Lehtimäki Kimmo K, Valonen Piia K, Griffin Julian L, Väisänen Tuula H, Gröhn Olli H J, Kettunen Mikko I, Vepsäläinen Jouko, Ylä-Herttuala Seppo, Nicholson Jeremy, Kauppinen Risto A
Department of Biomedical NMR and National Bio-NMR Facility, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland.
J Biol Chem. 2003 Nov 14;278(46):45915-23. doi: 10.1074/jbc.M306209200. Epub 2003 Sep 3.
Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.
单纯疱疹病毒胸苷激酶（HSV-tk）基因疗法可诱导大鼠BT4C胶质瘤细胞发生程序性细胞死亡，利用氢核磁共振波谱（1H NMR）在体内监测与细胞损伤相关的代谢物变化，通过高分辨魔角旋转（HRMAS）1H NMR在体外监测，以及在肿瘤的高氯酸提取物中进行体外监测。以水作为内参在体内定量以及在提取物中体外定量的代谢物浓度，与细胞密度相关。结果显示，体内和体外的甘氨酸和肌酸浓度均遵循体积平均细胞密度，而总含胆碱化合物的浓度在细胞损失接近60%时不受影响。同时，饱和和不饱和的可通过1H NMR检测到的脂质均增加。在14.1特斯拉下对肿瘤样本进行的HRMAS 1H NMR波谱分析表明，在体外胶质瘤样本中存在来自腺苷和尿苷核苷酸的核苷酸峰。通过肿瘤提取物的加标实验结合1H和31P NMR波谱，确认了7.95 ppm处的双峰归属于尿苷二磷酸（UDP）。HRMAS还将体内3.2 ppm处的含胆碱峰解析为胆碱（3.20 ppm）、磷酸胆碱（3.22 ppm）、甘油磷酸胆碱（3.24 ppm）和牛磺酸（3.26 ppm）的共振峰。这些共振峰与程序性细胞死亡的时间进程无关。我们的结果表明，1H NMR检测到的脂质和一些小分子代谢物对基因疗法有反应。然而，含胆碱化合物不受细胞密度严重下降的影响。后一观察结果支持了这样一种观点，即三酰甘油而非膜磷脂是1H NMR可见脂质的关键成分，并且也对含胆碱化合物的共振作为体内程序性细胞死亡诊断标志物的有效性提出了质疑。
