétérinaires dans les Denrées Alimentaires d'Origine Animale (ANSES) – Anses – 10B rue Claude Bourgelat, 35306 FOUGERES, FRANCE, France

Les biotoxines marines sont des composés toxiques produits par des microalgues. En raison du risque avéré qu'elles représentent pour le consommateur, leur présence dans les coquillages est réglementée et surveillée. La tendance actuelle est à l'abandon des tests biologiques au profit de méthodes chimiques pour des raisons éthiques notamment. Depuis 2010, la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) est la méthode officielle d'analyse des biotoxines lipophiles. Si cette méthode s'applique parfaitement aux toxines ciblées par la règlementation, elle ne permet pas de mettre en évidence la présence de toxines inconnues ou émergentes.

Cette étude vise donc à évaluer la pertinence et l'apport pour le contrôle sanitaire de nouvelles approches récentes d'analyses dites non-ciblées afin de mettre en évidence la présence de toxines émergentes. La stratégie analytique développée repose sur la comparaison d'empreintes biologiques obtenues via le criblage non ciblé (mode full-scan) par spectrométrie de masse à haut pouvoir résolutif (HRMS, QTOF) afin de discriminer des groupes de coquillages témoins de groupes de coquillages contaminés par des biotoxines. Cet essai de preuve de concept a été réalisé sur quatre lots différents de trois types de coquillages (moules, huîtres et coquilles StJacques) supplémentés avec un mélange de quatre toxines connues (13-déméthyl spirolide C, azaspiracide 1, gymnodimine A et pinnatoxine A) à trois niveaux de concentrations. Le workflow appliqué à partir des outils de la plateforme Galaxy Workflow4metabolomics et recouvrant les étapes principales d'analyse de données allant du prétraitement des signaux HRMS jusqu'à l'annotation seront décrits. Les résultats démontrent que cette approche semble constituer une alternative envisageable pour discriminer des coquillages contaminés de coquillages propres à la consommation au regard des biotoxines. Les limites ainsi que l'évaluation de la robustesse de cette approche à considérer seront abordées.

Mots-Clés: biotoxines marines, contrôle sanitaire, analyse non ciblée, LC, MS/MS, HRMS
<figure><img src="image_141.png" /></figure>

## Poster 27 – P27

# Hepatic metabolomics and transcriptomics provide valuable insights on inflammatory and metabolic features in rats fed on a high fat diet but showing similar clinical phenotyping alterations

Sergio Polakof<fnref n="1" />, Marie Tremblay-Franco<fnref n="2" />, Cécile Canlet<fnref n="2" />, Jérémie David<fnref n="1" />, Fabien Jourdan<fnref n="3" />, Nicolas Cabaton<fnref n="3" />, Nathalie Poupin<fnref n="3" />, Blandine Comte<fnref n="1" />, Daniel Zalko<fnref n="3" />

The complex processes of obesity development involve genetic and environmental factors. Genetic factors are considered to account for only a small fraction of the overall susceptibility to high fat (HF) induced obesity. Under certain conditions, foetal environment can trigger an adaptive response. This involves the further onset of insulin resistance to optimize the development of key organs (brain, heart...), to the detriment of others organs such as the liver. We studied the impact of a perinatal exposure to a HF diet on rats liver metabolome and transcriptome. Pregnant rats were fed with a normal fat (NF) or a HF diet during gestation and lactation. After weaning, males from the F1 generation were fed either the NF or the HF diet, until PND142, resulting in 4 groups depending on perinatal and post-weaning diet, respectively (NFxNF, NFxHF, HFxNF and HFxHF). H-NMR spectroscopy and mRNA (TLDA) analyses were performed on male livers extracts.

Clinical phenotyping were different according to post-weaning diet but not according to maternal diet. The multivariate OPLSDA models discriminated F1-animals based on F1 diet (NFxNF and HFxNF vs NFxHF and HFxHF). When dams were fed on a standard diet (NFxNF vs NFxHF)

HF-fed F1 animals showed an increased expression of genes participating to hepatic inflammation and a reduction in oxidative stress protectors (lower glutathione in the metabolome). In contrast, feeding dams and pups with a HF diet (HFxNF vs HFxHF) resulted in major changes at the metabolic level, including a reduced expression of lipogenic genes and several actors involved in branched-chain amino acid metabolism. The latter result was further supported by the increased hepatic levels of leucine (metabolome). The most extreme scenario compared to the control one (NFxNF vs. HFxHF) resulted in a more deleterious phenotype, which is the consequence of the combination of the inflammatory and metabolic features described above.

In conclusion, despite the fact that HF feeding at the F1 level induced similar changes in clinical phenotyping, metabolomics and transcriptomics analyses at the liver level were able to discriminate between animals, providing valuable information on the underlying inflammatory and metabolic processes specifically signing the consequence of different maternal feeding.

Mots-Clés: liver, metabolomics, transcriptomics, high fat, metabolism, maternal feeding, obesity

<footnote n="1">¹ INRA UMR1019 Unité de Nutrition Humaine – Université Clermont Auvergne, INRA, UNH – France</footnote>

<footnote n="2">² Plateforme MetaToul-AXIOM, Toulouse – Institut National de la Recherche Agronomique - INRA – France</footnote>

<footnote n="3">³ INRA Toxalim - Université de Toulouse – Institut national de la recherche agronomique [Toulouse] : UMR1331 – France</footnote>
<figure><img src="image_142.png" /></figure>

# Poster 28 – Associé FP9

## Syringol metabolites as biomarkers of smoked meat intake

Roland Wedekind¹, Pekka Keski-Rahkonen ¹, Vivien Viallon ¹, Pietro Ferrari ¹, Erwan Engel ², Marc Gunter ¹, Inge Huybrechts ¹, Augustin Scalbert¹

**Background:** Intake of processed meat has been associated with an increased risk of colorectal cancer but the estimation of intake of this heterogeneous food group in epidemiological studies is still challenging because of the lack of sufficient details in dietary questionnaires. **Objective:** The objective of this work is to identify in a dietary intervention study novel biomarkers for processed meat intake.

**Design:** A metabolomic approach based on high-resolution mass spectrometry was applied to 21 processed meat products previously digested *in vitro* and a randomized cross-over dietary intervention in which 12 volunteers consumed successively 3 processed meat products (bacon, salami, hot dog) and two other foods used as controls during 3 days. Identified biomarkers were tested for replication in 474 subjects from the European Prospective Investigation into Cancer and Nutrition (EPIC) cross-sectional study for which a detailed 24h dietary recall and food frequency questionnaires were available.

**Results:** Syringol and four derivatives of syringol were found to be characteristic of digests of smoked meat products. The same compounds present as sulfate esters in urine showed increased levels at 2 and 12 hours following consumption of smoked meat products (hot dog, bacon) in the intervention study. The same syringol sulfates also showed increased urinary excretion in participants of the EPIC cross-sectional study reporting recent or habitual consumption of smoked meat products. These markers showed good ability to predict smoked meat intake with receiver operator characteristic areas under the curve ranging from 0.78 to 0.86 and 0.74 to 0.79 for acute and habitual intake respectively.

**Conclusions:** The biomarkers of smoked meat intake identified in the present study may improve assessment of smoked meat intake in epidemiological studies.

**Mots-Clés:** Smoked meat, biomarker, metabolomics

<footnote n="3">³ Centre International de Recherche contre le Cancer - International Agency for Research on Cancer - Organisation Mondiale de la Santé / World Health Organization Office - France</footnote>

<footnote n="4">⁴ UR 370 Qualité des Produits Animaux, Equipe Micro-contaminants Arôme et Sciences Séparatives (MASS) (QuaPA, MASS) – Institut National de la Recherche Agronomique - INRA (FRANCE) – INRA Centre de recherches Auvergne-Rhône-Alpes Theix 63122 Saint-Genès-Champanelle, France</footnote>
<figure><img src="image_143.png" /></figure>

## Poster 29 – Associé FP3

# Impaired postprandial skeletal muscle metabolism in a minipig model of insulin resistance: insights from arteriovenous and biopsy-based metabolomics analyses

Marie Tremblay-Franco ¹,², Nathalie Poupin ¹, Florence Castelli, Jérémie David, Marianne Jarzaguet, Didier Rémond, François Fenaille ³, Dominique Dardevet ⁴, Sergio Polakof ⁴

5 UMR 1331 Toxalim - INRA-ENVT-INP-EIP-UPS - INRA [Toulouse] - France

6 Metatoul Axiom platform - Metabohub national Infrastructure - Toulouse - France

7 CEA-INRA UMR 0496/DRF/Institut Joliot/SPI/LEMM, Université Paris Saclay, MetaboHUB - CEA - Centre d'Etudes Nucléaires de Saclay, 91191 Gif-sur-Yvette, France, France

8 INRA UMR1019 Unité de Nutrition Humaine - Université Clermont Auvergne, INRA, UNH - France

The postprandial phase (PP) represents one of the most challenging phenomena in wholebody metabolism, as the metabolism must adapt to major changes in blood composition following meal intake. This capacity of adaptation is strongly dependent on the metabolic flexibility of the individual, which is compromised in insulin resistant (IR) conditions, particularly at the skeletal muscle level (1). We applied an untargeted LC/MS-based metabolomics approach to skeletal muscle biopsies obtained before (0) and 1.5, 3 and 6 h after a regular meal in a minipig (dexamethasone treated) model of IR (2). Biopsies analyses were complemented by the assessment of the incoming (arterial) and outgoing (venous) blood metabolomes. As the consequence of the anabolic effect of the meal, in healthy animals we observed increased PP muscle levels of metabolites related to glucose (lactate, glucose-1,6biP, lactoylglutathione), energy (citrate, malate), amino acids (AA; methionine, glutamate, aspartate) and nucleotide (GTP, inosine, guanosine) metabolisms. In contrast, these metabolites showed a blunted PP profile in IR minipigs, which illustrates the loss of response to the meal induced by such condition. These include the inability to uptake and use glucose through glycolysis, stimulate protein synthesis due to the lack of AA entry into the muscle and net loss of other nitrogen resources (nucleotides). For some of these metabolites (lactate, AA) the observed changes in the biopsies were in line with the arteriovenous differences across the muscle, which confirms that they were actively uptaken, while for other (nucleotides) the altered profiles in the muscle were the consequence of intramuscular metabolic changes. As a whole, our high-throughput arteriovenous metabolomics approach across the muscle allowed us to further explore the PP metabolic changes observed in the muscle biopsies from IR minipigs. This strategy could be therefore useful to determine in the systemic circulation metabolites (potential biomarkers) able to sign the IR condition without having access to the skeletal muscle biopsies.

(3) Galgani et al. Am J Physiol. 2008;295(5):E1009-17.

(4) Revel et al. PLoS ONE. 2017;12(10):e0186204.

Mots-Clés: insulin resistance, human nutrition, minipig, arteriovenous metabolome, biopsy, postprandial
<figure><img src="image_144.png" /></figure>

## Poster 30 – P30

# Characterization of GMO or glyphosate effects on the composition of maize grain and maize-based diet for rat feeding

Stéphane Bernillon ¹,², Mickaël Maucourt ¹,², Sylvain Chéreau ³, Catherine Deborde ¹,², Daniel Jacob ¹,², Nathalie Priymenko ⁴, Bérengère Laporte ⁴, Xavier Coumoul ⁵, Bernard Salles ⁴, Rogowsky Peter ⁶, Florence Forget ³, Annick Moing ¹,²

1 UMR 1332 Biologie du Fruit et Pathologie INRA : UMR1332, Université de Bordeaux – France

2 Plateforme Métabolome Bordeaux (PMB-MetaboHUB) – INRA – France

3 UR1264 MycSA INRA Bordeaux Nouvelle Aquitaine –INRA (FRANCE) : UR1264 MycSA – France

4 Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse –INRA

5 Université Paris Descartes - Paris 5 (UPD5) – Institut National de la Santé et de la Recherche Médicale - INSERM – 12, rue de l'École de Médecine - 75270 Paris cedex 06, France

6 Laboratoire Reproduction et Développement des Plantes – Université Claude Bernard et ENS Lyon – France

The yearly production of maize (Zea mays L.) grains is largely used for animal feed and human nutrition. In addition to classical breeding methods to improve yield, stress tolerance or quality traits, additional traits of agronomic interest were created by the insertion of foreign genes leading to genetically modified organisms (GMOs). Controversial studies about the potential impact of maize and other crop GMOs on human health and the environment have been published [1-3]. Studies about the effect of a given transgene on maize grain or leaf biochemical composition are not always confirmed and frequently result in contradictory conclusions [4-6] such as those related with herbicide tolerant maize.

To overcome these conflicting results, our objective was to combine NMR- and MS-based untargeted metabolomics analyses to assess possible compositional changes in maize grains induced by two bacterial gene-insertion events: a *Bacillus* toxin gene providing insect resistance, and a glyphosate-insensitive enzyme providing herbicide tolerance. Composition of GMO grains was compared with that of their closest non-GMO counterpart, each genotype pair cultivated in a given environmental condition. Untargeted metabolomic approaches were used to identify metabolite signatures and/or metabolites associated with the GMO event or with the glyphosate application in the case of the herbicide-tolerance event. We also checked if the changes observed in the grains were still visible in the feed containing 11 or 33% grain ingredient of GMO maize. This work on grain and pellet composition contributes to characterize the material used further in a rat feeding experiment [7] aiming at the detection of exposure or health effect biomarkers able to differentiate GMO and non-GMO or glyphosate-treated and control diets [8].

Mots-Clés: Maize composition, GMO, rat diet, Glyphosate
<figure><img src="image_145.png" /></figure>

## Poster 31 – P31

# LABERCA ANALYTICAL CHEMISTRY PLATFORM: METABOLOMIC AND STRUCTURAL ANALYSIS

Anne-Lise Royer<fnref n="1" />, **Yann Guitton**<fnref n="1" />, Sadia Ouzia<fnref n="1" />, Lauriane Rambaud<fnref n="1" />, Karinne Pouponneau<fnref n="1" />, Fabrice Monteau<fnref n="1" />, Bruno Le Bizec<fnref n="1" />

Metabolomics is an area of increased interest in many scientific fields such as chemical food safety and toxicology in order to investigate and identify (bio)markers of exposure, effect and/or susceptibility. However, the different parts of a metabolomic workflow are sources of many issues. Biological samples contain hundreds or even thousands of metabolites with different physicochemical properties limiting the analysis of the entire metabolome using a unique analytical technique. Therefore, the use of a multi-analytical platform in combination with appropriated sample preparation is required to enlarge the number of metabolites detected. Since 2007, our analytical chemistry platform is engaged in the development of tools and methods for untargeted metabolomics <fnref n="1" />. Through the years we’ve faced the different challenges of untargeted metabolomics studies (from experimental design to statistical analysis). Along the years we’ve worked with several matrices and developed dedicated sample preparation procedure 2,3. Our expertise also covers bioinformatics tools development through the workflow4metabolomics.org infrastructure 4 and more targeted tools for halogen (Cl, Br) compound retrieval 5. In 2017, our platform work was pushed a step further with the ISO17025 accreditation of an LC-HRMS methods derived from a metabolomics model dedicated to the detection of β-agonist administration in bovines that has been developed and fully validated. allowing for the very first official implementation of a metabolomics based strategy within French National Monitoring Plans 6.

Thanks to its environment, our platform has access to new technologies (IMS, APGC, ASAP, REIMS...) and has been able to investigate them in untargeted metabolomics studies 7–9.

Mots-Clés: Metabolomique, Accreditation, plate, forme

<footnote n="1">¹ LABERCA, Oniris, INRA, Université Bretagne-Loire, 44307, Nantes, France – Institut national de la recherche agronomique (INRA) : UMR1329, Ecole Nationale Vétérinaire de l’Alimentation et de l’Agroalimentaire, Nantes Atlantique - ONIRIS, route de Gachet, CS 40706, 44307 Nantes Cedex 03, France – France</footnote>
<figure><img src="image_146.png" /></figure>

## Poster 32 – P32

# Linking cocoa polyphenol composition to chocolate quality with average mass spectra fingerprints

Véronique Cheynier<fnref n="1" />, Noémie Fayeulle, Emmanuelle Meudec <fnref n="1" />, Jean-Claude Boulet <fnref n="2" />, Clotilde Hue, Renaud Boulanger, Nicolas Sommerer <fnref n="1" />

Chocolate quality is largely due to the presence of polyphenols, and especially flavan-3-ols that are present in large amounts in cocoa beans and contribute bitterness and astringency to the final product.

Prediction of chocolate quality from cocoa composition would be of great help for chocolate makers who currently base their sourcing on a time consuming and costly procedure involving sensory analysis of chocolates made using a standardized process.

The aim of the present work was to investigate the potential of polyphenol-fingerprints for discrimination of cocoa samples and their attribution to chocolate sensory groups.

Sixty cocoa bean samples have been processed into chocolates and attributed to four different sensory groups by sensory analysis. Cocoa polyphenol extracts have been prepared from the ground cocoa beans and analyzed by UHPLC-ESI-IT-MS. Mass spectra recorded on the whole UHPLC profile provided cocoa polyphenol fingerprints which were processed with chemometrics (PCA, PLS-DA) to select the most meaningful molecules for discrimination of the chocolate sensory groups.

Sixteen samples representative of the four sensory groups were used for selection of the most discriminant variables. Non supervised PCA analysis of the average mass spectra showed that IT-MS is sensitive and quantitative enough to discriminate the sensory groups without prior selection of target compounds, i.e polyphenolic markers. A supervised chemometrics treatment, PLS-DA, was applied to this data to select the most relevant molecules for the discrimination. A larger set of 44 cocoa samples was used to validate the results. The fingerprinting method proved to be quick and efficient and the chemometrics analysis highlighted 32 mass signals of known and unknown molecules that were finally targeted [1], enabling sensory-group discrimination. Most of these signals were attributed to flavan-3-ols, including 2 newly described ethyl-bridged flavan-3-ols arising from condensation of flavan-3-ols with acetaldehyde [2].

Mots-Clés: cocoa, chocolate, polyphenols, fingerprinting, mass spectrometry, chemometrics

<footnote n="1">¹ INRA - UMR1083 SPO - Plateforme d'analyse des polyphénols (INRA - PFP) – Institut National de la Recherche Agronomique - INRA - UMR1083 SPO 2 place Viala 34060 Montpellier, France</footnote>

<footnote n="2">² INRA, UMR Sciences pour l'Oenologie (INRA, UMR SPO) – INRA : UMR1083 – 2 Place Viala 34060 Montpellier cedex 2, France</footnote>
<figure><img src="image_147.png" /></figure>

## Poster 33 – P33

# Etude par spectroscopie 1H-NMR du profil métabolique du liquide allantoïque au cours du développement embryonnaire de deux lignées de poulets divergentes pHu- et pHu+.

