HRMAS NMR Studies Of Intact Biological Tissues

This dissertation is concentrated on the applications of high-resolution magic-angle spinning NMR on biological tissues. Some sensitivity-enhanced methods for ¹³C spectra of brain tissue and applications of HRMAS ¹H NMR on the metabolism of human hepatocellular carcinoma (HCC) and esophageal cancer (EC) tissues have been performed. ¹³C spectra of rat brain tissue with high resolution and sensitivity were obtained in NOE-CPMG experiment. The applications of HRMAS ¹H NMR on HCC and EC tissues may supply abundant biochemical information at molecular levels for tumor research.The poor sensitivity of ¹³C limits its extensive application in biological tissues because ¹³C nuclear only has 1% natural abundance of ¹H nuclear. Furthermore, the typical liquid-state nuclear magnetic resonance (NMR) methods are difficult to obtain high resolution spectra of biological tissues due to the semi-solid characteristics of tissues. Magic angle spinning (MAS), originally used in solid-state NMR, has been applied to the study of biological tissues to acquire high resolution spectra. In this study, several sensitivity enhancement techniques commonly used in liquid- and solid-state NMR, including CP, DEPT and NOE, were combined with MAS to acquire high resolution ¹³C spectra of intact rat brain tissue at natural abundance, and were compared for their performances. The results showed that different signal enhancement techniques were sensitive to different classes of molecules/metabolites, depending on their molecular weights and mobility. DEPT was found to enhance the signals of low molecular weight metabolites exclusively, while the signals of lipids, which often were associated with membranes and had relatively lower mobility, were highly sensitive to CP enhancement. The intensities of both small moleculars and metabolites with high molecular weight were all enhanced with NOE-CPMG method.High-resolution magic angle spinning (MAS) ¹H magnetic resonance spectroscopy has been an important tool for supplying detailed biochemical information of intact biological tissues. It has been extensively applied to many tumor researches, including brain, breast and prostate cancer. The purpose of this study was to explore the ability of ¹H HRMAS NMR technique for chemical characterization of human HCC and EC tumors. Principal component analysis (PCA) was used to extract detailed metabolic changes. The result showed that the profile of HCC spectrum was characterized by declined triglyceride and glucose/glycogen, and the increase of lactate, alanine, leucine, glutamine/glutamate, choline/PC/GPC and glycine, together with a slight elevation of bile acid. The results indicated that alteration of energy metabolism coupled with changes in TCA cycle were dominant in the HCC biochemistry. Moreover, application of HRMAS ¹H MRS to the study of esophageal cancer tissue also showed the biochemical change in cancerous tissue in contrast to adjacent non-involved tissue. The elevation of alanine, aspargine, glutamate, cholines, glycine, tyrosine and phenylalanine were found in esophageal cancerous tissue, while the concentration of creatine, myo-inositol and taurine decreased. HRMAS ¹H NMR is a useful tool for supplying more significant metabonomic information for tumor research.