| Diabetes is a group of common chronic endocrine diseases.According to etiology,diabetes can be divided into types 1 and 2 diabetes mellitus(T2DM).The latter is more common than the former and accounts for approximately 90%of cases.With the rising prevalence of diabetes and its tendency to afllict younger groups,such condition has become one of the most serious and critical health problems in the 21st century.The number of patients with diabetes is increasing,and the accompanying life-long diabtetic complications would contribute to high mortality.Diabetes has become one of the heaviest disease burdens in China and the world.How to prevent and control diabetes pandemic has become a major public health issue and global concern.The challenge of coping with T2DM should be addressed to achieve progress.Genetic and environmental factors determine the occurrence and development of diabetes.The 70%-90%risk of diabetes can be attributed to adverse environmental exposure,whereas diet also serves as an important factor.Reasonable dietary intervention has become one of the most effective means of curbing diabetes.Apart from beneficial nutrients in the dietary aspect,numerous pathogenic factors also exist.In food production and processing,hot processing is the most common method,which adds special flavors and attractive colors to food and extends its shelf life.However,the Maillard reaction during hot processing also produces several harmful substances,including advanced glycation end-products(AGEs).AGEs are widely available in everyday diets,exposure to such substances is unavoidable for humans.Current research suggests that long-term exposure to high-AGEs diet will promote inflammation and affect the cardiovascular and endocrine systems,leading to obesity,diabetes,and accelerate the development of diabetic complications.Considering that the current AGEs dietary database remains limited and lacks accurate quantitative methods,the development of large-scale epidemiology has been limited.The International Diabetes Federation(IDF)and the American Diabetes Association(ADA)provide no recommendations for dietary AGEs restriction,and the evidence for large populations of AGEs and T2DM remains lacking.With the development of chromatography and mass spectrometry,the use of ultra-high-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)to evaluate the level of AGEs in biological samples has been widely praised,thereby possibly enabling the investigation of AGEs biomarkers in large populations.The metabolism and signal transduction of AGEs in humans are affected by a variety of receptors,the most critical of which is the receptor for AGEs(RAGE),which may mediate a large part of the adverse health effects of AGEs.AGE-RAGE signal axis is also an important topic in current research.The single-nucleotide polymorphisms of the gene AGER encoding RAGE are associated with the function of RAGE and various metabolic diseases.In recent years,precision medicine has received extensive attention from various fields.Exploring the relationship between individual exposure factors and disease outcomes agrees with the concept of precision medicine in different genetic backgrounds.Considering that dietary exposure is an important source of AGEs in the body,their accumulation causes a series of cascade reactions through the AGE-RAGE signal axis.Consequently,the event promotes the development of various chronic diseases,such as diabetes.This study intends(1)to establish a stable and reliable UPLC-MS/MS detection method to simultaneously detect multiple AGEs in human plasma samples;(2)to explore the association of various AGEs biomarkers with T2DM and prediabetes;(3)to investigate the role of soluble RAGE(sRAGE),as an important clinical biomarker in the development of diabetes;(4)and to further explore the interaction of AGER gene polymorphisms and sRAGE in progression of diabetes.The main content of this study covers the following three parts.Part One Quantification of Advanced Glycation End-products in Human Plasma SampleObjective:To establish an UPLC-MS/MS method with simple pretreatment,high sensitivity,and high throughput,to quantify the concentration of five kinds of AGEs in human plasma.Methods:Agilent 1200SL UPLC system was used in tandem with Agilent 6460 triple tandem quadrupole mass spectrometer for targeted quantitative analysis of plasma AGEs.The ion source was electrospray ionization(ESI)with Agilent Jet Stream.The scan mode is multiple reaction monitoring(MRM).We optimized the sample preparation,chromatographic conditions,and mass spectrometry parameters to achieve the best sensitivity and separation and to validate the analytical method with reference to the European Medicine Agency's Bioanalytical Method Validation Guide.Results:Sample pretreatment was performed using acetonitrile-methanol(1:3,v/v)for protein precipitation.After optimization by mass spectrometry and liquid phase parameters,the analytes(CML,CEL,CMC,MG-H1,and Pyr)and corresponding internal standards were quantified via MRM mode with positive ESI.The optimized quantitation ion pairs(m/z)were 205.1?84.1(CML),219.1?84(CEL),180.0?89.0(CMC),229.1?70.1(MG-H1),and 255.1?148.1(Pyr).Chromatographic separation was performed using a ZORBAX Eclipse Plus C18 column(2.1 mm x 100 mm,1.8 ?m)with gradient elution of acetonitrile(Phase A)and 5 mM aqueous solution of nonafluoropentanoic acid(Phase B).The