Lydie Nadal-Desbarats<fnref n="1" />,<fnref n="2" />, Angélique Petit <fnref n="3" />, Sophie Réhault-Godbert <fnref n="3" />, Cécile Martias <fnref n="1" />, Frédéric Montigny <fnref n="2" />, Elisabeth Le Bihan-Duval <fnref n="3" />, Sonia Metayer-Coustard <fnref n="3" />

La sélection des souches de poulet de chair a porté sur l'augmentation de la croissance et du rendement en viande. Ceci conduit à un amenuisement des réserves énergétiques musculaires. Ce caractère, héritable, détermine génétiquement le niveau du pH ultime (pHu) de la viande, pour lequel deux lignées divergentes (pHu+ et pHu-) ont été sélectionnées. L'objectif de cette étude est d'identifier des biomarqueurs précoces du statut énergétique des animaux *via* la caractérisation du liquide allantoïque (LC) des lignées divergentes pHu- et pHu+ par 1H-NMR à 10, 14 et 17 jours de développement embryonnaire.

Les LC ont été prélevés, centrifugés, ultrafiltrés puis conservés à -80° C. Les échantillons ont été ajoutés à un tampon phosphate deutéré contenant du TSP. Les spectres 1H ont été réalisés sur un spectromètre Bruker 600MHz. Les intensités spectrales sont normalisées à l'aire de la référence pour une analyse statistique multivariée (SIMCA-P+, Umetrics, Umea, Sweden).

L'analyse conjointe des 3 stades de développement montre des profils spectraux très différents (PLS-DA, R²Y=0,9, Q<fnref n="2" />=0,84, X=26) à E10, E14, ou E17. Si le facteur temps de développement apparaît plus discriminant que le facteur " lignée ", les 2 souches se distinguent en fonction de leur génotype au stade E14 (PLS-DA, R²Y=0,65, Q<fnref n="2" />=0,6, X=19, CV-Anova p=0,003) mais pas aux autres stades. A E14, les principaux métabolites discriminants sont l'hypoxanthine (p=0,001), la xanthosine (p=7,12E-5), la 3-HOKynurenine (p=0,005), ainsi que certains acides aminés (tels que glutamate, valine, lysine, glycine, histidine).

Cette étude illustre le changement de composition du liquide allantoïque au cours du développement embryonnaire pendant lequel les sources de nutriment évoluent. C'est à E14, âge auquel les embryons changent de source de nutriments, qu'une signature métabolique différente est mise en évidence entre les 2 lignées. Ceci suggère des mécanismes d'utilisation métabolique différents en fonction de la génétique de l'embryon et ouvre des perspectives d'une détection précoce, in ovo, du statut énergétique de l'animal.

Mots-Clés: metabolomics, oeuf, SRM

<footnote n="1">¹ UMR 1253 iBrain, Université de Tours, Inserm, F-37000 Tours, France – Université de Tours – France</footnote>

<footnote n="2">² PST-ASB, Université de Tours, F-37000 Tours, France – Université de Tours – France</footnote>

<footnote n="3">³ UMR BOA, INRA, Université de Tours, F-37380 Nouzilly, France – INRA – France</footnote>
<figure><img src="image_148.png" /></figure>

## Poster 34 – Associé FP8

# Comparaison des profils métaboliques et des propriétés antimicrobiennes de produits formulés à base de piment (Capsicum)

Inès Remy-Larsonnier<sup>a</sup>, Marjolaine Rey<sup>a</sup>, Carine Commun<sup>b</sup>, Anne Doléans-Jordheim<sup>b</sup>, Gilles Comte<sup>a</sup>, Marie-Geneviève Dijoux-Franca<sup>a</sup>, Camille Rozier<sup>c</sup>

Réduire l'usage des antibiotiques en santé humaine et animale est devenue une absolue nécessité afin de limiter l'émergence de souches bactériennes antibio-résistantes. Les entreprises développant des additifs destinés aux animaux d'élevage tel qu'ID4FEED utilisent les propriétés biologiques des plantes comme base principale de leurs produits. Parmi les sources d'actifs ciblées par ID4FEED, le piment (Capsicum chinense) s'est révélé être un candidat majeur[1]. Les capsaïcinoïdes, responsables de la sensation de chaleur provoquée par le piment, sont la famille de métabolites très caractérisés jusqu'à présent chez le piment car ils possèdent une activité biologique d'importance en santé animale (antimicrobienne, antiinflammatoire, analgésique, radical-scavenging)[2]. Il a néanmoins été mis en évidence que d'autres classes de métabolites apportaient leurs activités dans le totum que représente la poudre de piment tels que des antioxydants et des antimicrobiens (vitamines A et E, caroténoïdes, polyphénols, phénols, terpènes).

L'objectif de ce travail a été de réaliser un suivi quantitatif et qualitatif des capsaïcinoides et des composés minoritaires de la poudre de piment et de comparer les profils métaboliques de la matière première et des produits commerciaux issus de cette matière première.

Une stratégie d'analyse globale par GC-QQQ et UHPLC-DAD-Q-TOF nous a permis de mettre en évidence des différences (quantitatives et qualitatives), notamment de certains flavonoïdes et volatiles contenus dans le piment. Le lien potentiel entre ces différences de profils métaboliques et les activités biologiques (antimicrobiennes et radical-scavenging notamment) de la matière première et des produits commerciaux est de même évalué en parallèle et nous permettra de renforcer notre compréhension des propriétés biologiques des préparations industrielles.

[1] Do Rêgo, E. R., Finger, F. L., & do Rêgo, M. M. (2012). Consumption of pepper in Brazil and its implications on nutrition and health of humans and animals.

[2] De Aguiar, A. C., Coutinho, J. P., Barbero, G. F., Godoy, H. T., & Martínez, J. (2016). Comparative study of capsaicinoid composition in capsicum peppers grown in Brazil. *International Journal of Food Properties*, 19(6), 1292-1302.

Mots clés : Capsicum ; additifs ; capsaïcinoides ; UHPLC-DAD-Q-TOF ; GC-QQQ

<footnote><sup>a</sup> Université Lyon 1, UMR CNRS 5557/INRA 1418, Ecologie Microbienne, CESN, Villeurbanne, France</footnote>

<footnote><sup>b</sup> Université Lyon 1, UMR CNRS 5557/INRA 1418, Ecologie Microbienne, Lyon, France</footnote>

<footnote><sup>c</sup> ID4FEED SAS square du Rhône, 12 avenue du Rhône, BP 10051, Annecy, France</footnote>
<figure><img src="image_149.png" /></figure>

**Communication Posters**

**Session Environnement - Plante**

<figure><img src="image_150.png" /></figure>
<figure><img src="image_151.png" /></figure>

## Poster 35 – P35

# Processus allélopathique et bio-herbicide : étude métabolomique de la diversité microalgale et caractérisation de substances allélochimiques assistée par les réseaux de similarités

Slimane Chaïb<fnref n="1" />, Delphine Raviglione<fnref n="1" />, Carole Vialleix<fnref n="2" />, Annabel Levert<fnref n="3" />, Jean-Paul Cadoret<fnref n="2" />, Cédric Bertrand<fnref n="1" />, Isabelle Bonnard<fnref n="1" />

Le glyphosate est la molécule active d'un des herbicides les plus connus au monde : le Roundup de Monsanto homologué en 1974. En mars 2015, l'évaluation du Centre international de recherche sur le cancer (CIRC), une agence de l'Organisation mondiale de la santé (OMS), classait le glyphosate dans la catégorie des produits à fort impact sur la biodiversité.

Dans ce contexte, la biomasse issue de la production de micro-organismes photosynthétiques est une alternative à mettre en avant. En effet, les micro-algues constituent, au sein du milieu marin, une source variée de molécules bioactives. Il est mentionné dans la littérature la présence de substances allélopathiques connues comme ayant divers effets inhibiteurs et/ou toxiques tel que la fischerelline A qui est fortement algicide et antifongique.

Le projet " ALL DREAM " a pour but de développer un outil innovant adapté à l'analyse de la diversité chimique des micro-algues et à l'évaluation de leur potentiel comme biocide. Dans ce cadre, 150 extraits de micro-algues issues de diverses familles ont été soumises à un test d'inhibition de la photosynthèse ainsi qu'à un test antioxydant. Les résultats sont couplés à une approche métabolomique et aux réseaux de similarités.

Les données sont tout d'abors traitées via la plateforme W4M et le logiciel R. Elles font ensuite l'objet d'une analyse comparative entre la plateforme GNPS et MzMine/MetGem avant d'être traitées sous Cytoscape pour l'annotation. Puis, les extraits actifs sont soumis à un fractionnement bioguidé via un couplage LC-PDA-ELSD avec collecteur de fractions et LC-HRMS pour obtenir une caractérisation du/des métabolites actifs. Les extraits actifs sont ensuite testés sur des jeunes plantules afin d'analyser leur effet herbicide.

Ce poster présente l'ensemble des résultats mettant en avant le potentiel de cette ressource " bleue " que sont les micro-algues ainsi que l'alternative " verte " qu'elles représentent pour l'agriculture du futur.

**Source de financement** : Bourse Région/FEDER, AKINAO, GREENSEA

Mots-Clés: Allélopathie, Micro, algues, Bio, herbicide, Métabolomique, Réseaux de similarités

<footnote n="1">¹ CRIOBE USR3278, Université de Perpignan Via Domitia – Univ de Perpignan Via Domitia – France</footnote>

<footnote n="2">² Greensea – Greensea – France</footnote>

<footnote n="3">³ Akinao – S.A.S. AkiNaO – France</footnote>
<figure><img src="image_152.png" /></figure>

## Poster 36 – P36

# Evidence of wheat plant ability to control Pseudomonas secondary metabolism

Laura Rieusset<fnref n="1" />, Marjolaine Rey<fnref n="1" />, Florence Gerin<fnref n="2" />, Daniel Muller<fnref n="2" />, Claire Prigent-Combaret<fnref n="2" />, Gilles Comte<fnref n="1" />

The plant root adhering soil houses an important microbial community. Roots exude a wide variety of secondary metabolites able to attract and/or control a large diversity of microbial species. In return, among the root microbiota, some bacteria are able to promote plant development, using plant growth-promotion and plant protection properties. These bacteria are named PGPR for Plant Growth-Promoting Rhizobacteria. Some PGPR belong to the *Pseudomonas* genus. *Pseudomonas* are known to produce a wide diversity of secondary metabolites that could have biological activity on the host plant and other soil microorganisms. But yet, the impact of host plant on *Pseudomonas* secondary metabolism is still poorly understood.

The aim of our project is to better understand the impact of the host plant on secondary metabolite production by fluorescent *Pseudomonas* strains.

A metabolomic approach was developed in order to decipher how plant may modulate secondary metabolites production in *Pseudomonas*. Five different fluorescent *Pseudomonas* strains were thus cultivated in the presence of root extracts of three wheat genotypes, at low concentration. This experimentation allows us to evaluate the impact of root metabolites on *Pseudomonas* secondary metabolism.

Analysis of our metabolomic workflow revealed that the production of several *Pseudomonas* secondary metabolites were significantly up- or down-regulated when bacteria were cultivated with root extracts. This shows that wheat root metabolites may act as signal compounds and modulate *Pseudomonas* secondary metabolism, including metabolites involved in plant stimulation and plant protection properties. Interestingly, root extract modulation differs according to wheat genotypes and *Pseudomonas* strains.

Mots-Clés: Wheat genotypes, Pseudomonas, secondary metabolites, metabolomics, plant/bacterial interactions

<footnote n="1">¹ Laboratoire d'écologie microbienne - CESN - CNRS : UMR5557, Institut national de la recherche agronomique (INRA) : UMR1418, Université Claude Bernard-Lyon I - UCBL (FRANCE) – France</footnote>

<footnote n="2">² Laboratoire d'Écologie Microbienne – CNRS : UMR5557, Institut national de la recherche agronomique (INRA) : UMR1418, Université Claude Bernard-Lyon I - UCBL (FRANCE) – France</footnote>
<figure><img src="image_153.png" /></figure>

## Poster 37 – Associé FP7

# Étude de la réponse métabolique de Pseudomonas syringae 32b-74 isolée des nuages à une exposition sub – létale au mercure

Christelle Ghaffar, Martine Sancelme¹, Binta Diémé², Marie Lagrée³, Cyril Jousse⁴,⁵, Pierre Amato³

Ces dernières décennies, les fortes activités industrielles ont conduit à une augmentation de la pollution aux métaux lourds de l'atmosphère, et notamment au mercure (1,7 ng d'Hg / m3 d'air) 1. Celui-ci est présent sous différentes formes dans l'environnement : élémentaire (Hg0), ionique (Hg2+) et méthylé (Me – Hg). Ce dernier agit sur les groupes thiols des protéines, siège de sa toxicité. Certaines bactéries présentent un mécanisme de résistance codé par l'opéron mer et sous le contrôle du régulateur transcriptionnel MerR 2.

Des études montrent que des microorganismes subsistent dans l'atmosphère et les nuages, avec des conséquences possibles sur les processus physico-chimiques atmosphériques 3 4. Ces derniers sont donc exposés à la pollution atmosphérique du mercure, et notamment à la forme ionique Hg2+.

Nous étudions ici l'impact du mercure à dose sub – létale sur le métabolisme d'une souche bactérienne précédemment isolée d'eau de nuage et dépourvue de l'opéron mer : *Pseudomonas syringae* 32b74 5. L'objectif est d'identifier des marqueurs métaboliques spécifiques de l'exposition au mercure, dans l'optique d'une utilisation des microorganismes atmosphériques comme traceurs environnementaux.

La souche *P. syringae* 32b74 est ainsi incubée en absence et en présence de mercure biodisponible Hg2+ (5 µg/mL). Un profilage métabolique est réalisé après 2h et 24h d'exposition. Le métabolisme des cellules est stoppé par immersion dans l'azote liquide, et les métabolites intracellulaires sont extraits par un mélange de solvant avant d'être analysés par LC-MS. Par la suite, une analyse en composante principale (ACP) est réalisée afin de discriminer les potentiels signaux biomarqueurs.

L'impact du mercure sur l'écosystème atmosphérique est une question environnementale importante. L'utilisation de la métabolomique nous permettra de l'observer plus finement sur cette souche isolée de l'atmosphère.

Mots-Clés: Mercure, Atmosphere, Pseudomonas syringae 32b74, Métabolomique

<footnote n="6">⁶ Institut de Chimie de Clermont Ferrand (ICCF) – Université Clermont Auvergne, CNRS, SIGMA Clermont – France</footnote>

<footnote n="7">⁷ Université François-Rabelais, INSERM U930 "Imagerie et Cerveau", CHRU de Tours, 10 Bv Tonnellé, 37044 Tours, France –</footnote>

<footnote n="8">⁸ Institut de Chimie de Clermont-Ferrand - Clermont Auvergne (ICCF) – SIGMA Clermont, Université Clermont Auvergne : UMR6296, Centre National de la Recherche Scientifique : UMR6296 – 24 Avenue des Landais / 63177 Aubière Cedex, France</footnote>

<footnote n="9">⁹ Institut de Chimie de Clermont-Ferrand (ICCF) – SIGMA Clermont, Université Clermont Auvergne : UMR6296, Centre National de la Recherche Scientifique : UMR6296 – 24 Avenue des Landais / 63177 Aubière Cedex, France</footnote>

<footnote n="10">¹⁰Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont-Ferrand, Clermont-Ferrand, France (PFEM) – Université Clermont Auvergne, INRA, Université Clermont Auvergne – France</footnote>
<figure><img src="image_154.png" /></figure>

## Poster 38 – P38

# Characterizing the effect of Zn contamination on variegated scallop, Mimachlamys varia, using metabolomics

Pascaline Ory <fnref n="1" />, Vincent Hamani <fnref n="1" />, Pierre-Edouard Bodet <fnref n="1" />, Emmanuel Dubillot <fnref n="1" />, Laurence Murillo <fnref n="1" />, Marianne Graber <fnref n="2" />,<fnref n="3" />

The variegated scallop (*Mimachlamys varia*) is a filter feeder bivalve of the *Pectinidae* family encountered in marine regions of the Atlantic coast. In particular, it is present in the La Rochelle marina (France), where it is used for the biomonitoring of marine pollution, due to its ability to strongly bioaccumulate pollutants. In this semi-closed environment, contamination generated by port activities leads to an accumulation of both organic pollutants and metals. Zinc has been identified as one of these pollutants. It arises, among others, from sacrificial anodes used to protect ship's hulls and engines from corrosions.

This study investigated the effects of 48 h zinc exposure upon the metabolic profiles of *Mimachlamys varia* using UHPLC/QTOF tandem mass spectrometry metabolomics. The objective was to determine the potential impairment of cell energy synthesis processes (glycolysis and respiratory chain pathways) by zinc in *M. varia* and highlight other metabolic pathways changes due to zinc exposure.

After acclimatization of a pool of individuals in mesocosms recreating *in situ* conditions, both controls and exposed bivalves with zinc chloride (nominal concentrations:150 µg/l) were shucked and deprived of the digestive gland and gills after 48h, and stored at -80 °C before metabolite extraction.

UHPLC/QTOF tandem mass spectrometry was performed to study metabolite composition of samples. The obtained data point out a sensible metabolic response of bivalves to 48 h metal exposure. Our study demonstrated that the metabolomics approach is a useful tool for obtaining comprehensive and novel insights into the molecular toxicity of environmental pollutants.

**Mots-Clés:** metabolomics, zinc contamination, bivalves, marine environment

<footnote n="1">¹ University of La Rochelle, UMR 7266 CNRS-ULR, LIENSs – Université de La Rochelle, CNRS : UMR7266 – France</footnote>

<footnote n="2">² Littoral Environnement et Sociétés [La Rochelle] (LIENSs) – CNRS : UMR7266, Université de La Rochelle – Bâtiment Marie Curie Avenue Michel Crépeau 17 042 La Rochelle cx1 - Bâtiment ILE 2, rue Olympe de Gouges 17 000 La Rochelle, France</footnote>

<footnote n="3">³ LIENSs, CNRS, Université de la Rochelle – Université de La Rochelle - CNRS – France</footnote>
<figure><img src="image_155.png" /></figure>

Poster 39 – P39

Metabolomics approach to study the environmental impact and
residues of biocontrol products (BP)

Mélina Ramos <fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Marie-Virginie Salvia, Raviglione Delphine <fnref n="4" />, Mercè Llugany Ollé <fnref n="5" />, Esther Badosa <fnref n="2" />, Emilio Montesinos <fnref n="2" />, Cédric Bertrand<fnref n="1" />

Biopesticides or Biocontrol Products (BP) represents an interesting alternative to the conventional pesticides. However, there is a need of technical efficacy studies and ecotoxicological profile references. The PALVIP project (local Mediterranean crops' alternative protection) aims to fill that gap while evaluating new BP developed by small local businesses partners. To reach that goal, the BP selected in the project are studied according to their efficiency through field experimentations, their effect on plants and their environmental impact.

In a first stage the CRIOBE / University of Perpignan Via Domitia will contribute to the part of the project regarding the evaluation of BPs' environmental impact. To date, the half-life, t1/2, was often used to study the fate of pesticides in environmental matrices. However, this value doesn't give any information regarding the formation of by-products and the effect on biodiversity. Consequently, an innovative approach based on metabolomics (LC-MS), the Environmental Metabolic Footprinting (EMF), was recently developed in the lab.

