TECHNICAL NOTE pH optimization for a reliable quantification of brain tumor cell and tissue extracts with 1 H NMR: focus on choline-containing compounds and taurine O. Robert & J. Sabatier & D. Desoubzdanne & J. Lalande & S. Balayssac & V. Gilard & R. Martino & M. Malet-Martino Received: 21 July 2010 / Revised: 3 October 2010 / Accepted: 10 October 2010 / Published online: 11 November 2010 # Springer-Verlag 2010 Abstract The aim of this study was to define the optimal pH for 1 H nuclear magnetic resonance (NMR) spectroscopy analysis of perchloric acid or methanol–chloroform–water extracts from brain tumor cells and tissues. The systematic study of the proton chemical shift variations as a function of pH of 13 brain metabolites in model solutions demonstrated that recording 1 H NMR spectra at pH 10 allowed resolving resonances that are overlapped at pH 7, especially in the 3.2– 3.3 ppm choline-containing-compounds region. 1 H NMR analysis of extracts at pH 7 or 10 showed that quantitative measurements of lactate, alanine, glutamate, glutamine (Gln), creatine+phosphocreatine and myo-inositol (m-Ino) can be readily performed at both pHs. The concentrations of glycerophosphocholine, phosphocholine and choline that are crucial metabolites for tumor brain malignancy grading were accurately measured at pH 10 only. Indeed, the resonances of their trimethylammonium moieties are cleared of any over- lapping signal, especially those of taurine (Tau) and phosphoethanolamine. The four non-ionizable Tau protons resonating as a singlet in a non-congested spectral region permits an easier and more accurate quantitation of this apoptosis marker at pH 10 than at pH 7 where the triplet at 3.43 ppm can be overlapped with the signals of glucose or have an intensity too low to be measured. Glycine concen- tration was determined indirectly at both pHs after subtracting the contribution of the overlapped signals of m-Ino at pH 7 or Gln at pH 10. Keywords In vitro 1 H NMR . Cell and tissue extracts . Brain tumor . Optimal pH . Choline-containing compounds . Taurine Introduction Histopathology is still the mainstay of the diagnosis and classification of gliomas, the most frequent tumors of the central nervous system, but information gained from cytoge- netics and molecular genetics [1] as well as imaging and spectroscopic techniques such as magnetic resonance imag- ing (MRI) or magnetic resonance spectroscopy (MRS) [2, 3] produce complementary data for their more precise charac- terization. Indeed, numerous studies have used the alterations of the biochemical pathways deduced from the 1 H MRS spectra to help the MRI-based diagnosis of brain tumors and assess their classification and grading [4–9]. However, at the low field strengths used for clinical in vivo 1 H MRS (typically 1.5 T and much less commonly 3 T), there is considerable resonance overlap in spectra due to limited resolution of the instruments and signal broadening origi- nating mainly from magnetic susceptibility variations in tissues, making the accurate determination of metabolic Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4321-4) contains supplementary material, which is available to authorized users. O. Robert : D. Desoubzdanne : J. Lalande : S. Balayssac : V. Gilard : R. Martino : M. Malet-Martino (*) UPS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique (SPCMIB), Groupe de RMN Biomédicale, Université de Toulouse, 118 route de Narbonne, 31062 Toulouse, Cedex 9, France e-mail: martino@chimie.ups-tlse.fr J. Sabatier UPS; Service de Neurochirurgie, CHU Purpan, Université de Toulouse, 31062 Toulouse, Toulouse, France Anal Bioanal Chem (2011) 399:987–999 DOI 10.1007/s00216-010-4321-4