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Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

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Abstract

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D 1H and 31P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. 1H–1H homonuclear and 1H–31P heteronuclear correlation experiments enabled the direct assessment of the 1H–31P spin systems for signals that suffered from overlapping in the 1D 1H spectra, and linked the information present in the 1D 1H and 31P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the 31P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from 31P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH3)3 signals of phosphocholine and choline in 1H spectra of the tissue in these tumour samples.

1H-31P HSQC (upper) and TOCSY (lower) spectra with the corresponding 1D 1H and 31P spectra for one GBM sample. The paths for homo and heteronuclear assignment are drawn as dashed lines (in blue for GPC, in red for GPE, in green for PC and in yellow for PE). The signals can be observed in the corresponding 1D 31P spectrum by the right margin of the HSQC spectrum, but not in the crowded 1D 1H spectrum above the HSQC spectrum

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Abbreviations

Ala:

Alanine

Cho:

Choline

Cr:

Creatine

ERETIC:

Electronic reference to in vivo concentrations

FA:

Fatty acids

GABA:

γ-Aminobutyric acid

GBM:

Glioblastoma multiforme

Gln:

Glutamine

Glu:

Glutamate

Gly:

Glycine

GPC:

Glycerophosphocholine

GPE:

Glycerophosphorylethanolamine

GSH:

Glutathione

HRMAS:

High-resolution magic angle spinning

MDPA:

Methylene diphosphonic acid

mI:

Myo-inositol

NAA:

N-Acetylaspartate

NAT:

Nonaffected tissue

PC:

Phosphocholine

PCr:

Phosphocreatine

PDE:

Phosphodiesters

PE:

Phosphoethanolamine

PME:

Phosphomonoesters

Tau:

Taurine

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Acknowledgements

The authors acknowledge the SCSIE-University of Valencia Microscopy Service for the histological preparations. They also acknowledge Martial Piotto (Bruker BioSpin, France) for providing the ERETIC synthetic signal. Furthermore, they acknowledge financial support from the Spanish Government project SAF2007-6547, the Generalitat Valenciana project GVACOMP2009-303, and the E.U.’s VI Framework Programme via the project “Web accessible MR decision support system for brain tumor diagnosis and prognosis, incorporating in vivo and ex vivo genomic and metabolomic data” (FP6-2002-LSH 503094). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

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Authors and Affiliations

  1. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 46100, Burjassot, Spain

    Vicent Esteve, Bernardo Celda & M. Carmen Martínez-Bisbal

  2. Physical Chemistry, University of Valencia, 46100, Burjassot, Spain

    Vicent Esteve, Bernardo Celda & M. Carmen Martínez-Bisbal

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  1. Vicent Esteve
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  2. Bernardo Celda
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  3. M. Carmen Martínez-Bisbal
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Correspondence to M. Carmen Martínez-Bisbal.

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Esteve, V., Celda, B. & Martínez-Bisbal, M.C. Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies. Anal Bioanal Chem 403, 2611–2625 (2012). https://doi.org/10.1007/s00216-012-6001-z

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  • DOI: https://doi.org/10.1007/s00216-012-6001-z

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