# Poster 014: P014 Study on Alterations of Metabolome by Blood Transfusion using LC-Q-TOF and GC-MS

Yoon Young YANG<fnref n="1" />; Haiping WANG<fnref n="1" />; Sang Jun YOON<fnref n="1" />, Jeohg Hill Park<fnref n="2" />, Sung Won Kwon<fnref n="1" />,<fnref n="2" />,*

## Introduction

Blood transfusions improve somatic state of patient for a while but it has effects on immune and non-immune systems. Allogenic blood transfusions during surgery are related with various dangers like allergic reactions, incompatibility and coagulopathy. Although, there were studies about side effect of blood transfusions, the global metabolic changes were almost unknown.

## Technological and methodological innovation

In our study, metabolite changes produced by allogenic blood transfusions were researched applying our metabolomics technology in Lewis rats and 14SD rats. We operated liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF) and gas chromatography-mass spectrometry (GC-MS) for profiling metabolome after allogenic and autologous blood transfusions.

## Results and impact

Allogenic blood transfusions accelerate microenvironment change connected with relatively high expression of GLUT1/GLUT4. To support this event, there was abnormal expression of IL-6 and IRS1 which is glucose metabolism related enzyme. Also, in ABT groups, LysoPC and PLA2 were meaningfully changed compared to autologous groups. Synthetically, difference between autologous and allogenic blood transfusions was demonstrated. And connection with cancer associated metabolic changes was proved.

## References

[1] Lee, Seul Ji, et al. "Metabolomics Approach Based on Multivariate Techniques for Blood Transfusion Reactions." Scientific reports 9.1 (2019): 1740.

<footnote n="1">¹ College of Pharmacy, Seoul National University, Seoul, 08826, South Korea</footnote>

<footnote n="2">² College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, South Korea</footnote>
# Poster 015: P015 Dried Blood Spots for the Determination of Reduced to Oxidized Glutathione Status

Isabel TEN-DOMÉNECH<fnref n="1" />; Álvaro SOLAZ-GARCÍA<fnref n="1" />; Guillermo QUINTÁS<fnref n="2" />,<fnref n="3" />; Máximo VENTO<fnref n="1" />,<fnref n="4" />; Julia KULIGOWSKI<fnref n="1" />

## Introduction

Glutathione (GSH) to glutathione disulfide (GSSG) ratio is considered a clear indicator of intracellular redox status. GSH/GSSG determination in whole blood can be accomplished by liquid chromatography-mass spectrometry (LC-MS) after derivatization of GSH with N-ethylmaleimide (NEM). We propose an alternative collection procedure based on dried blood spot (DBS) testing in order to postpone laboratory processing providing accurate and reproducible results for both, GSH and GSSG.

## Technological and methodological innovation

An experimental procedure was developed for the determination of GSH/GSSG in DBS samples after *in-situ* derivatization with NEM. In this protocol, filter paper was treated with NEM solution prior to blood collection, thus initiating GSH derivatization immediately after sample withdrawal. Using only 10 µL of blood and after up to 24 h of storage at RT, extraction of target species (GSH, GSSG) was carried out and further analyzed by means of LC-MS.

## Results and impact

Levels of GSH and GSSG measured in DBS samples were comparable to those obtained by a reference method [1]. Filter paper impregnated with NEM could be stored at least for one week and the studied biomarkers were stable during at least 24 h at RT. DBS testing reduces the immediate manipulation of samples by clinical staff. We expect that this method will prevent an overestimation of GSSG and in turn, provide a means for an accurate determination of GSH/GSSG values reported in clinical trials.

## References

[1] Escobar, J. et al. 2016. J. Pharm. Biomed. Anal. 123, 104–112.

<footnote n="1">¹ Neonatal Research Group; Health Research Institute La Fe; Valencia; Spain</footnote>

<footnote n="2">² Health & Biomedicine, Leitat Technological Center; Barcelona; Spain</footnote>

<footnote n="3">³ Analytical Unit, Health Research Institute La Fe; Valencia; Spain</footnote>

<footnote n="4">⁴ Division of Neonatology; La Fe University and Polytechnic Hospital; Valencia; Spain</footnote>
# Poster 016: P016 Preliminary investigations of alkaloid composition from Thalictrum isopyroides plant of Azerbaijan Flora

Ilkin NASIRLI¹; Eldar QARAYEV²; Gaëtan HERBETTE; Sunbul MUSTAFAYEVA;

The General and Toxicological Chemistry Department
Azerbaijan Medical University

## Introduction

Alkaloids are high biological active compounds and secondary metabolites with a high pharmacological sphere. One of the plant species compositions with alkaloids of the Azerbaijan flora is Thalictrum isopyroides C.A. Mey. This plant is widely spread around the Greater Caucasus Mountains. It has been used as a folk medicine. It shows pharmacological activity such as anti-inflammatory, diuresis and anticancer effects [1] [3]. That is why it is beneficial to identify alkaloids of this plant.

## Technological and methodological innovation

The air-dried roots, leaves and seeds of T. isopyroides were collected, powdered and extracted with 95% ethanol three times and after removal of ethanol in vacuum the extract was moistened with a 15% ammonia solution and extracted with dichloroethane. Extracts of different parts were collected using reference [2]. Thin layer chromatography (TLC) was carried with chromatography solvent system: chloroform-methanol-25% solution of ammonia (8:2:0.1), plate: Merck 254, and detector: Dragondorff's reagent.

## Results and impact

In the work consequence of this division of T. isopyroides with the application, TLC has resulted and the retention factor (Rf) parameters are defined. From leaves, stalks of herb alkaloid extracts of T. isopyroides allocated and divided. Experimental researches by TLC, we have been identified from the roots (Ext. 1) - 6 spots (Rf - 0,19; 0,29; 0,36; 0,45; 0,51 və 0,74), from the upper ground parts (Ext. 2) - 3 spots (Rf - 0,12; 0,18 və 0,27). T. isopyroides plant research continues in this direction.

## References

[1] Jiang J.et al. Benzophenanthridine alkaloids from the roots of Thalictrum microgynum Lecoy. ex Oliv //Natural product research. – 2019. – T.33. – № 20. – C. 2964-2969.

[2] Gurunathan A., Subramaniam P. Preliminary phytochemical studies in the leaf, stem and root extracts of the traditional medicinal plant species, Thalictrum Javanicum Blume //Ruhuna Journal of Science. – 2014. – T. 5.

[3] Li D. H. et al. Antiproliferative dimeric aporphinoid alkaloids from the roots of Thalictrum cultratum //Journal of natural products. – 2017. – T. 80. – № 11. – C. 2893-2904.
**Poster 017: P017 Serum targeted and untargeted metabolomics-based analyses in infants with Ureteropelvic Junction Obstruction**

Olga BEGOU¹,², Antigoni PAVLAKI³, Olga DEDA²,⁴, Evangelia FARMAKI³, John DOTIS³, Helen GIKA²,⁴, Nikolaos RAIKOS⁴, Anna TAPARKOU³, Fotios PAPACHRISTOU³, Georgios THEODORIDIS¹,², Nikoleta PRINTZA³

1 Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki

2 Biomic_Auth, Bioanalysis and Omics Lab, Centre for Interdisciplinary Research of Aristotle University of Thessaloniki, Innovation Area of Thessaloniki, Thermi, 57001, Greece 3 Pediatric Nephrology Unit, First

Department of Pediatrics, Hippokration Hospital, Aristotle University of Thessaloniki

4 Laboratory of Forensic Medicine and Toxicology, School of Medicine, Aristotle University of Thessaloniki

**Introduction**

Ureteropelvic junction obstruction (UPJO) constitutes the predominant cause of obstructive hydronephrosis/neuropathy in both neonates and infants. Fundamental questions regarding the mechanisms of the diseases in the specific cohort remain still unanswered. The aim of this study was to elucidate potential differences through serum metabolic profiling of surgical cases of infants with UPJO compared to both non-surgical cases and healthy matched controls.

**Technological and methodological innovation**

Serum samples were collected from 19 patients preoperatively, 19 patients with mild stenosis treated conservatively, and 17 healthy matched controls and subjected to both targeted and untargeted metabolomics analyses by HILIC-MS/MS and RPHRMS, respectively. The combination of the two analytical platforms provided a holistic approach of serum metabolic profile of neonates and infants with UPJO, revealing a larger panel of biochemical pathways.

**Results and impact**

Both univariate and multivariate statistical analysis managed to highlight significant differences in the metabolic profile of the studied groups, distinguishing patients who required surgery from those followed by systematic monitoring as well as from healthy controls. A panel of metabolites, including creatinine, tryptophan, choline, and aspartate, showed high specificity of UPJO and could be used as early diagnosis biomarkers complementary to clinical practice.

**References**

[1] Smith et al., 2007, Pedriatic transplantation, 11, 366-373
# Poster 018: P018 Investigating urinary and faecal metabolic differences among native Africans, African Americans and Alaskans using mass spectrometry

Grace F. Barker¹, Alex Pechlivanis¹, Adesola Bello¹, James Kinross¹, Joram M. Posma¹, Jeremy K. Nicholson¹,², Stephen J.D. O'Keefe³, Jia V. Li¹

¹Imperial College London, UK, ²Murdoch University, Australia, ³University of Pittsburgh, USA

## Introduction

Colorectal cancer (CRC) is the 3rd most commonly diagnosed cancer worldwide however, there is marked geographical variation in incidence rates [1]. Differences in diet may contribute to this variation, specifically the consumption of a ‘Western’, high-fat and low-fibre diet. This diet-associated risk is, in part, mediated by the colonic microbiota, specifically through the metabolism of undigested dietary residues, resulting in the production of either pro- or anti-carcinogenic metabolites [2].

## Methods

To investigate the impact of these metabolites in mediating CRC risk, semi-targeted bile acid profiling and untargeted global profiling methods based on liquid chromatography-mass spectrometry (LC-MS) were used to elucidate the faecal and urinary metabolic differences between high- and low-risk CRC populations, with a view to identifying metabolites that contribute to/ help prevent CRC development.

## Results and impact

Analyses of global profiling data revealed significant differences between the metabolic profiles of high- and low-risk groups, partly driven by increased faecal butenylcarnitine in high-risk groups. Semi-targeted bile acid analyses showed a general increase in carcinogenic secondary bile acids in high-risk groups, together with increases in lithocholic acid derivative, 3ketocholanic acid in those consuming a high-fibre, low-fat diet, suggestive of potential microbiota mediated detoxification.

## References

[1] Arnold M. et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017; 66(4):683-691

[2] O'Keefe S.J. et al. Fat, fibre and cancer risk in African Americans and rural Africans. Nature Communications. 2015;6: 6342
# Poster 019: P019 Alterations in plasmatic levels of metabolites involved in purine, urea cycle and tryptophan metabolic pathways during right ventricular remodeling in chronic thromboembolic pulmonary hypertension

Thaïs HAUTBERGUE<fnref n="1" /> ; Samantha GUIMARON<fnref n="2" /> ; Julien GUIHAIRE<fnref n="2" /> ; François FENAILLE<fnref n="1" /> ; Christophe JUNOT<fnref n="1" /> ; Olaf MERCIER<fnref n="2" /> ; Benoit COLSCH<fnref n="1" />

## Introduction

Chronic thromboembolic pulmonary hypertension (CTEPH) is a complication of acute pulmonary embolism, leading to right ventricular (RV) failure and premature mortality if left untreated [1]. In this context, metabolomic analyses were performed on plasma samples of CETPH patients in order to help for patient stratification according to the severity of the disease before surgery. In addition, histological characteristics were conducted on RV biopsies to confirm fibrosis and capillary depletion.

## Technological and methodological innovations

Among 62 screened CETPH patients, 10 were rigorously selected according to their clinical characteristics and compared with 10 controls. Untargeted metabolomic and lipidomic approaches were performed on plasma samples by LC-HRMS using a C18 and a ZIC-pHILIC Column coupled to an Exactive mass spectrometer for metabolomics [2] and a C8 column coupled to a tribrid Orbitrap Fusion mass spectrometer for lipidomics [3]. Data processing was achieved using the Workflow4Metabolomics platform [4].

## Results and impact

Metabolomic results highlighted a dysregulation of the urea cycle, tryptophan and purine metabolisms in addition to significant histological alterations of RV in CETPH patients such as macrophagic infiltration suggesting inflammatory processes compare to controls without RV dysfunctions. These preliminary results will serve as a basis of a longitudinal study of CETPH to determine prognostic biomarkers of right ventricle reshaping after surgery based on pulmonary thromboendarterectomy.

## References

[1] Galie, N., et al., 2016, Eur Heart J, 37(1): p. 67-119

[2] Boudah, S., et al. 2014, J. Chromatogr B Analyt Technol Biomed Life Sci, 966: 34-47.

[3] Seyer, A., et al. 2016, Metabolomics, 12: 91.

[4] Giacomoni, F., et al., 2015, Bioinformatics, 31(9):1493-5.

<footnote n="1">¹ Service de Pharmacologie et Immuno-Analyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments, CEA, INRA, Université Paris Saclay, MetaboHUB, F-91191 Gif-sur-Yvette, France</footnote>

<footnote n="2">² Research and Innovation Unit, Inserm UMR-S 999, Marie Lannelongue Hospital, Paris-Saclay University, Le Plessis Robinson, France</footnote>
**Poster 020: P020 Molecular networking-based mass spectrometry to decipher
tumor cells metabolism**

Johana REVEL<fnref n="1" />; Lionel MASSI<fnref n="1" />; Johanna CHICHE<fnref n="2" />; Mohamed MEHIRI<fnref n="1" />; Jean-Ehrland RICCI<fnref n="2" />

**Introduction**

The understanding of mechanisms that regulate the formation and evolution of tumor cells requires a careful study of its endogenous cellular metabolism. Recent advances in mass-spectrometry and related bioinformatics tools are opening new doors to the study of tumor cells metabolism.

This work focuses on mass spectrometry-based metabolomics coupled to molecular networking to highlight the metabolic adaptive mechanisms and expand the metabolic repertoire of tumor cells.

**Technological and methodological innovation**

Mouse primary Eμ-Myc lymphomas cells were cultured [1] and metabolites were extracted using methanol:acetonitrile:water (40:40:20, v/v/v). We developed an ion-pair ultra-high-performance-liquid chromatography coupled to high-resolution mass-spectrometry (IP-UHLPC-HRMS) method for fingerprinting the specific metabolism of Bcell lymphoma. Molecular networking tools (GNPS; [2]) will be used to reinforce the DLBCL metabolic repertoire and metabolic pathways.

**Results and impact**

A database of 34 chosen metabolites from 6 distinct metabolic pathways was set up to develop an IP-UHPLC-HRMS method in a SMART manner (Specific, Measurable, Attainable, Realistic, Timely). Detection of those metabolites in B-cell lymphoma extracts using single ion monitoring followed by data dependant MS2 serves as a proof of concept for further targeting a larger (100-150) metabolic cohort.

Molecular networking analyses will then be applied to detect all the metabolites within the same metabolic pathway.

**References**

[1] Chiche et al. 2019. Cell Metabolism. 29:1-15.

[2] Wang et al. 2016. Nature Biotechnology. 34.8:828-837.

<footnote n="1">¹ Université Côte d'Azur, Institut de Chimie de Nice, UMR CNRS 7272, Nice, France</footnote>

<footnote n="2">² Université Côte d'Azur, INSERM, C3M, Nice, France</footnote>
Poster 021: P021 "-Omics- sciences and therapeutic"

Gaysinski Marc(1), Massi Lionel(1), Fontaine Vive Fabien(1), Mehiri Mohamed(1)

¹ Université Côte d'Azur, UMR7272, ICN, 06106 Nice cedex 2

**Introduction**

The aim of our poster is to present the capabilities of the new MetaboUCA platform of University Côte d'azur (UCA) in the field of "omics sciences". To illustrate this, we present 3 research projects.

**Technological and methodological innovation**

MetaboUCA is a platform of the Cote d'Azur University (UCA). It aims to coordinate scientific experts and heavy equipment (Nuclear magnetic resonance (NMR), Mass Spectrometry (MS)) around "omics" sciences. PFTC is an actor of MetaboUCA and is located in the Institute of Chemistry, Nice (ICN). The available techniques are NMR (500MHz, 400MHz and 200MHz), LC/MS/MS (2 systems), GC/MSn and molecular modeling.

**Results and impact**

In the first one, we investigated the metabolic composition of Anemonia viridis and its photosynthetic dinoflagellate endosymbionts in order to find biomarker of thermal stress. In the second one, we investigated the metabolic finger print of marine sponges to find relevant chemical biomarker in order to be able to discriminate Ircinia species. In the third one, we investigated the abilities of a new series of C-nucleoside structure to bind The A site RNA.

**References**

Johana Revel et al (2016) Comparative Biochemistry and Physiology, Part A: Molecular&Integrative Physiology, 191, 140-151

Jean-Patrick Joly et al (2019) Chem. Commun, 55, 10432--10435
**Poster 022: P022 Metabolomic study of commercial
mistletoe extracts used in alternative anticancer
therapy**

Cécile VANHAVERBEKE<fnref n="1" />; Sylvie MICHELLAND<fnref n="2" />; Valérie CUNIN<fnref n="2" />; Cécile MEUNIER<fnref n="3" />; Michel SEVE<fnref n="2" />; Audrey LEGOUELLEC<fnref n="4" />; Bertrand TOUSSAINT<fnref n="4" />; David TOUBOUL<fnref n="5" /> and Florence SOUARD<fnref n="1" />,<fnref n="6" />.

**Introduction**

Mistletoe is a parasitic plant reported to have anticancer activity and used in Europe as a complementary therapy. However, its effectiveness in treating cancer is not understood. Various companies sell extracts of mistletoe for injection by the subcutaneous route or directly intra-tumoral. Therefore, we undertook a metabolome study of these commercial extracts sold as food supplements to explore their composition considering the totum interest.

**Technological and methodological innovation**

An untargeted LC-HRMS metabolomics analysis was performed on 18 extracts of mistletoe commercial products from 3 producers at different concentrations.

A classical workflow was conducted on the online and freely available Workflow4Metabolomics (W4M) platform. For a better interpretation of the chemical space, molecular networking has been undertaken, using the user-friendly Metgem software. This approach aids in the recognition of structurally related chemical derivatives.

**Results and impact**

Multivariate exploration highlights metabolomic profiles significantly different from a company to another. Differences between extracts from a host plant to another are also observed. Moreover, our results clearly show that the composition of these extracts is very rich. Molecular networks allowed to separate and identify many classes of metabolites, such as amino acids, carbohydrates, carotenoids, diterpene alkaloids. Thus, their status of food supplements should really be questioned.

**References**

[1] De Vos, RC et al. 2007. Nat. Protoc. 2, 778-791

[2] Guijas, C et al. 2018. Nat. Biotechnol. 36, 316

[3] Fisher, S et al. 1996. Immunol. Lett. 52, 69-72

<footnote n="1">¹ Univ. Grenoble Alpes, DPM, 38000 Grenoble, France</footnote>

<footnote n="2">² Univ. Grenoble Alpes, CHU Grenoble Alpes, IBP, LBFA and BEeSy, INSERM U1055, 38000 Grenoble, France</footnote>

<footnote n="3">³ Univ. Grenoble Alpes, CHU Grenoble, IBP, 38000 Grenoble, France</footnote>

<footnote n="4">⁴ Univ. Grenoble Alpes, CHU Grenoble, IBP, TIMC, 38000 Grenoble, France</footnote>

<footnote n="5">⁵ CNRS, Institut de Chimie des Substances Naturelles UPR 2301, 91190 Gif-sur-Yvette, France</footnote>

<footnote n="6">⁶ Department of Pharmacotherapy and Pharmaceutics (DPP), Université libre de Bruxelles, Campus de la Plaine, CP 207,
Boulevard du Triomphe, Brussels 1050, Belgium</footnote>
**Poster 023: P023 Optimization of the untargeted metabolomics workflow for studying the impact of dysbiosis on the host**

Valérie CUNIN<fnref n="1" />; Dalil HANANNI<fnref n="3" />; Michel SEVE<fnref n="1" />; Pieter DORRESTEIN<fnref n="4" />; Audrey LE GOUELLEC<fnref n="2" />; Bertrand TOUSSAINT<fnref n="2" />

...

**Introduction**

The gut microbiota is known for its metabolic and nutritional functions, and moreover influences host immunity (1,2,3). This
by direct interactions at the level of the intestinal mucosa, but also via soluble molecules, and in particular the bacterial
metabolites. In order to study the impact of bacterial metabolites on immunity, mice were fed either with antibiotics or
prebiotics to modulate their microbiota composition. Then, we developed a workflow of non-targeted metabolomics for this
study.

**Technological and methodological innovation**

Mice (n= 18 mice/group) were fed with water, antibiotics or prebiotics. 10uL of plasma were deproteinized and analyzed by LC-MS/MS in positive mode on a QExactive plus connected to a Vanquish UHPLC System (ThermoFisher Scientific). The column used for the chromatography separation was an accucore RP column. Raw data were converted in mzXML with Proteowizard, then LC-MS feature detection and alignment was done with MZmine2.0 and statistical analysis with MetaboAnalyst 3.0 software.

**Results and impact**

We demonstrated that we got a rich metabolomics fingerprint from only 10 µL of mouse plasma. We obtained more than 700 features after data curation. Each group was well distinguished with a predictivity of 96.3% in an OPLS-DA model. We annotated 118 metabolites with GNPS and highlighted VIP metabolites (Amino acids metabolism, Tryptophan catabolites, bile acids ...). Our workflow is suitable to understand modifications of the metabolomic activity caused by a modulation of gut microbiota.

**References**

Routy et al, 2018, Science. 359: 91-97

Frankel et al, 2017, Neoplasia. 19 (10): 848-855 [3] Johnson et al, 2016, Cancer Res. 76 (21): 6146-6152

<footnote n="1">¹ Univ. Grenoble Alpes, CHU Grenoble Alpes, LBFA and BEeSy INSERM U1055, Grenoble, France</footnote>

<footnote n="2">² Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, Grenoble, France</footnote>

<footnote n="3">³ Univ. Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, Grenoble, France</footnote>

<footnote n="4">⁴ Skaggs of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, San Diego, CA, USA</footnote>
# Poster 024: P024 Rapid extraction and High-Resolution Tandem Mass Spectrometry analysis of the acylCoA metabolome

Cécile MEUNIER<fnref n="1" />; Nour ZEAITER<fnref n="3" />; Saadi KHOCHBIN<fnref n="4" />, Patrice FAURE<fnref n="1" />, Audrey LE GOUELLEC<fnref n="2" />, Bertand TOUSSAINT<fnref n="2" />, Uwe SCHLATTNER<fnref n="3" />

## Introduction

Thioesters of Coenzyme A (CoA) carrying different acyl chains (Acyl-CoAs) may play a critical role in epigenetics and cell signalling. Their steady state concentrations and fluxes seem to reflect the metabolic state of a cell, determined by the genetic background and the environment, in particular fuel supply. One difficulty is the large concentration differences between acyl-CoA species and their varying chemical properties, ranging from very hydrophobic to rather hydrophilic chains. Here we attempt to obtain the acyl-metabolome of the lymphoblast cell line Reh, derived from acute lymphocytic leukemia, and to compare clones differing in their metabolism due to the knockdown of a mitochondrial gene.

