High resolution magic angle spinning hydrogen NMR spectroscopy on human tissues and cells

Experimental results in this thesis demonstrate that High Resolution Magic Angle Spinning (HRMAS) 1H NMR techniques, optimized to determine metabolite concentrations in ex vivo tissue and cell samples, offer improved spectral resolution over standard NMR spectroscopy techniques. Tissue and cell specific acquisition conditions were developed for HRMAS 1H NMR to: minimize tissue degradation resulting from, sample temperature, solvent volume, and acquisition time. Total Correlation Spectroscopy (TOCSY) experiments in which composite 90° and 180° pulses (MLEV-17 mixing scheme), were replaced with rotor synchronized, phase and frequency modulated, adiabatic pulses (WURST-8 mixing scheme), demonstrated a 1.5 to 10.5 times improvement in cross peak intensities.;Experimental studies were designed to, calibrate the analytical sensitivity of HRMAS 1H NMR spectroscopy, and to develop reproducible sample handling methods. Together, these experiments resulted in robust quantitation methods that were used to make pairwise comparisons of the metabolic profiles of three genetically engineered astrocytic cell lines. These cell lines were engineered to model some of the critical genetic alterations that occur during malignant transformation leading to Glioblastoma Multiforme (GBM). Pairwise comparisons of the metabolite levels in these cell lines showed significant differences in the taurine, choline, as well as the combined glycerophosphcoline+phosphocholine, and glutathione+glycine+cysteine+glutamate metabolite levels. In a separate study, HRMAS 1H NMR was used to evaluate metabolic changes that take place in tumor xenografts as a function of treatment with a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Experimental results of a comparison between a control (n=5) and treatment group (n=4) showed that the treatment group had significantly a higher lipid content and significantly lower levels of visible metabolites.;Experimental results on tissue and cell samples consistently showed limited changes in metabolite levels. Given that experimental results in this thesis indicate that some cell and tissue metabolite levels are undergoing, temperature dependent, changes as a function of time spent outside their respective in situ environments, it was hypnotized that although the work in this thesis demonstrated critical advances in HRMAS 1H NMR acquisition methodology, sample history, and sample handling remains a limiting factor to the biological sensitivity of HRMAS 1H NMR.