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.
Anal Bioanal Chem. 2011 Jan;399(2):987-99. doi: 10.1007/s00216-010-4321-4. Epub 2010 Nov 11.
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 overlapping 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 concentration 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.
本研究旨在确定最佳 pH 值，以便对脑肿瘤细胞和组织的高氯酸或甲醇-氯仿-水提取物进行（1）H 核磁共振（NMR）光谱分析。在模型溶液中对 13 种脑代谢物的质子化学位移随 pH 值变化的系统研究表明，在 pH 10 下记录（1）H NMR 谱可以分辨在 pH 7 下重叠的共振峰，特别是在 3.2-3.3 ppm 的胆碱化合物区域。在 pH 7 或 10 下对提取物进行（1）H NMR 分析表明，在两种 pH 值下都可以轻松进行乳酸盐、丙氨酸、谷氨酸、谷氨酰胺（Gln）、肌酸+磷酸肌酸和肌醇（m-Ino）的定量测量。只有在 pH 10 下才能准确测量甘油磷酸胆碱、磷酸胆碱和胆碱等对肿瘤脑恶性程度分级至关重要的代谢物的浓度。事实上，其三甲铵部分的共振完全清除了任何重叠信号，特别是牛磺酸（Tau）和磷酸乙醇胺的信号。四个非电离的 Tau 质子在非拥挤的光谱区域中以单峰共振，使得在 pH 10 下比在 pH 7 下更容易且更准确地定量此凋亡标志物，因为在 pH 7 下，3.43 ppm 的三重峰可能与葡萄糖信号重叠或强度太低而无法测量。在 pH 7 下减去 m-Ino 重叠信号或在 pH 10 下减去 Gln 重叠信号后，可以在两种 pH 值下间接确定甘氨酸浓度。