## Technological and methodological innovation

We first established a rapid extraction and metabolic quenching method to preserve endogenous acyl-CoA levels, followed by a novel high-resolution LC-MSMS method. The characterization of CoA and related substances was performed on a Vanquish UPLC system coupled to a Q Exactive plus Orbitrap mass spectrometer. The LC-MSMS method is based on a 24 min total run and using a reverse phase-chromatographic gradient. The dry cell pellets were dissolved into 30 µL of 30 mM ammonium acetate pH 6.5 containing 20% of methanol. Samples were centrifuged at 14000xg at 4°C during 3 min and the supernatant was transferred directly into LC vials. The injection volume was 10µL.

## Results and impact

In the present work, we aimed at establishing an useful LC-MS/MS method for the profiling of a set of different acyl-CoAs in the lymphoblast cell line Reh that differ in their mitochondrial metabolism. Moreover, the use of Parallel Reaction Monitoring (PRM) technique allowed us to identify with high resolution and high precision a panel from short to long chain of acyl-CoAs.

## References

[1] S.Neubauer et al. 2015. *Anal.Bioanal.Chem.* 407(22):6681-8

[2] X. Liu et al. 2015. *Molecular & Cellular Proteomics.* 14 and 1489-1500

This work is supported within the Idex CDP SYMER project by the French National Research Agency ANR (Investissements d'Avenir ANR-15-IDEX-02).

<footnote n="1">¹ Biochimie Hormonale et Nutritionnelle (BHN), Service de Biochimie, Biologie Moléculaire, Toxicologie Environnementale (SB2TE), CHUGA Grenoble, France</footnote>

<footnote n="2">² Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, Grenoble, France</footnote>

<footnote n="3">³ Univ. Grenoble Alpes and INSERM U1055, Laboratory of Fundamental and Applied Bioenergetics (LBFA) and SFR Environmental and Systems Biology (BEeSy), Grenoble, France</footnote>

<footnote n="4">⁴ Epigénétique et Signalisation Cellulaire, Institut Albert Bonniot (IAB) Grenoble, France</footnote>
# Poster 025: P025 Serum metabolic profiling following traumatic brain injury in rats using 1H Nuclear Magnetic Resonance spectroscopy

Flavia VALEO¹,², Rita CAMPOS-PIRES¹,³,⁴, Paul S. SOUMALIAS¹, Laura MARTINEZ-GILI², Julien CHILLOUX², Robert DICKINSON¹,³, Marc-Emmanuel DUMAS²

1 Anaesthetics, Pain Medicine and Intensive Care Division, Department of Surgery and Cancer, Imperial College London, United Kingdom

2 Systems Medicine Division, Department of Metabolism, Digestion and Reproduction, Imperial College London, United Kingdom

3 Royal British Legion Centre for Blast Injury Studies, Department of Bioengineering, Imperial College London, United Kingdom

4 Charing Cross Hospital Intensive Care Unit, Critical Care Directorate, Imperial College Healthcare NHS Trust, United Kingdom

## Introduction:

Traumatic brain injury (TBI) is a complex disorder associated with short- and long-term neurological dysfunction. Subtle symptomology is a challenge in the management of mild TBI, that is commonly misdiagnosed, hence the need for biomarkers of injury severity. Circulating blood metabolites may be markers of pathological pathways altered in response to TBI. Here we use a 1H Nuclear Magnetic Resonance (NMR) spectroscopy biomarker approach to assess the metabolome of varying TBI injuries in rats.

## Technological and methodological innovation:

Sprague-Dawley rats (n=28) were randomly assigned to naïve (n=4) or to TBI groups with different Controlled Cortical Impact (CCI) injury parameters (speed (m/s), depth (mm), tip shape): 6m/s, 2mm, flat (n=8); 8m/s, 1mm, flat (n=8); 8m/s, 2mm, flat (n=4); 8m/s, 2mm, round (n=4). Brains and serum samples were collected at 15min (n=12) or 24h (n=12) postCCI for histological and 1H NMR analysis respectively. Multivariate statistics was used to assess serum metabolic changes with injury severity.

## Results and impact:

Histological evaluation showed that different CCI injury parameter combinations result in different lesion sizes at 15min and 24h with differential injury development. Multivariate modelling of 1H NMR serum data showed that the metabolic signature of the injury groups correlates with lesion volume measurements as early as 15min post-injury. This data suggests a predictive ability of the metabolome for lesion size and development, relevant for the clinical diagnosis of TBI injury severities.
# Poster 026: P026 Surface analysis of lipids in rat mesenteric fat tissue sections by MALDI-MS imaging to understand the impact of obesity

Susana Alejandra PALMA DURAN<fnref n="1" />; Zoe HALL<fnref n="2" />, and Julian L. GRIFFIN<fnref n="1" />, <fnref n="2" />

...

## Introduction

In the obese state, excessive fat accumulation affects adipose tissue homeostasis, modifies the secretion of adipokines and cytokines by adipocytes, and favors the recruitment of immune cells [1]. The current interest in lipoinflammation has led to interest in understanding the heterogeneity and molecular remodeling of adipose tissue microenvironments. However, the spatial localization of lipids is lost when using traditional mass spectrometry as lipids are homogenized and extracted from tissue.

## Technological and methodological innovation

MALDI LTQ Orbitrap XL MS (Thermo Fisher Scientific) was used to characterize the distribution of lipids in mesenteric fat of Sprague-Dawley rats under PPAR-pan activator (0-1000mg/kg/d) treatment [2]. Tissue sections (15µm) were sprayed with matrix solution of 2,5-dihydroxybenzoic acid (DHB), norharmane, α-cyano-4-hydroxycinnamic acid (CHCA) and 2,4,6-trihydroxyacetophenone (THAP). Spectra between 200-1000 m/z were collected at 50µm step increments and 60,000 resolution.

## Results and impact

MALDI-MS imaging allowed the visualization and mapping of 40 to 90 lipids in positive mode, including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, lyso-phosphocholine, phosphatidic acids, diacylglycerides and triacylglycerols (TG). Adipocytes presented a characteristic distribution of lipids and were identified as a TG-rich region. The data will be displayed and discussed to derive knowledge of the lipid species and distribution within mesenteric fat tissue.

## References

[1] Caputo et al. 2017. FEBS lett. 591 and 3061-3088

[2] Ament et al. 2016. Free Radical Biology and Medicine. 95 and 357-368

<footnote n="1">¹ The Department of Biochemistry, University of Cambridge, Cambridge, UK.</footnote>

<footnote n="2">² The Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK</footnote>
**Poster 027: P027 Combined transcriptomics–metabolomics profiling of the heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus**

Ana M. ESTEVES<fnref n="1" />, Gonçalo GRACA<fnref n="1" />, Lindsay PEYRIGA<fnref n="2" />,<fnref n="3" />, Inês M. TORCATO<fnref n="1" />, Nuno BORGES<fnref n="1" />, Jean-Charles PORTAIS<fnref n="2" />,<fnref n="3" />,<fnref n="4" /> & Helena SANTOS<fnref n="1" />

**Introduction**

Microorganisms are frequently challenged by various environmental stresses that can result in structural damage and ultimately in disruption of vital cellular functions [1]. To survive, cells must be able to sense, respond, and adapt rapidly once sudden and adverse conditions arise. The archaeon Pyrococcus furiosus, which grows at 99°C, is regarded as a useful hyperthermophilic model. To better understand the adaptation strategies of this organism to thrive at such high temperatures, we used state-of-the-art omics approaches to investigate the metabolic response of P. furiosus to heat stress.

**Technological and methodological innovation**

To gain insight into how this model hyperthermophile copes with heat stress, we compared transcriptomic and metabolomic data of cells subjected to a temperature shift from 90 °C to 97 °C. In this study, we used RNA-sequencing to characterize the global variation in gene expression levels, while nuclear magnetic resonance (NMR) and targeted ion exchange liquid chromatography–mass spectrometry (LC–MS) were used to determine changes in metabolite levels.

**Results and impact**

At the transcriptomic level, 257 were upregulated and 295 were downregulated. At the metabolite level, 37 compounds were quantified. The level of di-myo-inositol phosphate, a canonical heat stress solute among marine hyperthermophiles [2], increased considerably (5.4-fold) at elevated temperature. Also, the levels of activated sugars were enhanced and it was concurrent with transcriptomics data. This work provides the first metabolomic analysis of the heat shock response of a hyperthermophile.

**References**

[1] Richter K et al. 2010. Mol Cell 40:253–266

[2] Esteves AM et al. 2014. Appl Environ Microbiol 80:4226–4233

<footnote n="1">¹ Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa Oeiras Portugal</footnote>

<footnote n="2">² TBI, Université de Toulouse, CNRS, INRA, INSAToulouseFrance</footnote>

<footnote n="3">³ MetaboHUB-MetaToul, National Infrastructure of Metabolomics and Fluxomics Toulouse France</footnote>

<footnote n="4">⁴ STROMALab – Université de Toulouse, CNRS ERL5311, EFS, INP-ENVT, Inserm U1031, UPS</footnote>
**Poster 028: P028** *Alternative carbon sources for the central metabolism of Trypanosoma brucei procyclic form*

Oriana VILLAFRAZ<fnref n="1" />, Nicolas PLAZOLLES<fnref n="1" />, Marc BIRAN<fnref n="2" />, Edern CAHOREAU<fnref n="3" />, Michael P. BARRETT<fnref n="4" />,<fnref n="5" />,
Emmanuel TETAUD<fnref n="1" />, Michael BOSHART<fnref n="6" />, Jean-Charles PORTAIS<fnref n="3" />, Frédéric BRINGAUD<fnref n="1" />

**Introduction**

The parasite Trypanosoma brucei adapts to the conditions encountered during the life cycle by remodeling its metabolism. In the glucose-free midgut of the insect vector, the procyclic form develops an elaborated energy metabolism with proline as a major carbon and energy source [1]. Intermediates from the TCA cycle like **α*-ketoglutarate (α-KG), succinate and to a lesser extent malate, can stimulate the oxygen uptake [2]. Here we explored in detail the metabolism of these three carbon sources.

**Technological and methodological innovation**

In this work, we used a dozen of T. brucei mutants impaired for enzymes of the TCA cycle, proline degradation and acetate production pathways in combination with isotopic profiling of the exometabolome by proton (1H) NMR analysis [3]. The assay allows distinguishing 13C-enriched from 12C-non enriched molecules. We used combinations of D-[U-13C]-glucose, L-[U-13C]-proline, succinate, malate and **α*-ketoglutarate to quantify the compounds consumed and the final products excreted by the parasites.

**Results and impact**

We found that T. brucei re-metabolize succinate excreted from glucose degradation. In addition, malate and α-KG are metabolized and stimulate growth. Strikingly, α-KG is consumed 10 times faster than glucose, probably due to maintenance of the mitochondrial redox balance at the substrate level. By testing the three metabolites using 1H NMR analysis with mutants of enzymes from the TCA cycle, we show for the first time evidence supporting the functionality of a complete TCA cycle in trypanosomes.

**References**

[1] Mantilla et al. 2017. PLoS Pathogens. 13 (1) art. no. e1006158.

[2] Ryley, J. F. 1962. Biochem. J. 85:211-23.

[3] Millerioux et al. 2013. Mol Microbiol. 90(1):114-29.

<footnote n="1">¹ Laboratoire de Microbiologie Fondamentale et Pathogénicité (MFP), Université de Bordeaux, CNRS UMR-5234,
Bordeaux, France</footnote>

<footnote n="2">²Centre de Résonance Magnétique des Systèmes Biologiques (CRMSB), Université de Bordeaux, CNRS UMR-5536,
Bordeaux, France</footnote>

<footnote n="3">³ LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, F-31077, Toulouse, France</footnote>

<footnote n="4">⁴Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</footnote>

<footnote n="5">⁵Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, Garscube Campus, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</footnote>

<footnote n="6">⁶Fakultät für Biologie, Genetik, Ludwig-Maximilians-Universität München, Grosshadernerstrasse 2-4, D-82152
Martinsried, Germany</footnote>
# Poster 029: P029 Characterization of bioherbicidal compounds stemming from microalgae diversity by differential metabolomic and spectral similarity network assisted dereplication (ALL DREAM)

Chaïb Slimane¹, Raviglione Delphine¹, Vialleix Carole³, Levert Annabel², Cadoret Jean-Paul³, Bertrand Cédric¹, Bonnard Isabelle¹

¹PSL Research University EPHE-UPVD- CNRS, USR 3278 CRIOBE, LabEx-CORAIL, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

²AKINAO, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

³GREENSEA, Promenade du Sergent Navarro, 34140 Mèze, France

Responding to public concerns about the impact of pesticides on health and the environment, European legislation has led to the development of new strategies to assess and limit the potential impact of different agricultural practices. Thus, the development of new "green" practices such as the use of biocontrol products are booming. In this context, biomass from the production of photosynthetic microorganisms can be an alternative to highlight. Indeed, micro-algae constitute, within the marine environment, a source of bioactive molecules. Micro-algae metabolites are active on a wide range of biological targets and can be recycled in various ways: bio-pesticide, cosmetic, pharmaceutical, etc.

"ALL DREAM" project is aimed to develop an innovative tool adapted to the analysis of the chemical diversity from the microalgae and evaluation of their potential as a bio-control product. This project proposes to combine, on the one hand, differential metabolic fingerprints and, on the other hand, analysis of the molecular diversity maps drawn by the study of "molecular networks. The "molecular network" is an innovative bio-informatics approach that allows characterizing classes of metabolites based on their spectral data and studying their degrees of similarity.

Through the use of molecular networks, data and complex mixtures can be compared in silico and annotated. This approach facilitates the study of the metabolome of living organisms and the characterization by dereplication of the studied active metabolites.

For this project, we selected 150 extracts of microalgae from various families that were subjected to a microplate photosynthesis inhibition as well as antioxidant assay. Their chemical fingerprints were analysed by a meabolomic approach and spectral similarity networking to facilitate the annotation and characterization of bioactive compounds. The data was first processed via W4M platform and R software, then subjected to a comparative analysis between the MetGem and GNPS platforms and finally processed under Cytoscape for their annotation and VIP search. Second, active extracts were subjected to bio-guided fractionation via an LC-PDA-ELSD coupled with a fraction collector and LC-HRMS to confirm the characterization of active metabolites. The active extracts are then tested on young seedlings to analyse their herbicide effect.

This poster presents all the results highlighting the potential of this "blue" resource that are microalgae as well as the "green" alternative they represent for the agriculture of the future.

## References:

* Idaira Jerez-Martel et al. 2017. Phenolic Profile and Antioxidant Activity of Crude Extracts from Microalgae and Cyanobacteria Strains (DOI: 10.1155/2017/2924508)

* Justine Chervin et al. 2017. Dereplication of natural products from complex extracts by regression analysis and molecular networking : case study of redox-active compounds from Viola alba subsp. (DOI: 10.1007/s11306-017-1227-6)

* John Berry, 2011. Marine and Freshwater Microalgae as a Potential Source of Novel Herbicides. (DOI: 10.5772/12942)

* Legrand C et al. 2003. Allelopathy in phytoplankton –biochemical, ecological and evolutionary aspects. (DOI: 10.2216/i00318884-42-4-406.1)

**Funding source:** Region/FEDER scholarship, AKINAO, GREENSEA
Poster 030: P030 Threonine metabolism in Leishmania

Chloé Morel¹, Marc Biran², Marc Ouellette³, Daniel Inaoka⁴, Michael Barrett⁵, Richard Burchmore⁵, Frédéric Bringaud¹, Emmanuel Tétaud¹

1 iMET team, Microbiologie Fondamentale et Pathogénicité, CNRS UMR5234, University of Bordeaux,
Bordeaux, France

2 Centre de Résonance Magnétique des Systèmes Biologiques, UMR5536 CNRS, Bordeaux, France

3 Centre de Recherche en Infectiologie du Centre de Recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Québec, Canada.

4 School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan; Graduate School of

Medicine, The University of Tokyo, Tokyo 113-0033, Japan

5 Institute of Infection, Immunity and Inflammation and Glasgow Polyomics, College of Medical, Veterinary & Life Sciences, University of Glasgow, UK.

The parasitic protozoan Leishmania is responsible for various clinical manifestations in humans. Deletion of the glucose transporter genes in the Leishmania ☐GT1/3 mutant1 induced a possible shift to amino acid metabolism, and seemed to point to threonine as a new carbon source. Indeed, serine hydroxymethyltransferase (SHMT) and aldehyde dehydrogenase (ALDH) are two

possible enzymes of threonine catabolism found overexpressed2 in the ☐GT1/3 mutant.

By combining different approaches, i.e. overexpression of SHMT and ALDH, gene inactivation using CRISPR-Cas9 technique and NMR analysis of metabolites excreted by the parasite using enriched carbons sources (threonine), we were able to study the involvement of these enzymes in the energy metabolism of the parasite.

We have demonstrated that ALDH is indeed involved in the catabolism of threonine by catalyzing the transformation of acetaldehyde into acetate, while SHMT does not seem to be involved in this metabolic pathway. However, we identified a third enzyme, threonine aldolase (TA), which converts threonine into acetaldehyde, thus allowing the reconstitution of threonine degradation pathway in Leishmania. Future studies will focus on the requirements of these enzymes over the parasite's life cycle.

Burchmore, R. J. S. et al. Genetic characterization of glucose transporter function in Leishmania mexicana. Proc. Natl. Acad. Sci. 100, 3901–3906 (2003).

Feng, X. et al. Remodeling of protein and mRNA expression in Leishmania mexicana induced by deletion of glucose transporter genes. Mol. Biochem. Parasitol. 175, 39–48 (2011).
# Poster 031: P031 Fluorinating Putida: Annotating Novel and Undesired Metabolites

Corey M. GRIFFITH<fnref n="1" />; Jean-François CONROTTE<fnref n="1" />; Mariela P. MEZZINA²; Emma L. SCHYMANSKI<fnref n="1" />; Carole L. LINSTER<fnref n="1" />

## Introduction

SinFonia, an EU H2020 project, aims to integrate the nonnative element fluorine into the metabolism of Pseudomonas putida to produce novel fluorinated biopolymers [1]. Metabolic rewiring may result in an accumulation of non-canonical metabolites that induce toxicity or cause poor yield [2,3]. To identify these byproducts, non-targeted mass spectrometry will be used to screen for fluorinated and undesirable metabolites in engineered bacteria.

## Technological and methodological innovation

Engineered systems may lack sufficient metabolite repair capacity, which we aim to counteract by screening for metabolite damages in our cell factories and envisaging strategies to repair them. Metabolite repair may be particularly important in SinFonia due to the load of adding heterologous pathways and a nonnative element to create new-to-nature products. If successful, SinFonia will provide a less hazardous and more sustainable solution to synthesizing fluorochemicals.

## Results and impact

Untargeted LC-MS and GC-MS was used to measure metabolic differences in engineered P. putida strains induced or not to produce polyhydroxyalkanoates, the chemical backbone in which we aim to incorporate fluorine. Prioritize features were annotated using internal and external libraries, metabolic models, and MetFrag [4]. These methods will be used to identify undesirable (fluoro)metabolites and construct metabolite repair modules to suppress toxicity and maximize polymer accumulation.

## References

[1] Thuronyi, B. et al. 2017. Angewandte Chemie. 44, 13825-13828

[2] Linster, C. et al. 2013. Nature Chemical Biology. 9, 72-80

[3] Sun, J. et al. 2017. Metabolic Engineering. 44, 150-159

[4] Ruttkies, C. et al. 2016. Journal of Cheminformatics. 8, 3

<footnote n="1">¹ University of Luxembourg, Luxembourg Centre for Systems Biomedicine</footnote>

<footnote n="1">¹ The Novo Nordisk Foundation Center for Biosustainability, Lyngby, Denmark</footnote>
**Poster 032: P032 Analysis of metabolome time- variations of an endophytic fungal strain of Botryosphaeria mamane through untargeted LC-MS profiling**

Romina PACHECO¹; Mohamed HADDAD¹; Patricia JARGEAT²; Billy CABANILLAS³; Carlos AMASIFUEN⁴; Nicolas FABRE¹; Marieke VANSTEELANDT¹

¹UMR 152 Pharma Dev, Université de Toulouse, IRD, UPS, France

²CNRS, IRD, France

³Unidad de Investigación en Productos Naturales, LID, UPCH, Lima, Peru

⁴Un (UNTRM), FICIAM, Chachapoyas, Peru

**Introduction**

The metabolome of all organisms change overtime as a result of all the dynamic biochemical processes involved in metabolism [1]. Nevertheless, fungi metabolome has not been extensively studied in terms of time- variations, which is an important factor to consider in gaining access to a greater chemical diversity [2]. This work aimed at studying the metabolome variations of an endophytic strain of Botryosphaeria mamane (BM) with a focus on 3 bioactive thiodiketopiperazines already isolated [3].

**Technological and methodological innovation**

Botryosphaeria mamane was cultured during 28 days at 27°C under natural light in 6-well plates containing malt extract agar. Each fungal culture (from day 1 to 28) was extracted with DCM-MeOH-H2O 64:36:8 (v/v/v) and crude extracts were then analyzed by UHPLC-DAD-LTQ Orbitrap XL. MZmine 2.51 and Metaboanalyst 4.0 software were used for data treatment and data analysis, respectively.

**Results and impact**

Chemical profiles highlighted global variations in the BM metabolome due to the production and metabolization of secondary metabolites. Incubation time is an important parameter to consider in the search for new compounds such as the maximum observed detection of the TKP's, between day 5 and 7. Moreover, the TKP's appeareance dynamic could give us insights to understand their pathways in order to optimize the production of these bioactive metabolites.

**References**

[1] Wignen, H. et al. 2006. Annual Review of Genetics, 40, 409-48

[2] Roullier, C. et al. 2016. Marine Drugs. 14, 103

[3] Barakat, F. et al. 2019. Phytochemistry. 158, 142-148
# Poster 033: P033 Study of the production of (phyto)hormones by mycorrhizal fungi

Simon PONS <fnref n="1" />,<fnref n="2" />, Sylvie FOURNIER <fnref n="1" />,<fnref n="2" />, Christian CHERVIN ³, Guillaume BÉCARD <fnref n="1" />, Nicolas FREI-DIT-FREY <fnref n="1" /> & Virginie PUECH-PAGÈS <fnref n="1" />,<fnref n="2" />

## Introduction

Arbuscular mycorrhizal fungi (AMF) live symbiotically with 72% of land plant species for which they have played and still play a major nutritional role.

Phytohormones are involved in the establishment of this symbiosis, but their role has been mainly examined from the plant side. Since other fungi were reported to produce phytohormones [1], and putative receptors of cytokinines and ethylene were identified in AMF [2], we wondered if AMF produce and/or perceive phytohormones.