On one hand, the EMF gives rise to a new integrative proxy, the resilience that corresponds to the time needed for the compound dissipation and its effects on the matrix (meta-metabolome). On the other hand, the EMF can be used in order to determine the preharvest interval (PHI) that corresponds to the time needed to have no residue (xenometabolome) difference between the treated sample and the control.

The degradation of the BP on vine leaves was monitored. Vines were treated against powdery mildew with 2 fungicides BPs. The vine leaves were sampled at different time points between 2 treatments in order to study the BP degradation (kinetics). The extraction steps were optimized and the extracts were analyzed using a UHPLC-HRMS instrument. For now, only the xenometabolome was studied and the preliminary results showed a degradation kinetics for the 2 fungicides BP with a PHI between 4 and 10 days. The endometabolome is currently analyzed. The same study will be performed for the 2019 field experiment (the project lasts 3 years (from 01/2018 to 12/2020) and sampling are done each year). Also, other matrices will be studied in the project: peach treated against brown rot, grape treated against grey mold and soil treated against weed development.

Mots-Clés: biocontrol compounds, environmental impact, LC, MS, metabolomics, resilience time

<footnote n="1">¹ CRIOBE USR3278, Université de Perpignan Via Domitia – Univ de Perpignan Via Domitia – France</footnote>

<footnote n="2">² Universitat de Girona [Girona] – Espagne</footnote>

<footnote n="3">³ Universitat Autònoma de Barcelona [Barcelona] – Espagne</footnote>

<footnote n="4">⁴ PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Bat T, Université de Perpignan,</footnote>

<footnote n="5">⁵ Universitat Autònoma de Barcelona (UAB) - Espagne</footnote>
<figure><img src="image_156.png" /></figure>

## Poster 40 – P40

# Analyse du métabolome d'une actinobactérie fixatrice d'azote :
Frankia

**Andrea Calhau**<fnref n="1" />, **Marjolaine Rey**<fnref n="1" />, **Hasna Boubakri**<fnref n="2" />, **Aude Herrera Belaroussi**<fnref n="3" />, **Anne-Emmanuelle Hay**<fnref n="1" />

*Frankia* est une actinobactérie saprophyte capable d'établir une symbiose racinaire avec près de 260 espèces de plantes dites actinorhiziennes, parmi lesquelles les Betulaceae, dont le genre *Alnus* (Benson *et al.*, 1993). 15% des entrées d'azote fixé biologiquement sur Terre proviennent de cette symbiose actinorhizienne. Cette interaction assure à ces plantes un avantage sélectif dans des écosystèmes où l'azote est limitant (e.g. moraines glaciaires, remblais miniers ...) et où elles constituent les premiers maillons d'une implantation pérenne d'un écosystème plus divers. Afin de mieux comprendre les interactions entre ces deux partenaires, une meilleure connaissance du métabolome de *Frankia* s'avère nécessaire. En effet, des approches *in-silico* ont permis de prédire une grande diversité de métabolites secondaires à partir du génôme de *Frankia* (Udwary *et al.*, 2011). Sous l'hypothèse que les métabolismes primaire et secondaire sont modifiés selon l'environnement de la bactérie, connaître le métabolome de *Frankia* en culture pure pourrait nous permettre d'interpréter les variations observées en conditions symbiotiques, dans le nodule. Cependant, malgré l'intérêt porté aux métabolites secondaires bioactifs produits par les actinobactéries (Solecka *et al.*, 2012), la métabolomique au sens propre est une approche encore peu utilisée chez les bactéries filamenteuses. L'étude présentée ici vise à mettre au point un protocole d'extraction à partir d'une culture pure de *Frankia* ACN14a (infective d'Alnus (Normand et Lalonde 1982), afin d'explorer les profils métaboliques obtenus par HPLC-FLD, UHPLC-QTOF et GC-QQQ. Pour commencer, nous nous sommes intéressés à l'effet temporel sur le métabolisme de la bactérie en optimisant le protocole sur deux cultures d'âge différent : 10 jours (fin de phase exponentielle) et 2 mois (phase stationnaire). Ce protocole a été testé sur cette même souche au cours d'une cinétique de croissance et permis d'observer des variations dans le métabolome en fonction de l'activité physiologique de la bactérie.

Mots-Clés: Métabolome, Frankia ACN14a, symbiose

<footnote n="1">¹ Laboratoire d'Écologie Microbienne - CESN - CNRS : UMR5557, Institut national de la recherche agronomique (INRA) :
UMR1418, Université Claude Bernard-Lyon I - UCBL (FRANCE) - France</footnote>

<footnote n="2">² Université Lyon 1 - CNRS : UMR5557 - Ecologie microbienne, Bâtiment Mendel, Villeurbanne cedex, France</footnote>

<footnote n="3">³ Laboratoire d'Ecologie Microbienne (LEM) - CNRS : UMR5557 - Villeurbanne, France</footnote>
<figure><img src="image_157.png" /></figure>

Poster 41 – P41

Metabolic foot-printing approach to study long term environmental
fate and impacts of biological and synthetic Biocidal products in
sediment

<u>Chandrashekhar Patil</u> <sup>1</sup>, <u>Amani Ben Jrad</u> <sup>1</sup>, <u>Hikmat Ghosson</u> <sup>1</sup>, <u>Raviglione Delphine</u> <sup>1</sup>, <u>Marie-Virginie Salvia</u> <sup>1</sup>, <u>Cédric Bertrand</u> <sup>1</sup>

1 PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Bat T, Université de Perpignan, 58 Avenue Paul Alduy, 66860 Perpignan Cedex, France – Univ de Perpignan Via Domitia – France

Mosquitoes can carry infectious diseases from person to person and from place to place. Presence and establishment of invasive mosquito species such as *Aedes aegypti* and *Aedes albopictus* are rapidly increasing in the European environment. The most efficient means to fight against mosquitos is the use of insecticides. However, most of the insecticides were removed from the market as they showed severe risks for human and animal health as well as for the ecosystem. The European Directive in 1998 led to the increasing use of biological insecticides such as cry proteins produced by the bacterium *Bacillus thuringiensis israelensis* (Bti) that kill mosquito larvae after being ingested. Considering the interest in Bti as more environmentally sustainable bioinsecticide, it is important to examine in detail environmental fate and impact of Bti. The available tool such as half-life, t1/2, does not consider the biodegradation and biotransformation phenomenon of complex formulations. To address this challenge, 'Environmental Metabolic Footprinting' (EMF), giving an idea of the resilience time was recently developed in the laboratory (Patil et al. 2016; Salvia et al, 2017) to evaluate the impact of synthetic, botanical and microbial insecticides on soil and sediment matrix respectively.

The project ‘EnvFate’ aims to employ an EMF approach, to dynamically characterize environmental markers of Bti pollution found among the sediment matrix meta-metabolome and to determine the resilience time upon exposure. Metabolome characterization will require to develop and optimize extraction and detection protocols using LC-MS platform. Emphasis will be placed on better standardisation, data interpretation and evaluation that will build confidence in the value of "omics technologies – this being essential to increase their (regulatory) use. Based on our preliminary results, we performed a year-long experiment to know the changes in the meta-metabolome and if resilience has reached after Bti and α-cypermethrin treatment in three different sediment matrices. We used climatic chambers to mimic natural conditions. These activities will advance our understanding of environmental risks associated with test insecticides, and pave the way for the development and adaptation of new environmental monitoring tools.

Mots-Clés: Natural products, biopesticides, Bti, metabolomics, environment, fate, LC, MS
<figure><img src="image_158.png" /></figure>

Poster 42 – P42

# Preliminary metabolomic approach on the microalgae Nitzchia palea exposed to herbicide diuron

Léa Gauthier¹, Nicolas Mazzella¹, Juliette Tison-Rosebery¹

¹Irstea-Bordeaux Unité EABX – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture
- IRSTEA (FRANCE) – France

Freshwater ecosystems are presently under a range of increasing pressures, some of which being directly due to anthropogenic activities such as agricultural practices or urban activities. Biofilms are communities of microorganisms (bacteria, algae, protozoa, fungi) and meiofaune integrated in an extracellular polysaccharide matrix fixed on immersed substrates. At the base of the trophic chain, biofilms play a key role in aquatic ecosystems. Due to their capacity to rapidly react to contamination, they are seen as efficient bioindicators and are increasingly used for water quality evaluation. Diatoms in particular dominate the algal biomass of biofilms and are routinely used in ecotoxicology to assess the impact of contaminations on freshwater ecosystems.
Traditional ecotoxicology approaches rely on quantitative concentration-response relationships highlighted on a batch of biological endpoints such as mortality, reproduction, growth and development, but all those approaches are materially and time expensive. We believe that metabolomics can enhance our knowledge of contaminant mode of actions, resulting in the identification of stress induced biomarkers.

The present study aims to highlight metabolome changes in the freshwater diatom *Nitzchia palea* exposed to an herbicide, the diuron (0.3 µg/L and 3 µg/L), during 4 hours. An extraction protocol as well as a non-targeted metabolomic approach using high resolution mass spectrometry (LC-TOF-MS) was developed in order to decipher the impact of contamination on a metabolic level.

Ongoing works will be dedicated to optimize both extraction and analysis methods to determine relevant biomarkers for risk assessment.

Mots-Clés: microalgae, diatom, environmental metabolomics, ecotoxicology
<figure><img src="image_159.png" /></figure>

Poster 43 – P43

# Etude de l'impact d'agents de contraste iodés sur deux organismes aquatiques modèles par des approches métabolomiques et métallomiques (projet ACTIONS)

**Gaelle Daniele**<sup>1</sup>, **Sandra Mounicou**<sup>2</sup>, **Carine Arnaudguilhem**<sup>2</sup>, **Alain Geffard**<sup>3</sup>, **Odile Dedourge-Geffard**<sup>3</sup>, **Anne Bado-Nilles**<sup>3</sup>, **Jean-Marc Porcher**<sup>3</sup>, **Emmanuelle Lebeau**<sup>1</sup>, **Aurélie Fildier**<sup>1</sup>, **Christelle Bonnefoy**<sup>1</sup>, **Emmanuelle Vulliet**<sup>1</sup>

La contamination des milieux aquatiques par les produits pharmaceutiques n'est plus à démontrer. Parmi ces composés, les agents de contraste iodés (ACI) sont utilisés depuis les années 50 en imagerie médicale pour augmenter artificiellement le contraste et ainsi mieux visualiser les organes, tissus, tumeurs,.... Ces composés sont présents ubiquitairement dans les eaux à des teneurs pouvant atteindre quelques µg/L voire mg/L. Bien que rapidement métabolisés et excrétés par l'organisme, les produits de contraste sont connus pour interférer avec la thyroïde, notamment par l'intermédiaire de l'iode qu'ils contiennent, et ainsi interagir avec les fonctions endocriniennes. De même, ces molécules peuvent induire des réactions immunitaires et inflammatoires ou encore une néphrotoxicité. Ainsi, une exposition en continu aux ACI pourrait provoquer des effets néfastes sur les organismes aquatiques, non seulement sur les poissons qui présentent un rôle prépondérant dans la structure et le fonctionnement des réseaux trophiques aquatiques, mais également sur les organismes filtreurs, dont le caractère filtreur en fait de bons bioaccumulateurs.

Le projet ACTIONS propose d'évaluer, sur la base d'expositions à des teneurs environnementales, l'impact de 2 ACI d'osmolalité différente régulièrement détectés dans les eaux, l'acide diatrizoïque et l'iohexol, sur deux espèces modèles de nos rivières : un mollusque bivalve, *Dreissena polymorpha*, et un poisson Téléostéen, *Gasterosteus aculeatus*.

Après l'étude de la répartition et de la spéciation de l'iode dans les différentes parties des organismes par imagerie élémentaire, la détermination de la bioaccumulation de l'iode et ses potentielles interactions avec les biomolécules seront menées par une approche métallomique combinant spectrométrie de masse élémentaire (ICP-MS) et moléculaire (ESI-MS/MS). En parallèle, l'étude des effets des ACI sur le métabolisme des organismes sera réalisée par une approche métabolomique en LC-HRMS. Cette approche favorisera une vue d'ensemble des voies de métabolisation des molécules tout en proposant des marqueurs pertinents d'exposition aux ACI.

Ce projet est financé par le PNREST Anses, 2018/1/222.

Mots-Clés: métabolomique, métallomique, espèces sentinelles, LC, HRMS, HPLC, ICP, MS, bioimagerie LA, ICP MS, environnement

<footnote n="1"><sup>1</sup> Institut des Sciences Analytiques – CNRS : UMR5280, Université Claude Bernard Lyon 1 – France</footnote>

<footnote n="2"><sup>2</sup> Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux – Université de Pau et des Pays de l'Adour, Centre National de la Recherche Scientifique : UMR5254 – France</footnote>

<footnote n="3"><sup>3</sup> Stress Environnementaux et BIOsurveillance des milieux aquatiques – Institut National de l'Environnement Industriel et des Risques : UMR-I 02, Université de Reims Champagne-Ardenne : UMR-I 02, Université Le Havre Normandie :</footnote>
<figure><img src="image_160.png" /></figure>

## Poster 44 – P44

# Etude métabolomique de la réponse au stress de lins déficients en lignanes

Kamar Hamade

Les lignanes sont des composés polyphénoliques largement distribués dans le monde végétal. Le lin, cultivé pour son huile ou sa fibre, renferme de grandes quantités d'un lignane de type dibenzylbutane : le sécoisolaricirésinol, qui est stocké dans le tégument de la graine sous forme diglucosylée, dans une macromolécule contenant de nombreux composés phénoliques assemblés par des liaisons esters (Ford et al., 2001). De nombreuses études s'intéressent aux effets bénéfiques sur la santé des lignanes et du sécoisolaricirésinol en particulier (Gutte et al., 2015), du fait de propriétés antioxydantes marquées, néanmoins le rôle de ces molécules in planta n'est pas clairement élucidé ; un rôle potentiel dans les mécanismes de réponse aux stress est toutefois proposé (Davin et al., 2008).

Une enzyme clé de la biosynthèse du sécoisolaricirésinol est la PLR (Pinorésinol Laricirésinol Réductase). Des lins RNAi PLR ont montré l'absence d'accumulation de SDG dans les graines (Renouard et al., 2014). Il est donc envisagé dans cette étude métabolomique de comparer le contenu en métabolites (semi-)polaires de lins accumulant du SDG ou non, en condition de stress osmotique ou en condition témoin. Les premières analyses réalisées en RMN et en LC-MS sur les extraits racinaires montrent une différence du profil métabolique due à la lignée de lin utilisée (avec ou sans le transgène RNAi PLR) ainsi que des variations métaboliques induites par l'application du stress osmotique.

**Mots-Clés:** Métabolomique, lins, lignanes, PLR, stress osmotique.
<figure><img src="image_161.png" /></figure>

## Poster 45 – P45

# Leaf specialized metabolome of Quercus pubescens exposed to amplified drought

Amélie Saunier <fnref n="1" />,<fnref n="2" />, Stéphane Greff<fnref n="2" />, Catherine Fernandez <fnref n="2" />, Elena Ormeno Lafuente <fnref n="2" />

The intensification of summer drought expected with climate change in the Mediterranean region can induce metabolism modifications in plants to help them cope with such conditions. In this experiment, we studied the specialized metabolome of *Quercus pubescens* leaves under natural (ND) and amplified drought (AD). An *in situ* experimental site, equipped with a rainfall exclusion device, allowed reduction of natural rainfall by 30% over the tree canopy. Leaves of ND and AD plots were collected in spring, summer and fall during 3 years, corresponding to the 2nd, 3rd and 4th years of drought application. We used a targeted approach to focus on phenolic metabolites and an untargeted approach to study a broader metabolome variation according to drought. Metabolic profiles (targeted approach) showed that the concentrations of quercetins, catechins and myricitrin were mostly decreased with drought stress, opposite to the concentrations of kaempferols that increased. Multivariate analysis of *Quercus* fingerprints (untargeted approach) demonstrated that metabolic contents remained almost unchanged under AD. Nevertheless, seven metabolites were highlighted as drought biomarkers using an univariate analysis (Venn diagrams). These biomarkers, still under study, likely belong to the phenylpropanoid pathway with one corresponding to kaempferol pentoside. Targeted and untargeted approaches permitted to demonstrate a time lag in leaf phenology when trees were exposed to drought with a modification of the phenylpropanoid pathway. Quercetins, catechins and myricitrin, specific to summer leaves were down-regulated to favor the biosynthesis of kaempferol, specific to fall leaves. Further studies would be necessary to understand how this time lag phenology may impact ecosystem functioning.

Mots-Clés: Quercus pubescens, amplified drought, Mediterranean forest, flavonoids, time lag phenology

<footnote n="1">¹ Department of Environmental and Biological Sciences – Finlande</footnote>

<footnote n="2">² IMBE – Aix Marseille Univ, Univ Avignon, CNRS, IRD, IMBE, Marseille, France – France</footnote>
<figure><img src="image_162.png" /></figure>

Poster 46 – P46

Molecular networking on wild "Tali"
(Erythrophleum, Fabaceae) from Central tropical Africa

Nausicaa Noret <fnref n="1" />, Cécile Vanhaverbeke <fnref n="2" />, Cédric Delporte <fnref n="3" />, Caroline Stévigny, Florence Mondeguer <fnref n="4" />, David Touboul <fnref n="5" />,
Florence Souard <fnref n="6" />

Plant secondary (or specialized) metabolites (SM) are essential for plants which rely on specific set of molecules to
handle basic life functions like reproduction (e.g. pollinator attraction) or defense (e.g. herbivore deterrence). As
metabolites are the result of a complex network of gene expression, protein expression and interactions and other
regulatory processes, they are therefore closer to the phenotype than transcriptome or proteome. Untargeted
metabolomics allows to investigate the metabolic phenotypes of plants including the diversity of SM. As SM are
produced by specific biosynthetic pathways, most metabolites have characteristic structural features and often
show a clear phylogenetic signature.

The tropical woody genus *Erythrophleum* (Fabaceae, Caesalpinioideae) contains original cassaine diterpenes, a subfamily of the cassane diterpenes restricted to various Fabaceae genera. The cassane skeleton is structurally characterized as a tricyclic diterpene with a substitution by ethyl group in C-13 position and a methyl group at C-14 position. Cassaine diterpenes are known for their non-steroidal inhibition of Na+/K+-ATPase in a way similar to that of the digitalis glycosides. In addition to this cardiotonic effect, the traditional use of *Erythrophleum* is based on emetic, antimalarial, antibacterial and anti-inflammatory properties.

In this study, we used two closely related species of *Erythrophleum*, *E. suaveolens* and *E. ivorense*, to investigate the metabolic diversity of their cassaine diterpenes. After a metabolomic exploration by LC-HRMS and multi-block correlation of neutral genetic diversity with metabolome, we have developed a molecular networking dereplication pipeline based on MS/MS focusing on the cassane diterpenes family. For molecular network construction the user-friendly Metgem software has been used. Principal results of dereplication will be presented in the poster and interpretations of the metabolome will be proposed for the different genotypes.

