| Objective: To develop a LC-MS method to measure plasma acrylamide(AA)concentration as internal dose,and to investigate the effects of 52-week chronic exposure to AA on glucolipid metabolism in rats.The aim of the research is to provide animal experimental evidence for determination of human AA exposure level and its health effects.Methods: Thirty six healthy SPF male Sprague-Dawley rats(80-100g)were random divided into Group H-AA(5mg/kg),Group L-AA(0.5mg/kg)and Group CON.Rats were exposed to AA by drinking water for 52 weeks.Observing rat behavior performance every day and their body weight was measured weekly,and the IPGTT test was applied on rat 52 weeks later.At the end of our study,we choose 4 rats from each group and each rat perfused with 4% paraformaldehyde.After the perfusion,the liver,kidney and pancreas were taken for paraffin section and HE staining was performed to observe histopathological changes.The remaining rats were sacrificed and the blood was collected.Some were used for the determination of glycated hemoglobin.While the rest were used to making serum and plasma.Plasma biochemical indicators and serum insulin levels were determined.The weight of the internal organs(brain,heart,liver,spleen,lung,kidneys and testicles)was measured at the end of the experiment after sacrifice.Developing an LC-MS with protein precipitation for determination of AA in rat plasma.Results:(1)The assay utilized a simple method of LC-MS with protein precipitation for determination of AA in rat plasma.This method achieved a lower limit of quantification of 20 ng/mL of AA for plasma.The mean intra and inter-day assay accuracy or precision were both lower than 15%.The process recovery and the matrix effect were both in 100±15% and the stability was in 100±15% when samples were storage in room temperature for 24 h.(2)The concentration of AA in the 5 mg/kg group and the 0.5 mg/kg group was significantly higher than that in the control(P < 0.001,P < 0.05).Compared with the 0.5 mg/kg group,the concentration of AA in 5 mg/kg group was also significantly increased(P <0.01).(3)In this study,the body weight of rats gradually increased with time,but there was no significant difference in body weight among the groups(P > 0.05),same as the organ coefficient(P > 0.05).(4)Effect on glucose metabolism: the result of IPGTT test showed that the blood glucose concentration of each group has the same changing model,and there was no significant difference in glucose concentration at the same time point(P > 0.05).The results of fasting blood glucose measurement showed that there was no significant difference among the groups(P > 0.05).The glycosylated hemoglobin assay showed that the concentration of glycated hemoglobin in the 5 mg/kg group and the 0.5 mg/kg group is significantly higher than that in control(P < 0.001,P <0.001).Compare to the 0.5 mg/kg group and the control group,higher serum insulin concentration was found in the 5 mg/kg group(P < 0.01,P < 0.05),and the insulin resistance index of 5mg/kg group is significantly higher than that of control.In the pancreas HE staining,scattered clusters of cord-like islets were observed in each group.Compared with the control,the cells of pancreatic islet in the 5 mg/kg group and the 0.5 mg/kg group were arranged more closely.(5)Effect on lipid metabolism: The measurement of TC,TG,HDL-C and LDL-C showed no significant difference among the groups(P > 0.05).Liver HE staining showed that the liver had a certain degree of hemorrhage and hepatic steatosis,but compared with the control group,hepatic hemorrhage and hepatic steatosis were more severe both in the 5 mg/kg group and the 0.5 mg/kg group.(6)The measurement of ALT,AST,CREA and UREA showed no significant difference among the groups(P > 0.05).And HE staining of renal showed visible structural glomeruli,proximal convoluted tubules,and distal convoluted tubules in each group with no abnormality in each group.Conclusions: We accomplished a reliable method of LC-MS for determination of plasma AA in rat.The 52-week AA exposure can increase the concentration of plasma AA in a dose-dependent manner.In addition,AA can also lead to increased glycosylated hemoglobin concentration,fasting insulin concentration and insulin resistance index,which indicates a higher risk of diabetes.AA has no harmful effects on lipid metabolism and liver and kidney function,but it can aggravate the degree of hepatic steatosis and hemorrhage. |
PDF Full Text Request