## Technological and methodological innovation

A protocol of global extraction and enrichment of phytohormones was adapted to different fungal samples, and their detection and identification by LC-MS was optimized. Isotopic labelling assays were performed to establish their fungal or plant biosynthetic origin. Ethylene production and biosynthesis pathways were investigated by GC-FID. Cytokinin and ethylene effects were assessed by bioassays and RNAseq.

## Results and impact

We identified for the first time an AMF production of ethylene, and an AMF exudation of several phytohormones. Identification of the metabolic origin(s) of these hormones and their role in AMF biology and in AM symbiosis still require further investigation.

This work is the first step toward the study of phytohormones as inter-kingdom mediators and genuine fungal hormones.

## References

[1] Chanclud & Morel. 2016. Molecular Plant Pathology 17: 1289 - 1297
[2] Hérivaux et al. 2017. mBio. e01739-16

<footnote n="1">¹ Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Castanet-Tolosan, France</footnote>

<footnote n="2">² Plateau MetaToul-Métabolites végétaux, Université de Toulouse, CNRS, UPS, Castanet-Tolosan, France ³ Génomique et Biotechnologie des Fruits, INRA/INP-ENSAT, Castanet-Tolosan, France</footnote>
**Poster 034: P034 Metabolites characterization of nine cyanobacteria isolated from thermal mud.**

Justine DEMAY¹; Anita REINHARDT²; Cécile BERNARD¹; Benjamin MARIE¹.

¹ UMR 7245 MCAM, Muséum National d'Histoire Naturelle-CNRS, Paris, 12 rue Buffon, CP 39, 75231 Paris CEDEX 05, France; justine.demay1@mnhn.fr; benjamin.marie@mnhn.fr; cecile.bernard@mnhn.fr
² Thermes de Balaruc-les-Bains, 1 rue du Mont Saint-Clair BP 45, 34540 Balaruc-Les-Bains, France; anita.reinhardt@thermesbalaruc.com

**Introduction**

Cyanobacteria are photosynthetic microorganisms that colonize diverse environments worldwide, ranging from ocean to freshwaters, soils, and extreme environments. Their adaptation capacities and the diversity of natural products that they synthesize support cyanobacterial success in the colonization of their respective ecological niches. Although cyanobacteria are well-known for their toxin production and their relative deleterious consequences, they also produce a large variety of molecules that exhibit beneficial properties with high potential in various fields [1].

**Technological and methodological innovation**

As part of my thesis project (CIFRE project in partnership with the thermal institute of Balaruc-Les-Bains, France), we decide to study the diversity of metabolites produced by 9 cyanobacteria isolated from thermal mud. We used two approaches: metabolites prediction by metagenomics analysis and identification of metabolites produced by LC-MS/MS. We decided to check 3 extraction methods (water, acetone and methanol) according to the large chemical diversity of cyanobacterial metabolites [2] [3].

**Results and impact**

We showed that the genomic potential for secondary metabolite is important for the 9 cyanobacteria. Surprisingly, there is a lot of unknown cluster into our metagenomes, despite the growing number of cluster describes until now [4]. As expected, we showed that there is a huge difference in metabolites extracted between the 3 methods used. Surprisingly, acetone extraction (often used in plant extraction) is not the most effective one, with a weak number of metabolites extracted. We also confirm the absence of cyanotoxins (through the use of standards to identify them) which is important for further development and exploitation of the cyanobacteria.

**References**

Max 5

[1] Demay J. et al. 2019. Marine Drugs. 17, 320

[2] Shah et al. 2017. Marine Drugs. 15, 364

[3] Kim Tiam et al. 2019. Toxins. 11, 498

[4] Dittmann et al. 2015. Trends in Microbiology. 23, 642-652
**Poster 035: P035 Metabolomics in plant-microbe interactions: "MetaboHUB-MetaToul-plant metabolites" platform facilities**

Sylvie FOURNIER¹, Justine CHERVIN¹, Salimata MABONGO DIARRASSOUBA¹, Marielle AGUILAR¹, Virginie DURAND¹, Simon PONS¹, Ophélie FRAISIER-VANNIER², Guillaume MARTI¹, Virginie PUECH-PAGES¹

¹ MetaboHUB-MetaToul-Plant Metabolites Platform, Laboratoire de Recherche en Sciences Végétales (LRSV)
UMR 5546, Université de Toulouse UPS CNRS, 24 chemin de Borde Rouge, Auzeville, BP 42 617, 31 326
Castanet-Tolosan, France ² IRIT, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France

The MetaboHUB-MetaToul-Plant Metabolites (MV) facilities hosted at the LRSV belong to the Metabolomics and Fluxomics platform of Toulouse and is part of the French resource core MetaboHUB. The MV platform provides research and development services dedicated to the characterization of metabolomes from plants, fungi or bacteria in the field areas of agrobiosciences and phytochemistry.

Targeted quantitative approaches are routinely available such as phytohormonomics which allows the detection and quantification of more than thirty phytohormones from plant (roots, aerial samples) or microbes in a single experiment. In addition, other classes of plant secondary metabolites are reachable or under development (signaling molecules, elicitors derived of amino acids, blumenol...), or specific fungal and bacterial metabolites (chitinic molecules).

A dedicated workflow for untargeted metabolomics has been developed in the platform to provide a global metabolite mapping from complex extracts and detect compounds of particular interest with the use of specific multivariate data analysis. This approach is endorsed to our in-house databases of more than 1000 standards and an in silico database of 500 000 unique metabolites to improve annotation rates.

**References**

www.lrsv.ups-tlse.fr/metatoul/

www.metatoul.fr

www.metabohub.fr
**Poster 036: P036 Investigating the effects of future climate scenarios in soft fruit: application of HPLC-PDA-MS metabolite profiling in blackcurrant cultivated under increased temperature and altered light regimes**

Introduction J. William Allwood¹; Tomasz L. Woznicki²,³; Yun Xu⁴,⁵; Alexandre Foito¹; Kjersti Aaby⁶; Julie

Sungurtas¹; Sabine Freitag¹; Royston Goodacre⁴,⁵; Derek Stewart¹,⁷; Siv F.

Remberg³; Ola M. Heide⁸; Anita Sønsteby²

¹ Environmental and Biochemical Sciences, James Hutton Institute, Invergowrie, Dundee, Scotland DD2 5DA, UK

2 NIBIO, Norwegian Institute of Bioeconomy Research, Pb 115, 1431 Ås, Norway

3 Department of Plant Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, 1432 Ås, Norway

4 School of Chemistry, Manchester Institute for Biotechnology, University of Manchester, Princess Street, Manchester M1 7DN, UK

5 Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK

6 Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, 1430 Ås, Norway

7 School of Engineering and Physical Sciences, Institute of Mechanical, Process and Energy Engineering, Heriot-Watt University, Edinburgh, Scotland EH14 4AS, UK

8 Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, 1432 Ås, Norway

**Introduction**

Blackcurrant (Ribes nigrum L.) is an excellent example of a super fruit with potential health benefits. Both genotype and cultivation environment (temperature, solar radiation and precipitation) are known to affect the chemical composition of blackcurrant, especially ascorbic acid and various phenolic compounds (e.g. anthocyanins amongst other quality traits). The objective was to deepen understanding of the effects of temperature and day length, on fruit quality and chemical composition. The studies relevance is accentuated by predicted and ongoing changes in global climate.

**Technological and methodological innovation**

A phytotron experiment with cultivation of single-stemmed potted plants of blackcurrant cv. Narve Viking was conducted at 12, 18 or 24 °C, and three different photoperiods (short day, short day with night interruption, natural summer conditions). Plants were also grown outdoors. Ripe berries were analyzed via HPLC-PDA-LTQOrbitrap XL MS to detect metabolites affected by the climatic factors. By applying online DDA- MS2 and MS3, 53 compounds were identified at MSI level 1, 103 and 221 at MSI levels 2 and 3, respectively.

**Results and impact**

Temperature had significant effect on a total of 365 metabolites. Tryptophan was upregulated as temperature increased, in turn impacting upon the regulation of Indole Acetic Acid and its control of fruit ripening. Phenylalanine, the major flavonoid precursor, also increased with temperature, as did its downstream metabolites including catechins, anthocyanins, anthocyanidins, flavones and flavanols. The levels of delphinidin- and cyanidin- 3-O-rutinosides (60% of total anthocyanins), were elevated between 12 - 18 °C, before being strongly reduced at 24 °C, suggesting that global warming will have large negative impacts on anthocyanin content. Interestingly, 34 annotated flavonoids were more abundant under outdoor conditions, illustrating the impact that UVA has on their synthesis. Based upon the most abundant anthocyanins, a comparison between targeted and untargeted analyses, revealed a close convergence of the methods, illustrating the precision of the metabolomics approach.
**Poster 037: P037 The role of food polyphenols in energy metabolism**

María de la Luz CÁDIZ-GURREA<fnref n="1" />,<fnref n="2" />; Álvaro FERNÁNDEZ-OCHOA<fnref n="1" />,<fnref n="2" />; María del Carmen VILLEGAS-AGUILAR<fnref n="1" />,<fnref n="2" />, Sandra PIMENTEL-MORAL<fnref n="1" />,<fnref n="2" />; Jesús LOZANO-SÁNCHEZ<fnref n="1" />,<fnref n="2" />; David ARRÁEZ-ROMÁN<fnref n="1" />,<fnref n="2" />; Antonio SEGURACARRETERO<fnref n="1" />,<fnref n="2" />

**Introduction**

Polyphenols are secondary metabolites naturally synthetized in plants and have demonstrated biological properties against disorders related to oxidative stress or inflammation, among others. Lippia citriodora, which is used as food seasoning or infusion, is rich in verbascoside, a potent anti-inflammatory phenylpropanoid. Theobroma cacao is worldwide consumed and common ingredient of many food products. Cocoa, containing proanthocyanidins, is a valuable source of antioxidant compounds.

**Technological and methodological innovation**

The antioxidant and anti-inflammatory properties of these extracts were tested in 2 mouse embryonic fibroblast (MEF) cell lines: a double knock-out mouse of paraoxonase-1, an important endogenous antioxidant enzyme, and a double knock-in mouse for monocyte chemoattractant protein 1, a pro-inflammatory cytokine. MEF wild-type were used as lean samples. Targeted metabolomics of energy metabolism was performed on gas chromatography coupled to a QTOF mass spectrometer and an electron impact source [1].

**Results and impact**

Energy metabolism changes drastically when MEF are incubated with LPS. *L. citriodora* and *T. cacao* have an important effect on energy metabolism in MEF PON-1 KO and MCP-1 KI incubated with LPS, leading metabolite values to these found in MEF wild-type. Special attention merits a-ketoglutarate since both plant extracts decrease strongly its levels. These results could be an approximation of how polyphenols act in oxidation or inflammation models.

**References**

[1] Cádiz-Gurrea et al. 2019. Food Research International. 118, 96-100

**Acknowledgements**

Ministry of Science, Innovation and Universities (RTI2018-096724-B-C22)

<footnote n="1">¹ Department of Analytical Chemistry, University of Granada, Spain</footnote>

<footnote n="2">² Research and Development of Functional Food Centre (CIDAF), Granada, Spain</footnote>
**Poster 038: P038 Study of dietary polyphenols metabolites by a top-down
metabolomics approach**

María del Carmen VILLEGAS-AGUILAR<sub>1,2</sub>; María de la Luz CÁDIZ-GURREA<sub>1,2</sub>; Álvaro FERNÁNDEZ-OCHOA<sub>1,2</sub>; Sandra PIMENTEL-MORAL<sub>1,2</sub>; Jesús LOZANO-SÁNCHEZ<sub>1,2</sub>; David ARRÁEZ-ROMÁN<sub>1,2</sub>; Antonio SEGURA-CARRETERO<sub>1,2</sub>

**Introduction**

Phenolic compounds from plant sources have been reported to have beneficial properties for the treatment of several human chronic diseases [1]. Traditionally, the study of these compounds has been carried out using a bottom-up approach (from *in vitro* to *in vivo* models). Therefore, our goal is to propose a top-down approach, starting from human *in vivo* assays, with the purpose of detecting metabolites derived from these compounds present in plants as well as knowing their mechanisms of action.

**Technological and methodological innovation**

The first stage corresponds to nutritional intervention assay in humans. In this trial, volunteers ingest supplements rich in phenolic compounds from *Lippia citriodora*, *Hibiscus sabdariffa*, *Olea europaea*, *Silybum marianum* and *Theobroma cacao*. After that, biological samples are collected at different times after intake and the identification of circulating metabolites is carried out using analytical techniques such as liquid chromatography coupled to mass spectrometry (HPLC-ESI-qTOF-MS).

**Results and impact**

The expected results of the use of this new approach will allow us to know which metabolites are really responsible for the bioactivity properties of different plant sources rich in phenolic compounds. In addition, a great advance of knowledge in relation to metabolic transformations and mechanisms of action of phenolic compounds will be achieved.

**References**

[1] Barrajon-Catalan, E., et al. (2014) *Adv Exp Med Biol* 824, 141-159.

Acknowledgments: Ministry of Science, Innovation and Universities (RTI2018-096724-B-C22)

<footnote n="1"><sup>1</sup> Department of Analytical Chemistry, University of Granada, Spain</footnote>

<footnote n="1"><sup>1</sup> Research and Development of Functional Food Centre (CIDAF), Granada, Spain</footnote>
**Poster 039: P039 NSP19 an original eco-extract from Dioscorea villosa with neuroprotective effects in in vitro Alzheimer's disease models**

Elnur GARAYEV<fnref n="1" />,<fnref n="2" />; Patricia FRANÇON<fnref n="1" />, Alexandre HENRIQUES<fnref n="1" />, Philippe POINDRON<fnref n="1" />, Noëlle CALLIZOT<fnref n="1" />

**Introduction**

Increasing evidence suggests that a loss in estrogen can increase the women's risk of developing Alzheimer's disease (AD)[1]. AD is a neurodegenerative disease characterized by a progressive loss of neurons associated with memory deterioration. Postmenopausal women receiving estrogen replacement therapy would exhibit a decreased risk of developing AD. Diosgenin, one of the most important steroidal saponins of *D. villosa*, has been shown to protect neurons via estrogen receptor pathway.

**Technological and methodological innovation**

Here, we have prepared an optimized ecofriendly extract from *D. villosa* rhizomes (NSP19) using microwave technology and green solvents, water and ethanol. NSP19 was tested on primary neuronal culture injured with beta Amyloid peptide (Aβ) or glutamate, cellular models of AD [2]. A deep analysis of the extract was performed using UHPLC-DAD-ELSD technology in order to evaluate Diosgenin content and associated actives.

**Results and impact**

NSP19 showed a significant neuroprotective effect. We proved that a) a synergistic effect between Diosgenin and phenolic compounds was involved in this neuroprotective action, b) a specific ratio of these compounds was needed to get the optimal neuroprotective efficacy of NSP19. Altogether, these results support the usage of NSP19, a steroidal natural product, as a food supplement that may prevent cognitive dysfunction and therefore reduce the risk of Alzheimer's disease in postmenopausal women.

**References**

[1] A. Paganini-Hillet al. 1994. Am. J. Epidemiol, 140, 3; 256-261

[2] N. Callizot, 2013, J. Neurosci. Res. 91;706-716

<footnote n="1">¹ Neuro-Sys SAS, R&D Department, 410 CD60 Parc de l'Oratoire de Bouc, 13120 Gardanne; France
elnur.garayev@neuro-sys.com</footnote>

<footnote n="2">² Neuralia SAS, R&D Department, 410 CD60 Parc de l'Oratoire de Bouc, 13120 Gardanne; France</footnote>
# Poster 040: P040 Digging out the terroir influence on bioactive polyphenols from grape stems: A correlation network driven approach to spatialize metabolomics data.

Kévin Billet<fnref n="1" />; Sébastien Salvador-Blanes<fnref n="2" />; Thomas Dugé de Bernonville<fnref n="1" />; Guillaume Delanoue<fnref n="3" />; Florent Hinschberger<fnref n="2" />; Audrey Oudin<fnref n="1" />; Vincent Courdavault<fnref n="1" />; Olivier Pichon<fnref n="1" />; Sébastien Besseau<fnref n="1" />; Nathalie Giglioli-Guivarc'h<fnref n="1" />; Arnaud Lanoue<fnref n="1" />

## Introduction

Grape stems are low-cost viticultural by-products rich in bioactive polyphenols. While their phytochemical composition highly depends on the genotype [1-3], the terroir influence is still unknown. In oenology, terroir defines all the interactions between genotype, environment and viticultural practices that give wine typicity [4]. Here, we hypothesized that change in environmental conditions, particularly in soil properties, may influence polyphenol composition.

## Technological and methodological innovation

A vineyard parcel planted with a unique grape clone covering four different geological layers was selected. Grape stems were harvested at the same GPS positions over 3 consecutive years and analysed by UPLC-DAD-MS-based metabolomics targeted on 43 polyphenols. Correlation-based networks were employed to select co-varying metabolites conserved over the three years and Geographic System Information (GIS) was used to study the spatialization of clustered metabolites.

## Results and impact

Beyond vintage effect, principal component analyses on intra-vintage data were reproducible and showed metabolites covariation by polyphenol subclasses. Flavonoids were spatialized on sandy soils when stilbenoid DP4 over-accumulated on loamy-silty soils. The unprecedented combination of metabolomics correlation-based networks and GIS mapping represents a powerful workflow to spatialize field omics data, a key challenge in precision agriculture.

## References

[1] Billet et al. 2018. Frontiers in plant Science. 9, 1-15

[2] Houillé et al. 2015. J. Agri. Food. Chem. 63(5), 1631-1638

[3] Lambert et al. 2013. J. Agri. Food. Chem. 61(47), 11392-11399 [4] Van Leeuwen et al. 2004. Am. J. Enol. Vitic. 55(3), 207-217

<footnote n="1">¹ EA2106 "Biomolécules et Biotechnologies Végétales", Université de Tours, UFR des Sciences Pharmaceutiques, 31 av. Monge, 37200, Tours, France</footnote>

<footnote n="2">² GéoHydrosystèmes Continentaux (GéHCO), EA6293, Université de Tours, Parc Grandmont, Bâtiment E, 37200, Tours, France</footnote>

<footnote n="3">³ Institut Français de la Vigne et du Vin (IFV), Tours, 37400, Amboise, France</footnote>
# Poster 041: P041 Development of a UHPLC-MS/MS targeted method in various matrices for the quantification of toxicologically relevant aldehydes produced from lipid peroxidation

C. ORLANDI<sup>1,2</sup>, S. CHEVOLLEAU<sup>1,2</sup>, M-H. MEIRELES<sup>1,2</sup>, I. JOUANIN<sup>1,2</sup>, A. BOUVILLE<sup>1,2</sup>, N. NAUD<sup>1</sup>, F. PIERRE<sup>1</sup>, F. GUERAUD<sup>1</sup>, L. DEBRAUWER<sup>1,2</sup>

<sup>1</sup> Toxalim, Toulouse University, INRAE, ENVT, INP-Purpan, Université de Toulouse 3 Paul Sabatier, F-31027 Toulouse, France

<sup>2</sup> Metatoul-AXIOM platform, MetaboHUB, National Infrastructure of Metabolomics and Fluxomics, INRAE, F-31027 Toulouse, France

## Introduction

Lipid peroxidation catalyzed by dietary heme iron leading to reactive aldehydes such as hydroxyl-alkenals (e.g. HHE and HNE) is a major hypothesis for linking red and processed meat-rich diets consumption with an elevated risk of colorectal cancer (CRC) <sup>[1]</sup>. The quantification of these representative hydroxyl-alkenals in different matrices is therefore of central importance to assess their occurrence and correlate it with other biological and biochemical markers of CRC promotion.

## Technological and methodological innovation

UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in fecal water <sup>[2]</sup>. Samples were derivatized in-situ using a brominated reagent (BBHA) in presence of deuterated internal standards (HNE-D<sub>11</sub>/HHE-D<sub>5</sub>), extracted by solid phase extraction, and then analyzed by LC-positive ESI-MS/MS in MRM mode. This method was then extended to be used in other matrices, such as plasma by adding a protein precipitation step to the sample pretreatment.

## Results and impact

BBHA allowed efficient stabilization of the reactive aldehydes <sup>[3]</sup> which detection is based on characteristic transitions monitored from bromine isotopic peaks and selective MRM mode. The method was validated according to the EMEA guidelines <sup>[4]</sup>, for selectivity, sensitivity, linearity, carry-over effect, recovery, matrix effect, repeatability, trueness and intermediate precision. The method is compatible with the detection of physiological HNE / HHE levels (ca. 100 nM) in the studied matrices.

## References

[1] F. Pierre et al. 2007. Carcinogenesis, 28, 321-327

[2] S. Chevolleau et al. 2018. Journal of Chromatography B., 1083, 171-179

[3] I. Jouanin et al. 2015. Synth. Commun., 45, 1585-1591

[4] Guideline on bioanalytical method validation. EMEA/CHMP/192217/2009/rev.1 Corr.2, 21 July 2011
# Poster 042: P042 LC-MS based untargeted metabolite profiling of wholegrain spelt (Triticum spelta) cultivated in Switzerland

Dan Zhu<fnref n="1" />; Fabio Mascher<fnref n="2" />; Laura Nyström<fnref n="1" />

## Introduction

Spelt is an ancient cereal related to common wheat, now being rediscovered due to their eco-friendly agronomical and valuable nutritional characteristics<fnref n="1" />. Wholegrain spelt is rich in dietary fiber and bioactive phytochemicals, which may reduce the risk of some chronic diseases<fnref n="2" />,³. We developed a novel robust high-throughput method based on untargeted metabolomics to evaluate phytochemical profiles in various spelt varieties from a wide range of breeder diversities.

## Technological and methodological innovation

A panel of 124 spelt varieties were cultivated under uniform conditions in the field in 2016. Phytochemicals of their grains were extracted with an automatic sample preparation device, and further analysed with a UPLC-ESI-Q-TOF-MS based untargeted metabolite profiling approach. The MS data from each variety was converted into markers for comparison. Multivariate statistical analysis was applied to find the characteristic markers, enabling us to find the discrimination among the varieties.

## Results and impact

Our research indicated that metabolite profiles were significantly different among spelt varieties. Characteristic markers were tentatively identified by in-house and public databases. Abundance of some compounds of interest can be compared efficiently among varieties with semiquantitative metabolomics data. The results of this study will provide insights for better selection of spelt varieties in agriculture with a high nutrient content and improvement of spelt food products in the future.

## References

[1] Starzyńska-Janiszewska et al. Spelt wheat tempe as a value-added whole-grain food product. 2019, LWT-Food Sciences and Technology. Volume 113, 108250.

[2] Gebruers, et al. Variation in the content of dietary fiber and components thereof in wheats in the HEALTHGRAIN diversity screen. 2008, Journal of Agricultural and Food Chemistry, 56(21), 9740-9749.