Mots-Clés: Erythrophleum, ecometabolomics, plantomics, environmental metabolomics : natural variation

<footnote n="1">¹ Laboratoire d'écologie végétale et de biogéochimie – Université Libre de Bruxelles Bâtiment BC - 5e niveau - aile C
Campus de la Plaine - CP244 Boulevard du Triomphe 1050 Bruxelles, Belgique</footnote>

<footnote n="2">² Département de Pharmacochimie Moléculaire, CNRS : UMR5063 – Université Grenoble Alpes – France</footnote>

<footnote n="3">³ RD3 - Pharmacognosy, Bioanalysis and Drug Discovery Unit Analytical Platform of the Faculty of Pharmacy, ULB -
Belgique</footnote>

<footnote n="4">⁴ Laboratoire Phycotoxines – Institut Français de Recherche pour l’Exploitation de la Mer IFREMER – Nantes, France</footnote>

<footnote n="5">⁵ Institut de Chimie des Substances Naturelles (CNRS-ICSN) – CNRS UPR2301, Univ. Paris-Sud, Université Paris-Saclay –,
Avenue de la Terrasse, 91198 Gif-sur-Yvette, France, France</footnote>

<footnote n="6">6 Département de Pharmacochimie Moléculaire, CNRS UMR5063 – Université Grenoble – France</footnote>
<figure><img src="image_163.png" /></figure>

Poster 47 – P47

Combining metabolomics and molecular networking as a tool to
target and identify phenolic compounds in flax leaves

Elvis Otogo Nnang<fnref n="1" />, Sébastien Rigaud <fnref n="2" />, Job Tchoumtchoua <fnref n="1" />, David Mathiron <fnref n="2" />, Emmanuel Petit <fnref n="1" />, François Mesnard <fnref n="1" />,
Roland Molinié <fnref n="1" />, Anthony Quéro <fnref n="1" />

Flax (*Linum usitatissimum*) is a crop that is widely cultivated as a source of fiber or oil1. It belongs to the genus *Linum* and family *Linaceae2*. The components from flax have diverse uses. Although flaxseed chemical composition is deeply studied, little is known about the phytochemistry of the flax leaves. This study aims at identifying the secondary metabolites contained in flax leaves using a metabolomics approach and the molecular networking as a dereplication strategy3. This computer-based approach is emerging as a promising method to visualize and organize tandem MS/MS data sets and to automate database searches for secondary metabolite identification within complex mixtures. This strategy led to the rapid detection of more than twenty flavonoids and lignans. After characterization, the metabolite profiling of 14 flax cultivars was performed by LC-MS. PCA analysis showed a clear discrimination between leaves of spring and winter flax. These results suggest the implication of phenolic compounds in flax cold tolerance.

Mots-Clés: Flax (Linum usitatissimum), metabolomics, molecular networking, PCA analysis

<footnote n="1">1 EA3900 BIOPI - Biologie des Plantes et Innovation, UFR de Pharmacie - Université de Picardie Jules Verne - France</footnote>

<footnote n="2">2 Plateforme Analytique – Université de Picardie Jules Verne, Chemin du Thil, 80000 Amiens – France</footnote>
<figure><img src="image_164.png" /></figure>

Poster 48 – P48

LC-MS and NMR metabolomics study of a tomato (Solanum lycopersicum L.) mutant affected in the fruit pericarp development

Constance Musseau <fnref n="1" />, Stéphane Bernillon <fnref n="1" />,<fnref n="2" />, Catherine Deborde <fnref n="1" />,<fnref n="2" />, Daniel Jacob <fnref n="1" />,<fnref n="2" />, Joana Jorly <fnref n="1" />, Stéphanie Gadin <fnref n="1" />, Annick Moing <fnref n="1" />,<fnref n="2" />, Frédéric Gévaudant <fnref n="1" />, Lucie Fernandez <fnref n="1" />

Metabolomics is widely used as a tool in plant genomic studies to decipher gene functions [1] and has been applied to the model species tomato to explore development and metabolism of fleshy fruit [2]. To improve metabolome coverage, the combination of LC-MS and NMR analysis has been shown to be very successful [3]. The aim of this study was to characterised the metabolome of tomato plant mutants for a gene coding for a large GTPase involved in the regulation of the fruit tissue morphology and whose function has not yet been described in plants. Two mutants for this gene were generated from two independent CRISPR-Cas9 transformation events. Mutant and control tomato plants were grown in greenhouse. Tomato pericarp samples were harvested at 4 fruit growth stages, i.e. 15, 20, 25 days post anthesis (DPA) and red ripe. They were analysed with both NMR and LCMS methodologies. For NMR analysis, 1H spectra were acquired with a 500 MHz Avance III NMR spectrometer (Bruker, France) and then processed using the software NMРProcFlow [4]. For LCMS analysis, data were acquired with a UHPLC-LTQ-Orbitrap-MS (Thermo Scientific, Germany) in both positive and negative ESI mode with a C18 separation. Acquisitions were processed using the software MS Dial [5]. 2D NMR and MS<fnref n="2" /> data were also acquired to enable annotation or identification of metabolites.

Processed data were explored with multivariate analysis that showed distinct metabolic trajectories for control and mutant pericarp tissues suggesting a deep perturbation of the whole metabolism in the mutants. Major modifications occurred at 25 DPA that correlated with pericarp cellular alterations. The identified metabolites belong to various classes of primary and specialised metabolisms such as sugars, organic and amino acids, phenolic compounds and glycoalkaloids. Besides, both processing softwares enabled visualisation and were interactive, making the data processing effortless while allowing the analyst to control and possibly correct automatic output.

Acknowledgements: MetaboHUB (ANR-11-INBS-0010) References:

Mots-Clés: LC, MS, NMR, tomato, processing

<footnote n="1">¹ UMR1332 Biologie du Fruit et Pathologie – Institut national de la recherche agronomique (INRA), Université de Bordeaux – France</footnote>

<footnote n="2">² Plateforme Métabolome de Bordeaux - MetaboHUB – (INRA), Université de Bordeaux (Bordeaux, France)</footnote>
<figure><img src="image_165.png" /></figure>

Poster 49 – P49

# Etude métabolomique des interactions chimiques dans le dépérissement de la lavande

Bénédicte Héral<fnref n="1" />, Florence Nicolé <fnref n="2" />, Xavier Fernandez <fnref n="1" />, Thomas Michel1

Les cultures de lavande (Lavandula angustifolia) et de lavandin (Lavandula x intermedia) sont, depuis une dizaine d'années, affectées par le phytoplasme du Stolbur (Candidatus phytoplasma solani), agent pathogène transmis par la cicadelle (Hyalesthes obsoletus) (FranceAgriMer, 2013). Cet insecte se nourrit de la sève de la lavande transmettant la bactérie de plant infecté en plant sain (Gasparich, 2010). Le déperissement de la lavande a des conséquences économiques importantes sur la filière lavandicole et les moyens de lutte sont actuellement inexistant ou restreints. Par ailleurs, les connaissances autour des interactions chimiques entre la cicadelle, le phytoplasme et la lavande sont encore limitées, c'est pourquoi il est important d'étudier les composés organiques non volatiles qui pourraient avoir une influence dans cette interaction tripartite.

Une approche métabolomique globale a été utilisée afin de déterminer les bio-marqueurs spécifiques :

- des variétés (Maillette et 7713 pour la lavande, et Grosso et Abrial pour le lavandin)

- de l'état symptomatique des plants (sain ou infecté).

Une campagne d'échantillonnage en champ a été mené sur le plateau de Sault (Vaucluse, France) en 2017, au cours de laquelle des échantillons de feuille et d'inflorescence ont été collectés. Les extraits eau-éthanol (50:50, v/v), obtenus par extraction assistée par ultra-sons, ont été analysés par UPLC-HRMS (XevoG2 QTOF, Waters). Les données ont par la suite été retraité sur la plateforme Workflow4Metabolomics 3.0. Le retraitement a nécessité un prétraitement comprenant la détection des ions (CentWave), l'alignement (PeakDensity), la correction des biais expérimentaux, tels que les dérives analytiques intra et inter séquence (loess). La qualité du jeu de données a ensuite été évalué grâce à des statistiques descriptifs (ACP), puis celui-ci a été réduit pour réaliser les analyses discriminantes (PLS-DA). Les premiers résultats obtenus sur les extraits de feuille seront présentés ici.

Mots-Clés: Lavande, phytoplasme, dépérissement, métabolomique

<footnote n="1">1 Institut de Chimie de Nice – CNRS UMR 7272, Université de Nice-Sophia Antipolis – France</footnote>

<footnote n="2">² Laboratoire BVpam – UMR 7272, 23 rue du Dr Michelon 42023 Saint Etienne Cedex 2 : Université de St Etienne – France</footnote>
<figure><img src="image_166.png" /></figure>

Poster 50 – P50

Metabolite profiling at the graft interface of grapevine in response to
grafting

Duyên Prodhomme <fnref n="1" />, Josep Valls Fonayet <fnref n="2" />, Cyril Hévin <fnref n="1" />, Céline Franc <fnref n="2" />, Ghislaine Hilbert <fnref n="1" />, Gilles De Revel <fnref n="2" />, Tristan Richard <fnref n="2" />, Nathalie Ollat <fnref n="1" />, Sarah Jane Cookson <fnref n="1" />

Grafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruit and vegetables. In viticulture, grafting is used to facilitate grapevine cultivation in soils infected with the Phylloxera, a soil-dwelling insect pest introduced to Europe from America at the end of the 19th century. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine one month after grafting. Table-top grafting was done on over-wintering stems of grapevine and the graft interface tissues were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses.

A total of 54 compounds from primary and secondary metabolism and the activity of two enzymes were measured using different metabolomic tools. The results revealed that graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Eight amino acids and six stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues. This study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes.

Mots-Clés: Grafting, grapevine, wood, stilbenes, flavanols, sugars, amino acids, HPLC, LC, MS/MS

<footnote n="1">¹ Ecophysiologie et Génomique Fonctionnelle de la Vigne – Institut National de la Recherche Agronomique : UMR1287, Université de Bordeaux, Institut des Sciences de la Vigne et du Vin (ISVV), Ecole Nationale Supérieure des Sciences Agronomiques de Bordeaux-Aquitaine – France</footnote>

<footnote n="2">² Unité de Recherche Œnologie USC 1366, EA 4577 – Institut National de la Recherche Agronomique INRA, Université de Bordeaux, Institut des Sciences de la Vigne et du Vin (ISVV) – France</footnote>
<figure><img src="image_167.png" /></figure>

## Poster 51 – P51

# Isoprostanoids quantitative profiling of macroalgae submitted to copper stress

Claire Vigor<fnref n="1" />, Guillaume Reversat<fnref n="1" />, Amandine Rocher<fnref n="1" />, Camille Oger<fnref n="1" />, Jean Marie Galano<fnref n="1" />, Joseph Vercauteren, Thierry Durand<fnref n="1" />, Thierry Tonon<fnref n="2" />, Catherine Leblanc<fnref n="3" />, Philippe Potin<fnref n="3" />

Macroalgae are living organisms inhabiting a broad range of particularly hostile aquatic environments that can cause them to undergo oxidative stress (OS). Considering that isoprostanoids derivatives (phytoprostanes, phytofuranes, isoprostanes) constitute the most relevant oxidative stress biomarkers, we are interested in this work to their qualitative and quantitative profile in six macroalgae submitted to a heavy metal exposure (copper). Thus, 9 phytoprostanes, 3 phytofuranes (observed for the first time in such matrices), and 3 isoprostanes are quantified through a new micro-LC-MS/MS method. The isoprostanoids profiles vary greatly among all the samples, the ent-16(RS)-9-epi-ST-14-10-PhytoF and the 5(RS)-5-F2t-IsoP being the major compounds for most of the macroalgae studied. Concentrations of these metabolites, at basal conditions range from 2.5 to 342.6 ng/g of algal tissue. After 24h exposure of macroalgae to cupric action, as expected, in response to copper-induced stress, an increase of lipid oxidation biomarkers is observed in the majority of cases, corroborating the original hypothesis. Only *L. digitata* seems to derogate from the rule with a reduction in the content of ent-9-L1-PhytoP.

Mots-Clés: PUFAs, oxidative stress, biomarkers, micro, LC, MS/MS

<footnote n="1">¹ Institut des Biomolécules Max Mousseron – CNRS : UMR5247 – France</footnote>

<footnote n="2">² Centre for Novel Agricultural Products, Department of Biologie, University of York – Heslington, York, YO10 5DD, Royaume-Uni</footnote>

<footnote n="3">³ Station biologique de Roscoff [Roscoff] (SBR) – Université Pierre et Marie Curie (UPMC) - Paris VI, CNRS : FR8227 – Place Georges Teissier - BP 74 29682 ROSCOFF CEDEX, France</footnote>
<figure><img src="image_168.png" /></figure>

Poster 52 – P52

EXPERIMENTAL STUDY OF THE
EFFECTS OF OXIDATIVE STRESS ON THE AQUATIC FLORA AND
ENVIRONMENT

Khaoula Menaiaia<fnref n="1" />

In recent decades, the increase and diversity of human activities have been accompanied by dispersion into the environment of sometimes considerable quantities of xenobiotics harmful to human health. In many countries, the accumulation of pollutants in ecosystems has led, over the years, to deteriorating environmental quality, forest decline, and declining agricultural productivity.

The accumulation in the soil of heavy metals, which are inherently non-degradable and potentially toxic, increases the risk of exposure of living beings: microorganisms, plants, animals, and humans, which is at the end of the food chain.

Our research is about the effect of zinc oxide on the behavior of *Phragmites australis*. For this purpose, samples of *Phragmites australis* were run at three concentrations based on ZnO (3, 6, 12 nmol/ml) for 7, 14 and 21 days. In order to evaluate the influence of the various concentrations of this nanometric molecule, metabolic and enzymatic parameters characteristic of the oxidative stress were measured.

Regarding the metabolites (proline, total sugars) and the enzymatic biomarker (CAT), no significant difference was observed, which reflects the tolerance of this species to zinc oxide (ZnO).

Mots-Clés: Phragmites australis, ZnO, Enzymatic activity, Oxidative stress.

<footnote n="1">¹Department of Biology, Faculty of Nature and Life Sciences, University of Mohamed Cherif Messaadia PO Box 1553. 41000, Souk-Ahras, Algeria – Algérie</footnote>
<figure><img src="image_169.png" /></figure>

## Poster 53 – P53

# Optimisation of 1D 1H-NMR profiling protocol for plant samples: from extract preparation, standardization, automation to spectra processing.

Catherine Deborde <fnref n="1" />,<fnref n="2" />, Jean-Xavier Fontaine <fnref n="3" />, Daniel Jacob <fnref n="1" />,<fnref n="4" />, Adolfo Botana ⁵, François Mesnard <fnref n="3" />, Annick Moing <fnref n="1" />,<fnref n="2" />, Roland Molinié <fnref n="3" />

1H NMR-based metabolomic profiling has a range of applications in plant sciences. In order to minimize uncontrolled variability in plant sample preparation before and during NMR metabolomic profiling, we provide advice and show examples for taking into account sample composition, including its specificity in terms of pH and paramagnetic ion concentrations, and NMR spectrometer performances.

A pH extract adjustment step reduces variations of pH-sensitive chemical shifts and addition of paramagnetic ion chelatant, like EDTA, improves citrate and malate resonance detection.

An automation of spectrometer preparation routine standardization before NMR acquisition campaign was implemented and tested on three plant sample sets (extracts of durum wheat, *Arabidopsis* and flax). We performed 1H-NMR spectroscopy in three different sites on the wheat sample set utilizing instruments from two manufacturers with different probes and magnetic field strengths. The three collections of spectra were processed separately with the NMRProcFlow web tool using intelligent bucketing, and the resulting buckets were subjected to multivariate statistical analysis. The comparability of large- (*Arabidopsis*) and medium-size (flax) datasets measured at 600 MHz and from the wheat sample set recorded at the three sites (400, 500 and 600 MHz) was exceptionally good in terms of spectral quality. The coefficient of variation of the full width at half maximum (FWHM) and the signal-to-noise ratio (S/N) of two selected peaks was comprised between 5 and 10% depending on the size of sample set and the spectrometer field.

A collection of 22 samples of wheat spikelet extracts, used as a proof of concept, showed that the data collected at the three sites on instruments of different field strengths and manufacturers yielded the same discrimination pattern of the biological groups.

We showed that standardization or automation of several steps from extract preparation to data reduction improved data quality for small to large collections of plant samples of different origins.

**Funding:** MetaboHUB (ANR-11-INBS-0010), CaDON (ANR-15-CE21-0001), VERTICIFRE (ANRT 2013/1259) and VERTILIN (Regional Council of Picardie).

**Mots-Clés:** Metabolomics, NMR acquisition, NMR spectra processing, Sample preparation, Plant

<footnote n="1">¹ Plateforme Métabolome Bordeaux, MetaboHUB, CGFB INRA, Université de Bordeaux, France</footnote>

<footnote n="2">² Biologie du Fruit et Pathologie – Institut National de la Recherche Agronomique - INRA : UMR1332, Université de Bordeaux : UMR1332, Université de Bordeaux : UMR1332 – France</footnote>

<footnote n="3">³ Laboratoire de Biologie des Plantes et Innovation – Université de Picardie Jules Verne : EA3900 – France</footnote>

<footnote n="4">⁴ JEOL-UK – Royaume-Uni</footnote>
<figure><img src="image_170.png" /></figure>

## Poster 54 – P54

# Utilisation des approches de métabolomique pour l'étude des interactions plantes-microorganismes

Virginie Puech Pages<fnref n="1" />, Sylvie Fournier<fnref n="1" />, Bernard Dumas<fnref n="1" />

L'environnement naturel des plantes est caractérisé par un microbiote extrêmement diversifié comportant des microorganismes bénéfiques pour leur développement, comme les bactéries fixatrices d'azote (bactéries en symbiose avec les légumineuses) et les champignons mycorhiziens (symbiose plante-champignon très ancienne et très répandue), ou pouvant provoquer des maladies (bactéries, champignons, oomycètes....). Dans ce contexte, les métabolites végétaux ou microbiens sont des acteurs essentiels, favorisant la mise en place puis la régulation des interactions symbiotiques et contribuant à la reconnaissance, et au rejet, des microorganismes pathogènes. Au Laboratoire de Recherche en Sciences végétales (LRSV, Castanet Tolosan) plusieurs équipes étudient le métabolome dans les interactions plantes microorganismes en tirant profit des avancées dans les approches de spectrométrie de masse (métabolomique ciblée et globale) qui sont développées sur la plateforme *MetaToul* " Métabolites végétaux " du LRSV (https://www.lrsv.upstlse.fr/metatoul/).

Une illustration de nos travaux sera faite avec les métabolites impliqués dans le dialogue moléculaire intervenant i) dans la mise en place de la symbiose mycorhizienne, avec les molécules d'origine végétale identifiées (strigolactones (Gomez-Roldan et al. 2008), blumenols (en cours), les signaux d'origine fongiques (lipochitooligosaccharides (Maillet et al. 2011), chitooligosaccharides (Genre et al. 2013), karrikines et hormones (en cours)) ii) dans la régulation de la symbiose mycorhizienne, avec des approches en métabolomique globales (Laparre et al., 2014 et travaux en cours).

