Screening Of Differential Biomarkers For Lethal Chlorpromazine Poisoning Based On Metabolomics

The determination of the cause of death due to iatrogenic drug poisoning has always been a difficult problem faced by forensic workers.According to the 2019 annual report of the American Poison Control Center,the number of deaths from sedative hypnosis/antipsychotic poisoning ranks first in the number of deaths from drug poisoning.Statistics in China also showed that sedative hypnosis/antipsychotics are common drugs in drug poisoning cases.Chlorpromazine,also known as Dongmianling,is the first-generation typical antipsychotic drug.It is mainly used for the treatment of refractory psychosis.However,due to irregular medication or suicide,death cases of chlorpromazine poisoning occur from time to time.Due to the lack of special pathological changes in acute chlorpromazine poisoning,the determination of the death of chlorpromazine poisoning is usually based on the chlorpromazine medication history of the deceased,the clinical manifestations of the poisoning,and the blood concentration of chlorpromazine.However,some cases of chlorpromazine poisoning still cannot reasonably explain the cause of death based on the above factors.Therefore,how to find more objective indicators to improve the judgment system of chlorpromazine poisoning death has become an urgent problem in forensic appraisal.The research object of metabolomics is small molecule compounds with a molecular weight of less than 1000,which is characterized by a general and comprehensive investigation of the body’s metabolic changes from the perspective of systems biology through high-throughput experiments and large-scale calculations.Since the metabolome is at the end of the biochemical regulation of the organism,it can accurately reflect the state of the organism.So far,it has been widely used in the research of disease mechanism,drug mechanism and biomarker screening.In the field of forensic toxicology research,metabolomics has been used to evaluate the toxicity of toxicants,determine the target organs of toxicants,screen biomarkers of poisoning,and study the toxicological mechanisms of toxicants.The purpose of this study is to facilitate the screening of specific metabolic markers of chlorpromazine poisoning death by metabolomics technology and explore the ability of discriminatory markers to identify the cause of death,so as to provide an objective auxiliary identification basis for chlorpromazine poisoning/fatality.Part One Screening of differential metabolic pathways and metabolites in the blood of dead mice poisoned by chlorpromazineObjective:Analyze the differential metabolic pathways and related differential metabolites in the blood of LCP animal models,and further screen the specific differential metabolites that are differential from other common NDRD.Method:1.Animal model preparation:The animal model uses 7-8 week old CD1 mice.Among them,the LCP model was given by intragastric administration at a dose of 1.5g/kg.After administration,some mice with static tremor and paroxysmal convulsions were included in the poisoning group,and the dead mice were included in the death group.Other common NDRD such as drowning,mechanical asphyxia,hemorrhagic shock,and high cervical spinal cord dissection model were used as a control group to screen for specific differences in LCP identification.2.Quantitative enrichment analysis method was used to analyze the differential metabolic pathways and related differential metabolites of LCPs and NLCPs.In the comparison model for distinguishing chlorpromazine poisoning death from a variety of common causes of death,this study uses marker meta-analysis combined with multiple chemometric methods(PLS-DA and RF)to verify the differential metabolites between LCP and other death cause models.ROC curve based on Monte Carlo cross-validation(MCCV)algorithm was used to show differential capacity,and differential metabolites with highest area under the ROC was the final differential metabolites combination.The verification of the differential metabolites was done by x-calibur and the MzValt module in Compound Discovery.Results:1.In the process of chlorpromazine poisoning and death,the synthesis and oxidation of fatty acids,ketone metabolism,degradation of valine,leucine,and isoleucine,and abnormalities in the metabolism of citric acid occur;2.A total of 21 differential metabolites between LCP and NDRDs(drowning,mechanical asphyxia,hemorrhagic shock,and high cervical spinal cord dissection)were screened out by marker meta-analysis,partial least square discrimination analysis and random forest modeling methods Metabolites.Among them,the combination of differential metabolites of acetyl-L-carnitine,propionyl-L-carnitine and succinic acid can clearly distinguish chlorpromazine poisoning deaths from the above-mentioned non-drug-related deaths(AUROC=0.978).Part Two Verification of the specificity and stability of the differential metabolites in the blood of mice killed by chlorpromazine poisoningObjective:To verify the specificity of three different metabolites against LCP and similar pharmacodynamic characteristics antipsychotics(olanzapine,clozapine,perphenazine)poisoning death ability to verify its specificity;and by observing different storage conditions The changes in the distinguishing ability of differential metabolites in the blood samples below verify its stability.Method:1.Animal