~1H NMR-based Metabonomics In Serum And Urine Of Methamphetamine-induced Acute Poisoning Death And Subacute Poisoning In Rats

Objective: 1H-NMR based non-targeted metabolomics was applied to analyze the changes of metabolites in serum and urine of methamphetamine(MA)acute poisoning death and subacute poisoning in rats,respectively.Their potential biomarkers and the related metabolic pathways were identified.It may provid new ideas for the forensic toxicological analysis and identification of MA acute poisoning death and subacute poisoning.Methods: Sixteen healthy male SD rats were randomly divided into acute poisoning death group(n = 8)and control group(n = 8).Acute poisoning death model(within 24h)was established by intraperitoneal injection of 40 mg / kg / 2h.The control group was intraperitoneally injected with the same volume saline by repeating the above method.Sixteen healthy male SD rats were randomly divided into subacute poisoning group(n = 8)and control group(n = 8).Subacute poisoning models were established by intraperitoneal injection MA at doses of 10 mg / kg,12 mg / kg and 15 mg / kg for 7 consecutive days,respectively.The control group was intraperitoneally injected with the same volume of normal saline.Urine and heart blood were collected after 21 days of MA injection.Heart blood was separated into serum,and the serum and urine were stored at-80 ° Crefrigerator until used.Serum and urine samples were detected using CPMG spin echo sequence and pre-saturated water peak suppression pulse sequence NOESYGPPR1 D respectively.SIMCA-P + software was used to analyze metabolic profiling of above-mentioned data,and screened the eigenvalues based on the importance of the value of the projection(VIP > 1)in combination with P < 0.05.The characteristic variables were assigned and quantified by Chenomx NMR Suite 8.2 software and related literature.Two independent samples t-test was used to examine the change between groups,and the difference metabolites of P < 0.05 were identified as potential biomarkers.Metabo Analyst 3.0 web analytic platform was used to analyze differential metabolites-related metabolic pathways.Impact values > 0.05 and P < 0.05 were used as criteria to identify metabolic pathways associated with differential metabolites.Results:1 MA acute poisoning death metabolomicsSeizures,paralysis,salivation and self-mutilation behavior in the experimental group were observed.The MA poisoning rats died within 24 h,indicating that the successful model of MA-induced acute poisoning rats was established.OPLS-DA was carried out pattern recognition model.R2 X,R2Y and Q2 of OPLS-DA model in serum were 0.704,0.93 and 0.507,respectively.R2 X,R2Y and Q2 of OPLS-DA model in urine were 0.596,0.907 and 0.475 respectively.OPLS-DA can distinguish serum and urine between experimental group and control group,and the clustering tendency was obvious.The OPLS-DA was verified by permutation test.The model has not been fitted and the model was effective.The four endogenous small molecules(betaine,lactate,succinate and glycine)in serum might be potential biomarkers in MA acute poisoning rats by OPLS-DA analysis screened.Urine 2-ketoglutarate,acetate,glutamate,lactate,succinate,taurine,creatinine and glycine eight endogenous small molecules may be potential biomarkers.Quantitative analysis showed that lactate was significantly increased in serum and urine of MA acute poisoning death rats as compared with the control group(P < 0.05).There were not significant differences in other metabolites between experimental group and control group.Pyruvate metabolism,citric acid cycle metabolism,glycine,serine and threonine metabolism,aspartate,alanine and glutamate metabolism,and primary bile acid anabolism were revealed by pathway analysis,which participated in the pathophysiology of MA acute poisoning death.2 MA subacute poisoning metabolomicsParoxysmal irritability and apathetic,dry yellow hair,hematuria and other symptoms were appeared in MA subacute poisoning rats.The OPLS-DA model of serum and urine of subacute poisoning rats can distinguish the experimental group and the control group well.The clustering tendency of the two groups was obvious.R2 X,R2Y and Q2 of serum OPLS-DA model were 0.529,0.967 and 0.856 respectively.The R2 X,R2Y and Q2 of urine OPLS-DA model were 0.433,0.976 and 0.68,respectively.All the models were reliable.The OPLS-DA was verified by permutation test,the model has not been fitted and the model was valid.Acetate and glycine two endogenous small molecules in serum of MA subacute poisoning rats might be potential biomarkers by OPLS-DA analysis.Urine screening 2-oxoglutarate,acetate,methylamine,creatinine,propionate,succinate and glycine endogenous seven small molecules may be potential biomarkers.Quantitative analysis showed that 2-oxoglutarate,glycine,methylamine and creatinine in the subacute poisoning rats were significantly higher than those in the control group(P < 0.05).Pathway analysis found that aspartate,alanine and glutamate metabolism and citric acid cycle two metabolic pathways involved in pathophysiological processes of MA subacute poisoning.Conclusion:1.Lactate in serum and urine might be used as a potential biomarker in MA acute poisoning death.Metabolism of pyruvate,citric acid and other metabolic pathways may be involved in the pathogenesis of MA acute poisoning death.2.2-oxoglutarate,glycine,methylamine and creatinine in urine might be used as potential biomarkers in MA subacute poisoning.Aspartate,alanine and glutamate metabolism and citrate cycle metabolic pathways may be involved in the pathogenesis of MA subacute poisoning.3.Metabolites and metabolic pathways were different between MA acute poisoning death and subacute poisoning.