[3] Biskup, et al. The potential role of selected bioactive compounds from spelt and common wheat in glycemic control. 2017, Advances in clinical and experimental medicine, 26.6: 1013-1019.

<footnote n="1">¹ Laboratory of Food Biochemistry, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, CH 8092 Zurich, Switzerland</footnote>

<footnote n="2">² Crop Plant Breeding and Genetic Resources, Agroscope, CP 1012, route de Duiller 50, 1260 Nyon, Switzerland</footnote>
**Poster 043: P043 Isotope Labelling and Molecular Networking to Identify New Fungal Secondary Metabolites**

Robin COSTANTINO ¹,², Thaïs HAUTBERGUE ¹,²,³, Souria TADRIST ¹,
Lauriane MENEGHETTI ¹,², Jean-Claude TABET ³,⁴, Laurent DEBRAUWER ¹,², Isabelle P. OSWALD ¹,
Olivier PUEL ¹ and Emilien JAMIN ¹,²

¹ Toxalim, Toulouse University, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France.

² Metatoul-AXIOM platform, MetaboHUB, Toxalim, INRAE, Toulouse, France. ³ Service de Pharmacologie et d'Immunoanalyse (SPI), Laboratoire d'Etude du tabolisme des dicaments, CEA, INRAE, niversit Paris Saclay, MetaboHUB, Gif-sur-Yvette, France.

⁴ Sorbonne niversit s, Campus Pierre et Marie Curie, IPCM, Paris, France.

**Introduction**

Characterization of fungal secondary metabolomes has become a great challenge in the last decades due to both the emergence of fungal threats leading to the destruction of food, and the industrial interest of many natural products. Fungal toxicity and the spreading of fungi into stock feed and food are caused by secondary metabolites. Therefore, it is crucial to characterize the metabolites produced by fungi and investigate the food contamination by potentially not yet known mycotoxins.

**Technological and methodological innovation**

A strategy based on double isotope labelling and untargeted metabolomic high resolution MS based analysis was adopted [1]. This strategy enabled the specific detection of metabolites naturally produced by a fungus, the unambiguous determination of their chemical formulas, and the generation of curated molecular networks using GNPS [2] and MetGem [3]. The combination of these 2 networks, curated using isotopes, allowed at the end the identification of unknown metabolites of Penicillium nordicum.

**Results and impact**

By this way, 92 metabolites were detected following infection of a natural substrate by P. nordicum, among which 69 were completely unknown. Two curated molecular networks of MS/MS data, specifically generated on the features of interest, highlighted 13 fungisporin-related metabolites previously unidentified in this fungus and 8 never observed in any fungus. The combination of the two molecular networks also allowed to identify some unknown fungisporins [4].

**References**

[1] Cano, P. . et al. 2013. Anal. Chem. 85 (17), 8412–8420.

[2] Wang, M. et al. 2016. Nat. Biotechnol. 34 (8), 828–837.

[3] Olivon, F. et al. 2018. Anal. Chem. 90 (23), 13900–13908.

[4] Hautbergue T. et al. 2019 Anal. Chem. 91 (19), 12191-12202.
**Poster 044: P044 Specific derivatization with isotopic labeling introduction and mass spectrometry to monitor lipid peroxidation carbonyl compounds in the intestinal lumen by both targeted and non-targeted approaches.**

Sylvie CHEVOLLEAU<fnref n="1" />,<fnref n="2" />; Maria-Héléna MEIRELES<fnref n="1" />,<fnref n="2" />; Isabelle JOUANIN<fnref n="1" />,<fnref n="2" />; Jean-François MARTIN<fnref n="1" />,<fnref n="2" />; Nathalie NAUD<fnref n="1" />; Fabrice PIERRE<fnref n="1" />; Françoise GUERAUD<fnref n="1" />; Laurent DEBRAUWER<fnref n="1" />,<fnref n="2" />

**Introduction**

Diets rich in red meat are associated with an elevated risk of colorectal cancer. A major hypothesis involves dietary heme iron which induces the formation of lipid oxidation products such as reactive aldehydes[1]. A strategy based on the simultaneous derivatization and stable isotope labeling of these carbonyl compounds is proposed, enabling both targeted monitoring of representative aldehydes and non-targeted carbonyl profiling to get an image of the lipid peroxidation in the intestinal lumen.

**Technological and methodological innovation**

Fecal water samples from rats fed various pro-oxidant diets were derivatized in situ using 1-[(aminooxy) methyl]-2bromobenzene hydrochloride (BBHA) [2] and analyzed by LC-ESI-MS after SPE extraction. High resolution MS (LTQOrbitrap) is used to filter the signals of the brominated compounds and derivatized aldehydes are identified by MS/MS by comparison with standards. LC-MS/MS in the MRM mode (TSQ) is used for the targeted quantification of two aldehydes (HHE and HNE) in internal calibration.

**Results and impact**

Derivatization by BBHA makes it possible to stabilize the reactive aldehydes and to use the characteristic isotopic signature of the bromine atom for specific detection by both non-targeted and targeted approaches. On the one hand, LC-HRMS allows specific signal filtering to obtain a qualitative image of lipid peroxidation in colon lumen without a priori [3]. On the other hand, targeted LC-MS/MS made it possible to quantify HNE and HHE using a method validated according to EMEA guidelines [4-5].

**References**

[1] F. Guéraud et al., 2015, Free Radic. Biol. Med., 83, 192-200

[2] I. Jouanin et al. 2015, Synth. Commun., 45, 1585-1591

[3] S. Chevolleau et al. (in preparation)

[4] S. Chevolleau et al. 2018. Journal of Chromatography B., 1083, 171-179

[5] Guideline on bioanalytical method validation. EMEA/CHMP/192217/2009/rev.1 Corr.2,21 July 2011

<footnote n="1">¹Toxalim, Toulouse University, INRAE, ENVT, INP-Purpan, Université de Toulouse 3 Paul Sabatier, F-31027 Toulouse, France</footnote>

<footnote n="2">²MetaToul-Axiom Platform, UMR 1331 INRAE Toxalim, MetaboHUB, National Infrastructure of Metabolomics and Fluxomics, F-31027 Toulouse, France</footnote>
**Poster 045: P045Characterization of A. thaliana responses to plant defence elicitors using untargeted metabolomics**

Aurélien Amiel<fnref n="1" />,<fnref n="2" />; Aziz Benamar<fnref n="2" />; Sylvie Fournier<fnref n="2" />,<fnref n="3" />; Virginie Puech Pagès<fnref n="2" />,<fnref n="3" />; Bernard Dumas<fnref n="2" />; Olivier André<fnref n="1" />,<fnref n="2" />

...

**Introduction**

The green revolution of the past century has led to an explosion in agricultural yields, supported by the intensive use of pesticides and chemical fertilizers. The limits of this model are gradually being revealed, leading to a change in the mindset from producing "more" to producing "better". Today the objective is to develop innovative solutions based on natural & biocontrol concepts. Among the proposed solutions, the elicitation of plant defense responses is a strategy for the control of diseases and pests. The concept of induced resistance is based on the application of a natural (plant extract, microorganisms, and mineral) or synthetic (hormone analogues) product, which triggers natural defence mechanisms and thus enables the plant to be in a state of resistance to a pathogen during an attack. Signaling pathways leading to the induction of plant defenses involved phytohormones and notably salicylic acid. Arabidopsis thaliana is a model organism for the study of physiological, cellular and molecular factors in plants. In this sense, it is also a good model to study the mechanisms involved in plant resistance and to better understand the mechanisms underlying elicitor protection. In our study 3 elicitors were tested: a commercialized synthetic product (Bion - Syngenta) containing a functional analog of the phytohormone Salicylic acid, an essential oil for Gaultheria procumbens (GEO) containing more than 95% of methylsalicylate2 and an extract of seeds of Fenugreek (Fen) known to activate A. thaliana defense through the salicylic acid signaling pathway3. The objective of our study was to characterize the plant response to these three compounds using an untargeted metabolomics approach.

**Technological and methodological innovation**

Obtaining precise information on the chemical composition of complex natural extracts is a challenging task that requires sophisticated methods. In this project we have developed, with the MetaboHUBMetaToul-Plant Metabolites platform, a method using liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) to compare the effect of plant defense elicitors on the metabolomic profile of Arabidopsis thaliana. Plant leaves were treated with solutions containing each of the three products and tissues were harvested 48 hours after the treatment. To analyse the results, a workflow including MS-Dial and MS-Finder softwares have been used to annotate and identify peaks with databases (PlantCyc, HMDB...)5. The identification of compounds whose accumulation was correlated with the treatments has been investigated by a multivariate and univariate statistical analysis combination.

**Results and impact**

This study showed common and specific plant metabolomic responses to the three treatment, Even if their activity relies to the salicylic acid signaling pathway. Globally, GEO treatment increases phenolic glycosides compounds whereas Bion treatment increases alkythiols and Fen treatment increase both flavonoids and fatty acids. This result highlights the significant role of downstream metabolic response regulation activated thought common signaling salicylic acid pathway.

<footnote n="1">¹ De Sangosse Bonnel, France</footnote>

<footnote n="2">² Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, France</footnote>

<footnote n="3">³ MetaboHUB-MetaToul-Plant Metabolites, LRSV, Université de Toulouse, CNRS, UPS, France</footnote>
**Poster 046: P046 Metabolomics implemented to the Equine Biologic Passport (EBP) for doping control.**

Benoit LOUP ; Agnès BARNABE ; François ANDRE ; Patrice GARCIA ; Vivian DELCOURT ; Marie-Agnès POPOT ; Ludovic BAILLY-CHOURIBERRY.

Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, 91370 Verrières-le-Buisson, France
...
...

**Introduction**

In the horseracing industry drug of abuse is an important concern and the doping control laboratories continuously develop new strategies to detect misuses of doping substances. In this context, the LCH began in 2009 the development of the Equine Biologic Passport (EBP) based on omics and biomarkers monitoring. The EBP is applied to the follow up of the best French trotters and combined different levels of monitoring including implementations of metabolomic approaches.

**Technological and methodological innovation**

LC-HRMS and GC-MS/MS metabolomics was conducted on horse urine samples collected after administration with growth promoters, erythropoiesis stimulating agents, immune castration. OPLS-DA models were developed allowing discrimination between treated and control animals[1-4]. These models are employed in the EBP and allow to routinely control French trotters at training. An analytical strategy based on quality controls enables to normalize data, manage analytical drifts and correct batch effects.

**Results and impact**

The results are routinely processed and the OPLS-DA models constitute new potential methods in the fight against doping. These models highlight metabolic disturbances due to the misuse of doping agents and provide a convenient method for result interpretation. Models are continuously improved with additional population studies. Moreover, annotation and structural identification of relevant features are in progress and could possibly find out new candidate biomarkers for doping control.

**References**

[1] Bailly-Chouriberry L et al. 2017. Drug Test. Analysis, 9, 1432-1440.

[2] Boyard-Kieken F et al. 2011. J Sep Sci., Dec;34(24):3493-501.

[3] Joré C et al. 2017. Journal of Chromatography A 1521:90-99. [4] Kaabia Z et al. 2014. Drug Test Analysis 6:376-388
**Poster 047: P047Unravelling the impact of the antidepressant venlafaxine on the metabolome of meagre fish**

Lúcia H.M.L.M. SANTOS<fnref n="1" />,<fnref n="2" />; Ana Luísa MAULVAULT<fnref n="3" />; Adrián JAÉN-GIL<fnref n="1" />,<fnref n="2" />; António MARQUES<fnref n="3" />; Damià BARCELÓ<fnref n="1" />,<fnref n="2" />,<fnref n="4" />; Sara RODRIGUEZ-MOZAZ<fnref n="1" />,<fnref n="2" />

**Introduction**

There is a growing interest on the application of metabolomics in environmental science to study how the exposure to xenobiotics affects the metabolism of aquatic organisms. The present work aimed to evaluate the changes in the metabolome of the juvenile fish Argyrosomus regius when exposed to the antidepressant venlafaxine (VLF). VLF was selected due to its ability of inducing toxic effects [1] and behavioral changes [2] in fish, as well as bioaccumulation in their tissues [3].

**Technological and methodological innovation**

Fish were exposed to 20 µg/L of VLF via water for 28 days followed by an elimination phase of 7 days. Metabolomics was applied to assess tissue-dependent changes in the profile of endogenous metabolites in different tissues (liver, brain and plasma) at 28 and 35 days. Analysis was performed using a non-target screening approach based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Data was processed using Compound Discoverer 3.0.

**Results and impact**

Tissue-dependent variations on endogenous metabolites of fish exposed to VLF were observed, showing that metabolomics allow to detect changes at an early stage of exposure. Several endogenous metabolites, altered after contaminant exposure, were tentatively identified and can be proposed as markers of contamination impact. In general, the metabolic pathways more significantly affected were phenylalanine, tyrosine and tryptophan metabolism; purine metabolism; and signaling pathway.

**References**

[1] Best et al. 2014. Aquatic Toxicology. 155, 190-198

[2] Maulvault et al. 2018. Science of the Total Environnent. 634, 1136-1147

[3] Maulvault et al. 2018. Chemosphere. 209, 286-297

**Acknowledgments**

Work funded by the Spanish State Research Agency of the Spanish Ministry of Science, Innovation and Universities and European Fund for Regional Development (project PLAS-MED; FEDER-MCIU-AEI/CTM2017-89701-C3-2-R). Authors thank Generalitat de Catalunya through Consolidated Research Group (ICRA-ENV 2017 SGR 1124 & 2017 SGR 1404). Lúcia H.M.L.M. Santos thanks the Juan de la Cierva program (IJCI-2017-32747) and Sara Rodríguez-Mozaz thanks the Ramon y Cajal program (RYC-2014-16707) from the Spanish State Research Agency of the Spanish Ministry of Science, Innovation and Universities (AEI-MCIU). A. Jaén-Gil thanks the predoctoral grant from the Agency for Management of University and Research Grants (AGAUR) (2019FI_B2_00202). A. Marques (IF program) and A.L. Maulvault (FishBudget project) thank the Portuguese Foundation for Science and Technology.

<footnote n="1">¹Catalan Institute for Water Research (ICRA), Spain, Carrer Emili Grahit 101, 17003 Girona, Spain</footnote>

<footnote n="2">²Universitat de Girona, Girona, Spain</footnote>

<footnote n="3">³Portuguese Institute for the Sea and Atmosphere (IPMA, I.P.), Portugal, Rua Alfredo Magalhães Ramalho, 1495-006 Lisbon, Portugal</footnote>

<footnote n="4">⁴IDAEA-CSIC, Spain, Jordi Girona 18-26, 08034 Barcelona, Spain</footnote>
# Poster 048: P048 Comparison of effects produced by the exposure of bisphenol A and estradiol in zebrafish embryos (Danio rerio) using an untargeted metabolomics approach

Miriam C. PÉREZ-COVA<fnref n="1" />,<fnref n="2" />, Laia NAVARRO-MARTIN<fnref n="1" />, Gabriel LEME<fnref n="3" />, Benjamin PIÑA<fnref n="1" />, Romà TAULER<fnref n="1" />, Dwight STOLL<fnref n="3" />, Joaquim JAUMOT<fnref n="1" />

## Introduction

Bisphenol A (BPA) is an exogenous endocrine disruptor, well known due to its estrogenicity [1], but it is also related to alterations in adipogenesis and obesity [2, 3]. Previous studies show disruption of lipid-related genes and in the metabolome of zebrafish embryos (Danio rerio) [4], but further characterization is desirable. Therefore, we aim to determine changes in the metabolomic profiles of zebrafish embryos exposed to BPA, and a natural hormone, estradiol (E2) during early development.

## Technological and methodological innovation

A HILIC-HRMS/MS workflow has been employed to assess the effects caused by the exposure of zebrafish embryos to BPA and E2. Zebrafish embryos were exposed at two concentration levels (sub-lethal doses), from 48 to 120 hours postfertilization (hpf) and collected at 120hpf. A TSK Amide 80 column and a QTOF mass spectrometer in ESI negative mode were used for the analysis. W4m online platform, PLS Toolbox and HMDB database were employed for feature detection, multivariate analysis and annotation.

## Results and impact

There is a need in deepening the knowledge of the consequences of BPA in the metabolism of exposed organisms. This study aims to achieve a better understanding of the metabolic consequences of its exposure, beyond its estrogenic effect, when compared to the effect produced by a natural hormone, as E2.

Preliminary results show that the main markers associated with the exposure correspond to different metabolomic pathways, including amino acid and sugar metabolisms, as previously reported [4].

## References

[1] Eramo, S., et al. 2010. Ann. Stomatol. 1: 14-21.

[2] Santangeli, S., et al. 2018. Sci. Total Environ. 636:641-655.

[3] vom Saal, F.S., et al. 2012. Mol. Cell. Endocrinol. 354:74-84.

[4] Ortiz-Villanueva, E., et al. 2018. Sci. Total Environ. 635:156-166.

<footnote n="1">¹IDAEA-CSIC, Department of Environmental Chemistry, Barcelona, Spain</footnote>

<footnote n="2">²Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, Barcelona, Spain</footnote>

<footnote n="3">³Department of Chemistry, Gustavus Adolphus College, Saint Peter, Minnesota 56082, United States</footnote>
*Photosynthetic Organisms from Atmospheric Ecosystem in a Multiscale Study*

Abélia PATRIGEON²,⁴; Hermine BILLARD²; Jonathan COLOMBET²; Céline JUDON¹; Marie LAGREE¹,⁴; Binta DIEME¹,⁴; Jean-Luc BARAY³; Delphine LATOUR²; Cyril JOUSSE¹,⁴

¹ Université Clermont Auvergne, CNRS, Sigma-Clermont, Institut de Chimie de Clermont-Ferrand UMR6296, F-63000 Clermont-Ferrand, France

² Université Clermont Auvergne, CNRS, Laboratoire Microorganismes : Génome et Environnement UMR6023, F-63000 Clermont-Ferrand, France

³ Université Clermont Auvergne, CNRS, Laboratoire de Météorologie Physique UMR6016, F-63000 Clermont-Ferrand, France

⁴ Université Clermont Auvergne, INRA, Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont, ClermontFerrand, France

**Introduction**

Atmosphere is a poorly understood compartment, ecologically speaking. Preliminary studies try to show impact of bioaerosols on climate changes and few of them try to understand physiological adaptations upon such harsh conditions [1, 2]. Among microorganisms from this compartment, several are photosynthetically active, from prokaryotic (e.g. cyanobacteria [3]) & eukaryotic phyla [4]. As the others, they come from – and fall to – all over the world, possibly influencing ecosystems of destination.

**Technological and methodological innovation**

Meta-metabolomics [5] aim to focus on environmental aspects and try to harvest evidences about how physiological plasticity leads to evolution among consortia or holobiontic contexts. However, the assumptions need to be reinforced / supported with other sources of information. Then, dealing with data from meteorological, ecological and omics is a challenge to reveal capabilities of bioaerosols to influence terrestrial reservoirs.

**Results and impact**

First results show on one way that atmospheric diversity is linked to several unpredictable parameters. On the other way, microbial lifespan and metabolome seem to be modified according to the recruited consortia. This project is at its alder and needs to mature methodological issues, collect more data and validate hypothesis with much more well-documented observations. Such meta-metabolomics breakthroughs will permit to open ecology field to much more accurate phenotypic assessments.

**References**

[1] Sesatic et al. 2012. Atmos. Chem. Phys. 12, 8645-61.

[2] Joly et al. 2015. Atmos. Env. 117, 92-98.

[3] Kourtev et al. 2011. Atmos. Env. 45(30), 5399-5405.

[4] Tesson et al. 2016. Appl. Environ. Microbiol. 82, 1978-91.

[5] O'Malley. 2013. In: Dubitzky W., Wolkenhauer O., Cho KH., Yokota H. (eds) Encyclopedia of Systems Biology. Springer, New York, NY
**Poster 050: P050 Developing a multi-omics approach for chemical grouping**

Hanna Gruszczynska<fnref n="1" />, Rosemary Barnett<fnref n="2" />, Doris Hirmann<fnref n="3" />, Ralf J. M. Weber<fnref n="1" />,<fnref n="4" />, Jiarui Zhou<fnref n="1" />, Elena Sostare<fnref n="2" />, Bram Versonnen<fnref n="3" />, Tomasz Sobanski<fnref n="3" />, John K. Colbourne<fnref n="1" />,<fnref n="2" />, Mark R. Viant<fnref n="1" />,<fnref n="2" />,<fnref n="4" />

**Introduction**

With the globally growing use of chemicals there is an ever-increasing demand for improved safety assessment. However, toxicity data is often lacking for substances[1]and their impacts on human and environmental health are poorly understood. One of the approaches frequently applied in regulatory toxicology for data gap filling is forming groups of substances and 'reading across' toxicity[2]. It is traditionally based on structural similarity and often does not fully consider toxicity mechanisms.

**Technological and methodological innovation**

Here, we evaluated the capabilities of high-throughput multi-omics approaches to substantiate or disprove the structure-based grouping of seven azo dyes, where the grouping was based on the molecular toxicity responses in Daphnia magna. This utilised direct infusion mass spectrometry (DIMS) non-targeted metabolomics[3] and the TempO-SeqTM platform for transcriptomics. The multi-omics data streams were integrated and analysed using hierarchical cluster analysis to derive mechanism-driven grouping.

**Results and impact**

The multi-omics investigation discovered sufficient metabolic and transcriptional responses to facilitate grouping of the azo dyes based on their toxicological fingerprints, demonstrating the potential of the approach. In addition, Ingenuity Pathway Analysis (IPA) of the gene expression data provided an independent mechanistic rationale for the multi-omics grouping. The groupings based on the two approaches of structural similarity vs. multi-omics responses were similar but not identical.

**References**

[1] European Chemicals Agency (ECHA), 2019. Registered substances database

[2] ECHA 2017, Read-Across Assessment Framework (RAAF)

[3] Southam A.D. et al., 2016. A complete workflow for high-resolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics. *Nat Protoc*, 12, 310-328

<footnote n="1">¹ School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK</footnote>

<footnote n="2">² Michabo Health Science, University of Birmingham Enterprise, Birmingham, B15 2SQ, UK</footnote>

<footnote n="3">³ European Chemicals Agency, Helsinki, Finland</footnote>

<footnote n="4">⁴ Phenome Centre Birmingham, University of Birmingham, Birmingham, B15 2TT, UK</footnote>
**Poster 051: P051 Microbiome and metabolome analyses of the medaka fish gut during a chronic exposure to toxic cyanobacterial blooms**

Alison GALLET¹; Hélène HUET²; Charlotte DUVAL¹; Benjamin MARIE¹; Sébastien DUPERRON¹

¹ UMR CNRS MNHN 7245 MCAM, Cyanobacteria Cyanotoxins and Environnement team ² Ecole Nationale Vétérinaire, BioPôle, Alfort, France

**Introduction**

Cyanobacterial blooms are freshwater natural events in which Microcystis is one of the most encountered genus. Those microorganisms are able to produce a large set of bioactive secondary metabolites [1], some of which are known to have antimicrobial activities [2]. Although metabolites effects on fish physiology have been well investigated, effects on microbiota are still lacking. Here, we assess potential interactions between the M. aeruginosa species and the gut microbiota of a model fish.

