MIMM • Isotopomics

Home MIMM team MIMM • Isotopomics

Isotopic analysis at natural abundance has been a core expertise of our team since it was created in the 1980s by Profs. Martin and Martin, who discovered an original process based on SNIF-NMR to detect the chaptalization of wines.

Since then, we have made pioneering developments in this field, leading to a novel “isotopomics” approach, defined as the measurement of a large number of isotopic information at natural abundance, whether it is position-specific information obtained by NMR or average molecular information obtained by Isotope Ratio Mass Spectrometry (IRMS).

The innovative isotopic NMR developments carried out within the team, combined with recognized expertise in high-precision IRMS, enabled our team to target particularly original applications of isotopomics in food science, environment, forensics, health or metabolism.

isotopomics-ceisam-mimm-team

Current team members: Lenny Hadda, Sophie Renoue, Vincent Portaluri, Anne-Marie Schiphorst, Virginie Silvestre, Mathilde Grand, Gérald Remaud, Serge Akoka*.

Past team members: Ghina Hajjar, Denis Loquert, Eliot Botosoa, Didier Diomande, Alexis Gilbert, Sophie Guyader, Flore Legrand, Noelle Merchack, Richard Robins, Katarzyna Romek. Collaborations: J. Bejjani, T. Rizk (Saint-Joseph University, Beirut, Lebanon), F. Thomas, E. Jamin (EUROFINS, Nantes, France), P. Lesot (University of Paris Sud, Paris Saclay)

Discrimination of the origin of vanillin samples from 13C position-specific composition obtained by isotopic 13C NMR.

 

This project concerns the characterisation of food matrices by the determination of the isotope profiles on targeted molecules or on the matrix itself (whole or a part). This isotopomics approach can be conveniently completed by other profiling such as metabolomics (see specific project). The interest of position-specific isotope analysis is demonstrated on ethanol and vanillin for which isotopic 13C NMR is enable to distinguish origins not discriminated by other methods. The geographical origins of olive oil or egg lipids are also separated on basis of relative 13C profiles measured on the whole matrix thank to the capability of modified INEPT pulse sequence.

References:

  • A. Gilbert, R.J. Robins, G.S. Remaud, G.Tcherkez. Intramolecular 13C-pattern in hexoses from autotrophic and heterotrophic C3 plant tissues. Proc. Natl. Acad. Sci. USA, 109, 18204-18209 (2012).
  • N. Merchak, J. Bejjani, T. Rizk, V. Silvestre, G. Remaud, S. Akoka. 13C isotopomics of triacylglycerols using NMR with polarization transfer techniques. Analytical Methods, 2015, 7(12) 4889-4891.
  • D. Diomande, E. Martineau, A. Gilbert, P. Nun, A. Murata, K. Yamada, N. Watanabe, I. Tea, R.J. Robins, N. Yoshida, G.S. Remaud Position-Specific Isotope Analysis of Xanthines: A C-13 Nuclear Magnetic Resonance Method to Determine the C-13 Intramolecular Composition at Natural Abundance. Anal. Chem., 87, 6600-6606 (2015).
  • K.M. Romeka, P. Nun, G.S. Remaud, V. Silvestre, G. Sotoing Taïwe, F. Lecerf-Schmidt, A. Boumendjeld, M. De Waarde, R.J. Robins. A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol. Proc. Natl. Acad. Sci. USA, 112, 8296-8301 (2015).
  • N. Merchak, V. Silvestre, L. Rouger, P. Giraudeau, T. Rizk, J. Bejjani, S. Akoka. Precise and rapid isotopomic analysis by 1H-13C HSQC: Application to triacylglycerol matrices. Talanta, 2016, 156-157, 239-244.
  • N. Merchak, V. Silvestre, D. Loquet, T. Rizk, S. Akoka, J. Bejjani. A strategy for simultaneous determination of fatty acid composition, fatty acid position, and position-specific isotope contents in triacylglycerol matrices by C-13-NMR. Analytical and Bioanalytical Chemistry, 2017, 409(1), 307-3015.
  • T. Texier-Bonniot, P. Berdague, RJ Robins, GS Remaud, P. Lesot. Analytical contribution of deuterium 2D-NMR in oriented media to H-2/H-1 isotopic characterization. Flavour Fragrance J, 33, 217-229 (2018).

Supported by: National Council for Scientific research, University of Nantes National Council for Scientific research of Lebanon and Saint-Joseph University of Beirut

Current team members: Anne-Marie Schiphorst, Virginie Silvestre, Serge Akoka, Gérald Remaud*.