Mots-Clés: champignons, symbiose, pathogènes, interactions plantes microorganismes

<footnote n="1">¹UMR5546, LRSV - CNRS: UMR5546, Université Paul Sabatier-Toulouse III - UPS - France</footnote>
<figure><img src="image_171.png" /></figure>

## Poster 55 – P55

# Comprehensive computational approaches to prioritize high-value natural products isolation from a chemodiverse collection.

Arnaud Gaudry¹Δ, Luis Quirós¹Δ, Adriano Rutz¹, Miwa Dounoue¹, Marcel Kaiser ², Laurence Marcourt¹, Bruno Davide³, Emerson Ferreira Queiroz¹, Pierre-Marie Allard¹* and Jean-Luc Wolfender¹*

1 Phytochimie et Produits Naturels Bioactifs, Section des Sciences Pharmaceutiques, Université de Genève, CH-1211 Genève 4, Switzerland.

2 Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland; University of Basel, Petersplatz 1, 4003 Basel, Switzerland.

3 Green Mission Pierre Fabre, Institut de Recherche Pierre Fabre, 3, Avenue Hubert Curien, BP 13562, 31562 Toulouse, France.

Nature is an inexhaustible source of chemical diversity and bioactive molecules. Given the complexity of natural extracts matrices, identification of novel natural products potentially bioactive continues as the hardest bottleneck in phytochemistry and pharmacognosy nowadays, and recurrent isolation of known compounds discourage further studies [1].

In this work, we report the dereplication of a large chemodiverse library composed by 1600 plants extracts (156 families, 533 genera, 783 species) by means of high resolution mass spectrometry profiling and multi-informative MS² spectral organization (Molecular Networks, MN) [2]. Integration of MS¹ identification, MS² in silico annotations [3] and taxonomical consistency of the annotations biosources [4] and bioactivity results mapping (3 different trypanosomatids and cytotoxicity assays), brings out potentially new bioactive scaffolds within the extracts.

We centered the prioritization from two perspectives: **Bioactive NP discovery**, approach based on the bioactivity mapping information from which we were able to highlight in the MN features and molecular families specific to active extracts. **Structure-focused discovery**, approach based on structure novelty considering combinations several criteria: MS¹ and MS² in silico annotations, annotation's taxonomical consistency, cluster's specificity at the taxa level and literature regarding the highlight species.

Using the bioactivity visualization, we were able to spot Melochia sp. quinoline alkaloids derivatives such as Waltherione G, a known trypanocidal compound, as a proof of concept. Selected species were subjected to a straightforward one-step isolation procedure using a gradient transfer combined with a dry-load injection of the sample at a semi-preparative HPLC scale [5]. This approach allowed the targeted isolation of a series of 14 original ß-agarofuran alkaloid derivatives (structural approach), which were fully characterized by 2D NMR and HRMS.

Our workflow is capable to spot novel natural products from a large and highly chemodiverse collection of plants, which are then efficiently isolated in the required amount for further bioactive analysis without extensive and time consuming purification steps.
<figure><img src="image_172.png" /></figure>

**Communication Posters**

**Session Développement Analytique**

<figure><img src="image_173.png" /></figure>

RÉSEAU FRANCOPHONE DE MÉTABOLOMIQUE ET FLUXOMIQUE
<figure><img src="image_174.png" /></figure>

## Poster 56 – P56

# Usefulness of retention time prediction for plant food bioactives

Dorrain Low<fnref n="1" />, Pierre Micheau<fnref n="1" />, Jan Stanstrup³, Claudine Manach<fnref n="1" />

Plant food bioactives and their metabolites are chemically diverse, and receive widespread interest for their protective health effects. However, the large chemical space of plant food bioactives and their host/microbial metabolites makes their identification in untargeted metabolomics profiles a challenging feat. Spectral libraries are incomplete for these compounds and standards are often costly or not commercially available. Retention time (RT) is valuable for assisting the certainty of identification of unknown features to MSI levels I and II. It also helps to narrow the number of hypotheses within an observed RT window to a manageable number of compounds to purchase or synthesize for confirmation. In the framework of the COST Action POSITIVe (https://www6.inra.fr/cost-positive, we evaluated the usefulness of PredRet (http://predret.org), an open access RT database, to predict retention times of plant food bioactives and their metabolites in a multi-laboratory test of 18 laboratories and 24 reversed-phase LC-MS or LC-PDA Chromatographic Systems (CS). Participants shared datasets of RT in their own CS for 29 to 104 compounds, covering a total of 471 chemically diverse chemicals, including highly polar to lipophilic aglycones, glycosides, as well as glucuronidated and sulfated metabolites and gut microbial metabolites of flavonoids, alkaloids, coumarins, phenolic acids, and others. Depending on its compatibility with other CSs (conditions and number of common compounds), every platform obtained predicted RTs for 67 to 667 compounds that were not yet analysed in their conditions. The predictions were very accurate, with a median prediction error ranging from 0.03 to 0.76 min, depending on the systems. Such level of prediction allowed distinguishing isomeric compounds. It also provided information in all CSs for rare standards. In conclusion, prediction of retention time with PredRet has proven very efficient and useful to facilitate annotation of plant food bioactives in metabolomics studies.

Mots-Clés: Plant food bioactives, Retention time, Prediction, PredRet, Multiplatform test

<footnote n="1">¹ Unité de Nutrition Humaine – Institut national de la recherche agronomique (INRA) : UMR1019, Université Clemont Auvergne – France</footnote>

<footnote n="2">² University of Copenhagen – Danemark</footnote>
<figure><img src="image_175.png" /></figure>

## Poster 57 – P57

# Fluxomic analysis of biomass production through a combination of analytical tools.

Arnaud Vigneron<fnref n="1" />, Patrick Jame<fnref n="2" />, Erik Bonjour<fnref n="2" />, Xavier Saupin<fnref n="2" />, Himaïji Chevrotee<fnref n="1" />

Biomass production set the flux of the main metabolic paths in proliferating cells. Precise knowledge of biomass production and nutritive distribution could thus highly constrain metabolic reconstruction. For these reasons, we developed new methods to determine how the atomic species of a nutrient distribute into biomass of cancer cells by using a combination of several analytical tools such as Total Organic Carbon analysis, Elementary analysis, Ionic Exchange chromatography coupled to Isotopic Ratio Mass Spectrometry (IRMS), and IRMS of macromolecular entities. Complete flux map can be generated from these analyses, tracing the fate of a particular nutrient into the cell biomass.

Mots-Clés: IRMS Cancer cell Metabolic Reconstruction

<footnote n="1">¹ Centre de Recherche en Cancérologie de Lyon – Centre Léon Bérard [Lyon], Université Claude Bernard Lyon 1, Institut National de la Santé et de la Recherche Médicale : U1052, Centre National de la Recherche Scientifique : UMR5286 – France</footnote>

<footnote n="2">² Institut des Sciences Analytiques – Centre National de la Recherche Scientifique : UMR5280, Université Claude Bernard Lyon 1, École Normale Supérieure - Lyon : UMR5280 – France</footnote>
<figure><img src="image_176.png" /></figure>

## Poster 58 – P58

# Comparaison inter-laboratoires de profils d'oxylipines par lipidomique ciblée dans le cadre du projet OXYGENATE

Céline Dalle ¹, Malwina Mainka ², Jessica Dalloux-Chioccioli ³, Annika Ostermann ⁴, Michael Rothe ⁵, John W. Newman ⁶, Justine Bertrand-Michel ³, Cécile Gladine ¹, Nils H. Schebb ²,⁴

1. Unité de Nutrition Humaine - Clermont Auvergne - Université Clermont Auvergne : UMR1019, INRA

2. University of Wuppertal - Allemagne

3. Plateforme MetaToul Lipidomique, I2MC - Centre de Recherche Inserm : UMR1048 - France

4. Institute for Food Toxicology, University of Veterinary Medicine Hannover - Allemagne

5. Lipidomix - Allemagne

6. USDA-ARS Western Human Nutrition Research Unit - États-Unis

Actuellement 20 à 25% de la population adulte mondiale présente un syndrome cardiométabolique. Ce syndrome est un exemple de maladie multifactorielle en lien avec l'alimentation et se caractérise par des troubles cardiovasculaires, métaboliques et inflammatoires. Ces troubles amènent au développement du diabète de type II et des maladies cardiovasculaires. Cependant le diagnostic de ce syndrome n'est pas satisfaisant car pas assez intégratif et précoce pour permettre une prise en charge nutritionnelle.

Les oxylipines, métabolites issus de l'oxygénation des acides gras polyinsaturés via différentes voies de biosynthèse, sont connues pour être des médiateurs lipidiques impliqués dans la régulation de processus biologiques liés au syndrome cardiométabolique et dont la biosynthèse est modulée par le statut cardiométabolique et aussi par l'alimentation. D'où l'hypothèse qu'un profilage complet des oxylipines pourrait révéler des perturbations précoces du statut cardiométabolique et pourrait être un nouvel outil d'évaluation de l'efficacité des préventions et interventions nutritionnelles.

Le projet OXYGENATE a pour but d'identifier et valider des oxylipines discriminant le statut cardiométabolique et la qualité de l'alimentation. Pour cela, il est nécessaire d'avoir une méthode fiable de profilage quantitatif des oxylipines permettant d'obtenir des profils comparables entre différents laboratoires. Une comparaison inter-laboratoires de notre méthode a été réalisée afin d'estimer les variabilités analytiques et d'identifier les oxylipines critiques. Cinq laboratoires ont préparé (en triplicat et sur 2 jours différents) et analysé 7 plasmas présentant des profils d'oxylipines très contrastés. Les variabilités intra- et inter-jour ainsi que la part de variabilité attribuable à la préparation et à l'appareillage sont évalués.

Au-delà de l'intérêt pour le projet, cette comparaison inter-laboratoires unique permettra d'harmoniser les profils d'oxylipines réalisés par l'ensemble de la communauté scientifique.

Mots-Clés: lipidomique ciblée, comparaison inter, laboratoires, oxylipines, syndrome cardiométabolique
<figure><img src="image_177.png" /></figure>

## Poster 59 – P59

### Have a break, Have a Robot!

**Julia Soullier**<sup>1</sup>, **Anthony Tournadre**<sup>1</sup>, **Pauline Le Faouder**<sup>1</sup>, **Amandine Hueber**<sup>1</sup>, **Jessica Dalloux-Chiccioli**<sup>1</sup>, **Anaelle Durbec**<sup>1</sup>, **Justine Bertrand-Michel**<sup>1</sup>, **Aurélie Batut**<sup>1</sup>

<sup>1</sup>MetaToul-Lipidomic Core Facility – Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Centre de Recherche Inserm – France

Lipids are ubiquitous biomolecules essential to all life, found in every cellular type, ranging from the human body and vegetal organisms, down to bacteria. They have many different functions in cell structuration, energy storage and signalling so they are natural biomarkers for different diseases like cancer, cardiovascular disease, neurodegenerative disease, lung disease, their study and quantification are then crucial. Mass spectrometry (MS) coupled with liquid chromatography or gas chromatography is mainly used for global and specific analysis of lipids. But before their analysis, there is an important and time consuming step of sample preparation. Due to their amphiphilic properties, there are usually two types of extractions: liquid-liquid extraction (LLE) and solid phase extraction (SPE). LLE and SPE are two very long protocols which are tedious when there are massive numbers of samples. They can also be the source of many errors, considering the experimenter-dependant possible repeated artefacts. To circumvent this point and increase the analytical service delivery, a TECAN robot has been acquired by MetaToul-Lipidomic facility to automate the sample preparation. Due to the specificity of lipid extraction, the robot needs a lot of optimization. This presentation will show part of adaptation we had to perform to validate fatty acid, neutral lipid and phospholipid profiling. That was related to the precision of solvent sampling which implies new adjustment accuracy for each of them, adding air gap, but also to modify the speed... First lipidomic's results obtained with a complete automated sample preparation will be presented for plasma sample.

Mots-Clés: sample preparation, lipidomics, automation, robot
<figure><img src="image_178.png" /></figure>

## Poster 60 – P60

# Development of lipidomic profiling by SFC-HRMS

Pauline Le Faouder<fnref n="1" />, Jean-François Martin<fnref n="2" />, Marion Regnier<fnref n="3" />, Hervé Guillou<fnref n="4" />, Justine Bertrand-Michel⁵

Lipids are essential cellular constituents that have many critical roles in physiological functions. They are involved in energy storage, cell signaling as second messengers, and are major constituents of cell membranes including lipid rafts1. Their crucial role is highlighted by their involvement in a large number of heterogeneous diseases such as cancer, diabetes, neurological disorders and inherited metabolic diseases2,3.

Due to the high structural diversity of lipid species arising from various combinations of fatty acyls and functional headgroups, the presence of isomeric and isobaric lipid species and their occurrence at a large concentration scale, a complete lipidomic profiling of biological matrices remains a challenge. In this context, the aim of our study presented in this poster is to develop an untargeted lipidomic approach by using supercritical fluid chromatography high resolution mass spectrometry to be able to propose a relative quantification of main lipids class in various biological sample.

To develop the method, the optimization of the separation and the detection of lipid species were performed on pure standards and then on liver lipid extract from 12-weeks-old C57BL6/J mice exposed to a control or an HFD (60% lipids in the diet) during 14 weeks. These analyses allowed the building of a homemade lipid data bank. An automatic process was then developed to produce the relative quantification of lipids species belonging to the 6 main class of lipids. The optimization of this method will be present with the first results obtained on liver disease model.

Mots-Clés: lipidomic profiling, Data processing, SFC, HRMS

<footnote n="1">¹ Institut des Maladies Métaboliques et Cardiovasculaires (I2MC) – Université Paul Sabatier - Toulouse
3, Hôpital de Rangueil, Institut National de la Santé et de la Recherche Médicale : U1048 – 1 avenue du Prof Jean Poulhes - BP
84225 - 31432 Toulouse Cedex 4, France</footnote>

<footnote n="2">² ToxAlim (ToxAlim) – Institut national de la recherche agronomique (INRA) : UMR1331 – 180 chemin de tournefeuille 31300
Toulouse, France</footnote>

<footnote n="3">³ ToxAlim (ToxAlim) – Institut National Polytechnique [Toulouse], Institut National de la Recherche
Agronomique : UMR1331, Université Paul Sabatier - Toulouse 3, Ecole Nationale Vétérinaire de
Toulouse – 23 ch. Capelles 31076 TOULOUSE, France</footnote>

<footnote n="4">⁴ INRA, Centre de Recherche en Toxicologie Alimentaire (INRA Toxalim) – Université de Toulouse,
INRA, ENVT, INP-Purpan, UPS – 180 chemin de Tournefeuille, 31027 Toulouse, France⁵ METATOUL – Inserm – France</footnote>
<figure><img src="image_179.png" /></figure>

Poster 61 – P61

" Less is More " : Analyse de données
multivariées issues de plans d'expériences multifactorielles

Víctor González-Ruiz <fnref n="1" />,<fnref n="2" />, Domitille Schvartz <fnref n="2" />,<fnref n="3" />, Jenny Sandström <fnref n="2" />,<fnref n="4" />, Julian Pezzati <fnref n="1" />, Fabienne Jeanneret <fnref n="1" />,<fnref n="2" />, David Tonoli <fnref n="1" />,<fnref n="2" />,
Julien Boccard <fnref n="1" />,<fnref n="2" />, Florianne Monnet-Tschudi <fnref n="2" />,<fnref n="4" />, Jean-Charles Sanchez <fnref n="2" />,<fnref n="3" />, Serge Rudaz <fnref n="1" />,<fnref n="2" />

Les plans d'expériences (Design of Experiments, DOE) permettent l'évaluation systématique des différents effets potentiels lorsque plusieurs facteurs expérimentaux sont étudiés simultanément. Toutefois, plusieurs sources de variabilités influencent les signaux mesurés et la décomposition de la variance présente une difficulté supplémentaire dans le cadre de données métabolomiques complexes. Dans cette étude, nous appliquons l'algorithme ANOVA Multiblock OPLS (AMOPLS), à l'étude des processus neuro-inflammatoires dans des cultures de cellules souches neurales 3D de rats.

Les cultures cellulaires ont été exposées au trimethylétain (TMT) et trois facteurs expérimentaux ont été considérés dans le DOE, à savoir (i) la maturité des cellules, (ii) le temps d'exposition et (iii) la concentration en TMT. Les métabolites ont été mesurés et identifiés à l'aide de quatre analyses LC-MS (RP+/- et HILIC+/- avec Q-TOF). L'analyse des données a été effectuée à l'aide d'un modèle consensus-AMOPLS sous MATLAB.

La méthode AMOPLS permet la décomposition et la quantification des sources de variabilité provenant de chaque effet expérimental. Les contributions les plus importantes proviennent de l'état de maturation, du temps d'exposition et de leur interaction (22.3%, 15.3% et 13.5% de la variabilité totale). Bien que faible (6.3%), la contribution de la dose de TMT est très significative (p-value < 0.01%). Une étape originale de déconvolution s'appuyant sur les valeurs des VIP2 permet de dévoiler la variabilité de chaque métabolite en relation avec chaque effet étudié. Les métabolites avec les plus grandes valeurs de VIP2 pour l'effet de la dose ont été utilisés pour l'analyse de voies métaboliques et mettent en évidence des altérations de (i) la différentiation neuronale, (ii) la signalisation nicotinique et (iii) le métabolisme du GABA en conséquence de l'exposition au TMT.

Dans cette étude, l'algorithme AMOPLS a permis l'analyse des données incluant un DOE. À partir d'un modèle global, les métabolites reliés à chaque facteur expérimental ont été mis en évidence. Nos résultats ont montré une forte perturbation de la neurotransmission GABAergique après l'exposition au TMT.

Mots-Clés: analyse multivariée, toxicologie, décomposition d'effets

<footnote n="1">¹ Analytical Sciences, School of Pharmaceutical Sciences, University of Geneva – Suisse</footnote>

<footnote n="2">² Swiss Centre for Applied Human Toxicology (SCAHT) - Suisse</footnote>

<footnote n="3">³ Translational Biomarker Group, Department of Internal Medicine Specialties, University of Geneva – Suisse</footnote>

<footnote n="4">⁴ Department of Physiology, University of Lausanne, Switzerland – Suisse</footnote>
<figure><img src="image_180.png" /></figure>

## Poster 62 – P62

# LipidQuan : A Targeted Lipid Profiling Solution Using UPLC/MS/MS

Mickael Hybois<fnref n="1" />, Nyasha Munjoma, Giorgis Isaac, Gethings Lee, Jimmy Hunk, Robert Plumb

Although advances in mass spectrometry (MS) have allowed for more in-depth lipidomics analysis, unambiguous identification and quantification has proven difficult as lipids exhibit a high number of isomeric and isobaric species. Furthermore, MS spectra often contain peaks and fragments from multiple compounds making confident identification and relative quantification of specific molecular species difficult and time consuming. As a result, the transfer of lipidomic data between laboratories is severely hindered, making multi-site study interpretation a challenge.

