Metabolic And Metalnomic Study On Subacute Single Chiral Methamphetamine Intoxication In Rats

In 2017,a total of 275 million persons with at least once drug using behavior around the world were identified and kept stable,accounting for 5.6%of the global population aged 15-64 years according to "2018 World Drug Situation Report" issued by Unitied National Office on Drugs and Crime(UNODC).Of those drug abusers,cannabis,opioids and amphetamine(AM)are the most widely abused drugs,up to 450 thousands persons have died related with abused drugs and is now on the upswing.Methamphetamine(METH)is one of the most abused AM stimulants.which has an asymmetric chiral center in its chemical structure.It has two enantiomers including S-(＋)-METH and R-(-)-METH.The two enantiomers have large differences in the effects,toxicity and metabolic mechanisms of the central nervous system(CNS).R-(-)-METH generally influences the sympathetic nervous system and is mostly used in medical treatment.However,S-(+)-METH,namely"ice",has a strong central nervous system stimulating effect with high toxicity.It also has a strong addictive nature and belongs to a narcotics control-substance in China.Hence,it is particularly important to distinguish the absolute configuration of METH and make a separate study on their organ damage and toxic mechanism.Studies have shown that METH subacute poisoning causes kinds of livers,brain neurological and cardiovascular injury.Previous studies demonstrated that the brain and bone metabolites changes in both METH abusers and animals.Besides,the trace element concentrations in the hair of METH abusers demonstrated significant disorder.With the rise and development of omics technology,diversified biomarkers related to disease,injury and poisoning mechanisms were discovered.However,a very limited number of-the overall metabolic profiling changes and related biomarker findings after METH-induced subacute poisoning have been reported.And no relevant manchanisms of combining differential metabolites,elemental markers,and target organ damage.were reported.Based on this,the qualitative and quantitative analysis methods of METH and AM chiral enantiomers in hair were first established and optimized by Liquid Chromatography-tandem Mass Spectrometry(LC-MS/MS).All the compounds showed great linear relationship(r>0.99).and the intra-day/inter-day precision were all<12%.Fifty drug abuser samples were determined by this method.Up to 70%of the cases were determined as S-(＋)-METH and S-(＋)-AM,while nine(18%)of the remaining 15 cases were S-(+)-METH,S-(+)-AM,R-(-)-METH and R-(-)-AM simultaneously.However,they are mainly based on the S configuration.The proposed method is rapid,simple,and provides analytical support for subacute single chiral methamphetamine toxicity injury studies.Secondly,a subacute poisoning model of Sprague Dawley(SD)rats was developed by using a single S-(+)-METH.24 SD rats were randomly divided into two groups and subacute poisoning caused by administrating with METH(15 mg/mL/kg,two times a day for 4 days)via intraperitoneal injections(i.p.).The Control group was injected with the same volume of normal saline(NA).The alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activity levels of METH group in serum increased significantly compared with the control group,suggesting obvious hepatic iinjury.The results were further confirmed by histopathological micro-observation.In order to further investigate the correlation between related metabolites,trace elements and hepatic injury,and to find out possible biomarker,a total of 18 small molecule metabolites and 19 elements with stable variability in animal plasma were selected for overall profiling analysis in this study and fully validated through assessments.The score plot was drawn and classified according to partial least squares discrimination analysis(PLS-DA)model through SIMCA-P program.Both Control and METH groups were completely separated.Five differential metabolic markers(valine,serine,alanine,biotin and nicotinamide)and 6 different elemental markers(B,Co,Se,Ni,K and As)were finally identified based on variable importance in projection(VIP,VIP>1)and t-test(p<0.05).The further metabolic pathway analysis using KEGG database and Metaboanalyst 4.0 program demonstrated that two pathways consisted of proline and serine biosynthesis and nicotinamide metabolism maybe serious interrupted after METH-induced subacute poisoning in rats.The above research provides scientific basis and technical support for forensic of methamphetamine enantiomers.Further studies on hepatic injury and metabolism,metalnomic provide a scientific explanation for the mechanism of subacute poisoning caused by METH,and the discovery of related metabolites and elements provides a theoretical basis for possible diagnosis of METH poisoning.