Application Of Metabonomics Techniques To Determining The Quality And Timing Of MDMA Use

Objective:The differences in metabolites of MDMA in rats after single,addiction and addiction were compared,and an exploratory study was conducted on the inference method of MDMA drug use and drug use time based on metabolites data.Methods:1.Single drug administration groupMale SD rats(weight 200 ± 20g)24,were randomly divided into low,medium,high dose group and blank control group,adaptive feeding were given by intraperitoneal injection of 5mg / kg,10 mg / kg,20 mg / kg MDMA,blank control group was given the same volume of saline,respectively,1h,12 h,5d,10 d,15d after administration by the inner canthus vein blood.2.Addiction rats administration group2.Addiction groupTwenty-four male SD rats(weighing 200 ± 20 g)were randomly divided into the addiction group and the blank control group.The acute addiction model was established after adaptive feeding.During the addiction period,MDMA was injected intraperitoneally in the addiction group,with the dose of 5 – 17 mg / kg,increasing by 2 mg / kg daily.The blank control group was given the same volume of normal saline for seven consecutive days.The behavioral changes of rats were observed during addiction and the weight was recorded.On the eighth day after administration,addictive groups were randomly divided into low,medium and high dose groups,respectively,intraperitoneal injection of 5mg / kg,10 mg / kg,20 mg / kg MDMA,blank control group was given the same volume of saline.Blood was taken from the medial canthus vein 1 hour and 12 hours after administration.3.Repeated administration group after addictionAddiction rats withdrawal7 days,build relapse model after addiction.Low,medium and high dose groups were given intraperitoneal injection of MDMA(5mg / kg,10 mg / kg,20 mg / kg)from the eighth day.The blank control group was given the same volume of normal saline.Blood was taken from the medial canthus vein 1h,12 h,5d,10 d,15d after administration.The serum metabolic profiles of all samples were detected by ultra-high liquid chromatography-time of flight mass spectrometry(UPLC-TOF-MS).The data were analyzed by principal component analysis(PCA)and cluster analysis by MPP,and the potential differential metabolites in serum of MDMA rats with different models were screened.Metabo Analyst 5.0 was used to analyze the metabolic pathways of different metabolites under different conditions.The results were qualitatively analyzed.The correlation between differential metabolites and drug use time was analyzed by PLS and LDA,and the inference discriminant function of drug use time was established.Results:1.Qualitative and timing of drug use after single drug use :A total of 46 metabolites in different dose groups compared with blank control group after single administration in rats They were yellow oleic acid,3-hydroxytetradecanedioic acid,octadecyl fumarate,sulfur-furan beehive diester,glyceryl sulfate butyrate heptane ester,(R)-10-hydroxystearate,4-diene-1-carboxylic acid ester,β-hydroxyarginine,arginine-proline,arginine-threonine,ursodeoxycholic acid glycine conjugate,paraxanthine,tryptophan,arginine,proline,sphingosine,plant sphingosine,ganglioside,pentyl oleate,β-hydroxyarginine,7bhydroxy-3-oxo-5b-cholic acid,7a-hydroxy-3-oxo-4-cholic acid,ursodeoxycholic acid 1.3,7,12-tetrahydroxycholic acid,3-Oxo-4,6-cholic acid,7α,24(S)-dihydroxycholesterol,docosatrienoic acid,cis-p-coumaric acid,sulfur-furan beehive diester,ethyl docosapentenoate,sulfur-furan beehive diester,glyceryl sulfate,methyl acetyl castor oil acid,and cis-9.10-epoxystearic acid,glyceryl tributyrate,butyrate,arginine-threonine,β-hydroxyarginine,dihydro(nerve)sphingosine,β-hydroxyarginine,all-trans-heptadienyl diphosphate,octadecyl fumarate,all-transheptadienyl diphosphate,all-trans-pentyl diphosphate,arginine-threonine,trans-4-hydroxycyclohexane carboxylic acid ester.These differential metabolites can be used for the qualitative study of drug use in normal rats.Different doses of differential metabolites : low dose group unique differential metabolites for six kinds,respectively,oleic acid,3-hydroxytetradecanedioic acid,glyceryl sulfate butyrate heptanoate,(R)-10-hydroxystearic acid,4-diene-1-carboxylic acid ester,arginine-proline.There were 13 kinds in the middle dose group,namely,7bhydroxy-3-oxo-5b-cholic acid,7a-hydroxy-3-oxo-4-cholic acid,ursodeoxycholic acid,3,7,12-tetrahydroxycholic acid,3-Oxo-4,6-cholic acid,7α,24(S)-dihydroxycholesterol,icosatrienoic acid,cis-p-coumaric acid,ethyl icosapentaenoic acid,glyceryl sulfate,methyl acetyl castor oil acid,cis-9,10-epoxy stearic acid,and glyceryl tributoxide.There were 6 kinds of high-dose group,namely dihydrosheatphine,pentyl oleate,trans-heptadienyl diphosphate,trans-heptadienyl diphosphate,transpentyl diphosphate,trans-4-hydroxycyclohexane carboxylic acid ester.The differential compounds in the three administration groups were selected and PLS models were established based on their peak areas and drug use time in the three groups to infer drug use time.The use of SPSS software within the group covariance matrix LDA to determine the drug use time.PLS model can well aggregate the same group of substances,and different groups of substances can well distinguish the accuracy of model