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In vivo cystathionine detection in gliomas by edited 1H magnetic resonance spectroscopy1Centre de NeuroImagerie de Recherche (CENIR), Institut du Cerveau et de la Moelle épinère (ICM), Paris, France, 2Sorbonne Université, UMR S 1127, Inserm U 1127, CNRS UMR 7225, Paris, France, 3Center for Magnetic Resonance Research and Department of Radiology, University of Minnesota, Minneapolis, MN, United States, 4AP-HP, Hôpital de la Pitié-Salpêtrière, Service de Neurologie 2, Paris, France, 5Department of Neurology, Foch Hospital, Suresnes, Paris, France, 6Onconeurotek tumor bank, Institut du Cerveau et de la Moelle épinère (ICM), Paris, France
Synopsis
This study reports the first measurement of cystathionine in vivo. Cystathionine was measured using the edited magnetic resonance spectroscopy (MRS). The identification of cystathionine was confirmed by comparing in vivo spectra acquired in gliomas with the cystathionine spectrum measured in a phantom at physiological pH and temperature and with a simulated spectrum generated using accurate chemical shifts and J-coupling constants. The noninvasive detection of cystathionine by MRS may represent a new in vivo marker of glioma subtypes which will benefit diagnosis and treatment of patients with gliomas.
Introduction
Oxidative stress and associated decrease of glutathione levels play key roles in the pathogenesis of neurodegenerative diseases1. Cystathionine is an immediate precursor of cysteine and glutathione in the transsulfuration pathway1, and it has been reported to be present at low concentrations in normal brain tissue ex vivo2. Higher cystathionine levels in brain tumors have been reported from ex vivo tissue analysis2,3. In particular tumors of glial origin showed the highest cystathionine concentrations, suggesting that cystathionine is preferentially synthesized in glial cells. No studies reported the detection of cystathionine in vivo in either diseased or normal human brain. Here, we report the first detection and quantification of cystathionine in human brain glioma. Cystathionine represents a new potential noninvasive marker of glioma subtypes, opening up the possibility for new diagnostic and therapeutic strategies.Methods
In vitro MRS. Cystathionine phantom ([cystathionine] = 1 mM, pH 7.2) was prepared and measured at physiological temperature. MRS acquisition was performed using a 3 T whole-body Siemens Prismafit system equipped with a 32-channel receive-only head coil. MR spectra were acquired with a single-voxel MEGA-PRESS4 sequence (TR = 2 s, TE = 68 ms) using previously described procedures and parameters5. 512 averages were acquired from a 3 cm3 volume of interest (VOI).
In vivo MRI/MRS. Acquisitions were performed using a 3 T whole-body Siemens Verio system equipped with a 32-channel receive-only head coil. Thirty-one patients with glioma were included in the study. 3D FLAIR images were acquired to position the spectroscopic VOI in the glioma (Figure 1A), keeping a minimum VOI size of 6 cm3. MEGA-PRESS acquisition (TR = 2 s, TE = 68 ms) was performed as previously described5 (256 scans). For 6 patients, spectra were acquired also from normal brain tissue outside visible lesions (Figure 1B). The acquired spectra were processed in Matlab (MathWorks Inc., Natick, MA). Single-shot spectra were frequency and phase aligned using the total choline signal at 3.22 ppm. All spectra were analyzed using LCModel6 with the basis sets simulated using the density matrix formalism7 taking into account RF duration and patterns for 90° and 180° pulses, slice-selective gradients during the 180° pulses, and timing used in vivo. The basis set included 2-hydroxyglutarate, cystathionine, γ-aminobutyric acid, glutamate, glutamine, glutathione, N-acetylaspartate, N-acetylaspartylglutamate, and the experimentally measured macromolecular spectrum.
