An Innovative Multicompartmental and multi-analytical Metabolomics Approach preserving Integrity of all Sample Types – IMAPIST

Part of our analyses will be done on animal or human tissue. One of the most appropriate analytical techniques to analyse this type of samples is the HRMAS NMR spectroscopy. Indeed, this method allows the analysis of intact tissues without extraction steps. However, due to its principle, it implies the rotation of the samples along the magic angle at a speed rate of several kHz (superior at 100 000g). Therefore, delicate samples are deteriorated during the analysis. In order to preserve the sample integrity, we wil use slow spinning HRMAS NMR (400Hz). The optimisation of the geometry of the rotor cavity, in which the sample is put, should lead to the acquisition of NMR spectra without spinning side bands or with very reduced ones. Regarding the other types of sampels (biofluids), the NMR and LC/MS protocols are well known.
For each sample type, a classical metabolomics approach will be used in order to determine the discriminant metabolites of i) the thyroid tumor malignancy, ii) the host/microbiote interactions in the mouse in function of the diet.
Then, all the data (for each case of study) will be combined in order to identify more relevant markers.

Slow HRMAS NMR: the use of 12ul rotor enables us to reduce the spinning side bands in the NMR spectra for the liquid samples (solution of phenylalanine) and fro the tissues (heifer liver). It has been possible to record slow-HRMAS «CPMG«-like spectrum using the PROJECT sequence at 400 Hz speed. However, a very specific sample preparaiton is compulsory but can be applied on 12ul rotors or 30ul disposable inserts.

HRMAS NMR on CaCO2 cells: the lysis of the CaCO2 cells are necessary to obtain a proton spectrum with high resolution. Ruminoccocus gnavu E1 supernantant must be in contact during 48h minimum with CaCO2 cells to affect their metabolism.

Effect of FOS on mouse metabolism : the use of FOS (prebiotic) enables the reduction of lipid storage in conventional mice. The feces samples showed that the prebiotic accelerates the bacterial activation increasing the acetate and butyrate level in the mouse gut. Moreover, we noticed an activation of the bile salt hydrolyse which is responsible for the tauro-beta-mucholic acid degradation, deconjugation of bile acids and alteration of lipid metabolism.

Metabolomics on fine-needle aspiration biopsies from patients with benin or malignant thyroid tumors: the OPLSDA metabolomic analysis of the samples led to a robust classification model with a predictivity of 89%, which is more predictive than those existing (genomics or proteomics)

The in vitro and in vivo models regarding the host/Ruminococcus gnavus E1 interactions have to be developed. the tissue samples from conventional mice exposed to high fat or high fat +FOS diet ave to be analysed using HRMAS NMR. The LC/MS data on the biofluids have to be statistically treated.

Publication
Torregrossa, L.*; Shintu, L*; Chandran, JN ; Tintaru, A; Ugolini, C ; Magalhaes, A ; Basolo, F ; Miccoli, P ; Caldarelli, S. Toward the Reliable Diagnosis of Indeterminate Thyroid Lesions: A HRMAS NMR-Based Metabolomics Case of Study Journal of Proteome Research, 2012, 11(6), 3317-3325

Marie Renault, Laetitia Shintu, Martial Piotto, and Stefano Caldarelli * Slow-spinning low-sideband HR-MAS NMR spectroscopy: delicate analysis of biological samples, Scientific Report, 2013

André, Marion; Dumez, Jean-Nicolas; Shintu, Laetitia; Rezig, Lamya; Piotto, Martial; Caldarelli, Stefano A complete protocol for slow-spinning High-Resolution Magic-Angle Spinning NMR analysis of fragile tissues Analytical chemistry, 2014, resoumis après révision

Conférence invité
L. Shintu – Conférence Invitée – Iode et Cancer – Nice -27 Juin 2014

Poster:
XXVth International Conference on Magnetic Resonance in Biological Systems
19-24 August 2012
NMR-based Metabolomics analysis of plasma samples for the diagnosis of thyroid cancer
L. Rezig, L. Shintu, L. Torregrossa, C. Ugolini, M. Minuto, F. Basolo, P. Miccoli, S. Caldarelli
Slow-spinning low-sideband HR-MAS NMR spectroscopy: delicate analysis of biological samples
M. Renault, L. Shintu, M. Piotto, S. Caldarelli

EUROMAR 2012, Dublin (Irlande)
1-5 July 2012
Metabolomics Analysis of targeted multi-compartmental samples for the diagnosis of thyroid cancer
L. Shintu, L. Torregrossa, J. Nambiath Chandran, M. Renault, C. Ugolini, M. Minuto, F. Basolo, P. Miccoli, S. Caldarelli

NMR based Metabolomics for the diagnosis of thyroid cancer - Lamya Rezig, , Adele Servadio, Liborio Torregrossa , Fulvio Basolo, Paolo Miccoli, Laetitia Shintu Stefano Caldarelli – Metabomeeting 2014 – 10-12 Septembre 2014

NMR based Metabolomics for the diagnosis of thyroid cancer - Lamya Rezig, , Adele Servadio, Liborio Torregrossa , Fulvio Basolo, Paolo Miccoli, Laetitia Shintu Stefano Caldarelli – 8ème journées du RFMF 2014 – 19-21 Mai 2014

This project aims at developing and testing an integrated protocol for the metabolomics search of potential biomarkers with an enhanced significance of its outcome. The protocol will be based on tissue/biofluid parallel analysis using NMR (HRMAS and liquid state) and Mass Spectrometry. These analytical techniques will rely on high-throughput tools, including the second refrigerated HRMAS sample changer installed worldwide.
The stability and performance of the protocol will be tested on two challenging case studies: the discovery of markers highlighting symbiotic gut/microbiota interactions in a gnotobiotic mouse model and the thyroid cancer malignancy assessment in a preclinical study. Both studies are well characterized by substantial preliminary works and the current project will contribute with specific information on relevant metabolites.
Metabolomics has been encountering a growing success, based on the maturity of the analytical and statistical tools and on the global approach of the methods, which requires minimal previous knowledge of the target metabolites. Most of Metabolomics studies have focused on the quantification of response-induced metabolic variations of a small part of the whole metabolism (one sample type) hypothesizing that it would suffice to provide relevant information. The discovery of markers is consequently carried out from a restricted set of detected metabolites, which may be poorly representative of the overall studied metabolism and consequently, this may lead to a truncated description of the response. However, the systemic connections have been invoked to unveil additional biomarkers.
The present project aims at enhancing the significance of the potential biomarkers singled out by metabolomics, thus likely simplifying the metabolic interpretation of the system response. This result will be achieved by devising the highest degree of integration on both analytical and sampling aspects. The first degree of integration will be by concurrently analyzing tissue materials, which provides a localized description of metabolic variations, and body fluids, for a systemic view. The second degree will concern multiple analytical techniques.

Methodological goals
1. We shall develop a slow-spinning HRMAS NMR protocol, compatible with high-throughput analysis of a large variety of biological tissues.
2. We shall implement a protocol of integrated tissue/biofluids analysis using NMR and MS spectrometry to enhance the significance of potential biomarkers and to simplify the identification of relevant metabolic pathways.

Application goals
1. We shall apply our integrated protocol to the search of markers of symbiotic gut/microbiota interactions.
2. We shall apply our integrated protocol to assess the degree of malignancy of thyroid cancer.