**Technological and methodological innovation**

Medaka fishes (Oryzias latipes) have been exposed during 28 days in microcosms to environmentally relevant and increasing concentrations of M. aeruginosa blooms. Both microbiome (16S rDNA) and metabolome have been analysed from gut samples to study respectively the composition of bacterial gut communities associated and the secondary metabolites diversity. Furthermore, multi-omics analyses allowed to determine variations and correlations between amplicon sequence variants and metabolic compounds.

**Results and impact**

Preliminary results for the microbiome revealed a shift in the composition of bacterial communities for fishes exposed to the highest concentration of M. aeruginosa, suggesting the existence of a tipping point. Additionally, the change in metabolic profiles with the increase in concentration is following a common continuous trajectory between samples and replicates. This suggests a possible progressive change in metabolite compositions.

**References**

[1] Dittmann et al. 2015. *Trends Microbiol.* 23, 642-652.

[2] Singh et al. 2001. *World J. Microbiol. Biotechnol.* 17, 15-22.
# Poster 052: P052 High-Throughput Targeted Lipidomics Analysis of Dihydroceramide Desaturase-1 (DES1) Knockout Mice

Mackenzie Pearson1; Santosh Kapil Kumar Gorti1; Cyrus Papan2; Marialuce Maldini2; Trevor S. Tippets3; Bhagirath Chaurasia3; Scott A. Summers3

1AB SCIEX, Framingham, MA; 2SCIEX, Darmstadt, Germany; 3University of Utah, Dept of

Nutrition and Integrative Physiology, Salt Lake City, UT...

## Introduction

Lipidomics is a fast-growing area of science, aiming to quantitatively define functions of lipid classes at the molecular species levels. There are about 150,000 different lipid molecular species present across the biological spectrum. This diversity reflects the different roles lipids play at the cellular, tissue, and organismal levels. Traditional approaches to lipid analysis in biological samples have relied on 'Shotgun' lipidomics, a high throughput infusion-based approach for screening studies, or IDA-based (i.e., data-dependent) discovery experiments. The challenge to both approaches is the need for extensive data analysis to identify molecules, and the quantitative results are limited to sum composition (e.g., PC 34:2) rather than molecular species composition (e.g., PC 16:0/18:2). The ability to identify and quantitate lipids at the molecular species level is essential to correlate lipidomics to metabolism and hence biology.

For studies requiring more sensitivity and specificity, a targeted lipidomics approach using liquid chromatography (LC) coupled with triple quadrupole based instrumentation in the multiple reaction monitoring (MRM) mode has advantages. Amide column separation is an attractive chromatographic strategy that effectively resolves both polar and non-polar lipids into classes and subclasses. This is an essential aspect of a column-based strategy due to the extensive isobaric overlap among different lipid classes that confounds identification and quantification. To validate this method, we selected DES1 knockout mice to measure lipid changes in liver and adipose tissues. The method was then adjusted to optimize window width and dwell weight to enhance sensitivity and coverage.

The QTRAP® 6500+ System offers unparalleled sensitivity even at high scan speed (2-5 msec transition time) with rapid positive/negative switching (< 5msec). Using qualified MRM transitions and commercially available lipid internal standard mixtures, this method provides quantitative measurement of over 1150 different lipid molecular species in a rapid, highly reproducible manner. The target list is customizable and expandable to include new lipid classes, or the list can be shortened for a class-specific study.

## CONCLUSIONS

Liver and epidymal white adipose tissue (eWAT) tissues were harvested from dihydroceramide desaturase-1 (DES1) knockout and control mice. DES1 is the enzyme responsible for inserting the 4,5-trans-double bond into the sphingolipid backbone causing dihydroceramides conversion to ceramides. While both of these classes of lipids are lower in abundance in the chosen tissues, this method enable the researcher to quantify them and show significant changes in these lipid classes in this study, however lipids from another 17 classes were not changing.

In this study we present an optimized method to target a broad array of different lipids at the molecular species level. An amide column-based chromatographic method was developed to minimize isobaric interference, which allows for a relatively rapid and specific lipid screening technique. The target list of lipids is comprehensive, covering most major lipid classes and categories, and MRMs were selected to cover lipids containing fatty acids with 14 to 22 carbons and 0 to 6 double bonds. The method is customizable, so new lipid categories, classes and molecular species can be added. In simple terms, this method is a specific, quantitative and comprehensive workflow to rapidly screen the lipid profile of any type of matrix and serves as an excellent starting point for further method development and optimization.
# Poster 053: P053 In Vivo Microdialysis of Endogenous and 13C-labeled TCA Metabolites in Rat Brain: Reversible and Persistent Effects of Mitochondrial Inhibition and Transient Cerebral Ischemia

Jesper F Havelund<fnref n="1" />; Kevin H Nygaard<fnref n="2" />,<fnref n="3" />; Troels H. Nielsen<fnref n="2" />,<fnref n="3" />; Carl-Henrik Nordström<fnref n="2" />,<fnref n="3" />; Frantz R. Poulsen<fnref n="2" />,<fnref n="3" />; Nils J. Færgeman<fnref n="1" />; Axel Forsse<fnref n="2" />,<fnref n="3" />; Jan Bert Gramsbergen<fnref n="4" />.

## Introduction

Cerebral microdialysis is routinely used, in neuro-intensive care patients after an ischemic insult, to monitor interstitial levels of glucose, lactate and pyruvate and thus mitochondrial function. Understanding the underlying mechanisms of disturbed energy metabolism are essential to improve treatments. Thus, we applied mitochondrial dysfunction rat models and measured tricarboxylic acid cycle (TCA)-centered metabolites and their incorporation of 13C upon perfusion with 13C-labeled succinate [1].

## Technological and methodological innovation

Two models of mitochondrial dysfunction were applied: a malonate-perfusion and endothelin-1-induced transient cerebral ischemia. The cerebral microdialysis fractions with a temporal resolution of 30 min were analyzed using a LC-MS to identify and relatively quantify TCA-centered metabolites. Moreover, as the rat brains were perfused with 13C-labeled succinate, compounds with 13C incorporated from 13C-succinate were also measured [1].

## Results and impact

Use of the malonate model confirmed the functionality of the approach. In the ET-1 model, increases in non-labeled TCA metabolites (reflecting release of intracellular compounds) and decreases in 13C-labeled TCA metabolites (reflecting inhibition of de novo synthesis) were observed. The analysis of 13C incorporation provides further layers of information to identify metabolic disturbances in experimental models and neuro-intensive care patients [1].

## References

[1] Havelund JF et al. 2019. Metabolites. 9, 204

<footnote n="1">¹ VILLUM Center for Bioanalytical Sciences, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark</footnote>

<footnote n="2">² Department of Neurosurgery, Odense University Hospital, University of Southern Denmark, Sdr. Boulevard 29, 5000 Odense C, Denmark</footnote>

<footnote n="3">³ BRIDGE - Brain Research - Inter-Disciplinary Guided Excellence, Institute of Clinical Research, University of Southern Denmark, Winsløwparken 19, 5000 Odense C, Denmark</footnote>

<footnote n="4">⁴ Institute of Molecular Medicine, University of Southern Denmark, 55, 5230 Odense C, Denmark</footnote>
**Poster 054: P054 Ion source optimization in liquid chromatography time-of-flight mass spectrometry for metabolomics studies using the design of experiments**

Hendri WASITO¹; Tim CAUSON¹; Stephan HANN¹

¹Department of Chemistry, Institute of Analytical Chemistry, University of Natural Resources and Life Sciences, Vienna
(BOKU), Muthgasse 18, Vienna 1190, Austria

**Introduction**

Metabolomics studies in the context of microbial cell factories are essential to assess influences on product yield and quality[1,2]. Consequently, analytical techniques for determining intra-cellular metabolites with high coverage and accuracy are required. The analysis using liquid chromatography mass spectrometry (LC-MS) represents a technical challenge due to the miss-annotation of in-source fragmentation[3] and requires careful optimization of the electrospray ionization (ESI) process.

**Technological and methodological innovation**

ESI parameters, including temperature and flow rate of drying gas and sheath gas, fragmentor, nozzle and capillary voltage, as well as nebulizer pressure were systematically investigated by experimental designs. The signal of several metabolites was determined using hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry. PlackettBurman and central composite experimental designs were employed for screening and optimizing ESI parameters, respectively.

**Results and impact**

The experimental design approach was a suitable method for evaluating the influence and interaction of ESI parameters for MS measurements. Our findings contribute significantly not only to explain ESI parameters on ion source suppression and fragmentation, but also to expand metabolome coverage and correct annotation in the intra-cellular metabolomics studies. First results addressing the non-targeted analysis of yeast cell extracts will be discussed.

**References**

[1] J. Nocon et al. 2014. Metab. Eng. 24: 129-138.

[2] A.F. Jozala et al. 2016. Braz. J. Microbiol. 47: 51-63.

[3] Y.-F. Xu et al. 2015. Anal. Chem. 87: 2273-2281.
**Poster 055: P055** *A novel multi-targeted quantitative approach for nutrimetabolomics research*

Raúl GONZÁLEZ-DOMÍNGUEZ¹,²; Cristina ANDRES-LACUEVA¹,²

¹ Biomarkers and Nutrimetabolomics Laboratory; Department of Nutrition, Food Sciences and Gastronomy; Food Technology Reference Net (XaRTA); Nutrition and Food Safety Research Institute (INSA); Faculty of Pharmacy and Food Sciences; University of Barcelona, 08028 Barcelona, Spain.

² CIBER Fragilidad y Envejecimiento Saludable (CIBERfes), Instituto de Salud Carlos III, Barcelona, Spain.

**Introduction**

Nowadays, metabolomics plays an essential role in nutritional research for accurate dietary assessment by means of the monitoring of food intake biomarkers, as well as for investigating the effect of dietary habits on health. To address the metabolome complexity, in this work we have developed a multi-targeted quantitative approach for comprehensive and rapid metabolomic fingerprinting of biological samples.

**Technological and methodological innovation**

A solid phase extraction method was optimized for the simultaneous extraction and pre-concentration of around 350 food-derived metabolites. Complementarily, simple extraction methods based on dilution and protein precipitation were also validated for the analysis of more than 500 endogenous metabolites from urine and plasma samples, respectively. Metabolomic fingerprinting is then conducted by reversed-phase UHPLC-MS/MS.

**Results and impact**

A novel multi-targeted approach was developed for the simultaneous quantitation of about 900 exogenous and endogenous metabolites in very short run times and using low volumes of biological sample, which facilitates its application to epidemiological studies. This metabolomic approach thus represents one-step further towards precision nutrition and dietary assessment, as well as for the investigation of the role of diet and nutrition in health status.

**References**

[1] González-Domínguez et al. 2019. J. Agric. Food Chem. DOI: 10.1021/acs.jafc.8b07023
# Poster 056: P056 FOBI: An ontology to represent food intake data and associate it with metabolomic data

Pol CASTELLANO-ESCUDER<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Raúl GONZÁLEZ-DOMÍNGUEZ<fnref n="1" />,<fnref n="3" />, David S. WISHART<fnref n="4" />, Cristina ANDRESLACUEVA<fnref n="1" />,<fnref n="3" />, Alexandre SÁNCHEZ-PLA<fnref n="2" />,<fnref n="3" />

## Introduction

Nutrition research can be conducted by using two complementary approaches: 1) traditional self-reporting methods or 2) via metabolomics techniques to analyze food intake biomarkers in biofluids. However, the complexity and heterogeneity of these two very different types of data often hinder their analysis and integration. To manage this challenge, we have developed a novel ontology that describes food and their associated metabolite entities in a hierarchical way.

## Technological and methodological innovation

The ontology presented is called FOBI (Food-Biomarker Ontology) and it is composed of two interconnected sub-ontologies. One is a "Food Ontology" consisting of raw foods and prepared foods while the second is a "Biomarker Ontology" containing food intake biomarkers classified by their chemical classes. These two sub-ontologies are conceptually independent but interconnected by different properties.

## Results and impact

This ontology uses a formal naming system, category definitions, properties and relations between both types of data. Potential applications of this ontology include the annotation of foods and biomarkers using a well-defined and consistent nomenclature, the standardized reporting of metabolomics workflows (e.g. metabolite identification, experimental design), or the application of different enrichment analysis approaches to analyze nutrimetabolomic data.

<footnote n="1">¹ Biomarkers and Nutritional & Food Metabolomics Research Group, Department of Nutrition, Food Science and Gastronomy. University of Barcelona, Barcelona, Spain.</footnote>

<footnote n="2">² Statistics and Bioinformatics Research Group, Department of Genetics, Microbiology and Statistics. University of Barcelona, Barcelona, Spain.</footnote>

<footnote n="3">³ CIBERFES, Instituto de Salud Carlos III. Madrid, Spain.</footnote>

<footnote n="4">⁴ Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E8, Canada.¹</footnote>
**Poster 057: P057 Comparison of different methods for lipidic extraction**

Anaëlle DURBEC¹, Justine BERTRAND-MICHEL¹, Anthony TOURNADRE¹

¹MetaToul-Lipidomic Core Facility, MetaboHUB, Inserm U1048, Toulouse, France

**Introduction**

On the MetaToul-Lipidomic facility, we usually use Bligh and Dyer (modified) method for lipidic extraction. During the automation (Tecan® robot), the results of experimentations revealed that lipids extractions yields were very low. Then to improve this step, we decided to compare different methods of liquid-liquid extraction (LLE) for the extraction of neutral lipids and phospholipids. In this study, the different methods were tested on liver, plasma and appropriate internal standards.

**Technological and methodological innovation**

The LLEs tested were a modified Bligh and Dyer method1, a Folch method2, a three phase liquid extraction (TPLE)3, the BUME method4 and a lipid extraction by methyl-tert-butyl ether (MTBE)5. Neutral lipids were analyzed on a GC-FID (Trace 1300, Thermo®) and phospholipids on a LC-MS/MS (1290 Infinity/6460 Triple quad, Agilent Technologies®).

**Results and impact**

The poster presents the results of the different extraction methods. Overall, they are similar, except for the TPLE, which has lower yields. We thus focused on the modified Bligh and Dyer, the BUME method, the MTBE method and the Folch method was replaced by a microBligh and Dyer method (using less solvent)6.

There are large variations in extraction yields from one method to another and from one lipid family to another. The prospects of this study would be to optimize sample preparation for each studied matrix and lipid family of interested.

**References**

E. G. Bligh et al. Can. J. Biochem. Physiol. (1959) 37, 911–917.

J. Folch et al. J. Biol. Chem. (1956) 226, 497–209.

G. Vale et al. J. Lipid Res. (2019) 60, 694–706.

L. Löfgren et al. J. Lipid Res. (2012) 53, 1690–1700.

V. Matyash et al. J. Lipid Res. (2008) 49, 1137–1146.

A. Seyer et al. Metabolomics (2016) 12, 1–14.
**Poster 058: P058 Development of an untargeted metabolomics workflow for investigating drug-induced cardiotoxicity using cardiac microtissues.**

Tara J. BOWEN<fnref n="1" />; Andrew R. HALL<fnref n="2" />; Amanda WILSON<fnref n="2" />; Amy POINTON<fnref n="2" />; Mark R. VIANT<fnref n="1" />,<fnref n="3" />

**Introduction**

Cardiac microtissues are a promising in vitro model system for exploring drug-induced cardiotoxicity, a major contributor to attrition during drug development[1]. Cardiac microtissues are 3D co-cultures of cardiomyocytes, fibroblasts and endothelial cells, consisting of ~500 cells[2]. Untargeted metabolomics offers an opportunity to gain molecular-level insight into drug-induced cardiotoxicity. The aim of this research is to develop an untargeted metabolomics workflow for cardiac microtissues.

**Technological and methodological innovation**

Sampling of cardiac microtissues for untargeted metabolomics must be rapid, effectively remove extracellular contaminants, and limit cell lysis and/or leakage. Presented here is a novel approach that uses cell strainers to meet these requirements. Additionally, a highly sensitive direct infusion mass spectrometry (DIMS) metabolomics method[3] has been applied to assess the minimum biomass required for sensitive and reproducible measurement of the cardiac microtissue metabolome.

**Results and impact**

Approximately 2000 and 3500 polar and non-polar features, respectively, were robustly detected (biological and technical median RSDs <30% and <20%, respectively) for 28 pooled microtissues using a rapid sampling approach and highly sensitive DIMS metabolomics method. The optimized workflow will be applied to investigate drug-induced cardiotoxicity in a physiologically relevant in vitro system, enabling greater mechanistic understanding of underlying molecular perturbations and toxicity pathways.

**References**

[1] Cook, D. et al., 2014. *Nat Rev Drug Discov*, **13**, 419-31

[2] Pointon, A., et al., 2017. *Toxicol Sci.* **155**, 444-457 [3]
Southam, A.D., et al., 2016. *Nat Protoc*, **12**, 310-328

<footnote n="1">¹ School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK</footnote>

<footnote n="2">² Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, CB4 0WG, UK</footnote>

<footnote n="3">³ Phenome Centre Birmingham, University of Birmingham, Birmingham, B15 2TT, UK</footnote>
**Poster 059: P059** *A metabolomics-like approach for chemical forensic: preliminary study and perspectives*

Grégoire DELAPORTE¹; Christine ALBARET¹; Anne BOSSEE¹

¹ DGA CBRN Defense, Analytical Chemistry department, 91710 Vert-Le-Petit, FRANCE

**Introduction**

The attribution of a chemical (or a mixture) to its source (i.e. synthesis path, production batch etc.) is the aim of the developing field called “Chemical forensic” [1]. It is of special interest in order to support the attribution of a chemical weapons use. Data analysis workflows coming from metabolomics were shown to have a high potential to address this issue [2]. However, more studies are needed to identify technological and methodological blocking points in the workflow.

**Technological and methodological innovation**

Two production batches of a nerve-agent precursor (methylphosphonic dichloride, DC) were analyzed by GC-MS after hydrolysis and extraction (both done in two separate laboratories for reproducibility assessment). An instrument-independent data treatment workflow based on R (peak-picking by XCMS) and Matlab (data analysis) was set up to differentiate samples according their impurity profiles and to detect potential technical bias (e.g. influence of operator or laboratory consumables).

**Results and impact**

A chemical profile was established for each sample. Interestingly, no major technical bias can be noticed regarding sample preparation. The following blocking points were identified in the chemical forensic workflow: (i) lack of complete coverage of MS data by peak-picking algorithms (ii) lack of flexible and validated variable selection strategies (iii) lack of harmonized analytical and data analysis strategy for inter-laboratory studies.

**References**

[1] Carlos G. Fraga, 2018, Talanta 186, 585

[2] Carlos G. Fraga et al., 2010, Analytical Chemistry, 82, 4165-4173
# Poster 060: P060 Simultaneous measurement of absolute metabolite concentration and isotope incorporation by mass spectrometry

Maud Heuillet<fnref n="1" />,<fnref n="2" />,†, Pierre Millard<fnref n="1" />,<fnref n="2" />,‡, Madi Y. Cissé³, Laetitia K. Linares³, Fabien Létisse<fnref n="1" />, Laurent Le Cam³, Jean-Charles Portais<fnref n="1" />,<fnref n="2" />,<fnref n="4" /> and Floriant Bellvert<fnref n="1" />,<fnref n="2" />

## Introduction

Studies of the topology, functioning and regulation of metabolic systems are based on two main types of information which can be measured by mass spectrometry: the absolute concentration of metabolites and their isotope incorporation in 13Clabeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the absolute concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified.

## Technological and methodological innovation

Here, we developed a generic method with a dedicated processing workflow to obtain these two information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of two both the number of cultivations to perform and the number of samples to collect, prepare and analyze. The proposed approach is based on an IS labeled with other isotope(s) which can be resolved from the 13C-mass fractions of interest.

## Results and impact

As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the PERK kinase, a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino-acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

<footnote n="1">¹TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, 31077, France</footnote>

<footnote n="2">²MetaboHUB-MetaToul, National Infrastructure of Metabolomics and Fluxomics, Toulouse, 31077, France ³IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier, Montpellier, F-34298, France</footnote>

<footnote n="4">⁴STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, Toulouse, France</footnote>
**Poster 061: P061 Evaluation of pure shift NMR method in metabolomics**

Cécile CANLET<fnref n="1" />,<fnref n="2" />; Marie TREMBLAY-FRANCO<fnref n="1" />,<fnref n="2" />; Lindsay PEYRIGA<fnref n="3" />; Edern CAHOREAU<fnref n="3" />; Roselyne GAUTIER<fnref n="1" />,<fnref n="2" />; Philippe PINTON<fnref n="1" />; Isabelle P. OSWALD<fnref n="1" />; Guy LIPPENS<fnref n="4" />

**Introduction**

Although 1H NMR spectroscopy is commonly used in metabolic profiling, the analysis of the spectrum is complex because of extensive signals overlap. To overcome this limitation, pure shift NMR, method through which multiplicities are collapsed into single lines, has shown to be a convenient tool to address crowded spectra [1,2] but has been rarely used for metabolomics [3]. In this study pure shift NMR was evaluated to explore hepatic metabolic disruptions of pigs exposed to a food contaminant.

**Technological and methodological innovation**

Aqueous liver extracts of pigs fed two different diets and exposed or not to Fumonisin B1 (FB1) were analyzed using standard 1D proton sequence with suppression of water signal (noesypr1d) and pure shift NMR sequence (SAPPHIRE-PSYCHE [4]) on a 800 MHz NMR spectrometer. After spectra bucketing, multivariate statistical analyses (Partial Least Squares Discriminant Analysis – PLS-DA) were performed on the two datasets. Discriminant metabolites were identified for each sequence and compared.

**Results and impact**

1H NMR spectra of aqueous liver spectra were simplified using the pure shift sequence, thereby allowing easier metabolite identification but at the expense of lower sensitivity than the standard proton NMR sequence. PLS-DA analysis showed a separation between the two diets, and between control and FB1 exposed animals for the first diet. In addition to the standard proton sequence, pure shift sequence enables the identification of additional discriminant metabolites such as betaine and lactate.

**References**

[1] Zangger K., Prog Nucl Magn Reson Spectrosc. 2015, 86-87:1-20.

[2] Castañar L, Parella T. Magn Reson Chem. 2015, 53(6):399-426.

[3] Lopez et al. 2019. Scientific Reports. 9:6900.

[4] Moutzourt et al. 2017. Chem. Commun. 53 and 10188-10191.