A hydrophilic interaction chromatography (HILIC) based approach for the separation of lipids by class prior to MS analysis is a proven method of reducing identification ambiguity. An additional benefit of separating lipid species by class is that fewer stable isotope labelled (SIL) standards are required for quantification, conferring a cost saving.

## Key Features :

* Streamlined and integrated lipidomics workflow (from sample preparation through to biological interpretations)

* Highly specific MRM transitions based on the fatty acyl chain fragments when applicable instead of the typical head group fragments to improve identification and specificity

* Routine targeted quantification of common lipids in plasma and serum

* Lipid class based separation reduces the number of stable isotope lipid standards (SILS) which results in significant cost saving

* Over 2000 lipid species MRMs

* Selection of screening method application notes available for download

Mots-Clés: Lipid, Lipids, LipidQuan, MetaboQuan, LC/MS/MS, lipidomics, HILIC, Waters

<footnote n="1">¹Waters Corporation – Waters Corporation – Saint Quentin en Yvelines, France</footnote>
<figure><img src="image_181.png" /></figure>

Poster 63 – P63

# Étude par RMN du monde caché d'un holobionte : à la recherche de la relation entre éponge et champignon marin

Aurore Michaud<sup>1,2,3</sup>, Jonathan Farjon<sup>2</sup>, Serge Akoka<sup>2</sup>, Yves-François Pouchus<sup>1</sup>, Emmanuel Gentil<sup>1</sup>, Olivier Grovel<sup>1</sup>, Patrick Giraudeau<sup>2,4</sup>

Les holobiontes sont la source de nombreuses questions en écologie chimique. L'une d'entre elles concerne l'intérêt de chacun des partenaires à vivre en cohabitation. Notre projet, s'intéressant à la souche fongique *Eurotium* sp. MMS1599 isolée de l'éponge marine *Tetilla* sp., n'échappe pas à cette interrogation et consiste en l'étude par métabolomique des dialogues moléculaires entre organismes.

Pour plonger au cœur de l'expression du métabolome de MMS1599 lorsqu'elle est en présence de son hôte originel, une double approche analytique associant spectroscopie de résonance magnétique nucléaire et spectrométrie de masse est employée. Ici, nous nous focalisons sur la démarche appliquée, comportant des outils rapides et sensibles en RMN multi-impulsionnelle. En effet, si la RMN du proton est couramment employée en analyse métabolomique pour sa rapidité et sa sensibilité, elle présente néanmoins un inconvénient majeur que sont les recouvrements de signaux lors d'analyses de mélanges complexes. Pour pallier cela, il faut alors envisager des séquences à deux dimensions permettant l'éclatement des signaux tout en gardant des temps d'analyse raisonnables. Nous présenterons ici une boîte à outils constituée d'expériences RMN récemment développées au laboratoire, ayant une excellente répétabilité et un temps d'expérience réduit. Trois types d'expériences ont été explorées : une INEPT 13C adiabatique, une 2D COSY 1H ultrarapide et une 2D HSQC 1H-13C accélérée et hautement résolue. Ces techniques optimisées pour le cas d'extraits de cultures de champignons marins permettent d'obtenir le meilleur compromis entre sensibilité, répétabilité, dispersion des signaux et temps d'expérience. Leur potentiel sera évalué pour le profilage métabolomique par RMN multi-dimensionnelle, une approche dont nous avons récemment démontré l'intérêt par rapport aux approches classiques. La combinaison des données RMN à celles de spectrométrie de masse permettra à terme, en utilisant des approches statistiques, de découvrir les mécanismes d'expression ou répression métaboliques en jeu chez *Eurotium* sp. lorsqu'il se trouve en présence de son organisme hôte *Tetilla* sp. et ainsi de comprendre les voies biosynthétiques mobilisées.

Mots-Clés: Ecologie chimique, Holobionte, éponge, souche fongique marine, RMN, méthodes rapides, INEPT, COSY ultrarapide, QUIPU, HSQC

<footnote n="1">¹ Mer, molécules et santé EA 2160, Université de Nantes, Nantes – Université de Nantes : EA2160 – France</footnote>

<footnote n="2">² Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation, (CEISAM), Université de Nantes, CNRS, UMR 6230, Nantes</footnote>

<footnote n="3">³ Plateforme CORSAIRE, Biogenouest, Université de Nantes – Université de Nantes, Plateforme Génomique Santé Biogenouest R,</footnote>

<footnote n="4">⁴ Institut Universitaire de France – Ministère de l'Éducation nationale, de l'Enseignement supérieur et de la Recherche, Ministère de l'Éducation nationale, de l'Enseignement supérieur et de la Recherche, Ministère de l'Éducation nationale, de l'Enseignement supérieur et de la Recherche, Ministère de l'Éducation nationale, de l'Enseignement supérieur et de la Recherche – France</footnote>
<figure><img src="image_182.png" /></figure>

Poster 64 – P64

Corsaire, plate-forme de métabolomique du Grand Ouest

Léa Cabioch<fnref n="1" />

Corsaire est une fédération de plateaux analytiques dédiée à l'analyse de petites molécules biologiques. Elle couvre les domaines de la métabolomique, la fluxomique, l'isotopomique et l'analyse structurale. La biologie marine, l'agronomie, la santé et l'écologie chimique sont le cœur de l'activité de Corsaire. Ses approches analytiques vont de l'approche non ciblée pour la recherche de biomarqueurs, à l'approche ciblée pour l'analyse qualitative et quantitative de molécules d'intérêt. Pour ce faire, la plate-forme dispose d'un large parc analytique, composé d'équipements de RMN et de spectrométrie de masse, associés à différentes techniques séparatives adaptées à des matrices biologiques de nature et d'origines variées. Un accompagnement de la procédure analytique est proposé depuis la prise en charge des échantillons biologiques, l'extraction, les phases séparatives, l'acquisition des données brutes et, au besoin, leur traitement chimiométrique.

Mots-Clés: métabolomique, plateforme, Biogenouest, RMN, Spectrométrie de Masse

<footnote n="1">¹Biogenouest – Biogenouest – France</footnote>
<figure><img src="image_183.png" /></figure>

## Poster 65 – P65

# Mass Spectrometry Imaging based on FTICR: application to Pt based anticancer drugs detection in human tissue

Isabelle Schmitz-Afonso<fnref n="1" />, Justine Ferey <fnref n="1" />, Marion Larroque <fnref n="2" />, Brice Bouyssière <fnref n="3" />, Sandra Mounicou <fnref n="3" />, Olivia Sgarbura <fnref n="4" />, François Quenet <fnref n="4" />, Sébastien Carrere <fnref n="4" />, Carlos Afonso <fnref n="1" />

Peritoneal carcinomatosis is a common extension of colon cancer. Platinum derivatives, such as oxaliplatin, are common metallodrugs applied during the Heated Intraoperative Chemotherapy (HIPEC), which consists in a local application of a high concentration of drug. During the HIPEC procedure, the metallodrug circulates into the peritoneal cavity and can affect other organs such as ovaries. Knowledge of penetration and distribution of this drug within the ovaries will help to better understand the effects of the drug on ovary functionality.

Mass spectrometry imaging (MSI) is a suitable tool for the evaluation of drug penetration. Previous work has been published based on MALDI-TOF instrument.<fnref n="1" /> Further developments are carried out within this study with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR-MS) in order to have a higher selectivity for precise image reconstruction and also for the unambiguous molecular formula attribution taking advantage of elemental fine structure. FTICR-MS is used to analyse extremely complex mixtures such as metabolome samples thanks to its high mass accuracy and ultrahigh resolution. This allows to confidently assign molecular formulas and to avoid any isobaric interference.<fnref n="2" />

Ovary tissue sections were mounted onto indium tin oxyde coated slides and coated with αcyano-4-hydroxycinnamic acid matrix. MALDI-FTICR MSI of metabolites was performed using a 12 T SolariX XR (Bruker) in positive ion mode to search for platinum containing metabolites. Transmission parameters were optimised for the *m/z* 150-1000 range. Internal mass calibration was implemented to get accuracies below 1 ppm for each scan. Data treatment was realised with SCILS software. One platinum derived compound was localised on the 6 ovary tissue sections analyzed. Its elemental composition was confirmed thanks to the high mass accuracy and isotopic fine structure.

Bianga J. et al *Metalomics*, 2014, 6, 1382.

Kihara M. et al *JASMS*, 2017, 28, 2469.

Mots-Clés: Mass Spectrometry Imaging, FTICR, metabolite

<footnote n="1">¹ Normandie Univ, FR 3038; IRCOF, (COBRA) - Université de Rouen, CNRS : UMR6014, INSA Rouen – Université de Rouen,</footnote>

<footnote n="2">² ICM/ Unité de Recherche Translationnelle, Campus Val d'Aurelle - ICM - 208 rue des Apothicaires, 34296 Montpellier, France, France</footnote>

<footnote n="3">³ CNRS/UPPA, UMR 5254, LCABIE - LCABIE - France</footnote>

<footnote n="4">⁴ ICM/ Service Chirurgie A2, Campus Val d'Aurelle - ICM - France</footnote>
<figure><img src="image_184.png" /></figure>

## Poster 66 – P66

# The Metaboscope : a metabolomic platform for the comprehensive profiling of metabolites

Florian Vasaï<fnref n="1" />, Raphaël Lugan<fnref n="1" />

The METABOSCOPE is a new metabolomic platform located at Avignon university, designed to meet the needs of scientists in a wide range of fundamental or applied researches. It is composed of 5 mass spectrometers hyphenated to liquid and gas chromatographs enabling untargeted or multi-targeted profilings, MS imaging and metabolite purification. Highly sensitive quantitative analysis of multiple targets can be performed by multiple reaction monitoring (MRM), with a triple quadrupole coupled to an ESI source and an UPLC UPLC-ESI-TQ. Non-targeted analyzes are achieved with an UPLC-Q-TOF ; this high resolution (40000) mass spectrometer is also implemented with an ion mobility cell for further metabolites separation, and comes with a MALDI source for in situ localization of compounds. A GC-TOF is dedicated to the profiling of primary metabolites (small carbohydrates, fatty acids, amino acids and organic acids), after a fully automated online chemical derivatization insuring high replicability; another GC-MS is fitted with a fully automated dynamic headspace collection system to analyze volatile compounds and a pyrolyzer for polymer structural characterization. Finally, molecules can be purified with a semi-preparative HPLC-MS enabling fraction collection based on mass signals. The Metaboscope provides analytical services but also method development, tips and practical training courses. It is opened to any collaboration with private or public partners.

Mots-Clés: metabolomic platform, analytic, collaboration

<footnote n="1">¹UMR Qualisud – Université d'Avignon – France</footnote>
<figure><img src="image_185.png" /></figure>

## Poster 67 – P67

# Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry-based metabolomics for kinetics tracking of natural herbicides' volatile residues: a simple non-destructive method

Hikmat Ghosson<fnref n="1" />,<fnref n="2" />, Delphine Raviglione <fnref n="1" />,<fnref n="2" />, Marie-Virginie Salvia <fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Cédric Bertrand <fnref n="1" />,<fnref n="2" />,<fnref n="3" />,⁴

Despite their known risks, herbicides are still essential for agriculture. Thus, natural herbicides are increasingly recommended to replace synthetic ones. They are supposed less harmful on human health and ecosystem. However, there are still limitations in studying the environmental fate of several of them. The main reason is the lack of methods dedicated for this type of herbicides. In fact, they usually consist of complex mixtures of substances, of which several are unknown.

Hence, in the framework of the Environmental Metabolic Footprinting (EMF) approach, the current work presents a method dedicated to analyze the volatile residues of herbicides applied on soil. The aim is to track the evolution of the volatile compounds issued from the herbicide by an untargeted metabolomics-based kinetics, in order to determine the "resilience time" of the gaseous phase above the soil. The approach aims to explore the environmental fate of these herbicides by combining it to other methods of the EMF. Moreover, it can estimate the exposure of farmers, insects and plants to potential toxic volatile substances.

The method is based on Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry (HS-SPME-GC-Q MS). The HS-SPME provides a non-destructive extraction. This allows reducing the number of samples. The GC separation technique provides high reproducible analysis for volatile compounds. In addition, it allows the calculation of the Retention Index (RI) as a tool for the molecular characterization. On the other hand, despite the low resolution of the Quadrupole mass analyzer, the Electronic Impact provides reproducible MS fragmentation patterns used for spectral library search and putative identification of unknown compounds.

The method was experimented for a pilot study. It was applied on a natural herbicide: the extract of *Myrica gale*, containing the Myrigalone A active substance. The setup of the analytical tool was performed by optimizing the system parameters. Then, a 38 days kinetics study was applied on control and spiked soil samples. Results proved the reliability of the method and demonstrated the robustness of the system and low matrix effect. Thus, new high scale experiments are planned. The integration of other natural and synthetic herbicides will be also considered.

Mots-Clés: Volatolomics, Headspace, Solid Phase Micro Extraction, GC, MS, Environmental Metabolic Footprinting, Natural herbicides

<footnote n="1">¹ UFR Sciences Exactes et Expérimentales, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan</footnote>

<footnote n="2">² PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Université de Perpignan,</footnote>

<footnote n="3">³ Laboratoire d'Excellence « CORAIL », Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France – EPHE-CNRS-UPVD – France</footnote>
<figure><img src="image_186.png" /></figure>

## Poster 68 – P68

# Exploration du métabolome d'un individu

Cécile Martias<fnref n="1" />, Raphaëlle Voisin, Sylvie Mavel², Antoine Lefèvre<fnref n="3" />, Frédéric Montigny<fnref n="4" />, Lydie Nadal-Desbarats<fnref n="1" />,<fnref n="4" />, Patrick Emond²

### Contexte :

L'approche métabolomique se concentre classiquement sur l'étude d'un type de prélèvement par une seule plateforme analytique. Une couverture métabolique la plus large possible (multicompartiments et multi-plateformes) permettrait l'élaboration d'une carte d'identité métabolique d'un individu.

### Objectif :

Standardiser pour la salive, les fèces et l'urine, le protocole d'extraction le plus adapté pour une exploration en RMN du 1H et en UHPLC-MS (HILIC et C18) afin d'avoir la couverture métabolique la plus exhaustive possible.

### Matériels et Méthodes :

Pour la salive, les fèces et l'urine différents protocoles d'extraction ont été testés (précipitation des macromolécules par ACN, MeOH, dilution) et analysés en 1H-RMN, UHPLC-MS (HILIC, C18) en détection positif et négatif.

Le nombre de composés, le CV moyen a été calculé pour chaque méthode d'extraction et chaque plateforme analytique afin de choisir le protocole d'extraction le plus adapté. La complémentarité des plateformes a été évaluée par diagramme de Venn.

### Résultats :

L'efficacité des protocoles d'extraction de métabolites pour chaque matrice est comparée par les analyses, en parallèle, 1H-RMN et UPLC-MS (HILIC, C18) en détection positif et négatif.

Pour la matrice salive, le solvant acétonitrile permet une plus grande extraction des métabolites soit 515 molécules potentielles. Une fois la redondance enlevée, la couverture métabolomique salaire d'un individu est de 323 molécules.

Pour la matrice fèces, le solvant acétonitrile/methanol/eau permet l'extraction de 642 molécules potentielles. Une fois la redondance enlevée, la couverture métabolomique fécale d'un individu est de 391 molécules.

Pour la matrice urine, le solvant méthanol permet l'extraction de 585 métabolites potentielles. Une fois la redondance enlevée, la couverture métabolomique urinaire d'un individu est de 395 métabolites.

### Conclusion :

Cette étude montre la complémentarité des plateformes 1H-RMN, UHPLC-MS (HILIC, C18) en détection positif et négatif pour l'exploration du métabolome d'un individu.

Mots-Clés: Multi, matrice, Multi, plateforme, UHPLC, MS, RMN, carte d'identité métabolique, urine, salive, fèces

<footnote n="1">¹ UMR 1253 iBrain, Université de Tours, Inserm, F-37000 Tours, France – Université de Tours – France
² Université François-Rabelais, INSERM U930 “Imagerie et Cerveau”, CHRU de Tours – Inserm : UMR930 – 10 Bv Tonnellé, 37044 Tours, France</footnote>

<footnote n="3">³ Université François-Rabelais, INSERM U930 “Imagerie et Cerveau”, CHRU de Tours – Inserm : U930, Inserm : U930 – 10 Bv Tonnellé, 37044 Tours, France</footnote>

<footnote n="4">⁴ PST-ASB, Université de Tours, F-37000 Tours, France – Université de Tours – France</footnote>
<figure><img src="image_187.png" /></figure>

## Poster 69 – P69

# Développements analytiques pour la caractérisation non-ciblée de l'exposome chimique dans des matrices biologiques humaines

Jade Chaker <fnref n="1" />, David M. Kristensen <fnref n="2" />, Bernard Jégou <fnref n="1" />, Arthur David <fnref n="1" />

L'exposition de l'homme aux mélanges complexes de xénobiotiques (i.e. exposome chimique) peut dans certains cas être un facteur environnemental d'importance contribuant à l'apparition de certaines maladies chroniques non-transmissibles. Il est donc primordial de pouvoir caractériser cet exposome chimique dans les matrices biologiques humaines afin d'évaluer son impact dans la survenue de ces maladies. Pour cela, les approches analytiques non-ciblées reposant sur la chromatographie liquide liée à la spectrométrie de masse à haute résolution (CL-ESI-SMHR) sont souvent utilisées afin de détecter sans à priori les mélanges de xénobiotiques accumulées dans ces matrices. L'utilisation de ces approches non-ciblées sur la matrice sang (plasma ou sérum) s'avère pertinent puisque cette matrice est couramment échantillonnée dans les études épidémiologiques ou de biosurveillance. Toutefois, ces méthodes non-ciblées reposant sur la CLSMHR sont souvent sujettes à des interférences analytiques importantes dues, entre autres, à la suppression d'ion. Elles nécessitent donc des développements analytiques afin d'améliorer leur sensibilité analytique mais également leur robustesse dans le but de pouvoir les appliquer à grande échelle. Afin de surmonter ces limites analytiques, onze méthodes de préparation d'échantillon reposant sur des méthodes de délipidation/déprotéination ou d'extraction en phase solide (SPE) ont été comparées avec une méthode traditionnellement utilisée pour les analyses non-ciblées (précipitation de protéine). Des critères quantitatifs (ex. recouvrement, effet de matrice) et qualitatifs (ex. facilité et rapidité d'implémentation) ont été utilisés pour l'évaluation de ces méthodes pour les matrices sérum et plasma. Ces résultats préliminaires ainsi que leur application sur des échantillons de cohorte mère-enfant seront présentés. Ces méthodes ont pour but d'être par la suite appliquées à grande échelle dans le cadre d'une étude épidémiologique (cohorte mère-enfant Danoise) pour renforcer la caractérisation de l'exposition humaine aux xénobiotiques et de mieux comprendre leurs impacts potentiels sur la santé humaine.

