Association Of Advanced Glycation End-products And Receptor For Advanced Glycation End-products With Gestational Diabetes Mellitus

Among the risk factors for gestational diabetes mellitus(GDM),dietary factor has received attention from domestic and foreign health guidelines as an important target for intervention.Advanced glycation end products(AGEs)are a complex and highly heterogeneous group of compounds formed by amino acids and reducing sugars,mainly produced during thermal processing of foods.The AGE-receptor for advanced glycation end-products(RAGE)axis promotes inflammation and oxidative stress through a series of cellular signaling cascades,leading to cellular dysfunction and the development of a range of chronic diseases.As a variable spliceosome of RAGE,soluble receptor for advanced glycation end-products(s RAGE)functions as a "decoy" to block the deleterious effects of the AGE-RAGE axis.Considering the widespread presence of AGEs in the daily diet and their potential harm to maternal and infant health,a method for the determination of AGEs based on ultra-high-performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)was established,and a case-control study was used to initially investigate the association of plasma AGEs and s RAGE with GDM.A nested case-control study based on prospective cohort was subsequently designed to elucidate the longitudinal association between early exposure levels of AGEs and s RAGE with GDM.And metabolomics was applied to further investigate the possible mechanisms by which AGEs affect the development of GDM.These results provide epidemiological evidence for the prevention of GDM and dietary guidance during pregnancy from the perspective of nutrition and food safety,which has important public health implications.Part I Establishment and optimization of a liquid chromatography tandem mass spectrometry method for plasma advanced glycation end productsObjective: Our team has previously established a complete method for the detection of free AGEs in plasma.Based on that technology,we propose to establish a relatively simple,high-throughput and high-sensitivity method for the detection of plasma protein-bound AGEs,which could effectively evaluate human plasma AGEs exposure levels in subsequent epidemiological studies.Methods: Based on Agilent 1200 SL ultra-high-performance liquid chromatography tandem 6460 triple quadrupole mass spectrometer,two representative protein-bound AGEs were selected to reflect the body AGEs load levels: carboxymethyl lysine and carboxyethyl lysine,and the two compounds were calibrated using lysine levels.The established new method was rigorously and comprehensively validated according to the European Medicines Agency guideline on bioanalytical method validation.Results: The best dissociation of protein-bound AGEs could be ensured by acid digestion at 110 ℃ for 18 h using 6 mol/L hydrochloric acid solution.The method was performed on a ZORBAX Eclipse Plus C18 reversed-phase column with gradient elution of 5 m M aqueous solution of nonafluoropentanoic acid and acetonitrile,and the mass spectrometry analysis was performed in electrospray ionization positive mode and multiple reaction monitoring scan.The results of the methodological validation showed that no significant endogenous interference was found for the target compounds and their internal standards at the corresponding retention times,and good linearity(r > 0.994)was presented in the given calibration curve range.The intra-day and inter-day precision of the protein-bound AGEs ranged from 0.44% to 11.12% and 1.55% to 12.62%,respectively,and the intra-day and inter-day accuracy ranged from-6.25% to 11.51% and-9.09% to10.25%,respectively,and the spiked recoveries ranged from 89.13% to 98.90%,all of which met the requirements of the bioanalytical validation guidelines.The stability experiment showed good stability of protein-bound CML,CEL and lysine in plasma.Conclusion: By optimizing the chromatographic and mass spectrometric parameters,our study established a UHPLC-MS/MS method for the determination of two representative protein-bound AGEs(CML and CEL)in plasma with a pretreatment-stable and high-sensitivity method.The methodological validation results demonstrated its good specificity and reliability,which can be adopted for the evaluation of human plasma AGEs exposure levels in subsequent epidemiological studies.Part II The association between plasma advanced glycation end products and gestational diabetes mellitusObjective: To investigate the association of plasma free and protein-bound AGEs with gestational diabetes mellitus.Methods: After 1:1 case-matching based on age,gestational age at blood sample collection,and parity,a total of 271 pairs of second-trimester case-control plasma samples were included in the case-control study,and a total of 189 pairs of first-trimester case-control plasma samples were included in the prospective cohort-based nested case-control study.Plasma free and protein-bound AGEs were quantified using an established ultra-high-performance liquid chromatography tandem mass spectrometry assay.The association between plasma advanced glycation end products and GDM was investigated by multifactorial conditional logistic regression analysis.Results: In the case-control study,age,pre-pregnancy BMI,family history of diabetes,fasting glucose,oral glucose tolerance test 1/2h,fasting insulin,homeostatic model assessment of insulin resistance and triglycerides were significantly higher in the GDM group than in the control group.Plasma free CML and CEL were positively associated with GDM in mid-pregnancy,and this association remained stable after adjusting for possible confounding factors,with odds ratio(OR)and 95% confidence interval(CI)of the highest tertile of 2.04(1.30,3.19),respectively 2.06(1.32,3.23)compared to the lowest tertile,while plasma