| Metabonomics, defined as "the quantitative measurement of the dynamic multiparametric response of a living system to pathophysiological stimuli or genetic modification", is an important branch of systems biology. It has become a hot topic of "Omics" research following genomics, transcriptomics and proteomics. Metabonomics provides a new concept for the understanding of biosystems by simultaneous monitoring the changes of metabolite pools in a relatively holistic way, which plays an important role on the illustration of the complicated life systems.Diabetes mellitus (DM) is a metabolic disorder characterized by a chronic hyperglycemia with disturbances of whole body metabolism resulting from defects in insulin secretion, insulin action, or both. The number of diabetic patients is increasing due to the changes of life style and an aging population. It has become the third noninfectious chronic disease (NCD) threatening the health of human life following tumor and cardiovascular disease. Type 2 DM (T2DM) which accounts for approximately 95% of patients with diabetes has been described as an "epidemic" of contemporary society. The complications associated with T2DM are the major causes of increased mortality and morbidity in T2DM patients. However, most of the patients have already had one or more compalicatons before they were comfirmly diagnosed as T2DM. Thus, comprehensive understanding of the mechanism and finding potential biomarkers for the early diagnosis of T2DM has become a hot topic in life sciences research. Currently, the effects of anti-diabetic drugs on the metabolic networks of diabetic patients are not fully understood and systematic understanding of the action mechanism of anti-diabetic drugs has not been achieved.In this research,1HNMR, UPLC/MS, HPLC/UV and GC/FID methods were developed and applied to the study on the metabolic alterations in impaired fasting glucose (IFG) and T2DM patients. Potential biomarkers associated with the development of T2DM were found using statistical analysis and bioinformatics. The methods were also applied to the study on metabolic alterations in T2DM patients after the treatment with Metformin or sulfonylureas. The results of this study are helpful for further understanding the mechanism and early detection of T2DM and also provide information on the action mechanism of anti-diabetic drugs, which will contribute to rational administration, safe medication and individualized treatment of T2DM.1. Analytical platform for human serum metabonomics study(1) An 1HNMR method was developed for human serum metabolic fingerprinting analysis. The serum samples were diluted with NaH2PO4-Na2HPO4 buffer solution and the 1HNMR data was collected by Carr-Purcell-Meiboom-Gill (CPMG) pulse. The metabolites in serum metabolite fingerprints were identified by comparing with the data in literatures. (2) A UPLC/MS method was developed for human serum metabolic fingerprinting analysis. The serum samples were pretreated by protein precipitation. Complicated components in serum were successfully separated by UPLC, and twenty metabolites were identified by MS/MS in the metabolite fingerprints. (3) Human serum amino acids profiling analysis was performed by pre-column derivatization with dansyl chloride. Seventeen amino acids were successfully separated and quantified by HPLC/UV. (4) Human serum free fatty acids profiling analysis was carried out by esterification with H2SO4-CH3OH. Ten free fatty acids were detected by GC/FID and qualified by GC/MS. (5) Human serum lysophosphatidylcholines target analysis was studied by UPLC/MS/MS. The serum samples were extracted with chloroform-methanol. Detection was performed in positive ion mode with multiple reaction monitoring (MRM). All the methods are well met the criterion of biological analysis and suitable for metabonomics research.2. Analytical platform for human urine metabonomics study(1) A UPLC/MS method was developed for human urine metabolic fingerprinting analysis. The urine samples were pretreated by diluting with deionized water and complicated components in urine were separated by UPLC. Eighteen metabolites in urine metabolite fingerprints were identified by MS/MS. (2) A GC/FID method was also developed for human urine metabolic fingerprinting analysis. Urine samples were derivatizated with ethyl chloroformate (ECF) and separated with HP-5 capillary column. By comparing with reference standards, thirteen metabolites were identified. The urine metabolic fingerprinting analyses based on UPLC/MS and GC/FID are well met the criterion of biological analysis and suitable for metabonomics research.3. The application of analytical platform for human urine metabonomics study to the pathogenesis and diagnosis research of T2DMThe developed UPLC/MS and GC/FID methods for human urine metabolic fingerprinting were applied to the pathogenesis and diagnosis research of T2DM. The following results were found:(1) Clear separations of urine metabolite fingerprints based on UPLC/MS and GC/FID were achieved among healthy controls, IFG and T2DM patients. (2) The urine metabolite fingerprints of IFG patients were between those of healthy controls and T2DM patients. (3) The concentrations of cresol sulfate, aspartic acid and glutamic acid were increased and those of hippurate, phenylacetylglutamine, creatinine, citric acid, tryptophan and nonanedioic acid were decreased in urine of IFG patients. In urine of T2DM patients, the concentrations of cresol sulfate, carnitines, glutamic acid, aspartic