Study On Biomakers For Type 2 Diabetes And Environmental Pollutants Exposure Based On Metabolomic Approaches

The health effects of type 2 diabetes and environmental pollutants exposure were investigated by LC-MS based metabolomics approaches, and biomonitoring of polycyclic aromatic hydrocarbons(PAHs) and benzene exposure in general population was carried out using LC-MS/MS methods in this thesis. Quantitative exogenous stimulations and variation of endogenous metabolites were integrated together by metabolomics approaches to aquire the early, dynamic and overall biochemical information, and find small-molecule biomarkers associated with type 2 diabetes and environmental pollutants exposure.Firstly, Lipidomics approaches suitable for analysis of serum lipid profile was developed based on LC-MS technology. Serum samples from populations with different stages of type 2 diabetes were analysed by global and targeted lipidomics approaches combined with multivariate statistical analysis, dynamic change trend analysis and ROC analysis to comprehensively investigate the change trends of lipids in the development of type 2 diabetes as well as find potential lipid biomarkers. As a result, significantly altered levels of 53 lipid metabolites related to metabolism of sphingolipids, glycerophospholipids, glycerides, and sterol esters were observed in the newly diagnosed type 2 diabetes and/or high risk population for type 2 diabetes. These disturbations may have an assoation with the altered state of insulin resistance and oxidative stess in the delepment of type 2 diabetes. Thirteen metabolites that showed regular change trends in populations with different stages of type 2 diabetes and had certain diagnostic power for type 2 diabetes were selected by dynamic change trend analysis and ROC analysis. These metabolites may be developed as lipid biomarkers for diagnosis and evaluation of therapeutic effects evaluation for type 2 diabetes. This study demonstrates that LC-MS based lipidomics approach is a useful tool to find lipid metabolites that closely associated with the development of type 2 diabetes, and provides key basis for the research on the molecular mechanism and development of more sensitive and reliable method for the early diagnosis of type 2 diabetes.In addition, to comprehensively evaluate the exposure levels of PAHs and benzene among a large population, LC-MS/MS methods for the quantitation of their 10 urinary metabolites were developed and validated based on a series of experiments. Biomonitoring and metabolic profiling of a large population from a coking-industrial polluted area and non-polluted control area were also carried out by LC-MS/MS methods. The dose-effects relationship between pollutants exposure and human metabolic response was intensively investigated by regression analysis, aiming to comprehensively evaluate the health effect of pollutants exposure and the underlying mechanism, and to find sensitive and reliable biomarkers associated with pollutants exposure. As a result, an incresded burden of PAHs and benzene caused significant changes in levels of many metabolites related to metabolism of amino acid, purine, lipid, and glucuronic acid in the populations living in a coking-industial polluted area. These changes were speculated to have a association with oxidative stress, indicating oxidative stress may be a underlying mechanism that linked the PAHs and benzene exposure with increased incidence rate of cancers, cardiopulmonary diseases, and diabetes in exposed populations. 1-Hydroxyphenanthrene and Carnitine C12:2 were found to have a potential for being sensitive and reliable biomarkers for PAH exposure and its metabolic outcomes in general population, respectively. This study demonstrates that, based on highly sensitive and highthrouput LC-MS/MS methods, biomonitoring and metabolomics approaches are useful tools to unveil the early molecular events induced by PAHs and benzne exposure, and provides new insight into the research on toxic mechanism of PAHs and benzne exposure as well as the development of preventive strategies for related diseases.1. LC-MS based lipidomics study for type 2 diabetesThe lipid metabolites in human serum showed remarkable diversity in structure and concentration, makeing it impossible to analyze all the lipids simultaneously using one of the currently available mass spectrometric ionization techniques. Three ionization methods, including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI), combining with reverse phase LC-MS operated in positive and negative ion modes were carried out to perform analysis on typical lipid standards from six categories as well as global serum lipidome of healthy volunteers, and the ionization ability, sensitivity and coverage of lipid compounds were explored systematically. The results showed that fatty acids(FFAs), phospholipids(PLs), sphingolipids(SLs), glycerolipids(GLs), and prenol lipids (PR) can be ionized efficiently under ESI source. APPI gave the best ionization efficiency for cholesterol esters (CEs). The ionization efficiency of APCI for lipid standards was less than ESI and APPI. APPI and APCI were not suitable for the detection of phospholipids. These results provides scientific basis