<footnote n="1">¹ Toxalim – Research Center in Food Toxicology, Toulouse University, INRAE UMR 1331, ENVT, INP-Purpan, Paul Sabatier University, F-31027 Toulouse, France</footnote>

<footnote n="2">² Metatoul-AXIOM platform, National Infrastructure for Metabolomics and Fluxomics, MetaboHUB, Toxalim, INRA UMR 1331, F-31027 Toulouse, France</footnote>

<footnote n="3">³ Metatoul-Metabolic Network platform, National Infrastructure for Metabolomics and Fluxomics, MetaboHUB, TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote n="4">⁴ TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>
# Poster 062: P062 A high throughput fluxomic workflow for exploration of metabolic phenotypes

Cécilia BERGES<fnref n="1" />,<fnref n="2" />, Edern CAHOREAU<fnref n="1" />,<fnref n="2" />, Hanna BARBIER<fnref n="1" />,<fnref n="2" />, Lara GALES<fnref n="1" />,<fnref n="2" />, Noémie BUTIN<fnref n="1" />,<fnref n="2" />, Matthieu GUIONNET<fnref n="1" />,<fnref n="2" />, Maud HEUILLET<fnref n="1" />,<fnref n="2" />, Mickael DINCLAUX<fnref n="1" />, Lindsay PEYRIGA<fnref n="1" />,<fnref n="2" />, Stéphanie HEUX<fnref n="1" />, Brice ENJALBERT<fnref n="1" />, Pierre MILLARD<fnref n="1" />, Floriant BELLVERT<fnref n="1" />,<fnref n="2" /> and Jean-Charles PORATAIS<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

## Introduction

High-throughput (HT) omics approaches enable the large-scale analysis of biological molecules and try to get a comprehensive understanding of living organism and their regulation. Among those, metabolic flux analysis aims at measuring the actual rates of biochemical reactions in metabolic networks. It is a powerful and growing approach to get comprehensive understanding of cellular metabolism in many fields of investigation, ranging from biotechnology to medical sciences.

## Technological and methodological innovation

However, a 13C fluxomic experiment requires several complex experimental and computational steps as well as large input of manual steps which may lower robustness of flux analyses. These make classical fluxomics a low throughput approach, which limits the number conditions which can be investigated at once. To increase fluxomics throughput and robustness while reducing experimental costs and human efforts, we designed a complete workflow in order to automatize, parallelize, integrate and optimize each step of fluxomics experiments.

## Results and impact

Thanks to this development, we are able to quantify flux responses to environmental or genetic perturbations in complement to other omics approaches. It opens the way to very large-scale fluxomics screening experiment. Thanks to our HT-Fluxomics workflow, 240 flux maps were generated in 3 months; this workflow can now be applied to large-scale studies in many fields: biotechnology, biomarker, drug development, medical science, toxicology or pharmacology.

## References

Wiechert W – Metabolic Engineering (2001). 13C Metabolic Flux Analysis, Volume 3, Issue 3, July 2001, Pages 195-206.
Heux.S & al – Metabolic Eng. (2014). A novel platform for automated high-throughput fluxome profiling of metabolic variants.

Millard P & al – Biotech. Bioeng (2014). IsoDesign: a software for optimizing the design of 13C-metabolic flux analysis experiments.

Sokol & al. – Bioinformatics (2012). Influx_s: increasing numerical stability and precision for metabolic flux analysis in isotope labelling experiments

Millard P & al – Bioinformatics (2019). IsoCor: isotope correction for high-resolution MS labeling experiments.

<footnote n="1">¹ TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote n="2">² MetaToul-MetaboHUB, National Infrastructure of Metabolomics & Fluxomics (ANR-11-INBS-0010)</footnote>

<footnote n="3">³ STROMALAB, Université de Toulouse, CNRS ERL5311, EFS, INP-ENVT, Inserm U1031, UPS</footnote>
**Poster 063: P063 Metabolomic fingerprint of aging on red blood cells**

Inés DOMINGO-ORTÍ<fnref n="1" />, José Raúl HERANCE<fnref n="2" />, Rubén LAMAS-DOMINGO<fnref n="3" />, Andrea CIUDIN<fnref n="4" />, Rafael SIMÍO<fnref n="4" />, Martina PALOMINO-SCHÄTZLEIN<fnref n="3" />*, Antonio PINEDA-LUCENA<fnref n="5" />*

**Introduction**

Increases in the lifespan due to better life conditions, together with the drop in the birth rates are leading to a more aged society<fnref n="1" />. The aging process leads to many changes in the human organism and has been associated with the development of many conditions<fnref n="2" />,<fnref n="3" />. Since these changes are reflected in the human metabolome, the evaluation of the metabolomic alterations occurring in the human body during aging is an attractive strategy to address diseases.

**Technological and methodological innovation**

Metabolomics is a relatively novel and personalized strategy<fnref n="4" /> that has been widely used in human blood samples. However, Red Blood Cells (RBCs) have been poorly assessed<fnref n="5" />. RBCs are abundant, readily available and they contain many metabolites, making them a potential tool to obtain information about the health condition of each person. Therefore, the goal of this work was to perform an NMR metabolic study of RBCs to assess aging alterations and the effect of lifestyle factors, such as BMI.

**Results and impact**

Aging showed a clear fingerprint on RBCs with alterations on aminoacid, carbohydrate, lipid and energy metabolism. BMI also presented metabolite patterns in RBCs and altered the age-related metabolic changes, whereas gender had a less significant impact on its metabolic profile. Metabolomic profiling of RBCs by NMR proved to be a fast and robust method to detect the fingerprint of age and determine relationships between the biological processes that take place in aging and obesity.

**References**

[1] World Population Ageing 2019: Highlights. United Nations

[2] Norman R. Lazarus *et al*. 2018. Front. Physiol. 9:1135

[3] Efraim Jaul *et al*. 2017. Front. Public Health. 5:335

[4] Xiaohua Douglas *et al*. 2015. J Pharmacogenomics Pharmacoproteomics. 6:2 [5]
Romanas Chaleckis *et al*. 2016. PNAS. 113:4252

<footnote n="1">¹ Drug Discovery Unit, Instituto de Investigación Sanitaria La Fe, Av. Fernando Abril Martorell, Torre 106 A, 46026, Valencia, Spain</footnote>

<footnote n="2">² Medical Molecular Imaging Research Group, Vall d'Hebron Research Institute, CIBBIM-Nanomedicine, Pg. Vall d'Hebron 119-129, Barcelona, Spain</footnote>

<footnote n="3">³ NMR Facility, Centro de Investigación Príncipe Felipe, C/ Eduardo Primo Yúfera, 3, 46012 Valencia, Spain.</footnote>

<footnote n="4">⁴ Diabetes and Metabolism Research Unit, Valld'Hebron Research Institute, Pg. Vall d'Hebron 119-129, Barcelona, Spain; and CIBERDEM (Instituto de Salud Carlos III), Madrid, Spain.</footnote>

<footnote n="5">⁵ Medicinal Chemistry Laboratory, Molecular Therapeutics Program, Centro de Investigación Médica Aplicada, Universidad de Navarra, Avda. Pío XII, 55, 31008-Pamplona, Spain</footnote>
# Poster 064: P064 Intelligent Acquisition for Comprehensive Metabolome Coverage in Plants, Mammals, and Bacteria

Tatjana TALAMANTES¹; Sven HACKBUSH¹; Ioanna NTAI¹; Amanda SOUZA¹

¹ Thermo Fisher Scientific, San Jose, CA

**Introduction (500/500 characters spaces included)**

Unknown identification remains a challenge in LC/MS based untargeted metabolomics where thousands of features from a single sample are routinely detected. However, not all features represent metabolites of biological origin resulting in fragmentation spectra of background ions and degenerate signals. Thermo Scientific™ AcquireX intelligent data acquisition was applied to plant, mammal and bacterial samples to deliver an efficient workflow for deeper metabolome coverage and increased annotations.

**Technological and methodological innovation (490/500 characters spaces included)**

Data-dependent acquisition often provides information for the most abundant ions. Data-independent acquisition can get fragmentation data for all features, but results in convoluted MS/MS spectra complicating identification. The AcquireX acquisition software determines on-the-fly features corresponding to background contaminants and compound degeneracy, like isotopes, adducts, and dimers, enabling more efficient precursor selection to target unique metabolites of biological relevance.

**Results and impact (496/500 characters spaces included)**

Samples of varying complexity were used to demonstrate the utility of AcquireX acquisition. By excluding background and degenerate signals, the total number of fragmentation targets was reduced without losing metabolite coverage. Over 10,000 features were detected in E. coli extracts, but after data reduction on-the-fly, only 1,040 features corresponded to unique compounds. A similar 10-fold reduction in precursors to target was seen in extracts of yeast, tea, human urine, and human plasma.
# Poster 065: P065 Novel MALDI imaging solution empowered by a dual-source Q-TOF and a dedicated bioinformatics pipeline for identification of peaks from tissue

Sabine Jourdain⁵, Janina Oetjen¹, Arne Fuetterer¹, Juergen Suetering¹, Niels Goedecke¹, Stephanie Kaspar-Schoenefeld¹, Scarlet Koch¹, Shannon Cornett², Christian Marsching³, Carsten Hopf⁵, Nikolas Kessler¹, Wiebke Timm¹; Aiko Barsch¹; Heiko Neuweger¹, Jan H. Kobarg⁴, Dennis Trede⁴, Sören-Oliver Deininger¹, Alice Ly¹, Jens Fuchser¹, Lucy Woods¹, Oliver Raether¹, Jens Höhndorf¹

1 Bruker Daltonik GmbH, Bremen, Germany

2 Bruker Daltonics Inc., Billerica, MA, USA

3 Mannheim, Germany

4 SCiLS, Bremen, Germany

5 Bruker Daltonics, France

## Introduction

MALDI Mass Spectrometry Imaging (MALDI-MSI) has emerged as a technique with a broad range of applications in Omics research. We present initial results from the timsTOF fleX system; a timsTOF Pro QTOF mounted with a high-throughput, high spatial resolution MALDI source and stage. The instrument allows for the robust, high speed acquisition of both MALDI and LC-MS/MS data. When combined with a software processing pipeline for automatically annotating measured ions, this enabled generation of annotated images from MALDI-imaging data.

## Technological and methodological innovation

ESI mode performance on the timsTOF fleX was evaluated by analyzing a commercially available HeLa digest (Pierce) using DDA PASEF. For MALDI-MSI experiments, tissue sections were mounted on conductive glass slides, and coated with matrix using standard protocols or a specialised sublimation device. Ion mobility imaging experiments were acquired at 150 1/K0 mobility resolution. SCiLS Lab was used for visualising and statistical analysis of MALDI-MSI data. MetaboScape was used for feature extraction, de-isotoping, ion deconvolution and automatic annotations.

## Results and impact

MALDI Imaging data was acquired at a rate of up to 20 pixels/second in positive and negative mode. Rat testis sections measured at 10µm zoom-mode showed different seminiferous tubule structures. In experiments designed to stress the system, 20 hours of image acquisition showed no decline in imaging dataset quality and a mass deviation of RMS 2.06 without lock mass. Trapped ion mobility imaging measurements removed isobaric interferences in lipid imaging.

MetaboScape was used for lipid annotation using untargeted (raw formula generation) or targeted (e.g. HMDB, LipidMaps) methods.
**Poster 066: P066 Establishing a Spectral Library and Accurate Mass Retention Time (AMRT) Database for Pediatric Metabolomics Analysis**

Anas KAMLEH<fnref n="1" />, Chiara LAVARELLO<fnref n="2" />, Sebastiano BARCO<fnref n="2" />, Igor FOCHI<fnref n="1" />, Martina BATOLUCCI<fnref n="2" />, Gino TRIPODI<fnref n="2" />, Giuliana CANGEMI<fnref n="2" />, Andrea PETRETTO<fnref n="2" />

**Introduction**

Metabolomics is an established discovery tool for biomarker discovery, disease diagnosis and novel mechanistic insights of pathophysiological processes. Liquid-chromatography mass spectrometry (LCMS) is currently the method of choice for metabolome analysis, however, the wide variety of chromatographic columns and conditions is presenting a challenge in sharing and interpretation of metabolite identities.

**Technological and methodological innovation**

We have established a spectral library of compounds in the (IROA) kit for metabolite identification. Data was acquired using flow injection analysis (FIA) which allowed the fast acquisition of spectra at a wide range of collision energies (0-200, normalized collision energy NCE). Reference standards were injected on an Vanquish™-Q-Exactive™ LCMS system. Two libraries were established, one spanning all NCEs and one restricted to a carefully selected NCE for each compound.

**Results and impact**

The libraries were used as a training set to select generalized NCE values for unknowns. We provide a scheme to produce the optimal number of mixtures for IROA metabolites or similar reference libraries. The mixtures were used to establish an accurate mass/retention time (AMRT) databases on the optimized chromatographic conditions (reversed-phase and HILIC). Metabolite identification in matrices (plasma, urine and sweat) were further validated using the established AMRT databases and libraries.

<footnote n="1">¹ Thermo Fisher Scientific, Europe</footnote>

<footnote n="2">² Gasilini Hospital for Children, Genoa, Italy</footnote>
## Poster 067: P067 Investigation of mevalonate pathway by quantitative metabolomics and isotopologue profiling

Hanna Kulyk<fnref n="1" />, Sara Castano-Cerezo<fnref n="1" />, Pierre Millard<fnref n="1" />, Jean-Charles Portais<fnref n="1" />,<fnref n="2" />, Stéphanie Heux<fnref n="1" />, Gilles Truan<fnref n="1" />, Floriant Bellvert<fnref n="1" />,<fnref n="2" />

Mevalonate pathway is an important metabolic pathway which plays a key role in multiple cellular processes. In recent years the mevalonate pathway has become a challenging and, in the meantime, fascinating topic, when a large number of experimental and clinical studies suggested that inhibition of nonsterol isoprenoids might have valuable interest in human pathology. These molecules that are essential for cell growth and differentiation appear to be potentially interesting therapeutic targets for many areas of ongoing research: oncology, autoimmune disorders, atherosclerosis, and Alzheimer disease. Thus, functional understanding of their biosynthesis is of high importance. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors (mevalonate intermediates and prenyl pyrophosphates). We present a complete methodology for functional analysis of isoprenoids biosynthesis, with a novel quantification method based on liquid chromatography coupled to high-resolution mass spectrometry. This workflow covers all the experimental and computational steps from sample collection and preparation to data acquisition and processing. It ensures accurate, absolute quantification (RSD < 20 %) of all mevalonate and prenyl pyrophosphates intermediates with a high sensitivity over a large linear range (from 0.1 to 50 pmol). Determination of their isotopologue distributions in isotopic labeling experiments was performed, opening the way for 13C-metabolic flux analysis of isoprenoids biosynthesis.

In conclusion, the described methodology fills one of the last technical gaps for functional studies of isoprenoids biosynthesis and should be applicable to other eukaryotic and prokaryotic (micro)organisms after adaptation of some organism-dependent steps.

<footnote n="1">¹TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France</footnote>

<footnote n="2">²MetaToul-MetaboHUB, National infrastructure of metabolomics and fluxomics, Toulouse, France</footnote>
**Poster 068: P068 High-throughput metabolite profiling of cell media for improved antibody production utilizing a dual separation/mass spectrometry system**

Ioanna NTAI¹; Amanda SOUZA¹; Andreas HUHMER¹

¹Thermo Fisher Scientific, San Jose, CA, US

**Introduction**

Chinese hamster ovary (CHO) cells are often used for commercial production of antibodies. Optimization of cell culture medium and feeds is required to obtain maximum product yield. Metabolomics analysis allows for quick determination of nutrient limitations or metabolite buildup during the cell culture. A semi-targeted workflow was designed to confidently measure critical nutrients, while allowing for the discovery of previously unidentified metabolites that affect growth and antibody yield.

**Technological and methodological innovation**

A dual UHPLC separation system coupled to an Orbitrap™ mass spectrometer was used to increase analysis throughput of a time-course study of 5 different feeds, while also maximizing metabolome coverage. This dual LC system combines RP and HILIC separation for the detection of hydrophobic and polar metabolites from the same sample. AcquireX intelligent acquisition maximized the number of metabolites interrogated by MS/MS, resulting in more confident metabolite annotations.

**Results and impact**

By combining RP and HILIC, we reproducibly analyzed a larger number of metabolites. Efficiently overlapping the two separations resulted in 30% decrease in analysis time. This increased throughput did not compromise reproducibility. Nutrient depletion could be easily measured and correlated to improved titer. This dual LC/MS system was successfully used for the analysis of cell media and provided robust metabolic indicators of cell growth and antibody yield and possible optimization strategies.
**Poster 069: P069** *The development of selective sorbent for isolation of catecholamines and their metabolites from biological fluids*

Anton Podjava¹; Peteris Mekss¹

¹ Laboratory of Chromatography and Mass Spectrometry, University of Latvia Academic Centre, University of Latvia, Riga, Latvia

**Introduction**

The role played by catecholamines (CAs) and their main metabolites (homovanillic and vanillylmandelic acid) in different physiological processes and various disorders [1, 2] attracts interest in many areas of medical research. Thus, simultaneous determination of CAs and their metabolites in biological fluids using fast and reliable analytical procedure is the efficient way for diagnosis, therapy and prognosis of related diseases [3, 4].

**Technological and methodological innovation**

Taking into account several challenges usually encountered during isolation and detection of CAs and their metabolites, the aim of this study is the development of solid-phase extraction sorbent for simultaneous separation and enrichment of CAs together with corresponding acidic metabolites from biological fluids in single step. The development of the sorbent is based on combination of non-covalent and semi-covalent molecular imprinting [5] method to form binding sites for CAs and the acids.

**Results and impact**

Despite the fact that the study is in progress, the optimization of molecular imprinting procedure has been completed by varying cross-linking monomers, solvent system and the ratio of functional monomers to cross-linker. As a result, eight polymers based on methylenebisacrylamide as cross-linker have been prepared and tested. Preliminary single point batch extraction experiments have shown rather high capacity of these polymers for the analytes in comparison to control polymers (up to 7:1).

**References**

[1] D. Goldstein. 2010. Clinical Autonomic Research. 20, 331-352

[2] L. Liu et al. 2011. Journal of Separation Science. 34, 1198-1204

[3] C. Lv et al. 2015. Journal of Mass Spectrometry. 50, 354-363

[4] Y. He et al. 2016. Journal of Chromatography A. 1458, 70-78
**Poster 070: P070 Data processing workflow to study the exposome by suspect screening**

Emilien JAMIN<fnref n="1" />,<fnref n="2" />, Jean-François MARTIN<fnref n="1" />,<fnref n="2" />, Robin COSTANTINO<fnref n="1" />,<fnref n="2" />, Alyssa BOUVILLE<fnref n="1" />,<fnref n="2" />, & Laurent DEBRAUWER<fnref n="1" />,<fnref n="2" />

**Introduction**

The characterization of all factors, chemical or not, to which an organism is exposed during his life, could be in part addressed by non-targeted metabolomics approaches. Biomarkers detected from this kind of methodology could be particularly interesting to better characterize the exposure to xenobiotics. However, one of the main challenges of nontargeted approaches to study the chemical exposome is to differentiate metabolites of xenobiotics from all other metabolites.

**Technological and methodological innovation**

One solution to highlight xeno-metabolites is suspect screening, as previously applied for pesticides [1]. It is based on a nontargeted acquisition of data, followed by a screening of suspect compounds that are expected to be present in samples. Annotation is achieved using databases of known xenobiotics, their known or putative metabolites but for which no standard is available. To deal with the potentially huge amount of matches, an advanced data processing workflow has been developed.

**Results and impact**

A large but representative database was built from metabolites of xenobiotics. Annotated features from this database are curated according to several parameters: chromatographic signal, signal/noise, blank comparison, potential annotation of an endogenous metabolite, isotopic pattern (mono-charged, monoisotopic, halogenated ions, etc.), targeted MS/MS, MS3 for conjugated metabolites. Following identifications at least at level 3, statistics can be performed on features of identified metabolites.

**References**

[1] Jamin, E.L. et al. 2014. Analytical and Bioanalytical Chemistry. 406 1149-1161

<footnote n="1">¹ Toxalim, Toulouse University, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France.</footnote>

<footnote n="2">² Metatoul-AXIOM platform, MetaboHUB, Toxalim, INRAE, Toulouse, France</footnote>
**Poster 071: P071 Development of an untargeted approach based on isotope profiling of metabolic networks by high resolution mass spectrometry**

Noémie Butin<fnref n="1" />,²,<fnref n="3" />, Maud Heuillet<fnref n="1" />,², Samuel Naudi<fnref n="1" />,², Matthieu Guionnet<fnref n="1" />,², Cecilia Bergès<fnref n="1" />,², Lindsay Peyriga<fnref n="1" />,², JeanCharles Portais<fnref n="1" />,²,<fnref n="3" /> and Floriant Bellvert<fnref n="1" />,²

**Introduction**

The study of metabolism has become a major research challenge for understanding the operation of biological systems (cells, tissues, organisms). A relevant approach is to study the topology and operation of metabolic networks mainly based on 13 C 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.

These current targeted approaches are very effective but do not allow to highlight unsuspected metabolic pathways not initially targeted. 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 a biological organism without a priori knowledge.

**Technological and methodological innovation**

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.

**References**

[1] Kiefer et al. 2013. Bioinformatics. 29:963-964

[2] Capellades et al. 2016. Anal. Chem. 88:621-628

[2] Huang et al. 2014. Anal. Chem. 86:1632-1639

<footnote n="1">¹MetaboHUB-MetaToul, national infrastructure for metabolomics and fluxomics, 31400 Toulouse, France ²TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France.</footnote>

<footnote n="3">³STROMALAB, Université de Toulouse, CNRS ERL5311, EFS, INP-ENVT, Inserm U1031, UPS</footnote>
**Poster 072: P072 Improving lipid annotation coverage using intelligent precursor selection software on an Orbitrap-based mass spectrometer**

Sven Hackbusch<fnref n="1" />, Claire Dauly<fnref n="2" />, David Peake<fnref n="1" />, Reiko Kiyonami<fnref n="1" />

...

Lipid profiling provides valuable information to identify disease states and other physiological changes. A common approach used in lipid profiling by LC-MS is to identify lipid species by their MS/MS spectra prior to extraction of precursor information for relative quantitation between conditions. The novel intelligent data acquisition strategy, AcquireX, on the Thermo Scientific™ Orbitrap ID-X™ Tribrid™ mass spectrometer automatically excludes background ions from the MS/MS acquisition and prioritizes MS/MS precursor selection on sample relevant compounds, resulting in deeper lipidome coverage. Here, Bovine liver total lipid extract (Avanti Polar Lipids) was used to demonstrate the utility of the workflow. Samples were separated using reversed-phase conditions on a C30 column with mass spectral data acquired on an Orbitrap ID-X Tribrid MS. Data analysis was performed using Thermo Scientific™ LipidSearch™ 4.2 software to identify lipid molecular species based on acquired MSn fragmentation spectra.

The AcquireX data acquisition strategy was used to automatically generate background exclusion and/or compound inclusion lists that were updated iteratively for replicate injections, reducing redundant data collection and triggering more unique lipids for fragmentation. As a result, a significantly larger number of lipids (>40%) could be detected compared to a conventional data-dependent MS/MS approach.