Results
In a subset of gliomas (16 patients), unexpected signals observed in the edited MR spectrum at ~2.7 ppm and ~3.9 ppm were identified as cystathionine based on the chemical shifts of the resonances as well as the J-couplings needed for those signals to appear in the editing scheme used (Figure 1B). As shown in Figure 2, the resonance at ~2.2 ppm is within the bandwidth of the editing pulse and is J-coupled to resonances at ~2.7 and ~3.9 ppm. In vivo, the cystathionine pattern is clearly visible at ~2.7 ppm, while the multiplets at ~3.9 and ~2.2 ppm partially overlap with the glutamate, glutamine, and N-acetylaspartate signals. The excellent agreement between the in vivo spectrum, the cystathionine spectrum measured at physiological pH and temperature, and the simulated spectrum used in the basis set affirms the correct assignment of the in vivo signal and the accuracy of the chemical shifts and J-coupling constants used to generate the basis spectra. Cystathionine was not detectable in normal brain tissue (Figure 1B). The cystathionine concentration in gliomas ranged from 0 mM to 4.1 mM.Discussion and conclusion
MRS enables in vivo detection and monitoring of molecules that accumulate specifically in certain diseases. The detection of cystathionine in brain glioma in vivo by edited MRS, herein reported for the first time, paves the way for an accurate and noninvasive identification of glioma subtypes with potential monitoring of specific treatment responses. Previous ex vivo tissue analysis reported higher cystathionine concentration in the brain than in other organs in human8. Significant regional differences were observed within the brain9, with the lowest concentration found in the cerebellum and cortex and the highest concentration found in the thalamus, ranging from 4.71 to 55.34 nmol/mg protein10. One previous study reported higher cystathionine levels in glioma and correlated it with tumor grade3. In our study, cystathionine levels in the normal brain regions investigated were too low to be detected, while abnormal accumulation of this metabolite was observed only in a subset of gliomas. The biological mechanisms of cystathionine accumulation in gliomas and its clinical relevance are under investigation.Acknowledgements
FB and SL acknowledge support from Investissements d’avenir [grant number ANR-10-IAIHU-06 and ANR-11-INBS-0006]. DD and MM acknowledge support from following National Institutes of Health grants: BTRC P41 EB015894 and P30 NS076408. The authors would like to thank Edward J. Auerbach, Ph.D., for implementing MRS sequences on the Siemens platform.References
1. Vitvitsky V, Thomas M, Ghorpade A et al. A Functional Transsulfuration Pathway in the Brain Links to Glutathione Homeostasis. J Biol Chem. 2006;281(47):35785–35793.
2. Lefauconnier J-M, Portemer C, Chatagner F. Free amino acids and related substances in human glial tumours and in fetal brain: comparison with normal adult brain. Brain Res. 1976;117(1):105–113.
3. Wróbel M, Czubak J, Bronowicka-Adamska P et al. Is Development of High-Grade Gliomas Sulfur-Dependent?. Molecules 2014;19(12):21350–21362.
4. Mescher M, Merkle H, Kirsch J, Garwood M, Gruetter R. Simultaneous in vivo spectral editing and water suppression. NMR Biomed. 1998; 11:266-272.
5. Branzoli F, Di Stefano AL, Capelle L, et al. Highly specific determination of IDH status using edited in vivo magnetic resonance spectroscopy. Neuro-Oncol. 2018; 20(7):907-916.
6. Provencher SW. Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med. 1993; 30(6):672-679.
7. Henry P-G et al. Proton-observed carbon-edited NMR spectroscopy in strongly coupled second-order spin systems. Magn. Reson. Med. 2006;55(2):250–257.
8. Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J. Biol. Chem. 1958;230:707–716.
9. Gjessing LR, Torvik A. Distribution of cystathionine in the human brain. Scand. J. Clin. Lab. Invest. 1966;18:565.
10. Bronowicka-Adamska P, Zagajewski J, Czubak J, Wróbel M. RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain. J. Chromatogr. B 2011;879(21):2005–2009.
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