Mots-Clés: Exposome, Non ciblé, Xénobiotiques

<footnote n="1">¹ Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMRs1085, F-35000 Rennes, France -- UnivRennes -- France</footnote>

<footnote n="2">² Department of Neurology, Danish Headache Center, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark – Danemark</footnote>
<figure><img src="image_188.png" /></figure>

Poster 70 – P70

Profiling and annotation of flavonoids using a product ion-
dependent MSⁿ data acquisition method on a Tribrid Orbitrap
mass spectrometer

Marie-Pierre Pavageau¹, Reiko Kiyonami¹, Iwao Sakane², Seema Sharma¹, Graeme Mcalister¹, Caroline Ding¹ and Andreas Huhmer¹

¹Thermo Fisher Scientific, San Jose, CA, USA, ²ITO EN, LTD, Tokyo, Japan

The untargeted profiling of flavonoids provides insights into their biological functions and potential health benefits for humans. However, comprehensive identification of flavonoids from real samples remains challenging because of the limited availability of authentic standards and the structural diversity of this class of compounds. Previous studies relied upon extensive expert knowledge about fragmentation rules, a priori knowledge of the structures of flavonoids, and simple MS2 based analyses that are often not sufficient for complete structural characterization. Here we present a new flavonoid profiling workflow that uses comprehensive fragment ion information from HCD MS-MS and higher order FTMSⁿ for rapid flavonoid identification and quantitation on a Tribrid Orbitrap mass spectrometer.

As the proof of concept of the workflow, flavonoid extracts from different types of natural products were tested. A C18 column was used for flavonoid separation, and a modified Orbitrap Fusion™ Tribrid™ mass spectrometer was used for collecting HRAM MS and MSⁿ (up to MS⁵) data. The collected data were processed using Mass Frontier 8.0 and Compound Discoverer™ 3.0 software. A novel structure ranking algorithm included in the Compound Discoverer 3.0 software was applied to the MS and MSⁿ data for confident structure elucidation of the unknown flavonoids based on ChemSpider database and custom flavonoids database. The MSⁿ data were critical, especially for the identification of flavonoid glycocnjugates.

Brief summary: The new LC-MSⁿ workflow enables improved throughput, identification coverage and confidence for flavonoids profiling experiments
<figure><img src="image_189.png" /></figure>

Communication Poster

Session Traitement du signal - Statistiques -

Intégration des données

<figure><img src="image_190.png" /></figure>
<figure><img src="image_191.png" /></figure>

Poster 71 – P71

R FiBiCo script : In silico screening of bioactive compounds from extracts

Lucie Ory <fnref n="1" />, El-Hassane Nazih <fnref n="1" />, Yves-François Pouchus <fnref n="1" />, Samuel Bertrand <fnref n="1" />,<fnref n="2" />, Catherine Roullier <fnref n="1" />,<fnref n="2" />

In natural product drug discovery, several strategies have emerged to highlight specifically bioactive
compound(s) within complex mixtures (fractions or crude extracts) using metabolomics tools. In this area, a great
deal of interest has raised among the scientific community on strategies to link chemical profiles and associated
biological data, leading to the new field called "biochemometrics". The project presented here falls into this emerging
research by proposing a complete workflow, which was divided into three major steps. The first one consists in the
fractionation of the same extract using four different chromatographic stationary phases and appropriated elution
conditions to obtain five fractions for each column. The second step corresponds to the acquisition of chemical
profiles using HPLC-HRMS analysis, and the biological evaluation of each fraction. The last step evaluates the links
between the relative abundances of molecules present in fractions (peak area) and the global bioactivity level
observed for each fraction. To this purpose, an original bioinformatics script (encoded with R Studio software) using
the combination of four statistical models (Spearman, F-PCA, PLS, PLS-DA) was here developed leading to the
generation of a "Super list" of potential bioactive compounds together with a predictive score. This strategy was
validated by its application on a marine-derived *Penicillium chrysogenum* extract exhibiting antiproliferative activity
on breast cancer cells (MCF-7 cells). After the three steps of the workflow, one main compound was highlighted as
responsible for the bioactivity and identified as ergosterol. Its antiproliferative activity was confirmed with an IC50
of 0.10 µM on MCF-7 cells. The script efficiency was further demonstrated by comparing the results obtained with a
different recently described approach based on NMR profiling and by virtually modifying the data to evaluate the
computational tool behaviour. This approach represents a new and efficient tool to tackle some of the bottlenecks
in natural product drug discovery programs.

Mots-Clés: metabolomics, biochemometrics, natural products, Liquid Chromatography, Mass Spectrometry, R script

<footnote n="1">¹ Mer, molécules et santé, MMS-EA 2160 – Université de Nantes – France</footnote>

<footnote n="2">² Corsaire-ThalassOMICS Metabolomics Facility – Université de Nantes, Université de Nantes – France</footnote>
<figure><img src="image_192.png" /></figure>

## Poster 72 – P72

# Comparison of PLS-derived sparse methods to find accurate biomarkers in NMR spectra.

Manon Martin<fnref n="1" />, Bernadette Govaerts <fnref n="1" />

The search for biomarkers in metabolomics is a vast topic with a multitude of methodological procedures that are suggested in the literature, and where some of them are rather intricate. Furthermore, the true biomarkers' location is usually unknown, and some uncertainty remains in the selected biomarkers' validity. This contribution compares the performances of common multivariate analysis methods, (O)PLS-DA, and their sparse extensions in a simulation study. The aim is to decipher pros and cons of the different approaches to help the researchers opt for the best method to apply, given their dataset attributes and the scope of their study. The dataset on which is based this simulation study consists in a two-class problem with simulated NMR spectra, where the exact location of biomarkers is known. Hence, the accuracy of the selected biomarkers can be precisely evaluated, in particular with ROC curves. Different dataset characteristics are stressed (size, degree of noise) to mimic the variability in the experimental datasets characteristics. Additionally, other performance metrics, such as feature stability and prediction accuracy, are also studied.

Mots-Clés: (O)PLS, sparse, simulated data, feature selection

<footnote n="1">¹ISBA, UCLouvain – Belgique</footnote>
<figure><img src="image_193.png" /></figure>

## Poster 73 – P73

# " Less is More " : Analyse de données multivariées issues de plans d'expériences multifactorielles

Víctor González-Ruiz ¹,², Domitille Schvartz ²,³, Jenny Sandström ²,⁴, Julian Pezzati ¹, Fabienne Jeanneret ¹,², David Tonoli ¹,², Julien Boccard ¹,², Florianne Monnet-Tschudi ²,⁴, Jean-Charles Sanchez ²,³, Serge Rudaz ¹,²

Les plans d'expériences (Design of Experiments, DOE) permettent l'évaluation systématique des différents effets potentiels lorsque plusieurs facteurs expérimentaux sont étudiés simultanément. Toutefois, plusieurs sources de variabilités influencent les signaux mesurés et la décomposition de la variance présente une difficulté supplémentaire dans le cadre de données métabolomiques complexes. Dans cette étude, nous appliquons l'algorithme ANOVA Multiblock OPLS (AMOPLS), à l'étude des processus neuro-inflammatoires dans des cultures de cellules souches neurales 3D de rats.

Les cultures cellulaires ont été exposées au trimethylétain (TMT) et trois facteurs expérimentaux ont été considérés dans le DOE, à savoir (i) la maturité des cellules, (ii) le temps d'exposition et (iii) la concentration en TMT. Les métabolites ont été mesurés et identifiés à l'aide de quatre analyses LC-MS (RP+/- et HILIC+/- avec Q-TOF). L'analyse des données a été effectuée à l'aide d'un modèle consensus-AMOPLS sous MATLAB.

La méthode AMOPLS permet la décomposition et la quantification des sources de variabilité provenant de chaque effet expérimental. Les contributions les plus importantes proviennent de l'état de maturation, du temps d'exposition et de leur interaction (22.3%, 15.3% et 13.5% de la variabilité totale). Bien que faible (6.3%), la contribution de la dose de TMT est très significative (p-value < 0.01%). Une étape originale de déconvolution s'appuyant sur les valeurs des VIP2 permet de dévoiler la variabilité de chaque métabolite en relation avec chaque effet étudié. Les métabolites avec les plus grandes valeurs de VIP2 pour l'effet de la dose ont été utilisés pour l'analyse de voies métaboliques et mettent en évidence des altérations de (i) la différentiation neuronale, (ii) la signalisation nicotinique et (iii) le métabolisme du GABA en conséquence de l'exposition au TMT.

Dans cette étude, l'algorithme AMOPLS a permis l'analyse des données incluant un DOE. À partir d'un modèle global, les métabolites reliés à chaque facteur expérimental ont été mis en évidence. Nos résultats ont montré une forte perturbation de la neurotransmission GABAergique après l'exposition au TMT.

**Mots-Clés:** analyse multivariée, toxicologie, décomposition d'effets

<footnote n="5">⁵ Analytical Sciences, School of Pharmaceutical Sciences, University of Geneva – Suisse</footnote>

<footnote n="6">⁶ Swiss Centre for Applied Human Toxicology (SCAHT) – Suisse</footnote>

<footnote n="7">⁷ Translational Biomarker Group, Department of Internal Medicine Specialties, University of Geneva – Suisse</footnote>

<footnote n="8">⁸ Department of Physiology, University of Lausanne, Switzerland – Suisse</footnote>
<figure><img src="image_194.png" /></figure>

Poster 74 – P74

MIMOSA: A mass spectrometry application for processing and
analysis of metabolomics data – Application to lipidomics

Joséphine Abi-Ghanem ¹, Djomangan Adama Ouattara ¹

¹ BIOASTER – Omics Hub – France

MIMOSA (Mass spectrometry processIng of MetabOlomicS DatA) is a transversal computational framework for
metabolomics. MIMOSA allows building simple to complex pipelines to process and analyze metabolomics data
with a high reproducibility and traceability. MIMOSA is ready-to-use, fast and extendable. It is specially designed to
handle large metabolomics data (up to hundreds of raw files) in a reasonable time laps on a classical laptop. It
allows interfacing available software like ProteoWizard, OpenMS and built-in functions for data normalization,
statistical analysis and the identification of metabolites using Sirius, Metfrag and LipidMatch. As well, other tools
can easily be interfaced into the MIMOSA to build customized workflows. MIMOSA is available as open Matlab
scripts and a standalone application to non-developer users. Its extension to more open programming languages is
planned. MIMOSA is also presented in a workshop at the 12th RFMF. This workshop introduces the main
fundamentals of the bioinformatics tools developed at BIOASTER for the processing and analysis of mass
spectrometry data in metabolomics, with a focus on lipidomics.

Mots-Clés: MIMOSA, Lipidomics, Computational metabolomics, Analysis of metabolomics data, Metabolite identification, Workflow traceability
<figure><img src="image_195.png" /></figure>

Poster 75 – P75

NMR-based metabolomic analysis of young St.John's Wort plants
grown under different color LED lighting

Paul Shipley <fnref n="1" />, Francois Miros <fnref n="1" />

The use of metabolomics techniques to classify unrelated species, in particular for the detection of adulteration, has proven to be robust and relatively method-independent. In contrast, metabolomics analysis of more closely related samples, such as highly related species or different growing conditions, both classification and identification of significant metabolites can approach the false discovery rate. *Hypericum perforatum*, or St. John's wort, is a flowering plant native to parts of Asia and Europe, and invasive to several other regions including North America. St. John's wort has a long history of use in traditional medicine for the treatment of depression. In this study, clonally propagated St. John's wort were grown in culture with different light conditions. NMR data for the lyophilized and extracted plants were acquired and investigated using uni- and multivariate statistical methods. Two methods of feature reduction were used and compared, and metabolites that drove variance between plants grown in the different lighting conditions identified using VIP identification from multiple models, coupled with STOCSY analysis to identify peaks covarying with the identified peaks. It was found that primary metabolites varied most strongly in relationship to the ratio of red to blue light, corresponding to chlorophyll A and B absorbances.

Mots-Clés: Medicinal plants, NMR, magnetic resonance, model validation, significant metabolite identification, untargeted metabolomics

<footnote n="1">¹ University of British Columbia – Canada</footnote>
<figure><img src="image_196.png" /></figure>

Poster 76 – P76

CANPA: Computer-Assisted Natural Products Anticipation

Alexander Enrique Fox Ramos, Coralie Pavesi, Marc Litaudon <fnref n="1" />, Vincent Dumontet, Erwan Poupon <fnref n="2" />, Pierre Champy <fnref n="3" />,
Grégory Genta-Jouve <fnref n="4" />, Mehdi Beniddir <fnref n="5" />

Traditional natural products discovery workflows implying a combination of different targeting strategies including structure- and/or bioactivity-based approaches, afford no information about new compound structure until late in the discovery pipeline. By integrating a MS/MS prediction module and a collaborative library of (bio)chemical transformations, we have developed a new platform, coined MetWork, that is able of anticipating the structural identity of metabolites starting from any identified compound. In our quest to discover new monoterpene indole alkaloids, we demonstrate the utility of the MetWork platform by anticipating the structures of five previously undescribed sarpagine-like N-oxide alkaloids that have been targeted and isolated from the leaves of *Alstonia balansae* using a molecular networking-based dereplication strategy fueled by computer-generated annotations. This study constitutes the first example of a natural product discovery workflow, termed CANPA, in which the targeted structures were initially generated and therefore anticipated by a computer prior to their isolation.

Mots-Clés: chemical prospecting, natural products, alkaloids, structure elucidation, anticipation, in silico metabolization, MetWork

<footnote n="1">¹ Institut de Chimie des Substances Naturelles – Centre National de la Recherche Scientifique : UPR2301 – France</footnote>

<footnote n="2">² BioCIS – Université Paris Sud - Paris XI – France</footnote>

<footnote n="3">³ CNRS UMR 8076 BioCIS - BIOMolécules : Conception, Isolement et Synthèse (BioCIS) – CNRS : UMR8076 - CNRS UMR 8076 BioCIS - BIOMolécules : Conception, Isolement et Synthèse Bureau D5 309 - Bâtiment D5 - 5, rue J.B. Clément 92296 Châtenay-Malabry, France, France</footnote>

<footnote n="4">⁴ Université Paris Descartes (COMETE) – CNRS : UMR8638 – 4 Avenue de l'observatoire, France</footnote>

<footnote n="5">5 Université Paris Sud, Laboratoire de Pharmacognosie associé au CNRS (UPS, BioCIS UMR CNRS 8076) – Université Paris XI - Paris Sud - 5, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France</footnote>
<figure><img src="image_197.png" /></figure>

## Poster 77 – P77

# Overview of multi-organ lipid profile alteration in pre-diabetic mice through multiblock modeling

Florence Mehl<fnref n="1" />, Hervé Lestunff<fnref n="2" />, Kelly Meneyrol<fnref n="3" />, Jessica Denom<fnref n="3" />, Céline Cruciani-Guglielmacci<fnref n="3" />, Leonore Wigger<fnref n="1" />, Ana Rodriguez Sanchez-Archidona<fnref n="4" />, Christian Klose⁵, Mathias J. Gerl⁵, Kai Simons⁵, Bernard Thorens<fnref n="4" />, Christophe Magnan<fnref n="3" />, Mark Ibberson<fnref n="1" />

Hyperlipidemia is a common feature of the metabolic syndrome and has been linked with type II diabetes and insulin resistance. It is thus of high importance to study how the lipid profile is affected when metabolism is subjected to high-energy dietary stress conditions leading to pre-diabetic phenotype.

In this work, three mice strains were involved. Although the mouse strain C57BI/6 has been considered the strain of choice to study type II diabetes and the metabolic syndrome in mouse models, a recent comparison of the phenotypic and metabolic response to high fat (HF) diet of multiple strains in parallel through out different conclusions; highlighting not only C57BI/6 but also DBA/2J and BALB/cJ as the strains that developed a clear pre-diabetic phenotype under high-energy dietary stress conditions with different features that may be relevant for the study of diabetes in both mouse models and human (Cruciani-Guglielmacci C, et al. 2017).

In order to find a common underlying mechanism regardless the different phenotypes of these genetically different strains under high fat diet; liver, adipose and soleus muscle tissues as well as plasma were sampled and shotgun lipidomic analyses were realized.

To better understand how lipidome reacts under high-energy dietary stress conditions it is mandatory that all lipid profiles from the different tissues and biofluid are integrated in a global model. A Common Component and Specific Weights Analysis (CCSWA) allowed us to integrate all lipid profiles from the 4 different biological matrices in a single model (Qannari, EM, et al. 2000). This unsupervised multiblock analysis showed that the most important and common source of variation of the dataset is the diet. This method enables the evaluation of consequences of a high fat diet on the lipid profiles of liver, muscle, adipose tissue and plasma.

Cruciani-Guglielmacci C, et al. Molecular phenotyping of multiple mouse strains under metabolic challenge uncovers a role for Elov12 in glucose-induced insulin secretion. Mol Metab. 2017 Jan 26;6(4):340-351. Qannari EM, Wakeling I, Courcoux P, MacFie HJH. Defining the underlying sensory dimensions. Journal of Food Quality and Preference. 2000:11, 151–154.

data integration, lipidomics, diabetes, chemometrics, multiblock

<footnote n="1">¹ Swiss Institute of Bioinformatics (SIB) – Suisse</footnote>

<footnote n="2">² Institut des Neurosciences de Paris-Saclay – Université Paris-Sud - Paris 11, Centre National de la Recherche Scientifique : UMR9197 – France</footnote>

<footnote n="3">³ Biologie Fonctionnelle Adaptative" (BFA) UMR 8251 - Université Paris Diderot - Paris 7 – France</footnote>

<footnote n="4">⁴ Center for Integrative Genomics - University of Lausanne – Suisse ⁵ Lipotype GmbH, Tatzberg 47, 01307 Dresden – Allemagne</footnote>
<figure><img src="image_198.png" /></figure>

**Communication Poster**

**Session Développements bioinformatiques -
Outils d'intégration**

<figure><img src="image_199.png" /></figure>

RÉSEAU FRANCOPHONE DE MÉTABOLOMIQUE ET FLUXOMIQUE
<figure><img src="image_200.png" /></figure>

## Poster 78 – Associé FP5

# Analytic correlation filtration: A new tool to reduce analytical complexity of metabolomic datasets

Stéphanie Monnerie¹, Mélanie Pétéra², Bernard Lyan², Pierrette Gaudreau³, Blandine Comte¹, Estelle Pujos-Guillot²,¹

Metabolomics generates massive and complex data that need dedicated workflows to extract the meaningful information and to enrich our knowledge of biological systems. For biological interpretation, experts are mainly focusing on metabolites rather than on the redundant different analytical species. Moreover, the high degree of correlation in datasets is a constraint for the use of data mining and statistical methods.

In this context, we developed a new tool to detect analytical correlation into datasets without confounding them with biological correlations that may exist within samples. The algorithm principle is to group features coming from the same analyte and to propose one single representative per group. The user can define grouping criteria with various options including correlation coefficient, retention time, mass information from a reference list of isotopes, adducts and fragments. Thresholds for all these criteria can be fixed and the representative feature can be determined following four methods according to the user needs and the analytical technology.

