| Metabolism is the essence of life. The occurrence and development of any disease will affect body metabolism, and then lead to changes of metabolites. A set of diagnosis-related biomarkers might be discovered by comparison of the physiological states between the diseased and the health, As biological characteristics to assist the diagnosis and typing of diseases. Metabolites are the end products of gene expression, which are related with physiology, pathological conditions. Therefore, metabonomics provides a feasible method for disease diagnosis. In this thesis, NMR-based metabonomics has been applied to study the changes of metabolic profiles in two kinds of disease animal models, with further discussions about the pathogenesis. In order to understand the quantitative changes of metabolites in body fluids, quantitative 1H NMR method was established to determine the content of metabolites in plasma. This research mainly includes the following parts:1. NMR-based and GC-MS-based metabonomics were applied to study the db/db mouse model of typeâ…¡diabetic nephropathy. 1H NMR spectra of plasma, urine and the extracts of renal tissues were collected and analyzed using pattern recognition. Metabonomics analysis indicated that the kidney extracts of db/db mice were distinguished clearly from those of control db/m mice, with significantly decreased concentrations in a range of metabolites including glutamate, acetate, choline and glycine, and slightly decreased levels in valine, creatinine, nicotinamide, phenylalanine and tyrosine, while the levels of lactate and sugars were significantly increased. GC/TOF-MS and multivariate statistical analysis showed that lactate, urea, phosphoric acid, glucose, alanine, serine, glycine, stearic acid, palmitic acid and 11-cis-octadecenoic acid are differential metabolites for the two groups. The results demonstrated that abnormal metabolism occurred in glycolysis, gluconeogenesis and related pathways like amino acid metabolism regulated by leptin receptor gene, which might be due to the autosomal recessive defect in the leptin receptor gene of db/db mouse.2. Quantitative 1H NMR method was established for the determination of plasma metabolites. STD spectra was used to select suitable internal standard not binding with proteins. The result showed that the six selected internal standards can all bind protein, but with varied extent. The protein-binding properties of these chemicals were further investigated based on line broadening and peak area reduction. The results showed that formate had the minimum binding with proteins. The quantitative method was also applied to determine the contents of ketobodies in the plasma samples of db/db and db/m mice. The results indicated that there was no significant distinction between two groups.3. NMR-based metabonomics were applied to study the changes of metabolites at different time courses of myocardial infarction in animal models of chronic heart failure. The results of heart tissues showed that when compared with controls, the model groups had higher levels of choline and LDL/VLDL and lower levels of lactate, glutamine, phosphocholine, taurine, creatine, inosine and NA at 3 weeks after operation. At 6 weeks after operation, the metabolic differences between the two groups were similar as at 3 weeks after operation except for the higher LDL in model group. Choline was significantly increased again at 9 weeks after operation. At 12 weeks after operation, the increase in VLDL was not as much as that at 3 weeks. The results showed that there was no tendency of metabolic changes in disease model toward the control, suggesting that it is difficult to recover for heart after myocardial damage. This was also demonstrated by the continuously decreased levels of glutamine, which has the growth-promoting effect on myocardial cells, and taurine, which has the protective effect on myocardial cells. Metabolism was affected in the beginning and then recovered owing to general stress response. |
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