for the chose of ionization method in LC-MS analysis of lipidomics and lipid metabolites.Based on the research mentioned above, LC-(±)ESIMS based global and targeted lipidomic approaches combined with multivariate statistical analysis were used to investigated the viriation in serum lipids in populations with different stages of type 2 diabetes. As a result, a total of 44 lipid metabolites and 29 lipid metabolites were found to have significantly changed levels in the newly diagnosed type 2 diabetes and high risk population for type 2 diabetes, respectively. These discriminating metabolites were generally associated with metabolism of spingolipids, glycerophospholipids, glycerolipids and sterol esters. The results of dynamic change trend analysis and ROC analysis revealed that thirteen lipid metabolites showed regular change trends with the development of type 2 diabetes and had certain diagnostic power for type 2 diabetes. Three of the thirteen lipid metabolites, including TG(16:0/1 8:1/18:0), TG(16:0/18:1/22:6) and CE(18:2-OH) were up-regulated in the newly diagnosed type diabetes and high risk population; The other ten lipid metabolites, including C16 sphingosine, C16 sphinganine, phytosphingosine, sphinganine, SM(dl8:1/24:1), SM(d 18:2/24:1), lysoPC(18:0), lysoPC(18:1), PC(18:0/18:0) and TG(16:0/16:0/18:0) were down-regulated in the newly diagnosed type 2 diabetes and high risk population. The change trends of most of these metabolites in the type 2 diabetes diagnosed for more than two years and treated with drugs were less significant than in the newly diagonosed type diabetes and high risk population. These metabolites may be developed as biomarkers for the disease’s progression and therapeutic effects of type 2 diabetes.2. Biomonitoring and metabolomics study for populations exposing to PAHs and benzeneTo evaluate the exposure level of PAHs and benzene in general population, LC-MS/MS methods for the quantitation of 10 urinary metabolites of PAHs and benzene, including S-phenylmercapturic acid(SPMA), 1-hydroxynaphthalene(1-OHN), 1-hydroxynaphthalene (2-OHN),2-hydroxyfluorene (2-OHF),1-hydroxyphenanthrene (1-OHPH),2-hydroxyphenanthrene(2-OHPH),3-hydroxyphenanthrene (3-OHPH), 4-hydroxyphenanthrene (4-OHPH),1-hydroxypyrene (1-OHP), and 6-hydroxychrysene (6-OHC) were developed and validated. These methods showed good performance in terms of linearity, accuracity and precision, and can be used for large-scale screening of PAHs and benzene exposure in general population. The urinary concentrations of 10 metabolites of PAHs and benzene in 238 elderly nonsmokers,114 elderly smokers and 214 children from a coking-industrial polluted area and non-polluted control area were determinated using the established methods. The results showed that the urinary concentrations of most of PAHs and benzene metabolites were significantly higher in the exposed populations than in the corresponding controls. The mean urinary 1-OHP concentration was 1.24 μg/g creatinine for children,0.88 μg/g creatinine for elderly nonsmokers, and 1.11 μg/g creatinine for elderly smokers in the pollued area. The mean urinary SPMA concentration was 0.68 μg/g creatinine for children,0.49 μg/g creatinine for elderly nonsmokers, and 3.04 μg/g creatinine for elderly smokers in the pollued area. This study prodives comprehensive information for the exposure level of PAHs and benzene in elders and children from a polluted aera and a non-polluted control area, and will benefit the health risk assessment of environtal pollutants exposure for these populations.To investigate the early biological effects induced by PAHs and benzene exposure, urine samples from the same populations as mentioned above were analysed by a LC-MS based metabolomics approach. As a result, increased levels of PAHs and benzene exposure significantly changed the levels of many endogenous metabolites in the populations from the polluted area,18 of which were identified. These discriminating metabolites are related to metabolism of amino acid, purine, lipid, and glucuronic acid. These metabolic outcomes of PAHs and benzene exposure may be associated with oxidative stress. One possible explanation for these results is that the metabolic activation of PAHs and benzene by the phase Ⅰ and phase Ⅱ enzymes can result in the generation of semiquinone radicals and ROS, which can then cause extensive biological effects, including the depletion of antioxidant, increased lipids perioxidation, dysfunction of mitochondrial lipids metabolism, and the up-regulation of phase Ⅱ metabolizing enzymes.Based on the results mentioned above, correlation of 10 metabolites of PAHs and benzene with 18 endougenous discriminating mtebaolites was performed by linear and logistic regression analysis to investigate the dose-response relationship between PAHs and benzene exposure with its metabolic outcomes, and find more reliable biomarkers closely associated with PAHs and benzene exposure. As a result,1-OHPH may be more sensitive and reliable biomarker than 1-OHP to evaluate total environmental PAHs exposure and its metabolic outcomes, while carnitine C12:2 was found to have a potential for being new biomarker for oxidative stress and related diseases induced by PAHs and benzene exposure.