Exposure Assessment Of CML In Grain Products And Its Effect On Lipid Metabolism Of HepG2 Cells

Backgrounds:N~ε-carboxymethyl-lysine(CML)is one of the advanced glycation end products(AGEs)in maillard reaction process.In addition to CML synthesized by organisms themselves,dietary CML is widely found in foods rich in lipids,proteins and carbohydrates,and different food processing methods will also affect the generation of CML.Dietary CML is a low-toxicity or non-toxic substance,which has long-term cumulative toxic effects.Studies have shown that abnormal accumulation of CML in the body is directly involved in the pathogenesis of cardiovascular and metabolic diseases,including diabetes,kidney disease and atherosclerosis.In this study,CML concentrations in different grain products were detected,and the influence of different processing methods on it was analyzed.Based on the results of CML exposure in grain products,the effects of lipid metabolism in human hepatoma cells(HepG2)were investigated in vitro,detection of cell biochemical indexes,oil red O staining and lipidomics analysis.Method:(1)Based on National standard of grain(GB 2715-2016),rice products,wheat products,potato products and beans products were selected as research objects for sampling,and LC-MS/MS methodwas applied to detect the CML exposure levels of grain products and affected by different processing methods.(2)Based on the results of CML exposure in grain products,HepG2 cells were exposed to five CML solutions with different concentrations(0 ppm,0.01 ppm,0.1 ppm,1ppm and 10 ppm)for 24 h,and the changes in cell viability were detected by MTT assay.According to the results of cell viability,3 exposure doses were finally selected for the determination of cell biochemical indexes,oil red O staining and follow-up experiments.(3)Lipid metabolites of HepG2 cells exposed to different doses of CML(0.1 ppm,1ppm,and 10 ppm)for 24h were detected by Orbitrap Exploris 120.Potential differential lipid metabolites were screened by multivariate statistical analysis and pathway enrichment analysis was performed.Based on the established LC-MS/MS targeting semi-quantitative analysis method,the changes in lipid metabolite content co-regulated by each dose group in the metabolic pathway were verified by targeting.Result:(1)In 124 samples of grain products,except rice products,the detection rate of CML in wheat products,potato products and beans products reached 100%.CML exposure levels in rice products were 0-50.227 mg/kg,wheat products were 1.019-108.587 mg/kg,potato products were 0.124-43.163 mg/kg and beans products were 1.052-88.91 mg/kg.Under the influence of different processing methods,the average concentration of CML showed the following trends:baking(27.717 mg/kg)>frying(20.030 mg/kg)>raw grain(18.173 mg/kg)>fermentation(17.982 mg/kg)>drying(14.262 mg/kg).(2)HepG2 cells were treated with five doses of CML solution(0 ppm,0.01 ppm,0.1ppm,1 ppm,10 ppm)for 24 h,and the overall viability of HepG2 cells showed a downward trend(P>0.05).Based on cell viability results,0.1 ppm,1 ppm,and 10 ppm were selected as the three final exposure doses.After 24 h of CML treatment in HepG2cells,the contents of TC,TG,GSH,LDL-C and MDA were significantly increased in CML exposure group compared with control group,while the contents of HDL-C were significantly decreased(P<0.05).According to the results of oil red O staining,HepG2cells were treated with 0.1 ppm,1 ppm and 10 ppm CML solution for 24 h,and the accumulation of lipids in HepG2 cells was significant.It indicated that CML had a lipid accumulation effect on HepG2 cells.(3)Through multivariate statistical analysis,Metabo Analyst 5.0 and MS-DIAL,it was found that under the conditions of VIP value>1,P-value<0.05,FC value>1.5 or<0.5,a total of 61 potential differential metabolites were screened in the CML exposure group under the positive and negative ion mode.It was found that the effect of different concentrations of CML solution on lipid metabolism in HepG2 cells is mainly achieved by Glycerophosphlipid metabolism.Phosphatidylethanolamine(PE),phosphatidylcholine(PC)and lysophosphatidylcholine(LPC)are biomarkers of this pathway.Based on the enrichment results of glycerophosphlipid metabolism pathway,lipid metabolites co-regulated by each dose of CML exposure group were PC(16:0/20:5),PC(22:1/0:0),PE(16:1/16:1),PE(20:4/0:0)and PE(16:1/22:6).And the five lipid metabolites were validated by semi-quantitative analysis.The results showed that there was no significant difference in PE(20:4/0:0)content,and the contents of the other 4 lipids were significantly up-regulated compared with the control group,which was consistent with the results of non-targeted lipidomics.