Proceedings of the meeting - Department of Physics - University of ...

Preliminary Data for Quantitative Two-Dimensional HRMAS Spectroscopy of Human Brain TumoursAlan WRIGHT 1 , Kirstie Opstad 1 , John Griffiths 1 , B. Anthony Bell 2 and Franklyn Howe 11 CRUK Biomedical Magnetic Resonance Research Group , Division of Basic Medical Sciences and 2 Department of Neurosurgery, StGeorge’s (University of London), SW17 0RE.P32IntroductionHigh resolution magic angle spinning (HRMAS) NMR spectroscopyis being used for metabolomic analysis of tumour biopsies and hasadvantages of minimal sample preparation and subsequenthistopathological analysis. Despite the improvement in 1D spectralresolution achieved by performing MAS on tissue samples (1), 2DTOCSY has even greater peak separation compared to 1D spectra(Figure 1) thus improving peak assignment and measurement.However, quantitation using a 2D cross-peak volume must accountfor: J coupling; relaxation during the mixing time; T1 saturation dueto the short TR used in 2D acquisitions. A method of quantitative 2DTOCSY HRMAS is proposed and demonstrated for phosphocholine(PCh) and glycero-phosphocholine (GPC), two metabolites of interestin phospholipid metabolism.levels, including creatine of metastases and meningiomas, over a 3hour period. Changes in choline compounds are summarised in Table2. These show that PCh and GPC concentrations decrease during the2D experiment time while free Cho levels increase.Tumour Quantification PCh GPC RatioGPC/PChMeningioma 1D LCmodel 0.43±0.4 0.59±0.5 1.382D TOCSY 0.56±0.3 0.29±0.3 0.52Metastases 1D LCmodel 1.76±1.3 0.37±0.1 0.212D TOCSY 1.59±1.2 0.53±0.3 0.34Table 1: Comparison of estimated concentrations of PCh and GPC,and the GPC/PCh ratio, as quantified from 1D and 2D spectra4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0Ω−ppmFigure 2: An overlay of two 1D presaturation spectra from differentmetastases. Peak heights are normalised to the highest peak in eachspectrum. The differences in relative peak heights between the twospectra is typical of the variation seen between differrent metastasisbiopsies.Figure 1: Regions of 1D presaturation and 2D TOCSY spectrashowing the improved separation of PCh and GPC peaks with theextra dimensionMethodsTumour samples were taken from histopathologically-verified humantumour biopsies (4 meningiomas and 3 metastases) that had beenremoved and snap-frozen in liquid N2 during routine surgicalexcision. HRMAS spectra were acquired at 4°C using a Bruker600MHz system. For each sample a 1D presaturation spectra wasacquired at the start and end of the NMR protocol (3hrs total time)which consisted of: a 2D TOCSY experiment, CPMG spectra at shortand long TR to estimate T1 saturation; a pulse-acquire experiment tomeasure tissue water signal for a concentration reference. 2D TOCSYof standard metabolites solutions were acquired and used for crosspeakcalibration referenced to Cr. Metabolite-concentration estimateswere made from the 1D data using LCmodel. The change inmetabolite concentration over time was quantified from the 1Dpresaturation spectra acquired at the beginning and end of the NMRexperiment set.ResultsEstimated metabolite concentrations, for meningiomas and metastases,as measured from the 1D presaturation and 2D TOCSY spectrum, aregiven in Table 1. These data show reasonable agreement between thetwo methods though these average results show high standarddeviations about the mean. These large standard deviations betweentissue samples reflect the ‘heterogenous’ nature of the NMR spectrawithin each tumour classification (see Figure 2). The 1D presaturationspectra indicated changes of