We chose to compare the present tool to one of the most commonly used free package proposing a grouping method: 'CAMERA', using its Galaxy version 'CAMERA. annotate' also available in Workflow4Metabolomics (W4M; http:// workflow4metabolomics.org). To illustrate the 'Analytic correlation filtration tool' functionalities and the results obtainable on typical experimental data, a published dataset available on W4M was used as an example. This dataset named 'Sacurine' was obtained from human urine samples analysed by LC-HRMS in negative ionization mode (Thevenot et al., 2015).

Within the 3,120 ions of the urine dataset, the tool allowed creating 2,651 groups, meaning that 15% of ions are proposed to be filtered because of analytical redundancies. While CAMERA generated more than 20 groups of more than 10 ions, the proposed tool subdivided them into smaller ones corresponding to individual annotated metabolites, thus demonstrating the efficiency and relevance of the present approach.

As a key element in metabolomics data analysis, the tool will be available via the web-based galaxy platform W4M with different output files for vizualisation for further data analysis within workflows.

Mots-Clés: analytical redundancies, filtration, workflow

<footnote n="4">4 Université Clermont Auvergne, INRA, UNH, Mapping – (INRA) : UMR1019 – France</footnote>

<footnote n="5">5 Université Clermont Auvergne, INRA, UNH, Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont - INRA
Clermont-Ferrand-Theix : UMR1019 – France</footnote>

<footnote n="6">6 Centre de Recherche du Centre hospitalier de l'Université de Montréal ; Département de médecine, Université de Montréal – Canada</footnote>
<figure><img src="image_201.png" /></figure>

Poster 79 – P79

mlomic, an R package and Shiny application for automated reporting
of metabolomics data analysis

Yasmina Adimy <fnref n="1" />, Florence Castelli <fnref n="1" />, Thaïs Hautbergue <fnref n="1" />, François Fenaille <fnref n="1" />, Benoit Colsch <fnref n="1" />

Mass-spectrometry-based metabolomics is widely used in several clinical studies in order to correlate human, animal or microbial metabolic profiles with internal or external factors such as diseases or environmental parameters. However, large datasets produced by mass spectrometry are not directly readable and interpretable by medical and biological staff. Before sending metabolomics results, analytical data need to be translated into relevant biological information, presented under a clear and visual organization. In this context, we aim to automate the generation of analytical reports, integrating interactive visualizations of statistical analysis as well as annotations and providing guidelines to highlight pertinent results and guide further investigations.

We have implemented *mlomic*, a package usable from a shiny user-friendly interface, allowing users without programming experience to easily analyze their data. First steps of data analysis, including preprocessing, normalization and quality control, are done using XCMS implemented in the Workflow4Metabolomics platform. Then, in order to identify significant variables, package's workflow includes two steps: annotation and statistical analysis. Annotation consists in matching variables with either an in-house spectral database gathering more than one thousand compounds (essentially of human origin but also some xenobiotics) or with public databases. Then, we provide univariate and multivariate (PCA, PLS-DA) statistical analysis to identify significant features. All relevant and understandable results are gathered by automated reporting into HTML, PDF or Word documents, integrating interactive visualizations to give more direct insights into data. Finally, reports also include supporting information to help clinicians interpret their results. It allows reproducible and unbiased data treatment in a time-efficient manner with various relevant data visualization options.

This toolbox will afterward include metabolite projection into metabolic pathways and be extended to lipidomics data analysis. We could then make these tools available to the metabolomics community by implementing them in Workflow4Metabolomics.

Mots-Clés: Bioinformatics, shiny, R-markdown, Automated reporting, Annotation, Statistical analysis, Interactive visualizations

<footnote n="1">¹ Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Etude du Métabolisme des Médicaments, CEA, INRA, Université Paris Saclay, MetaboHUB, F-91191 Gif-sur-Yvette, France – CEA, CNRS, Université Paris-Saclay, CEA Saclay 91191 Gif sur Yvette France – France</footnote>
<figure><img src="image_202.png" /></figure>

Poster 80 – P80

# Intégration d'outils de LC-MS/MS et de visualisations interactives sur l'infrastructure Galaxy Workflow4Metabolomics

Julien Saint Vanne, Romain Dallet, Erwan Corre, Yann Guitton<fnref n="1" />, Gildas Lecorguille

L'analyse de données métabolomiques est un processus complexe, à multiples étapes, et en évolution constante avec le développement de nouvelles techniques d'analyse, de nouvelles méthodes mathématiques, de nouveaux outils bioinformatiques ainsi que de nouvelles bases de données. L'infrastructure Galaxy [1] de workflow4metabolomics.org [2,3] (W4M) fournit un environnement unique, centralisé, simple et performant pour créer, exécuter et partager des flux de travail métabolomique pour les technologies de LC-MS, GC-MS et de RMN.

Un des verrous actuels de l'approche métabolomique est l'identification des composés. La spectrométrie de masse en tandem (MS/MS) permet d'apporter une précision sur la structure des composés recherchés. Pour faciliter cette étape d'identification, cette technologie est de plus en plus utilisée par les laboratoires, mais l'analyse de ces données MS/MS reste complexe et fastidieuse. Afin de faciliter cette étape, différents outils d'aide à l'identification ont été développés par la communauté scientifique. Un workflow d'analyse de ces données MS/MS a été créé dans W4M en se basant sur 3 outils reconnus : un outil de sélection de données, msPurity [4], ainsi que deux outils d'identification, metFrag [5] et Sirius-CSI:FingerID [6].

Afin de faciliter l'utilisation des outils Galaxy, un ensemble de visualisations interactives ont été ajoutées à l'infrastructure W4M Galaxy. Le développement récent d'un ensemble d'applications Shiny exécutable dans des environnements interactifs ("Galaxy Interactive Environments") permet désormais une interaction (zoom, sélection d'échantillons, ...) avec des sorties graphiques telles que des chromatogrammes, des "heatmaps" ou des ACP. Toutes ces illustrations étaient classiquement figées au format pdf ou png, sans réelle possibilité d'interaction. Les utilisateurs de W4M peuvent à présent bénéficier de tous ces nouveaux développements.

Mots-Clés: Galaxy, Metabolomique, Interactivité, MS/MS, Identification, MS, NMR, Statistiques

<footnote n="1">¹ Laberca, Oniris, INRA, Université Bretagne Loire, 44307, Nantes, France – Institut National de la Recherche Agronomique : UMR1329, Ecole Nationale Vétérinaire, Agroalimentaire et de l’alimentation Nantes-Atlantique : UMR1329 – France</footnote>
<figure><img src="image_203.png" /></figure>

## Poster 81 – P81

# Mapping lipidomics data on metabolic networks with MetExplore

Florence Vinson <fnref n="1" />, Arthur Moreau <fnref n="1" />, Pauline Le Faouder <fnref n="2" />, Justine Bertrand-Michel <fnref n="2" />, Fabien Jourdan <fnref n="1" />, Nathalie Poupin <fnref n="1" />

Lipidomics has become a powerful tool for investigating alterations in lipid metabolism in various biological contexts (metabolic diseases, xenobiotics exposure ...). MS-based lipidomics analyses allow detecting changes in concentrations of lipids in tissues and cells. To go further in the interpretation of lipidomics profiles and identify the metabolic pathways and reactions that are affected, it is necessary to put lipids in their metabolic context. Genome-scale metabolic network reconstructions, such as the human metabolic network Recon2 [1], gather all the metabolic reactions and their associated metabolites that can occur in a cell or tissue. However, mapping lipids measured in lipidomics experiments on metabolic networks is challenging because of the differences in identifiers and in precision between lipidomics data and metabolic networks. Indeed, in metabolic networks, for many lipids (e.g., triacylglycerols), all specific molecular species are not detailed but only the generic class is represented. To help linking lipids from lipidomics datasets with lipids in metabolic networks, we developed a new mapping method, freely available on the MetExplore webserver (http://www.metexplore.fr) [2]. It allows performing matching based on identifiers from public databases, such as CHEBI and LipidMaps, and uses the ontology defined in those 2 databases to retrieve generic lipid "class" in network for specific lipid species in dataset when these species are not detailed in the network. We applied our method to the list of lipids that can be measured by the MetaboHub-MetaToul lipidomics platform [3], including lipids from different classes (fatty acids, phospholipids, cholesterol esters ...). Among these 611 detectable lipids, only 38 (mostly fatty acids and eicosanoids) could be retrieved in Recon2 metabolic network when performing usual exact mapping, but we were able to map 290 of these lipids when using our new method, additional mapped lipids being mostly glycerolipids and sphingolipids. This new method opens the way for better interpretation of lipidomics data in terms of lipid metabolism modulations. [1]Thiele I, et al. Nat Biotechnol. 2013;31: 419-425. [2] Cottret L, et al. Nucleic Acids Res. 2018. [3] Khoury S, et al. Biomolecules. 2018;8: 174.

Mots-Clés: metabolic networks, lipidomics

<footnote n="1">¹ UMR1331 Toxalim – Université de Toulouse, INRA, ENVT, INP-Purpan, UPS – France</footnote>

<footnote n="2">² Institut des Maladies Métaboliques et Cardiovasculaires – Hôpital de Rangueil, Université Paul Sabatier (UPS) - Toulouse III, Inserm : U1048 – France</footnote>
<figure><img src="image_204.png" /></figure>

Poster 82 – P82

MetExploreViz : Nouvelles fonctionnalités et stratégie de visualisation

Maxime Chazalviel <fnref n="1" />, Nathalie Poupin<fnref n="2" />, Florence Vinson <fnref n="2" />, Clement Frainay <fnref n="2" />, Ludovic Cottret <fnref n="3" />, Adrien Rohan <fnref n="2" />,
Fabien Jourdan<fnref n="2" />

Le réseau métabolique d'un organisme regroupe toutes les réactions biochimiques qui peuvent se produire dans cet organisme, quelles que soient les conditions. Afin de visualiser ces réseaux, nous avons développé MetExploreViz, une bibliothèque javascript gratuite et open source. Nous avons implémenté de nouvelles fonctionnalités et proposons une stratégie améliorée pour interpréter des signatures métaboliques avec MetExploreViz.
Une visualisation plus complète et modifiable. MetExploreViz a été amélioré afin de produire des visualisations de réseaux métaboliques plus proches des visualisations conventionnelles des voies métaboliques et du format SBGN, un standard de visualisation de réseaux biochimiques. L'outil permet notamment de visualiser les cycles sous forme de cercles, de courber les arêtes et de prendre en compte les notions de substrats et de produits dans le dessin des réactions.

D'autres fonctionnalités permettent de mettre en évidence des éléments du réseau ou d'intégrer des données omics à la visualisation. Pour ce faire, MetExploreViz permet de modifier le style et la position des labels des nœuds de manière à les mettre en évidence ou d'éviter le chevauchement des nœuds. Il est également possible de mettre en évidence les voies métaboliques sur les arêtes pour apporter des informations fonctionnelles au réseau et d'importer et lier des images à un nœuds pour faire apparaître des informations sur les données.
Il est également possible de remplacer l'ensemble des métabolites et des réactions d'une voie métabolique par un seul nœud représentant la voie métabolique choisie de manière à alléger la visualisation. Cette fonctionnalité permet également de mettre en évidence les résultats du " pathway enrichment " de MetExplore.

Mots-Clés: Visualisation, Réseaux métaboliques, Interprétation

<footnote n="1">¹ MedDay Pharmaceuticals – MedDay Pharmaceuticals – France</footnote>

<footnote n="2">2 UMR1331 Toxalim – Université de Toulouse, INRA, ENVT, INP-Purpan, UPS – France</footnote>

<footnote n="3">³ Laboratoire des interactions plantes micro-organismes (LIPM) – CNRS : UMR2594, Institut national de la recherche agronomique (INRA) : UMR441 – Chemin de Borde-Rouge - BP 27 31326 CASTANET TOLOSAN CEDEX, France</footnote>
<figure><img src="image_205.png" /></figure>

Poster 83 – P83

# Annotation de nouvelles classes de glycérolipides par l'approche des réseaux moléculaires au sein du métabolome de la macroalgue brune *Taonia atomaria*

Nathan Carriot<fnref n="1" />, Benoît Paix <fnref n="1" />, Stéphane Greff <fnref n="2" />, Jean-François Briand <fnref n="1" />, Gérald Culioli <fnref n="1" />

Dans le milieu marin, toute surface immergée est rapidement colonisée par des bactéries, ainsi que par d'autres micro-organismes, qui forment des structures tridimensionnelles appelées biofilms. L'algue cosmopolite *Taonia atomaria* est un modèle biologique peu colonisé et dont certains métabolites de surfaces présentent une activité anti-adhésion vis-à-vis de bactéries marines (Othmani et al 2016). Dans le but de réaliser des études en écologie chimique visant à mieux comprendre les mécanismes de défense chimique de cette algue, une annotation exhaustive de sa production métabolique est donc essentielle. Lorsqu'elle est couplée à une approche phytochimique " classique ", l'annotation par réseaux moléculaires (via des données obtenues par LC-HRMS/MS) constitue un outil puissant qui peut conduire à l'identification potentielle d'un grand nombre de métabolites et ce, même dans le cas d'organismes peu étudiés. Dans leur globalité, les réseaux moléculaires obtenus via le serveur GNPS s'articulent en clusters (groupements de métabolites) représentant des familles de molécules formées du fait de la similarité de leurs spectres de masse. L'identification d'un métabolite décrit lors de l'analyse phytochimique permet ensuite d'annoter les autres métabolites au sein d'un même cluster. Dans le cas de *T. atomaria*, la construction initiale d'un réseau simplifié a conduit à l'identification de plusieurs dizaines de métabolites répertoriés en 14 familles chimiques, dont certaines se sont avérées constituées de lipides originaux tels que des glycérolipides portant un groupement de type géranylgéraniol, farnésol ou acide méthacrylique. La construction de réseaux de plus en plus complexes a permis finalement de caractériser plus de 200 métabolites. Cette première annotation détaillée du métabolome de *T. atomaria* permettra par la suite de mieux appréhender, lors d'études menées par métabolomique, les mécanismes de défense chimique et d'adaptation métabolique de cette algue vis-à-vis de microorganismes colonisateurs.

Mots-Clés: Réseaux moléculaires, Lipidomique, Glycérolipides, Algues brunes, Biofilms marins

<footnote n="1">¹ Laboratoire Matériaux Polymères Interfaces Environnement Marin (MAPIEM), EA 4323 – Université de Toulon : EA4323 – France</footnote>

<footnote n="2">² Institut méditerranéen de biodiversité et d'écologie marine et continentale (IMBE) – Institut de recherche pour le développement [IRD] : UMR237, Aix Marseille Université, CNRS : UMR7263, Université d'Avignon – France</footnote>
<figure><img src="image_206.png" /></figure>

## Poster 84 – P84

# Development of an untargeted approach based on isotope profiling of metabolic networks by high resolution mass spectrometry

Noemie Butin¹,², Maud Heuillet ¹,², Samuel Naudi-Fabra ¹,², Cécilia Bergès ¹,², Lindsay Peyriga ¹,², Jean-Charles Portais ¹,², Floriant Bellvert¹,²

¹ MetaToul-MetaboHUB, National infrastructure of metabolomics and Fluxomics, Toulouse, France –

² LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France – LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France – France

The study of metabolism has become a major research challenge for understanding the operation of biological systems (cells, tissues, organisms). Relevant approaches to study the topology and operation of metabolic networks are based on 13C isotope labelling strategies. For this purpose, the MetaToul-MetaboHUB platform has developed different approaches for a wide range of applications, including biotechnology and human health, such as measuring metabolic fluxes and the isotopic profiling based to follow the incorporation of 13C atoms into metabolites through targeted analysis of central and energetic metabolisms and amino acid metabolism.

Based on the analysis of a limited number of metabolites, current targeted approaches are very effective, however, they do not allow to highlight untargeted and by extend unsuspected metabolic pathways. For this reason and to increase the coverage of metabolome and the understanding of the biological systems, the platform wishes to develop an untargeted approach based on isotopic profiling. This approach aims at the characterization of the metabolic networks of biological organisms without a priori knowledge.

The implementation of this workflow is based in particular on the development of a software for automated data integration adapted to non-targeted analyses with 13C labelling. For this purpose, the preliminary step consists of an evaluation of pre-existing tools allowing the establishment of specifications. Then, the software developed by the platform according to these specifications will be integrated in emzed, an open source toolbox for rapid and interactive development of LCMS data analysis workflows in Python language in collaboration with ETH Zürich.

Mots-Clés: untargeted, 13C isotope labelling, automation
<figure><img src="image_207.png" /></figure>

## Poster 85 – Associé FP4

# MetGem Software for the Generation of Molecular Networks Based on the t-SNE Algorithm

Nicolas Elie<fnref n="1" />, Florent Olivon<fnref n="1" />, Gwendal Grelier<fnref n="1" />, Fanny Roussi<fnref n="1" />, Marc Litaudon<fnref n="1" />, David Touboul<fnref n="1" />

Metabolomic studies based on MS produce large amount of data requiring dedicated chemo/bioinformatics tools to explore them. Whereas many algorithms are available for the data treatment at MS1 level, methodologies for MS2 data are scarce. One of the most popular is the generation of molecular networks (MNs) through the calculation of cosine score on a collection of MS2 spectra available on the online GNPS platform. We introduce here dedicated software, called MetGem (https://metgem.github.io), allowing the generation of GNPS-like MNs together with a t-distributed stochastic neighbor embedding (t-SNE) based visualization of the cosine score matrix. Starting from the .mgf file, all spectra detected are compared to each other using the GNPS-based cosine score calculation system.

One way to represent these results is to conceive a square matrix gathering together high-dimensional objects, i.e. MS2 spectra, whose dimensions contain the similarities (cosine score values), taken pairwise, between all the spectra of the dataset. As it is difficult to apprehend these high-dimensional objects and visualize them in a meaningful manner, several manifold learning algorithms have been developed for dimensionality reduction purposes and pattern recognition. The idea developed herein was to feed the t-SNE algorithm with the pairwise similarity matrix. Considering that only small distances are reliable in high-dimensional spaces, t-SNE aggregates local data points closer in the lower-dimensional space. By focusing preferentially on the local structure of the data, t-SNE tends to extract better clustered local groups of point. Thus, it allows distinguishing easily patterns lying in different manifolds by simple visual analysis and in an unsupervised way.

Performances and advantages of the t-SNE output have been evaluated on two datasets, i.e. LC-MS2 analyses of fractions from a supercritical CO2 extraction of S. lineata leaves and a bark extract of C. peltatum. In both cases, t-SNE allows us to annotate more nodes compared to the GNPS-based cosine score calculation system and thus a deeper exploration of the experimental datasets was demonstrated.

Finally, in terms of network calculation and visualization, our interface running on laptop computer is up to 20 times faster for small and medium datasets (< 1000 MS2 spectra) and competes equally with GNPS for larger files (up to 35000 MS2 spectra).

Mots-Clés: cosine, similarity, MS2, t, SNE, molecular networking, python, desktop, chemo, informatics, bioinformatics, mass spectrometry, algorithm, metgem, mgf, GNPS, manifold, machine learning

<footnote n="1">¹Institut de Chimie des Substances Naturelles – Centre National de la Recherche Scientifique : UPR2301 – France</footnote>