In addition, the acquisition of neutral loss-triggered CID-MS3 and product ion-triggered CID-MS2 improved the confidence in the lipid annotations. For example, the use of fatty acid neutral loss-triggered MS3 fragmentation with triglycerides allowed to distinguish multiple co-eluting isomers with different fatty acid chain lengths based on their respective MS3 data.

<footnote n="1">¹ Thermo Fisher Scientific, San Jose, CA, USA</footnote>

<footnote n="2">² Thermo Fisher Scientific, France</footnote>
**Poster 073: P073** *Comparison of normalization techniques for untargeted metabolomics of urine samples.*

L.Mervant¹, ², M.Tremblay-Franco¹, ², J-F.Martin¹, ², E.Jamin¹, ², F.Gueraud¹, L.Debrauwer¹, ²

¹ Toxalim, Toulouse University, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France. ² Metatoul-AXIOM platform, MetaboHUB, Toxalim, INRAE, Toulouse, France

**Introduction**

Urine is one of the most used matrixes for metabolomics, in particular in toxicology since it represents one of the main detoxification routes. To overcome the natural and high variability of urine concentration that can hamper the results interpretation, many normalization strategies have emerged. Since there is no consensus on which normalization technique should be used or not, we evaluated the most commonly used techniques on a metabolomics dataset.

**Technological and methodological innovation**

Urine samples of 40 healthy volunteers (20 women and 20 men) were analysed by UPLC-HRMS. Before the analysis, samples were diluted to normalize creatinine or osmolality level or diluted by a common factor. Postacquisition normalization methods based on these parameters (in combination or not with PQN or MSTUS) were also assessed. Comparison of normalization techniques relied on their ability to reduce unwanted variability and to improve the performance of generated statistical models.

**Results and impact**

Compared with non-normalized results, data obtained from either osmolality or creatinine normalized samples yielded better results. Pre-acquisition normalization led to a better statistical discrimination compared to nonnormalized or post-acquisition normalized samples especially with specific dilution factors according to osmolality levels. MSTUS and PQN normalization did not improve statistical discrimination due to the suppression of part of the biological variability of interest.
**Poster 074: P074 Comparison of two sampling and extraction protocols to perform
metabolomic studies on human adherent cells**

Joran Villaret-Cazadamont<fnref n="1" />, Noémie Butin³,⁴, Jean-François Martin<fnref n="1" />,<fnref n="2" />,³, Marie Tremblay-Franco<fnref n="1" />,<fnref n="2" />,³, Cécile Canlet<fnref n="1" />,<fnref n="2" />,³, Emilien Jamin<fnref n="1" />,<fnref n="2" />,³, Roselyne Gautier<fnref n="1" />,<fnref n="2" />,³, Daniel Zalko<fnref n="1" />, Nicolas Cabaton<fnref n="1" />, Floriant Bellvert³,⁴, Nathalie Poupin<fnref n="1" />.

**Introduction**

Different methods of cell sampling and metabolites extraction can be used when performing metabolomics analyses on adherent cells. However, these steps are critical and can notably impact the results. An appropriate design of the experiment is therefore necessary, particularly as regards the quenching of metabolic activity and the washing steps. These steps have to be fast and reproducible to prevent biases and unwanted variability in the detection of metabolites due to different factors (temperature change, cell stress...).

**Technological and methodological innovation**

We compared two sample collection workflows for performing targeted and untargeted metabolomics on adherent cells. The first one is a standard protocol involving 1 operator, widely used for such application and consisting in the washing and scraping of the cells within wells [1]. The second is a fast-quenching (FQ) protocol requiring 5 operators, which aims to block cell metabolism as fast as possible to minimize metabolic status disturbances, and optimizes the washing step to avoid a contamination by cell media [2]. Sampled were analyzed by LC-HRMS, IC-HRMS and NMR.

**Results and impact**

Samples obtained with the FQ workflow were shown to display less variability, suggesting that stopping cells metabolism fast enough allows to lessen experimental cells metabolome disruption, especially as regards central carbon metabolites which are prone to fast metabolic reactions. Blank samples obtained with the FQ method were also found to be less polluted, providing a valuable alternative to prevent metabolites concentration overestimation in targeted metabolomics, and preserve a maximal number of variables during the step of blank filtration, in untargeted metabolomics.

**References**

[1] Cabaton et al. 2018. Front. Endocrinol. 9:571

[2] Martano et al. 2014. Nat. Protoc. 10:1-11

<footnote n="1">¹ UMR1331 Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31300 Toulouse, France</footnote>

<footnote n="2">² MetaToul-AXIOM platform, INRAE, 31300 Toulouse, France ³ MetaboHUB-MetaToul, national infrastructure for metabolomics and fluxomics, 31027 Toulouse, France ⁴ TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France.</footnote>
**Poster 075: P075 Development of a System Suitability QC Sample for Nano-Electrospray Direct Infusion Mass Spectrometry Metabolomics**

Judith B. NGERE<fnref n="1" />; Martin R. JONES<fnref n="1" />,<fnref n="3" />; Ralf J. M. WEBER<fnref n="1" />,<fnref n="2" />; Mark R. VIANT<fnref n="1" />,<fnref n="2" />

**Introduction**

Nano-electrospray direct-infusion mass spectrometry (nESI-DIMS) based metabolomics is well recognized for its capability for high throughput screening, high analytical sensitivity and low selectivity. In order to be adopted for applications other than academic research, for example into regulatory or clinical practice, standardised and robust quality assurance and quality control (QA/QC) procedures are required, as described in the MERIT guidelines<fnref n="1" />.

**Technological and methodological innovation**

Presented is the development and testing of a system suitability QC (SSQC) comprising of a mixture of standards analysed using our current DIMS workflow<fnref n="2" />. For added confidence, in-source fragments were considered alongside multiple adducts, isotopes and database matching. Repeated analyses of the SSQC were then carried out in a multi-batch DIMS metabolomics study, facilitating in-depth exploration of the m/z accuracy, and m/z and intensity precision to allow acceptability thresholds to be set.

**Results and impact**

Of the ca. 77 standards analysed, 10-25 chemicals were reproducibly detected and annotated per assay<fnref n="3" />. Repeat analyses showed expected m/z precision to be <0.5ppm and intensity RSDs of 5-30%. Mass errors were higher than predicted with a range of +/-10ppm, though the observed trend between mass error and m/z was strikingly repeatable across replicates. Future work will include tandem DIMS analysis of SSQC for level 1 identification<fnref n="3" />.

**References**

[1] Viant, M. R., et al., 2019. Nat Commun, 10, 3041

[2] Southam, A.D., et al., 2016. Nat Protoc, 12, 310-328

[3] Sumner, L. G., et al., 2007. Metabolomics, 3, 3, 211-221

<footnote n="1">¹School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK</footnote>

<footnote n="2">²Phenome Centre Birmingham, University of Birmingham, Birmingham, B15 2TT</footnote>

<footnote n="3">³Eawag, Überlandstrasse 133, 8600 Dübendorf (new address)</footnote>
**Poster 076: P076 Evaluation of U-¹³C Spirulina (Arthrospira platensis) for stable isotope assisted untargeted metabolomics and liquid chromatography-isotope dilution mass spectrometry**
(LC-IDMS)

Salah ABDELRAZIG\*, Bruna DE FALCO\*, Laudina SAFO, Dong-Hyun KIM

Centre for Analytical Bioscience, Advanced Materials and Healthcare Technology Division, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K

\* Salah ABDELRAZIG and Bruna DE FALCO are joint first authors.

**Introduction**

The use of commercially available microorganisms grown on labelled substrates has become an accessible source of multiple isotopes for the use in stable isotope assisted untargeted metabolomics (SIAM) and LC-IDMS [1]. However, in most cases the enrichment profile of the metabolites is not provided and therefore, the presence of unlabelled metabolites in the labelled extract introduces more variations in sample analysis, leading to false identification and inaccurate quantification.

**Technological and methodological innovation**

Commercially available U-¹³C spirulina was investigated using untargeted metabolomics approach. ZIC-pHILIC-HRMS/MS was used for the analysis of the methanolic extracts of U-¹³C, U-¹²C spirulina (n=6) and 268 authentic standards in a single analytical run. Metabolite identification and SIAM were performed using Compound Discoverer by interrogating a species specific BioCyc database, mzCloud database and RTs of the standards and the use of stable isotope labelling algorithm.

**Results and impact**

226 metabolites were identified including amino acids, nucleotides, carbohydrates, organic and fatty acids and the relative exchange ratio [¹³C/¹²C] showed that 42% were fully labelled, 33% were partially labelled and 25% were unlabelled. The characterisation of spirulina has an added impact on its use; the labelled metabolites in the U-¹³C extract enhance SIAM and LC-IDMS analyses, while the unlabelled isotopologues restrict their use as internal standards thus an adjustment method is needed.

**References**

[1] Schatschneider et al. 2018. Analytical Chemistry. 90, 4470–4477
**Poster 077: P077 Increased throughput and coverage for the annotation of Saponins using a structurebased MSn approach on a Tribrid Orbitrap mass spectrometer**

Reiko Kiyonami, Caroline Ding, Seema Sharma, Marie-Pierre Pavageau and Andreas Huhmer Thermo Fisher Scientific, San Jose, CA, USA

**Introduction**

Saponins are major components of Chinese medicines and exert various pharmacological effects such as cardiovascular protective activity and anticancer activity. The comprehensive annotation of saponins remains challenging because of the limited availability of authentic standards and the structural diversity of this class of compounds.

**Technological and methodological innovation**

Taking advantages of high resolution MS and MSn capability offered by the tribrid Orbitrap mass spectrometer (Thermo Scientific™ Orbitrap ID-X™ Tribrid™), we developed a product ion-dependent MSn data acquisition method in which MS² data is constantly collected and further followed by higher order FTMSn if sugar neutral loss are detected from the MS² data. The MSn (up to ⁴) spectral tree data were processed using Thermo Scientific™ Mass Frontier™ 8.0 and Thermo Scientific™ Compound Discoverer™ 3.1 softwares. Multiple databases were employed in the processing workflow including mzCloud™ spectral library, ChemSpider database, and custom databases for unknown saponin structure annotation.

**Results and impact**

As the proof of concept, methanol extracts from Sanqi were analysed. The collected MSn tree data were first searched against the mzCloud MSn spectral library in batch mode using the subtree search tool of Mass Frontier 8.0 software for identifying the saponin class compounds. The unknown compounds with exact MSn spectral tree match against the library references were annotated. However, for most unknown compounds, only partial MSn tree matched with the library references, resulting in only partial saponin basic structure annotation of these unknown compounds. The partial structure of unknown compounds could be annotated as the precursor structure which generated the MS3 spectrum of the saponin library reference and the unknown compound was annotated as one of saponin class compounds.

In addition, the novel structure ranking tools included in the Thermo Scientific™ Compound Discoverer™ 3.1 software together with Chemspider and custom database was used for final structure annotation of identified saponin class compounds. The structure-based MSn workflow was able to annotate 127 saponin compounds.
**Poster 078: P078 Application of ¹³C flux analysis in diagnosis of metabolic disorders**

Marek J NOGA<fnref n="1" />, Raisa VEIZAJ<fnref n="1" />, Hanneke KWAST<fnref n="1" />, Liesbeth WINTJES<fnref n="1" />, Frans van der BRANDT<fnref n="1" />, Richard RODENBURG<fnref n="1" />, Dirk J LEFEBER<fnref n="1" />,<fnref n="2" />

**Introduction**

Significant cost reduction of DNA sequencing results in discovery of multiple new gene variants. However, their pathogenicity and molecular phenotypes needs to be uncovered and verified by functional tests. Labeling with stable isotopes and targeted metabolomics can be used to measure metabolic fluxes within central carbon and sugar metabolism providing a unified functional assay capable of diagnosing multiple genetic defects.

**Technological and methodological innovation**

Targeted LC-MS/MS methods were optimized for detection of incorporation of ¹³C labels into intermediates of sugar and central carbon metabolism. In silico MS/MS fragmentation was performed to predict mass of fragment ions for selective detection of isotopomers. Patient-derived fibroblasts were cultured until near confluency. After a shift to a medium supplemented with ¹³C glucose the incorporation of isotopic label was followed for up to 48 hours.

**Results and impact**

The method allowed detection of ¹³C incorporation into TCA cycle intermediates on positions consistent with action of pyruvate dehydrogenase and citric acid synthase. Significantly lower PDH-dependent flux into TCA cycle was observed for cells derived from patient with prior diagnosis of PDH deficiency.

The results show potential to use MS/MS to distinguish positional isotopomers of metabolites of central carbon metabolism and deconvolute fluxes through alternative pathways.

**References**

[1] Anke P Willems et al. 2019. Biochimica et Biophysica Acta (BBA) - General Subjects. Vol 1863, 1471-1479

<footnote n="1">¹ Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands.</footnote>

<footnote n="2">² Dep. of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands.</footnote>
**Poster 079: P079 Study on Intracellular Tracking of 13C isotope - labeled Volatile Cancer Markers**

Gyu Hwan HYUN<fnref n="1" />; Dong-Kyu LEE<fnref n="2" />; Jeong Hil PARK<fnref n="1" />; Johan LIM³; Sung Won KWON<fnref n="1" />*

**Introduction**

Intracellular volatiles generated during the development or progression and intensification of cancer have the potential to be a significant indicator of cancer diagnosis. But it is difficult to track changes in intracellular metabolites simultaneously, especially when they are volatile. Therefore, we established a novel intracellular detection method of volatile cancer markers through isotopic labeling and estimation of the pseudo-steady state of intracellular metabolite flux.

**Technological and methodological innovation**

Targeted cancer cell was supplied with ¹³C labeled glucose and confirmed that the internal metabolic system was sufficiently saturated to ¹³C. This process was measured by nanoSIMS imaging of ¹³C/¹²C compared to ¹²C/¹⁴N, and the isotopic pseudosteady state was calculated by extrapolating carbon flux in the cells. ¹³C-saturated volatile metabolites were traced to find significant cancer markers that were significantly increased in cancer cells.

**Results and impact**

Metabolic flux analysis results indicate that 2-pentadecanone is derived from metabolic cascade from glucose to fatty acid biosynthesis. 2-pentadecanone was firstly identified as a potential pan-cancer marker. We also applied a novel analytical platform to trace intracellular metabolism-derived volatiles and established the relationship between volatiles and cancerspecific metabolism.

**References**

[1] DK Lee et al. 2018. ACS Central Science. 4(8), 1037-1044.

[2] Millard P et al. 2012. Bioinformatics. 28 (9), 1294-1296.

[3] Colon A et al. 2010. Plant J. 62 (1), 64-76.

[4] Sekine R et al. 2017. ACS Nano. 11 (11), 10894-10902. [5] Gates S. D et al. 2018. Anal. Chem. 90 (3), 1613-1620.

<footnote n="1">¹ College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea</footnote>

<footnote n="2">²College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul 08826,</footnote>

<footnote>Korea ³Department of Statistics, Seoul National University, Seoul 08826, Republic of Korea</footnote>
# Poster 080: P080 Metabolomics profiling urine by Benchtop NMR

Hélène FREICHELS, Jürgen KOLZ, Federico CASANOVA

Magritek GmbH, Philipsstrasse 8, 52068 Aachen, Germany

## Introduction

NMR is routinely used for metabolomics investigations of biofluids in order to detect and quantify potential disease biomarkers. The direct proportionality of the NMR signal amplitude to the concentration of the molecules in the sample allows for an absolute quantification without the need for demanding calibrations. Furthermore, the calibration of the method is independent of the composition of the sample or the solvent used for the preparation. [1]

## Technological and methodological innovation

Benchtop NMR is a fast-developing field, where the main advantages compared to standard high field systems are portability, no maintenance requirement, easy-to-use and robustness. Moreover, benchtop NMR does not require deuterated solvents to work, making the sample preparation of biofluids very convenient. Therefore, the use of benchtop NMR for fingerprinting metabolomics in biofluids area is an emerging field of interest. [2-4]

## Results and impact

The aim of this work is to show the potential of benchtop NMR for the detection of metabolomics in urine samples of common markers [3], in particular we will provide examples to demonstrate the resolution and sensitivity that can be expected from spectra measured in this type of equipment.

<figure><img src="image_51.png" /><figcaption>1</figcaption></figure>

H NMR of urine sample.

## References

[1] S. K. Bharti et al. 2012 Trends Anal. Chem., 2012, 35, 5

[2] J. L. Izquierdo-Garcia et al 2019 European Respiratory Journal, 2019, 54, 553

[3] B. C. Percival et al. High-Throughput 2019, 8, 2

[4] S. Korchak et al. 2019 Physical Chemistry Chemical Physics 21, 22849
**Poster 081: P081 Multi-platform and multi-matrix analytical development**

C. Martias¹, R. Voisin², S. Mavel¹, A. Lefèvre², F. Montigny², L. Nadal-Desbarats¹,², P. Emond¹,²,³

¹ - UMR 1253, iBrain, University of Tours, Inserm, Tours, France

² – PST-ASB, University of Tours, Tours, France

³ – CHRU Tours, Nuclear medecine in vitro departement, Tours, France

**Introduction**

The untargeted metabolomic approach, focuses on the study of a type of sampling by several analytical platforms [1] or by several samples by an unique analytical platform [2] . NMR and MS coupling to UHPLC require a specific sample preparation. Because clinical studies need a huge number of samples, for multi-platform and multi-matrix approaches, the preparation can be time consuming.

The goal of this study is to achieve a suitable and common sample preparation for NMR and MS in order to detect the maximum number of metabolites. Saliva, urine, and feces are considered in this study.

**Technological and methodological innovation**

Different solvents (acetonitrile, methanol, water) and solvent ratios were tested using 1H-NMR and UHPLC (HILIC and C18) coupling to Q-Orbitrap (ESI +/-), each protocol were evaluated. To evaluate the most suitable extraction protocole, metabolite numbers and coefficient of variation were calculated. Analytical method validation, precision, linearity and limit of detection, are performed. Venn diagram are drawn in order to show the multi-platform complementary.

**Results and impact**

The suitable sample preparation for urine matrix is water dilution, acetonitrile for saliva matrix and methanol/acetonitrile/water solvent for faeces matrix. Those extractions allow to detect 395 identified molecules (urine), 299 identified molecules (saliva) and 382 identified molecules. Analytical platforms are complementary around 40% of metabolites are detected by C18, 50% with the HILIC phase and 10% with 1H-NMR. Validation method parameter, linearity, precision have been evaluated in 1H-NMR and UHPLC-MS.

**References**

[1] : Johnson et al. 2010

[2] De Paepe et al. 2018
**Poster 082: P082** *A comparison between a targeted and an untargeted approach in the frame of a pilot biomonitoring study*

Gianfranco FRIGERIO<fnref n="1" />, Rosa MERCADANTE<fnref n="1" />, Laura CAMPO<fnref n="2" />, Elisa POLLEDRI<fnref n="1" />, Luca BONIARDI<fnref n="1" />, Luca OLGIATI<fnref n="2" />, Pasquale MISSINEO<fnref n="2" />, William J. NASH<fnref n="3" />, Warwick B. DUNN<fnref n="3" />,<fnref n="4" />,<fnref n="5" /> and Silvia FUSTINONI<fnref n="1" />,<fnref n="2" />

**Introduction**

Exposure to environmental toxicants can be assessed through the determination of metabolites of chemicals in biofluids.

Targeted and untargeted approaches can be applied to characterize the “exposome”, both with advantages and disadvantages [1]. The aim of this work was to compare the results of a pilot biomonitoring study obtained both with a targeted quantification of urinary mercapturic acids, metabolites of volatile organic compounds, and with an untargeted approach.

**Technological and methodological innovation**

Urine samples were collected from 67 adults: 38 non-smokers, 7 electronic cigarette smokers, and 22 traditional smokers. 17 mercapturic acids were quantified with a validated isotope dilution LC-MS/MS method, using a triple-quadrupole mass spectrometer [2]. Using the same chromatographic conditions, the untargeted approach was performed with a time-of flight operating in data dependent mode; data were processed using XCMS and the ANOVA test was used to detect significant features among groups.

**Results and impact**

Most analytes were quantified in all samples with the targeted approach. 3613 features were identified in the untargeted assay, among which only five mercapturic acids were present; seven mercapturic acids were not identified as a feature but detected inspecting the chromatograms; and four mercapturic acids were not detected at all in the untargeted approach. Despite its promising ability to characterize exposome, the untargeted approach may not detect chemicals present at low levels.

**References**

[1] Dennis et al. 2017. Environ Health Perspect. 125, 502-510

[2] Frigerio et al. 2019. J Chromatogr B Analyt Technol Biomed Life Sci. 1117, 66-76.

<footnote n="1">¹ Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan, Italy</footnote>

<footnote n="2">² Environmental and Industrial Toxicology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy</footnote>

<footnote n="3">³ School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK</footnote>

<footnote n="4">⁴ Phenome Centre Birmingham, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK</footnote>

<footnote n="5">⁵ Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK</footnote>
**Poster 083: P083** *Development and validation of a RPLC-MS/MS method for the quantification of ceramides in human serum of patients with CAD*

Olga BEGOU<fnref n="1" />,<fnref n="2" />, Olga DEDA<fnref n="2" />,<fnref n="3" />, Helen GIKA<fnref n="2" />,<fnref n="3" />, Georgios THEODORIDIS<fnref n="1" />,<fnref n="2" />

**Introduction**

Ceramides are key-role compounds that regulate numerous central cellular processes, such as apoptosis, inflammation, etc. A plethora of studies have showed that changes on the concentrations of these bioactive lipids in serum are associated with different pathophysiological states, namely obesity, cardiovascular diseases, ovarian cancer, metabolic disorders. Four specific molecular ceramides are highly linked to coronary artery disease (CAD), enhancing the prediction of fatal outcomes.

**Technological and methodological innovation**

Different sample preparation methodologies were tested, with supportive liquid extraction (SLE) providing the optimum analytical results for the quantification of ceramides in human serum. Here, we present a targeted metabolic profiling for the rapid quantification of ceramides C16:0, C18:0, C24:0 and C24:1 in serum with RPLC-MS/MS.

**Results and impact**

SLE was found to be an effective sample preparation approach for the accurate and reproducible determination of molecular ceramides in human serum. The validated method is considered of having potentially clinical significance for the rapid diagnosis of CAD subjects enabling the establishment of valid references values in the specific population and the understanding of ceramides' biochemical role. The proposed method can be used in routine clinical analyses.