Past team members: Valentin Joubert, Ugo Bussy, Didier Diolmande, Maxime Julien, Richard Robins.

Collaborations: F. Thomas, E. Jamin (EUROFINS, Nantes), Patrick Hohener (), F. Besacier, V. Ladroue (INPS, Lyon), J.R. Desmurs (CDP Innovation)

Information obtained from isotope data can be of significant value for Law Enforcement Agencies. In this respect, deducing the source can be the major issue for determining the liable party in forensic investigations of pollutants, drugs and active pharmaceutical ingredients. We demonstrated that isotope profiling collected from NMR experiments is of great interest in forensic investigations: in environment (ANR ISOTO-Pol), in counterfeited medicines (ANR IS-o-TOP) and in drug trafficking (ANR FRIIME).

References:

  • U. Bussy, C. Thibaudeau, F. Thomas, J.-R. Desmurs, E. Jamin, G.S. Remaud, V. Silvestre, S. Akoka. Isotopic finger-printing of Active Pharmaceutical Ingredients by 13C NMR and polarization transfer techniques as a tool to fight against counterfeiting. Talanta 85, 1909-1914 (2011).
  • M. Julien, J. Parinet, P. Nun, K. Bayle, P. Hoehener, R.J. Robins, G.S. Remaud. Fractionation in position-specific isotope composition during vaporization of environmental pollutants measured with isotope ratio monitoring by C-13 nuclear magnetic resonance spectrometry. Environ. Pollut., 205, 299-306 (2015).
  • M. Julien, P. Nun, P. Höhener, J. Parinet, R.J. Robins, G.S. Remaud. Enhanced forensic discrimination of pollutants by position-specific isotope analysis using isotope ratio monitoring by 13C nuclear magnetic resonance spectrometry. Talanta, 147, 383–389 (2016)
  • V. Joubert, V. Silvestre, M. Grand, D. Loquet, V. Ladroue, F. Besacier, S. Akoka, G. S. Remaud. Full Spectrum Isotopic 13C NMR Using Polarization Transfer for Position-Specific Isotope Analysis. Analytical Chemistry, 90(14) 8692-8699. (2018).

Supported by: ANR, INPS

Current team members: Anne-Marie Schiphorst, Virginie Silvestre, Mathilde Grand, Serge Akoka, Gerald Remaud*. Collaboration: K. Freeman, C. House (PSU, USA), J. Eiler, A. Sessions (CalTech, USA), J. Kubicki (UTEP, USA), J. McDermott (Lehigh, USA), G. Love (Univ. of CA, USA), A. Gilbert, N. Yoshida (Tokyo Tech, Japan)
Isotopomics-13

Comparison of PSIA measurements on ethanol obtained by isotopic NMR or by Mass spectrometry coupled to pyrolysis device.

The Astrobiology Center for Isotopologue Research (ACIR) brings together an international team of top scientists and their cutting-edge observational and computational tools in order to find out: to what degree do the abundance and positions of isotopes within molecules explicitly reveal the origins and history of organic compounds? The consortium seeks predictive understanding of how substrates, processes, and environments are encoded in the isotopes of organic compounds in diverse matrices, environments, and organisms. EBSI team is particularly involved in the improvement of precision, trueness and sensitivity in PSIA.

Website : https://www.isotopologues.org

Supported by: NASA, USA, National Council for Scientific research and University of Nantes

Current team members: Anne-Marie Schiphorst, Mathilde Grand, Gérald Remaud, Illa Tea*.
Past team members: Estelle Martineau, Julie Lalande, Maxime Julien, Richard Robins.

Collaborations (past and present): Sophie Barillé-Nion (CRCNA Nantes), Estelle Martineau (CEISAM, Univ Nantes), Anneke Blackburn (JCSMR, ANU, Canberra), Jane Dahlstrom (Canberra Hospital), Mikael Croyal (CRNH Nantes), Guillaume Tcherkez (RSB, ANU Canberra)

MIMM-Isotopomics-14a

MIMM-Isotopomics-14b

The natural 13C and 15N abundance can discriminate normal and cancerous biopsies, major metabolic pathways are responsible for changes in the natural 13C and 15N abundance in tumors and cancerous cultured cells.

Breast cancer (BrCa) is the most prevalent cancer in women worldwide and is amongst the most important cause of mortality. There are important challenges: finding new treatments, improving diagnosis and cancer classification so as to anticipate treatment sub-type and life prognostic, and finding good markers to follow the response of cancer to treatment and anticipate cancer reactivation. Using stable (non-radioactive) isotopes at natural abundance (13C/12C and 15N/14N ratios), we have been the first to show that natural isotope abundance reflects cancer cell metabolism and that the isotope signature is a potential biomarker to differentiate normal and cancer cells and tissues.