injection volume was 10 ?L,and the single sample analysis time was 14 min.Method verification showed the considerable separation of each target substance,and no notable endogenous interference was observed.The calibration curve of each AGEs exhibited good linearity.The intra-and inter-day precision of four different concentrations of quality control samples were within 15%,whereas the accuracy was within±15%.Mean extraction recoveries were between 85%and 115%for all AGEs at three different concentrations.All AGEs in the quality control samples of the three concentration levels stabilized when placed at room temperature for 24 hours,repeatedly freeze-thawed thrice,or stored at low temperature(-80 ?)for one year.Conclusions:We established a UPLC-MS/MS quantitative detection method for free AGEs quantification in human plasma sample.The method was characterized by simple pretreatment,high sensitivity,and good stability and reliability,laying the foundation for subsequent epidemiological research.Part Two Association of Plasma Advanced Glycation End-products with Type 2 Diabetes Mellitus and Impaired Glucose RegulationObjective:To examine the association of plasma free AGEs adducts with risk of T2DM and IGR.Methods:On the basis of a case-control study design,836 newly diagnosed T2DM,515 IGR,and 1214 controls with normal glucose tolerance(NGT)were included according to the World Health Organization's 1999 Diabetes Diagnostic Criteria.Sociodemographic data,lifestyle habits,and history of diseases were obtained through a standardized questionnaire.professionals detected the body composition and biochemical indices.Plasma free AGEs adducts were quantified using established UPLC-MS/MS methods.The association of multiple AGEs adducts and AGEs score with T2DM and IGR was explored using multivariate logistic regression analysis.Results:The distribution of plasma AGEs adducts(CML,CEL,CMC,and MG-H1)in NGT,IGR,and T2DM showed an increasing trend,whereas no significant difference was observed in pyrraline levels among NGT,IGR,and T2DM groups.After adjusting for potential confounders,the odds ratios(ORs)and 95%confidence intervals(CIs)for T2DM with per standard deviation(SD)increment of AGEs were CML:1.40(1.26-1.56),CEL:1.54(1.38-1.73),CMC:1.81(1.60-2.06),and MG-H1:1.14(1.02-1.27).Similar results were obtained for the association between AGEs and IGR.The ORs and 95%CIs for IGR with per SD increment of AGEs were CML:1.21(1.08-1.36),CEL:1.09(0.97-1.22),CMC:1.40(1.25-1.57),and MG-H1:1.05(0.94-1.18).Compared with the relationship between AGEs and T2DM,the association between AGEs and IGR was somewhat attenuated.With per 1-point increment of AGEs score,the ORs(95%CIs)were 2.70(2.20-3.32)and 1.52(1.22-1.88)for T2DM and IGR,respectively.Conclusions:The current study suggested that multiple AGEs adducts and AGEs score are positively associated with T2DM and IGR in a dose-dependent manner.Part Three Circulating Concentration of Soluble Receptor for Advanced Glycation End-products,AGER Gene Polymorphisms,and Risk of Type 2 Diabetes MellitusObjective:To investigate the association of plasma sRAGE and AGER gene variants with T2DM risk.Methods:We conducted a large case-control study,including 1360 newly diagnosed T2DM patients,859 subjects with IGR,and 2495 controls with NGT.T2DM and IGR were diagnosed according to the World Health Organization's 1999 Diabetes Diagnostic Criteria.All subjects were enrolled with oral glucose tolerance test from the endocrine clinic of Tongji Hospital,which is affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei Province from 2012 to 2015.Basic information was obtained through a standardized questionnaire,and professionals detected the body composition and blood parameters.Plasma sRAGE was detected by an enzyme-linked immunosorbent assay kit,and genotyping was performed using a matrix-assisted laser desorption ionization time-of-flight MS system(Agena MassArray system).Results:Plasma sRAGE levels measured 879.2(643.0-1214.9),908.3(678.6-1198.3),and 1075.7(772.3-1436.8)pg/mL in the T2DM,IGR,and normal controls,respectively(P<0.001).In the restricted cubic spline regression model,plasma sRAGE concentration was negatively correlated with T2DM and IGR risk in a linearly dose-response manner.After adjusting for sociodemographic profile,lifestyle,family history of diabetes,and history of diseases,the ORs of T2DM and IGR reached 0.70(95%CI:0.64-0.76)and 0.79(95%CI:0.73-0.87)with per SD increment of plasma sRAGE.In the stratified analysis,robust associations were detected between elevated plasma sRAGE levels with lower odds of newly diagnosed T2DM in all subgroups,whereas an inverse sRAGE-T2DM association was pronounced in non-smokers(P for interaction = 0.04).The OR of AGER rs2070600 homozygous genotypes TT compared with CC was 0.65(0.43-0.97)for T2DM.Per SD increment of plasma sRAGE was associated with 65%[OR:0.35(0.15-0.73)]lower odds of T2DM in TT carriers,44%[OR:0.56(0.45-0.68)]lower odds in CT carriers,and 38%[OR:0.62(0.55-0.69)]lower odds in CC carriers(P for interaction = 0.02).Conclusions:Higher plasma sRAGE concentrations were associated with lower odds of T2DM and prediabetes.The inverse association of plasma sRAGE concentrations with T2DM was modified by AGER rs2070600.Further studies are warranted to confirm our findings and to clarify the underlying mechanisms. |
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