**References**

[1] Laaksonen et al., 2016, European Heart Journal, 37, 1967-1976

<footnote n="1">¹Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki</footnote>

<footnote n="2">²Bionic_Auth, Bioanalysis and Omics Lab, Centre for Interdisciplinary Research of Aristotle University of Thessaloniki, Innovation Area of Thessaloniki, Thermi, 57001, Greece</footnote>

<footnote n="3">³Laboratory of Forensic Medicine and Toxicology, School of Medicine, Aristotle University of Thessaloniki</footnote>
**Poster 084: P084 AlpsNMR: an R package for fully untargeted NMR-based
metabolomics workflow**

Francisco MADRID-GAMBIN<fnref n="1" />; Sergio OLLER-MORENO<fnref n="1" />; Luis FERNANDEZ<fnref n="1" />; Simona BARTOVA<fnref n="3" />; Christopher JOYCE<fnref n="3" />; Francesco FERRARO<fnref n="3" />; Ivan MONTOLIU<fnref n="3" />; Sofia MOCO<fnref n="3" />; Santiago MARCO<fnref n="1" />,<fnref n="2" />

**Introduction**

NMR-based metabolomics is widely used to explore metabolic fingerprints of biological systems [1]. While there are various computational tools developed for targeted metabolomics, there is a lack of open-source tools for fully untargeted NMR-based metabolomics, able to handle datasets from raw data to metabolite lists [2].

**Technological and methodological innovation**

Here, we present AlpsNMR (Automated spectraL Processing System for NMR), an R package that offers an automated workflow for untargeted NMR-based metabolomics analysis. AlpsNMR includes spectra loading, metadata handling, signal processing, integration, statistical analysis, and metabolite identification based on the Human Metabolome Database [3]. This tool allows non-experienced users to include this workflow in their metabolomics routines.

**Results and impact**

The AlpsNMR R-package, along with a guided tutorial including a test case [4], is freely available to download from
https://github.com/sipss/AlpsNMR/blob/master/vignettes/tutorial.pdf under the MIT license.

**References**

[1] Vignoli et al. 2019. Angew. Chemie Int. Ed. 58, 968–994.

[2] Stanstrup et al. 2019. Metabolites. 9(10), 200.

[3] Wishart et al. 2018. Nucleic Acids Res. 46(D1), D608–D617.

[4] Gralka et al. 2015. Am. J. Clin. Nutr. 102, 1313–1322.

<footnote n="1">¹ Signal and Information Processing for Sensing Systems, Institute for Bioengineering of Catalonia (IBEC), the Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain.</footnote>

<footnote n="2">² Department of Electronics and Biomedical Engineering, Universitat de Barcelona, Marti i Franqués 1, Barcelona 08028, Spain.</footnote>

<footnote n="3">³ Nestlé Research, EPFL Innovation Park, H, 1015 Lausanne, Switzerland.</footnote>
**Poster 085: P085 Costs and benefits of switching from vendor-based to open source pipelines for untargeted LC-MS metabolomics**

Álvaro Fernández-Ochoa<fnref n="1" />,<fnref n="2" />, María del Carmen Villegas-Aguilar<fnref n="1" />,<fnref n="2" />, María de la Luz Cádiz-Gurrea<fnref n="1" />,<fnref n="2" />, Isabel Borrás-Linares<fnref n="2" />, Rosa Quirantes-Piné<fnref n="2" />, Sandra Pimentel-Moral<fnref n="1" />,<fnref n="2" />, Jesús Lozano-Sánchez<fnref n="2" />,<fnref n="3" />, David Arráez-Román<fnref n="1" />,<fnref n="2" />, Carl Brunius<fnref n="4" />, Antonio Segura-Carretero<fnref n="1" />,<fnref n="2" />

**Introduction**

Data pre-processing of the LC-MS data is a critical step in untargeted metabolomics studies in order to achieve correct biological interpretations. The purpose of data pre-processing is to reduce the complexity of the raw data, extract the main features and transform them in order to subsequently perform adequate statistical tests [1]. Several tools have been developed for pre-processing, and these can be classified into either commercial or open source software [2].

**Technological and methodological innovation**

In the present work, two methodologies, vendor vs open-source, for data pre-processing in untargeted metabolomics studies were compared. We highlight differences in these two pipelines using 369 plasma samples analyzed by LC-MS. Specifically, we have processed the data using Agilent software, representing a vendor pipeline. On the other hand, an open source methodology based on R was represented by the combination of several packages, namely IPO/XCMS [3], batchCorr [4] and RAMClustR [5].

**Results and impact**

Both vendor and open source methodologies have strength and weaknesses. However, we have shown that the open source methodology is the most suitable option for metabolomic studies with larger number of samples in multiple batches. However, this environment is also less intuitive, frequently with lower quality graphical output and with a distinctly steeper learning curve. We provide a detailed tutorial to help users of commercial software to start processing data through R-based methodology.

**References**

[1] S. Castillo et al. 2011. Chemom. Intell. Lab. Syst. 108, 23-32.

[2] L. Hao et al. 2018. Sci. Rep. 8, 9291, 1-10.

[3] G. Libiseller et al. 2015. BMC Bioinformatics. 16, 118.

[4] C. Brunius et al. 2016. Metabolomics. 12, 1-13.

[5] C.D. Broeckling et al. 2014. Anal. Chem. 86. 6812-6817.

**Acknowledgments**

Ministry of Science, Innovation and Universities (RTI2018-096724-B-C22)

<footnote n="1">¹ Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Av Fuentenueva s/n, Granada 18071, Spain.</footnote>

<footnote n="2">² Research and Development of Functional Food Centre (CIDAF), Health Science Technological Park, Avda. del Conocimiento, n° 37, s/n, Granada 18016, Spain.</footnote>

<footnote n="3">³ Department of Nutrition, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, Granada 18071, Spain.</footnote>

<footnote n="4">⁴ Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.</footnote>
**Poster 086: P086 Volatile Biomarker discovery in flatus by GC-MS and GC-IMS to diagnose digestive disorders and diseases.**

Rafael Teixeira FREIRE<fnref n="1" />; Marianela MEGO<fnref n="2" />; Fernando AZPIROZ<fnref n="2" />; Santiago MARCO<fnref n="1" />

**Introduction**

Little is known about the organic volatiles (VOCs) in flatus and their relation to health implications. Patients and healthcare professionals have observed that feces change its smells during gastro-intestinal diseases, suggesting that the identification of VOCs biomarkers could offer the potential for rapid identification of gastrointestinal diseases and disorders. The flatus complexity and the difficulties in the identification of minor metabolites still provide great analytical obstacles. [1 2 3 4 5]

**Technological and methodological innovation**

The aim of this work is to propose new general methodologies to investigate digestive disorders using VOCs found in human flatus. These methodologies are based on GC-MS and GC-IMS measurements focusing on the creation of analytical methodologies and bioinformatics pipelines for data pre-processing and multivariate analysis of VOCs. The flatus analysis is so new, that even the exploratory analysis of the composition for healthy individuals may be of interest for publication.

**Results and impact**

This study potentially offers novel and significant insights to understand the volatiles from flatus and any advance in this area may have a large impact if any association between VOCs and health is found. Such use would lead to improved diagnoses and the development of tailored therapies with potential clinical benefits to the patients. Using the volatiles expelled from the human body, opens a new frontier in medical diagnostics due its inexpensive potential and being a non-invasive technique.

**References**

[1] Aman A. et al. 2007. Mini Rev Med Chem. 7 115-129

[2] Garner G. et al. 2007 YEAR. Journal of Chromatography A. 1449: 89-99

[3] Di Lena, et al. 2013 World J Gastroenterol. 19(12): 1855-60

[4] Mazzone, Peter J. 2008. Journal of thoracic oncology. 3(7): 774-80.

<footnote n="1">¹Signal and Information Processing for Sensing Systems, Institute for Bioengineering of Catalonia (IBEC), the Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain.</footnote>

<footnote n="2">²Vall d'Hebron University Hospital Passeig de la Vall d'Hebron, 119, 129, 08035 Barcelona</footnote>
**Poster 087: P087 MetExplore: Omics data analysis in the context of metabolic networks**

Maxime Chazalviel¹, Florence Vinson², Nathalie Poupin², Adrien Rohan², Arthur Moreau², Clément Frainay², Ludovic Cottret³ and Fabien Jourdan²

¹ MedDay Pharmaceuticals, Paris, France

² ¹UMR1331, Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAe, ENVT, INP-Purpan, UPS, 31300 Toulouse, France

³ LIPM, Université de Toulouse, INRAe, CNRS, Castanet-Tolosan, France ...

**Introduction**

Metabolism of an organism is composed of hundreds to thousands of interconnected biochemical reactions responding to environmental or genetic constraints. This metabolic network provides a rich knowledge to contextualize omics data and to elaborate hypotheses on metabolic modulations. Nevertheless, performing this kind of integrative analysis is challenging for end users without sufficiently advanced computer skills since it requires the use of various tools and web servers.

**Technological and methodological innovation**

MetExplore [1] is a well-established sustainable freely available project maintained since 2010. It is a web server (www.metexplore.fr) dedicated to the analysis of genome scale metabolic networks, with special care taken to allow analyzing metabolomics data in the context of these networks.

MetExplore offers a free all-in-one online solution composed of interactive tools for metabolic network curation, network exploration/visualization [2] and omics data analysis.

**Results and impact**

Today, MetExplore contains 297 public metabolic networks and 1196 private networks representing 404 organisms. MetExplore has 846 registered users and was used for 39 national and international trainings.

The server is used to tackle many scientific questions, particularly MedDay Pharma uses MetExplore to study neurological diseases such as hepatic encephalopathy [3] and CAFSA syndrome [4].

**References**

[1] Cottret L. et al. MetExplore: Manage and Explore metabolic networks. (2018) Nucelic Acids Research.
[2] Chazalviel M. et al. MetExploreViz: web component for interactive metabolic network visualization. (2017) Bioinformatics. 34 (2). 312-313.
[3] Frainay C. et al. MetaboRank: network-based recommendation system to interpret and enrich metabolomics results. (2018). Bioinformatics.
[4] Del Mar Amador M. et al. Targeted versus untargeted omics - the CAFSA story. (2018) J Inherit Metab Dis.;41(3):447456
Poster 088: P088 BARSA: BidimensionAl nmR Spectra Annotation

Marie TREMBLAY FRANCO<sup>1,2</sup>; Coline GARDOU<sup>1,2</sup>; Florence VINSON<sup>1</sup>; Lindsay PEYRIGA<sup>3</sup>; Edern CAHOREAU<sup>3</sup>; Franck GIACOMONI<sup>4</sup>; Laurent DEBRAUWER<sup>1,2</sup>; Cécile CANLET<sup>1,2</sup>;

**Introduction**

Metabolomic studies usually aim at comparing metabolic profiles of organisms exposed to different stimuli (e.g. control vs xenobiotic-exposed animals). NMR is one of the main analytical techniques used to generate metabolic profiles. Automated identification of discriminant metabolites based on 1D spectra is a major bottleneck for biomarker discovering, mainly because of peaks overlapping of many metabolites, whereas two-dimensional (2D) NMR can provide more specific structural information.

**Technological and methodological innovation**

2D NMR spectra provide resonance separation along a second dimension, facilitating identification of individual chemical components [1]. We developed an automated algorithm for metabolite identification based on several 2D NMR sequences using the R language. BARSA compares peak lists of 2D spectra of biological samples to those of pure compounds included in an in-house cured database. Criteria can be applied to exclude metabolites for highly confident minimizing of false positive metabolites.

**Results and impact**

BARSA algorithm was assessed using a mix of 23 standard compounds. Annotation was based on individual or combined sequences (2 to 5). Sensitivity and specificity were computed for several thresholds based on presence probability and unicity of peak annotation. Highest values were obtained by combining 4 sequences and a 0.5-threshold on presence probability. BARSA overcomes 1D NMR limitations and enables automated annotation of complex NMR spectra with high confidence on identified metabolites.

**References**

[1] Robinette et al. 2008. *Anal Chem.* 80, 3606–361.

<footnote n="1"><sup>1</sup> Toxalim – Research Center in Food Toxicology, Toulouse University, INRAE UMR 1331, ENVT, INP-Purpan, Paul Sabatier University, F-31027 Toulouse, France</footnote>

<footnote n="2"><sup>2</sup> Metatoul-AXIOM platform, National Infrastructure for Metabolomics and Fluxomics, MetaboHUB, Toxalim, INRAE UMR 1331, F-31027 Toulouse, France</footnote>

<footnote n="3"><sup>3</sup> Metatoul-Metabolic Network platform, National Infrastructure for Metabolomics and Fluxomics, MetaboHUB, TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote n="4"><sup>4</sup> Université Clermont Auvergne, INRAE, UNH, Plateforme d'Exploration du Métabolisme, MetaboHUB Clermont, F-63000 Clermont-Ferrand, France</footnote>
**Poster 089: P089 Assessing the impact of physicochemical parameters on the predictive capabilities of thermodynamics-based stoichiometric approaches under mesophilic and thermophilic conditions**

Claudio TOMI-ANDRINO<fnref n="1" />,<fnref n="2" />,<fnref n="3" />; Rupert NORMAN<fnref n="2" />; Thomas MILLAT<fnref n="2" />; Philippe SOUCAILLE <fnref n="2" />,<fnref n="4" />; Klaus WINZER<fnref n="2" />; David A. BARRETT<fnref n="1" />; John KING<fnref n="3" />; Dong-Hyun KIM<fnref n="1" />*

**Introduction**

Metabolic engineering in the post-genomic era exploits novel methods in metabolomics and fluxomics. Flux distributions are either experimentally measured (metabolic flux analysis, ¹³C-MFA [1]) or simulated (flux balance analysis, FBA [2]), by using constraint-based models. However, the optimal solutions do not guarantee thermodynamically-feasible metabolic fluxes – applying thermodynamic constraints has been suggested, but in-depth comparisons and evaluation of these approaches are lacking.

**Technological and methodological innovation**

Towards this end, the recently published matTFA toolbox [3] was extended to include a broader range of physicochemical parameters and more suitable adjustment equations (2⁶ combinations). Additionally, an approach based on max-min driving forces (MDF) [4] was used for comparison. Available experimental results (bioprocessing parameters, ¹³C-MFA-derived fluxes and metabolomics data) for Escherichia coli and Thermus thermophilus were used to assess the predictive capabilities of these methods.

**Results and impact**

Whilst a high level of reliability at the fluxomics level (similarity to ¹³C-MFA) was achieved for all the combinations, the quality of predicted metabolite concentrations values varied significantly. Adjusting the calculations to the growth conditions (e.g. t = 37 °C) improved the correlation with metabolomics data from 5% to 41%. In addition, frequently neglected limitations of 13C-MFA/FBA were analysed, suggesting the need for standardisation and cross-validation in the field.

**References**

[1] Antoniewicz MR. 2015. J Ind Microbiol Biotechnol. 42(3):317-25.

[2] Orth JD et al. 2010. Nat Biotechnol. 28(3):245-8.

[3] Salvy P et al. 2018. Bioinformatics. 1(3).

[4] Flamholz A et al. 2012. Nucleic Acids Res. 40:D770–D5.

<footnote n="1">¹ Centre for Analytical Bioscience, Advanced Materials and Healthcare Technology Division, School of Pharmacy, The University of Nottingham, Nottingham, United Kingdom.</footnote>

<footnote n="2">² BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, Centre for Biomolecular Sciences, The University of Nottingham, Nottingham, United Kingdom.</footnote>

<footnote n="3">³ BBSRC/EPSRC Synthetic Biology Research Centre, School of Mathematical Sciences, The University of Nottingham, Nottingham, United Kingdom.</footnote>

<footnote n="4">⁴ LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077, Toulouse Cedex, France.</footnote>

<footnote>*Correspondence to: Email: dong-hyun.kim@nottingham.ac.uk Tel: +44 1157484697</footnote>
**Poster 090: P090 Uniting metabolomics data processing and highly confident annotation across six MS instrumental set ups: MetaboScape 5.0**

Nikolas KESSLER¹; Wiebke TIMM¹; Sascha WINTER¹; Ulrike SCHWEIGER-HUFNAGEL¹; Sven MEYER¹; Aiko BARSCH¹; Heiko NEUWEGER¹

¹Bruker Daltonik GmbH, Bremen, Germany

**Introduction**

By combining different platforms, complementing their respective strengths, the gap between high-throughput and in-depth analysis strategies can be closed. MetaboScape 5.0 incorporates the feature extraction and ion deconvolution algorithms TReX 2D, 3D and 4D, integrates the processing-, dereplication- and unknown annotation-workflows for FIA-MRMS, LCMRMS, LC-ESI-TOF, and LC-ESI-TIMS-TOF in a single software. T-ReX² additionally covers MALDI-MRMS and MALDI-QTOF imaging data.

**Technological and methodological innovation**

The T-ReX feature extraction algorithms transform raw data from different instrument types into compound matrices of putative compounds, comprising ions and isotope patterns. Various tools for metabolite annotation seamlessly adapt to the annotation quality criteria each instrumental platform provides (accurate mass, isotope pattern (incl. fine structure), retention time, MS/MS spectra, and/or collisional cross section). Data acquired in different polarities can be merged for joint evaluation.

**Results and impact**

MetaboScape allows for molecular formula generation, in-silico fragmentation, CCS prediction, MSMS spectral and retention time matching across different platforms. This allows uniting high throughput workflows with the maximum of in-depth exploitation of all the mass spectral information for specific metabolites. As an example, it also enables to transport metabolite annotations from LC-ESI-TIMS-PASEF, confirmed by all 5 quality criteria, into MALDI imaging measurements for spatial metabolomics.

**References**

[1] Wolf *et al.*; BMC Bioinformatics 2010, 11: 148

[2] Ruttkies *et al.*; Journal of Cheminformatics 2016, 8:3
**Poster 091: P091 Integrated workflow with quality control for large cohort and clinical metabolomics research using robust hardware and signal correction**

Sebastian GOETZ<fnref n="1" />; Ulrike SCHWEIGER-HUFNAGEL<fnref n="1" />; Matthias SZESNY<fnref n="1" />; Aiko BARSCH<fnref n="1" />; Sven W. MEYER<fnref n="1" />; Matthew R. LEWIS<fnref n="2" />; Nikolas KESSLER<fnref n="1" />

**Introduction**

Metabolomics research relies on precision measurement of statistically powered sets of hundreds or thousands of samples. First, this requires robust analytical hardware with long term stability, capable of generating high precision data. Second, processing of large datasets may require additional mathematical correction to compensate for systematic changes in observed signals as samples interact with the analytical system affecting its performance.

**Technological and methodological innovation**

We present a fully automated software workflow for the automated feature-wise correction of intensity drifts based on quality control samples, improving data precision and statistical reliability. An interactive and intuitive visualization provides rapid review of intensity drifts and their corrections as well as detection of statistical outliers.

**Results and impact**

We investigated the long-term stability of an LC-HR-QTOF system by measuring a batch of more than 1000 urine samples and monitoring the effect of data acquisition on MS ion source contamination, detector aging and sample degradation. A new run-order signal drift correction reduced the relative standard deviation of feature intensities within sample groups measured across replicate quality control measurements. This increased the number of analytes which meet the requirements of an RSD below 20%.

<footnote n="1">¹Bruker Daltonik GmbH, Bremen, Germany</footnote>

<footnote n="2">²Imperial College London, London, United Kingdom</footnote>
**Poster 092: P092 Automatic annotation in untargeted metabolomics: a proof of concept protocol based on recurrent experiments**

Mar GARCIA-ALOY; Vinicius VERI; Johannes RAINER

Institute for Biomedicine, Eurac Research, Bozen (Italy)

**Introduction**

Untargeted LC-MS metabolomics detects thousands of signals in each experiment. Some of them refer to real measured metabolites, but others, known as non-biological signals, can be associated to a wide range of factors, such as background ions, chemical contaminants or informatic artifacts, among others. It is broadly recognized that one of the major bottlenecks in the metabolomics workflow is metabolite identification, which is time-consuming and usually has a considerable failure rate.

**Technological and methodological innovation**

A large number of metabolites are detected recurrently in different studies on the same sample type when using the same analytical method. Knowledge acquired in previous experiments thus allows to accelerate the identification process of new studies and enables to focus on novel and/or still unknown metabolites. We propose to implement functionality required to annotate signals in a specific system and to build a reference database facilitating annotations of subsequent studies.

**Results and impact**

Currently we are listing signals associated with specific compounds, but also yet unidentified ions detected in the system. We think that in the future we will be able to provide a catalog of metabolites detected with a specific analytical method in a specific biological matrix. The effectiveness of this pipeline is now evaluated using a series of different standard mixtures and next we plan to apply this on complex biological matrices.
**Poster 093: P093 Metabolomics results interpretation using recommender system embedded in metabolic network visualization**

Clément FRAINAY<fnref n="1" />; Maxime CHAZALVIEL<fnref n="2" /> and Fabien JOURDAN<fnref n="1" />

**Introduction**

The visual and interactive exploration of metabolic networks, usually aiming to reconstruct metabolic scenarios explaining experimental data, is mainly driven by expert knowledge not necessarily modelled in the network. It is thus difficult to fully automatize the reconstruction of metabolic scenarios and the selection of the most-relevant ones. On the other hand, manual exploration is usually tedious without a priori filtering, given the complexity and size of genome-scale metabolic networks.

**Technological and methodological innovation**

In order to reduce the information overload, we recently developed a recommendation system, highlighting relevant compounds to investigate, based on their connections to markers identified by metabolomics [1]. In order to ease its use in the reconstruction of metabolic scenarios, we embedded it in an interactive network visualization [2]. We shifted from the traditional overview browsing model for large networks to an incremental expansion of users' initial focus [3], empowered by AI support.

**Results and impact**

Our approach, focused on expert interaction with their data, is freely available through MetExplore [4]. By highlighting parts of a compound's neighborhood that can lead to known markers and by providing integrated document retrieval from PubMed, our metabolic exploration framework assists the reconstruction in a comprehensive way. We used it to extend recently published mechanistic scenarios, adding parts of the network which relation to the condition under study is supported by the literature.

**References**

[1] Clément Frainay, Sandrine Aros, Maxime Chazalviel, Thomas Garcia, Florence Vinson, Nicolas Weiss, Benoit Colsch et al. MetaboRank: network-based recommendation system to interpret and enrich metabolomics results. Bioinformatics, (35):274-283, 2018.

[2] Maxime Chazalviel, Clément Frainay, Nathalie Poupin, Florence Vinson, Benjamin Merlet, Yoann Gloaguen, Ludovic Cottret, and Fabien Jourdan. MetExploreViz: web component for interactive metabolic network visualization. Bioinformatics, (34):312-313, 2017.

[3] Frank Van Ham and Adam Perer. "Search, show context, expand on demand": supporting large graph exploration with degree-of-interest. In IEEE Transactions on Visualization and Computer Graphics 15, no. 6 pages 953-960. 2009.

[4] Ludovic Cottret, Clément Frainay, Maxime Chazalviel, Floréal Cabanettes, Yoann Gloaguen, Etienne Camenen, Benjamin Merlet et al. MetExplore: collaborative edition and exploration of metabolic networks. Nucleic acids research, (46):W495-W502, 2018.

<footnote n="1">¹ UMR1331, Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31300 Toulouse, France</footnote>

<footnote n="2">² Medday Pharmaceuticals SA, 24-26 rue de la Pépinière, 75008, Paris, France</footnote>