protein-bound AGEs were not significantly associated with GDM.Furthermore,a nested case-control study was applied to investigate the temporal relationship between plasma AGEs and GDM,and significant differences existed in age,fasting blood glucose,and oral glucose tolerance test 1/2h in the GDM group compared with the control group,but there were no differences in pre-pregnancy BMI,family history of diabetes,gestational age at blood sample collection,parity,and smoking and drinking status.There was no significant association between plasma free AGEs and the risk of GDM in the first trimester.The risk of GDM was significantly higher in the second tertile for plasma protein-bound CML and CEL,with OR(95% CI)of2.97(1.64,5.38)and 3.10(1.74,5.50),respectively,while the significance disappeared in the third tertile.The fitted curves based on generalized additive model showed a non-linear relationship between plasma protein-bound CML and CEL with the prevalence of GDM,which tended to increase and then decrease.Conclusion: Two plasma free AGEs(CML and CEL)were positively associated with GDM,and this association remained stable after adjusting for relevant confounders.There was no significant association between plasma protein-bound AGEs and the risk of GDM in mid-pregnancy,while the plasma protein-bound AGEs and the risk of GDM showed a non-linear relationship in early pregnancy,with a potential time-series relationship.Part III The association between plasma receptor for advanced glycation end products and gestational diabetes mellitusObjective: To investigate the association between plasma s RAGE levels and the risk of GDM.Methods: Based on 1:1 matched case-control study and a nested case-control study design,the levels of soluble receptor for advanced glycation end products in plasma samples were measured by enzyme-linked immunosorbent assay.The association between plasma s RAGE levels and GDM was elucidated by multifactorial conditional logistic regression analysis,and the association between plasma protein-bound AGEs level,and the interaction between AGEs and s RAGE on gestational diabetes mellitus was further explored.Results: In the case-control study,plasma s RAGE had a significant protective effect against GDM in mid-pregnancy,which remained stable after adjusting for possible confounding factor,with an OR(95% CI)of 0.62(0.39,0.98)in the highest tertile compared with the lowest tertile.The association between plasma s RAGE and GDM was further investigated in a nested case-control study,and there was no significant association between plasma s RAGE and risk of GDM before and after adjusting for confounding factors.Then the interaction between plasma AGEs and s RAGE on the risk of GDM was analyzed.In mid-pregnancy,only free CMC had a significant interaction with s RAGE(P= 0.013)among the five free AGEs,and none of the protein-bound AGEs had a significant interaction with s RAGE.In early pregnancy,only free CEL had a significant interaction with s RAGE(P = 0.024)among the five free AGEs,and no significant interaction between protein-bound AGEs and s RAGE was revealed.Conclusion: Plasma s RAGE was negatively associated with the risk of GDM in mid-pregnancy,whereas plasma s RAGE was not significantly associated with the prevalence of GDM in the first trimester,suggesting that this association may not be causal.Plasma free AGEs(CMC,CEL)had significant interaction with s RAGE.Part IV A metabolomics-based study of potential mechanisms of advanced glycation end products and gestational diabetes mellitusObjective: To explore the possible mechanisms by which advanced glycation end products promote the development of GDM.Methods: Twenty-five pairs of 1:1 matched second-trimester and first-trimester case-control samples were selected and analyzed by Thermo Scientific Q Exactive HF quadrupole-Orbitrap mass spectrometer combined with high performance liquid chromatography.The compounds were identified by multiple databases,and further screened for differential metabolites.Enrichment analysis and network analysis was adopted to search for changes in metabolic pathways.Results: In the metabolomic analysis of the second trimester,plasma free CML and CEL concentrations in the GDM group were significantly higher than those in the control group.The PCA(principal component analysis)score plots and expression heatmaps of plasma metabolites showed significant changes in several metabolite groups between the two groups.Based on the small molecule pathway database,a total of 16 major metabolic pathways were identified,mainly including methylhistidine metabolism,glycine and serine metabolism,methionine metabolism,lysine degradation,arginine and proline metabolism.In the metabolomic analysis of the first trimester,plasma protein-bound CML concentration in the GDM group was significantly higher than that in the control group.The PCA score plots and expression heatmaps of plasma metabolites showed significant changes in few metabolite groups between the two groups.Based on the small molecule pathway database,a total of 15 major metabolic pathways were identified,mainly including methylhistidine metabolism,beta oxidation of very long chain fatty acids,methionine metabolism,fatty acid metabolism,histidine metabolism,glycine and serine metabolism,tryptophan metabolism.Conclusion: There were differences in multiple amino acid metabolism-related pathways between the GDM group and healthy controls.In the second trimester,as lysine and arginine are the main precursors for the derivation of AGEs,the lysine degradation and arginine metabolic pathways may be directly related to the relationship between AGEs and GDM,while alterations in other amino acid pathways may be one of the mechanisms by which AGEs affect the progression of GDM.Cellular experiments are desirable to further validate the resolved differential metabolic pathways.