prediction by 94.44 %.LDA model classifies and predicts each group.LDA model can correctly classify 96.7 % of the original groups,and crossvalidate the cases in the analysis.Both of the two methods have high accuracy and can accurately infer the drug use time.2.Qualitative analysis and time inference of addiction and drug use :After administration,there were 37 different metabolites in different doses compared with the blank control group.They were methyl glycernate,linolenic acid,glycerol 1-phosphate,oleic acid glycerol ester,β-D-glucosyl o-aminobenzoic acid ester,trimethylolpropane-trimethacrylate,-testosteronephenylpropionate,-16-dehydropregnenolone acetate,tributyl acetocitrate,soybean phenolic lactone,ethyl docosahexaenoate,ganglioside,methionine,tryptophan,arginine,proline,tyrosine,N(Alpha)-T-butoxycarbonyl-L-leucine,N-hydroxy-L-phenylalanine,histidine-glycine,paraxanthine,uric acid,tributyl acetocitrate,γ-glutamyl-valine,thiocholineglycine,N-(1-deoxy-1-fructoseyl)leucine,N-(1-deoxy-1-fructoseyl)-glycine,N-(1-deoxy-1-fructoseyl),1,3,7-trimethyl uric acid,uric acid glycerol methyl nicotinate,acetyl tributyl citrate,1,3,7-trimethyl uric acid.Different doses of differential metabolites : low dose group unique differential metabolites have 11 kinds,respectively,glycerol 1-phosphate,β-D-glucosyl oaminobenzoate,trimethylolpropane trimethacrylate,testosterone propionate,16-dehydropregnenolone acetate,acetyl citrate tributyl ester,soybean phenol lactone docosahexaenoic acid ethyl ester,N(Alpha)-T-butoxycarbonyl-L-leucine,Nhydroxy-L-phenylalanine,histidine-glycine.There were three kinds of middle dose group,namely methyl glyceronicotinate,acetyl tributyl citrate and thioglycine.There were 11 kinds in the high-dose group,namely,N-(1-deoxy-1-fructose)leucine,N-(1-deoxy-1-fructose)tryptophan,N-(1-deoxy-1-fructose)phenylalanine,γ-glutamyl-valine,N-(1-deoxy-1-fructose)tyrosine,thiocholineglycine,N-glycineL-glutamyl-L-methionine,N2-fructose arginine paraxanthine,1,3,7-trimethyl uric acid,glycerylnicotinic acid methyl ester,acetyltributyl citrate.The differential compounds in the three administration groups were selected and the intra-group covariance matrix in SPSS software was used for LDA to determine the drug use time.LDA model classifies and predicts each group.LDA model can correctly classify 86.7 % of the original groups,and cross-validate the cases in the analysis.LDA model has high accuracy in inferring drug use time,which can be more accurate in inferring drug use time.3.Qualitative and time estimation of relapse after addiction :After addiction,there were 21 different metabolites in different dose groups compared with the blank control group.They were leucine,hard amide,creatine,allopurinol,deoxycortisol,purine alcohol,cytosine,thymine,paraxanthine,oleic acid glyceride,tryptophan,oleic acid amide,ganglioside,stearic acid glyceride,nicotinic acid methyl ester,bufalin O-glucoside,oleic acid amide,D-pantothenic acid,adenine,urea.Different doses of differential metabolites : low dose group unique differential metabolites have 5 kinds,respectively,leucine,hard amide,creatine,allopurinol,deoxycortisol.There were 5 kinds of cytosine,stearic amide,glyceryl stearate,methyl nicotinate,bufalin O-glucoside in middle dose group and 4 kinds of D-pantothenic acid,adenine,tryptophan and urea in high dose group.According to the different metabolites of low,middle and high dose groups,the dose of MDMA can be determined.The differential compounds in the three administration groups were selected and the intra-group covariance matrix in SPSS software was used for LDA to determine the drug use time.LDA model classifies and predicts each group.LDA model can correctly classify 79.4 % of the original groups,and cross-validate the cases in the analysis.LDA model has high accuracy in inferring drug use time,which can be more accurate in inferring drug use time.4.Analysis of metabolic pathways :The metabolic pathways of single dose group were mainly concentrated in glycerol phospholipid metabolism,arginine and proline metabolism,primary bile acid synthesis pathway,purine metabolism,glycosylphosphatidylinositol-anchored biosynthesis,phosphopentose pathway,pantothenic acid and coenzyme a biosynthesis.The metabolic pathways in the administration group after addiction were mainly concentrated in caffeine metabolism,biosynthesis of unsaturated fatty acids,amino acid metabolism,and glycerol phospholipid metabolism.The metabolic pathways of relapse group after addiction were mainly concentrated in caffeine metabolism,purine metabolism,β-alanine metabolism,glycerol phosphate metabolism,glycosylphosphatidylinositol-anchored biosynthesis,pentose phosphate pathway,pantothenic acid and coenzyme a biosynthesis.Conclusion:In this study,a method for screening endogenous biomarkers related to MDMA drug use and drug use time based on high-resolution mass spectrometry metabolomics technology was established,and the inference method of drug use time was preliminarily explored through its learning method.The toxicological mechanism of MDMA addiction and relapse after addiction based on metabolomics characteristics was studied.The results can provide a scientific basis for the qualitative analysis of drug use and the inference of drug use time in suspected MDMA cases.