Since cancer cells exchange nutrients and waste products with blood, we anticipate that isotopic differences should be detectable in some plasmatic metabolites. Therefore, we intend to carry out a biomarker discovery process by which we will elaborate on our preliminary findings, and identify candidate isotopic biomarkers. The ultimate aim is to find avenues to develop a blood test for BrCa detection and thus patient screening, and to monitor BrCa patients for the recurrence of disease, detecting it earlier and improving their treatment outcomes.

The project adopts a step-wise process to develop a plasmatic isotopic assay for primary or metastatic BrCa. By using innovative isotopic techniques to measure the natural abundance of isotopes 13C/12C and 15N /14N (isotopic signatures) in targeted compounds from normal and BrCa samples, we aim to: (i) reveal isotopic signatures in paired normal and cancerous breast tissues and its subtypes as a retrospective study; (ii) characterize metabolism and thus identify best candidate isotopic metabolites; (iii) investigate the potential of candidate biomarkers in plasma to define a non-invasive biomarker. We are now working on 450 samples in total, which are provided by Australian Breast Cancer Tissue Bank (ABCTB). The impact of our project is expected to be a new diagnostic tool for BrCa detection that may be adapted to clinical settings.

Key References:

  • Tea I, Martineau E, Giraudeau P, Akoka S and Barillé-Nion S. “Method to characterize the origin and/or the state of pathological or healthy cells, and its applications in biology”: International patent WO2012/123886(A1)
  • I. Tea, E. Martineau, I. Antheaume, J. Lalande, C. Mauve, F. Gilard, S. Barillé-Nion, A. C. Blackburn, G. Tcherkez 13C and 15N natural isotope abundance reflects breast cancer cell metabolism Scientific Reports 6 (2016), 34251

Supported by: Région Pays de la Loire (PLAISIR project Association for International Structure), Canberra Hospital (Private Practice Fund), French Funding from University Technology Transfer Services, Australian National University ANU

Current team members: Anne-Marie Schiphorst, Mathilde Grand, Gérald Remaud, Illa Tea*

Collaborations: José Hureaux (CHU Angers, MINT team), Guillaume Tcherkez (RSB, ANU Canberra)

MIMM-Isotopomics-15

Lung cancer biopsies are 15N-depleted: Δ between tumor and adjacent tissue (P<0.01, n=5)

Lung cancer (LC) is amongst the worst cancers, causing 40,000 deaths each year in France. Furthermore, LC is associated with short life expectancy after diagnosis since it is often discovered at late stages. It is therefore critical to avoid late diagnostics and increase our capability for LC screening. However, no genetic or protein blood test has proved reliable and there is presently no clinically-implementable LC biomarker. In the present project, we intend to exploit a new technique based on the natural abundance in stable (i.e., non-radioactive) isotopes 13C and 15N.

In fact, differences in metabolic fluxes between healthy and cancerous cells should in principle lead to a difference in natural isotope redistribution. It is now a long time since stable isotopes at natural abundance have been implemented to other fields (such as plant biology), but their use in medicine has mostly been overlooked. Here, we hypothesize that the natural isotope abundance is a new type of biomarker that can be easily implemented to detect LC, and we are exploring this possibility using a set of samples from a tumour collection from CHU Angers and clinical trials.

Supported by: French Funding for international cancer research ‘Fondation de France’, CHU Angers, Australian National University ANU

Current team members: Anne-Marie Schiphorst, Mathilde Grand, Gérald Remaud, Illa Tea*

Collaborations : José Hureaux (CHU Angers, MINT team), Guillaume Tcherkez (RSB, ANU Canberra)

MIMM-Isotopomics-16

Overview of instrumental coupling for compound specific isotopic analysis (CSIA), performed using off-line (A) & on-line methods (B).

The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of 2H, 18O, 13C, and 15N isotopes in metabolites. Therefore, compound specific isotopic analysis (CSIA) and intra is crucial to biological and medical applications of stable isotopes. We are exploring CSIA extended to other molecules (such as Arginine which is a good candidate for breast cancer biomarkers) and elements such as oxygen and hydrogen (still in its infancy in biology and medicine) and sulfur (nearly nonexisting currently).

Key References:

  • Tea & G. Tcherkez, Natural Isotope Abundance in Metabolites: Techniques and kinetic isotope effect measurement in plant, animal, and human tissues Methods in Enzymology 596 (2017),113-147

Supported by: Australian National University ANU, University of Nantes