| Objective: 1.To observe the forensic toxicokinetics of organophosphorus pesticides intoxication albumin adducts,screen the biomarkers which would determine the before death or after death invade body in the case of suspected organophosphorus pesticides intoxication.2.To screen the potential biomarkers of organophosphorus pesticides intoxication by metabolomics,observe the forensic toxicokinetics of potential biomarkers and determine the biomarkers of organophosphorus pesticides intoxication.Methods: 1.Synthesis of O-(O-alkyl phosphonyl)tyrosine adducts N-carbobenzyloxy-L-tyrosine benzyl ester were prepared from Z-Tyr-OH and benzyl bromide in refluxing THF.Then N-carbobenzyloxy-L-tyrosine benzyl ester was reactive with alkylchlorophosphate.At last,O-(O-alkyl phosphonyl)tyrosine adducts were synthesized which the benzyl and N-carbobenzyloxy protecting groups were removed from N-carbobenzyloxy-O-(O-diethyl phosphonyl)tyrosine benzyl eater.2.The forensic toxicokinetics of OPs-albumin adducts The OPs-albumin adducts in blood were analyzed at acute intoxication group,chronic poisoning group,administrated OPs after sacrificed group and the blood was incubated with OPs at37℃ for 24 h group by LC-MS-MS.The stability of OPs-albumin adducts at different storage conditions which included room temperature,4℃,-20℃ and with 1%Na F.The blood of OPs poisoning cases were analyzed to compared with the results of anminal experiment.3.Screen the potential biomarkers by metabolomics Metabolic fingerprint and potential biomarkers were screened based metabolomics.Multivariate statistical analysis such as PCA and PLS-DA were employed to analyze the MS data to discriminate between the organophosphorus pesticides group and control group.The mechanism of OPs acute poisoning was interpreted at the metabolic level.4.Forensic toxicokinetics of potential biomarkers The concentration of potential biomarkers which included guanidineacetic acid,creatine,phosphatidylcholine,glycerophosphoryl choline and diethylphosphorodithioate in organophosphorus pesticides group and control group rats’ blood were analyzed by LCMS-MS and then determine the metabolic biomarkers of OPs acute intoxicant.Results: 1.Synthesis of O-(O-alkyl phosphonyl)tyrosine adducts The molecular weight of diethyl phosphonyl tyrosine was 317.1 and the major ion was MH+.The product ion was 272.1,244.0 and 216.0,respectively.The molecular weight of dimethyl phosphonyl tyrosine was 289.2.The major ion was MH+ and the product ion was 228.2.The molecular weight of diethylthiophosphonyl tyrosine was 333.3 and the major ion was MH+.The product ion was 187.2,217.1,245.1 and 120.2,respectively.2.The forensic toxicokinetics of OPs-albumin adducts Dimethyl phosphonyl tyrosine in blood which collected from acute intoxication group,chronic poisoning group and mixture group were identificated by LC-MS-MS.There was not detected any dimethyl phosphonyl tyrosine in vitro incubation with blood group and the OPs poisoning cases.It seems that dimethyl phosphonyl tyrosine was unstability at different storage conditions.Addition of Na F in blood should be avoided due to the accelerated degradation of dimethyl phosphonyl tyrosine.Diethylthiophosphonyl tyrosine in blood which collected from acute intoxication group,chronic poisoning group,mixture group,administrated OPs after scarificed 0.5h group,in vitro incubation with blood and the OPs poisoning cases were identificated by LC-MS-MS.Addition of Na F in blood and storage at room temperature should be avoided due to the accelerated degradation of diethylthiophosphonyl tyrosine.Specimens containing diethylthiophosphonyl tyrosine should be stored at-20 °C and analyzed as early as possible.3.Screen the potential biomarkers by metabolomics The eighteen potential biomarkers were identificated by metabolic fingerprint.The impact of stearamide,octylamine,N,N-dimethylaniline,ceramide,diethylthiophosphate,pyruvate and gluconic acid were notable in organophosphorus pesticides acute poisoning.Compared with control group,the concentration of ceramide was decreased and gluconic acid was increased in all poisoning groups.The diethylthiophosphate was detected in all poisoning group except methamidophos poisoning group.The sixteen metabolic pathways were influenced by eleven metabolics and energy metabolism,liver,kidney and nervous system functions disorders were the major mechanism of OPs acute poisoning.4.Forensic toxicokinetics of potential biomarkers Diethylphosphorodithioate should be a biomarker of phorate poisoning.The concentration of creatine in blood was increased and the ganidineacetic acid was decreased in all poisoning groups.The concentration of creatine was decreased in sacrificed by asphyxia for 1/2h group and 1h group compared with the acute poisoning group.It is proved that it is a very useful method to determine the OPs poisoning by analyzed the level of guanidineacetic acid and creatine in blood.The level of glycerophosphoryl choline was no difference in all groups.The concentration of phosphatidylcholine was decreased in sacrificed by asphyxia for 1/2h group and 1h group compared with the acute poisoning group.Conclusion: 1.The methods of synthesis of O-(O-alkyl phosphonyl)tyrosine adducts,identificated metabolics by LC-Q-TOF-MS,analyze OPs-albumin adducts,guanidineacetic acid,creatine,phosphatidylcholine,glycerophosphoryl choline and diethylphosphorodithioate in blood by LC-MS-MS could be used for the study of forensic toxicokinetics.2.Although dimethyl phosphonyl tyrosine should be a biomarker to identificated the before death or after death invade body,it was unstable at different storage conditions.Diethylthiophosphonyl tyrosine couldn’t be a biomarker to identificated of before death or after death invade body,but it could be a biomarker to prove ingested OPs.3.The eighteen potential biomarkers were identificated by metabolic fingerprint.The sixteen metabolic pathways were influenced by eleven metabolics.The changes of metabolites indicated that it may cause energy metabolism disorder especially TCA cycle after acute exposure OPs,as well as liver,kidney and nervous system functions.4.Diethylphosphorodithioate should be a biomarker of phorate poisoning and ingested before death.The OPs poisoning should be identified though analyzed the level of guanidineacetic acid,creatine,phosphatidylcholine and glycerophosphoryl choline in blood.Phosphatidylcholine in blood should be a biomarker to determine the poisoning by OPs before death. |
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