acid, palmitic acid were increased and those of hippurate, phenylacetylglutamine, creatinine, citric acid, tryptophan and nonanedioic acid were decreased. Alterations of these metabolites in urine indicate disturbances of amino acids metabolism, energy metabolism, gut microflora metabolism and fatty acids oxidation in T2DM patients. (4) Receiver operating characteristic (ROC) curve was applied to evaluate the diagnostic accuracy of potential biomarkers. Creatinine, phenylacetylglutamine and glutamic acid were found to be potential biomarkers which may be valuable in the early diagnosis and possibility prediction of T2DM (AUC> 0.8). Glutamic acid, aspartic acid, tryptophan, hippurate, creatinine, carnitine C8:1, nonanedioic acid and phenylacetylglutamine were found to be potential biomarkers which may be applied to the dianogsis of T2DM (AUC> 0.8).4. The application of analytical platform for human serum metabonomics study to the pathogenesis and diagnosis research of T2DMThe developed 1HNMR and UPLC/MS methods for human serum metabolic fingerprinting analysis and human serum amino acids, free fatty acids profiling analysis, human serum lysophosphatidylcholine target analysis were applied to the pathogenesis and diagnosis research of T2DM. The following results were found:(1) Significant differences of serum metabolite fingerprints obtained by 1HNMR were observed between healthy controls and T2DM patients. Clear separations of serum metabolite fingerprints obtained by UPLC/MS and amino acids, free fatty acids profiles obtained by HPLC/UV and GC/FID respectively were observed among healthy controls, IFG and T2DM patients. (2) The serum metabolite fingerprints, amino acids and free fatty acids profiles of IFG patients were between those of healthy controls and T2DM patients. (3) Compared with healthy controls, the concentrations of serum lysophosphatidylcholines, phenylalanine, tetradecanoic acid, hexadecanoic acid,9-hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid and 9,12-octadecenoic acid were increased while those of arachidonic acid, aspartic acid, alanine, profline and cysteine were decreased in IFG patients. In serum of T2DM patients, the concentrations of serum lysophosphatidylcholines, phenylalanine, lipoprotein, lactate, NAC, acetoacetate, unsaturated lipids, tetradecanoic acid, hexadecanoic acid,9-hexadecenoic acid, octadecanoic acid,9-octadecenoic acid and 9,12-octadecenoic acid were increased and those of alanine, aspartic acid, argnine, threonine, glycine, proline, tryptophan, tyrosine, cysteine, arachidonic acid, citric acid, creatinine, histate and formate were decreased. Changes of these metabolites in serum indicate that besides the disturbances in glucose, lipid and amino acids metabolism, the disorders of oxidative stress, energy and gut microflora metabolism were also observed in T2DM patients. (4) Serum (16:0/0:0) LPC, phenylalanine, aspartic acid, tetradecanoic acid, hexadecanoic acid,9-hexadecenoic acid, octadecanoic acid,9-octadecenoic acid and 9,12-octadecenoic acid were found to be potential biomarkers which may also have application values in the early diagnosis and possibility prediction of T2DM (AUC> 0.8). Serum lysophosphatidylcholine, phenylalanine, lipoprotein, citric acid, creatinine, unsaturated lipids, aspartic acid, histate, formate, threonine, glycine, cysteine, tetradecanoic acid, hexadecanoic acid,9-hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid,9,12-octadecenoic acid, NAC and arachidonic acid were found to be potential biomarkers which may also be valuable in the diagnosis of T2DM (AUC> 0.8). (5) Three LPC potential biomarkers were quantified by UPLC/MS/MS analysis. The sensitivity and specificity of (16:0/0:0) LPC in the early diagnosis of T2DM were 63% and 95% respectively.5. The application of analytical platform for human serum metabonomics study to the treatment mechanism research of T2DMThe developed 1HNMR and UPLC/MS methods for human serum metabolic fingerprinting and human serum amino acids, free fatty acids profiling methods were applied to the treatment mechanism research of T2DM. (1) Clear separations of serum metabolite fingerprints, amino acids and free fatty acids profiles were observed between T2DM patients and patients after the treatment of Metformin or sulfonylureas. (2) Compared with T2DM patients, the concentrations of serum lysophosphatidylcholine, phenylalanine, hexadecanoic acid, octadecanoic acid,9-octadecenoic acid, 9,12-octadecenoic acid, lipoprotein and lactate were decreased, while those of 3-hydroxybutyrate, citric acid, creatinine, TMAO, formate, histate, aspartic acid, serine, threonine, glycine, alanine, proline, tryptophan, tyrosine, lysine and cysteine were increased after the treatment of Metformin. The concentrations of serum lysophosphatidylcholine, phenylalanine, tetradecanoic acid, octadecanoic acid, 9-octadecenoic acid and 9,12-octadecenoic acid were decreased, while those of arachidonic acid, phosphatidylcholine, aspartic acid, glutamic acid, proline, methionine, tryptophan, tyrosine and cysteine were increased in sulfonylureas treated patients. (3) Using the analytical platform for, human serum metabonomics study in treatment mechanism research of T2DM, potential effects of Metformin and sulfonylureas on oxidative stress, gluconeogenesis and lipid decomposition were observed. An improvement of gut microflora metabolism was also found in T2DM patients after the treatment of Metformin. |
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