model preparation:The preparation of LCP animal model is the same as the first part.The dose of perphenazine,olanzapine,clozapine and promethazine lethal poisoned mice was 5 folds of LD50.Immediately after the death of the animal,blood samples were drawn and its metabolites were analyzed by untargeted detection methods.2.The preparation of LCP,drowning and hemorrhagic shock mouse models and the method of obtaining blood samples are the same as the first part,and then 6 samples(70ul each)are drawn from each whole blood sample and stored in the refrigerator at 4℃.Take a portion at 0 day,1 day,2 days,5 days,10 days,and 20 days,and transfer to-80℃ refrigerator for later use.The processing and detection methods of whole blood samples are the same as before.3.In the above death model,the area under the ROC curve(AUROC)is used to show the ability of acetyl-L-carnitine,propionyl-L-carnitine and succinic acid to distinguish chlorpromazine poisoning death from other death models.The closer its value is to 1,it indicates this The prediction result of the discrimination model is more realistic.Results:1.Based on the AUROC value,the plasma concentration of chlorpromazine and the ability of acetyl-L-carnitine,propionyl-L-carnitine and succinic acid to distinguish between LCP and NLCP were compared in parallel.The results showed that the three different metabolite combinations were better than blood.The drug concentration has a higher degree of discrimination,and the overall discrimination ability is higher after the two sets of indicators are combined(AUROC=1);2.The three different metabolites cannot distinguish LCP from death caused by olanzapine,clozapine,and perphenazine poisoning,but they can distinguish the above three antipsychotics from non-drug-related death models(AUROC>0.84);3.The AUROC value of the ability of the three different metabolites to distinguish LCP from NDRD(drowning,hemorrhagic shock)was higher than 0.86 under storage conditions of 0-5 days and 4℃,suggesting that the three different metabolites pair The two groups had high and stable discrimination ability;but after 5 days,the AUROC value decreased(AUROC≤0.77),indicating that their discrimination ability decreased.Part Three Analysis of Differential Metabolites in the Liver of Chlorpromazine Poisoned MiceObjective:Screening the different metabolites of three antipsychotics(chlorpromazine,olanzapine and quetiapine)poisoning deaths and non-drug-related deaths in the liver samples,and discussing the influence of their temporal changes on the identification ability.Method:1.Animal model preparation and sample treatment:chlorpromazine,olanzapine,and quetiapine poisoning deaths and 4 non-drug-related death models are the same as before.The dead mice were then placed in a temperature box at 15℃ and 40%humidity for 30 days.During this period,approximately 200 mg of liver samples were cut at 0 hours of death and 1,2,10,20,and 30 days after death,quenched in liquid nitrogen,and transferred to a-80℃ refrigerator for storage for later use.2.The differential metabolites of chlorpromazine,olanzapine,and quetiapine were screened for the differential metabolites of the three antipsychotics and non-drug-related death models with reference to the analysis process of the differential metabolites of blood samples.Results:1.A total of 33 differential metabolites between lethal antipsychotics(chlorpromazine,olanzapine,quetiapine)poisoning and other non-drugrelated deaths(drowning,mechanical asphyxia,Hemorrhagic shock and high cervical dislocation)were screened out by biomarker meta-analysis,partial least squares discrimination analysis and random forest models.Among them,the combination of ascorbic acid,gluconic acid and succinic acid can clearly distinguish the three antipsychotic deaths from the above-mentioned non-drug-related deaths(AUROC=0.926).2.Under constant environmental conditions of 15℃ and 40%humidity,the relative content of the same different metabolites in the liver tissues of chlorpromazine poisoning and high cervical spinal cord dissection mice showed similar general trends with the time of death.3.Under the above constant environmental conditions,within 48 hours of death,the ability of the three different metabolites to distinguish three antipsychotic deaths from non-drug-related deaths(drowning,mechanical asphyxia,hemorrhagic shock,and cervical dislocation)by AUROC value is higher than 0.87,indicating that the three different metabolites have a higher ability to discriminate between the two groups;but in the late stage of death,the AUROC value decreases(AUROC<0.52),indicating that its discriminating ability is poor.Conclusion:Chlorpromazine poisoning can cause abnormal fatty acid synthesis and oxidation,ketone metabolism,leucine,isoleucine metabolism,and citric acid metabolism in mice.Detection of acetyl-L-carnitine,propionyl-L-carnitine and succinic acid in plasma by metabonomic analysis method can clearly distinguish LCP from a variety of NDRD,but cannot distinguish chlorpromazine from olanzapine and chlordiazepine death,perphenazine poisoning.In an environment of 15℃ and 40%humidity,ascorbic acid,succinic acid and gluconic acid in the liver tissue in the early death period(within 48 hours)have higher differential capacity between chlorpromazine,olanzapine and quetiapine poisoning deaths and nondrug-related deaths;but in the late stage of death,its ability to discriminate decreases.