Study On Toxicokinetics And Metabolomics Of Cyanide In Animals

Objective:1.Based on the theories and methods of toxicokinetics,the amount of CN^-,SCN^-and ATCA in arterial blood of the model animals were quantified,and the time of cyanide entering the body through digestive tract was inferred,combined with gas chromatograph,ion chromatograph and liquid chromatography tandem mass spectrometry.2.Using the technologies and methods of metabonomics,cyanide was introduced into the digestive tract,the activated metabolic pathways and the different metabolites were initial researched.3.The external conditions that promoted the conversion of ITCA to ATCA in blood samples,were preliminary studied.Methods:1.The animal model of cyanide poisoningthe entry time inference model of cyanide:A total of six healthysix healthy male rabbits were fed adaptively for about one week,which were fasted 12 h to adapt to the environment at 1/2 LD₅₀dose lavage(LD₅₀=5mg/m L,Chem IDplus,https://chem.nlm.nih.gov/),after to 0,0.25,0.5,0.75,1,2,3,4,6,8,10,12,24 hours different time points for carotid artery blood,to-80℃under the condition of preservation.metabonomics model of cyanide:A total of 12 healthy Sprague-Dawley（SD）rats with a body weight of（220±20）g were randomly divided into control group and model group with six rats in each group and fed adaptively for about one week.Control group rats were fasted and water abstained overnight.Blood samples（500μL）were collected from orbit and stored in a centrifuge tube containing heparin sodium at-80℃.Model group rats were intragastrically given potassium cyanide solution at a dose of 5 mg/kg（LD50,Chem IDplus,https://chem.nlm.nih.gov/）.After 20 minutes,blood samples（500μL）were collected from the orbit and placed in a centrifuge tube containing heparin sodium.The samples were stored at-80℃.The conditions of promoting the conversion of ITCA to ATCA:The healthy rabbits blood were collected from ear margin vein as blank blood,and then gavaged at2LD₅₀dose of cyanide.After 15 minutes,blood collected from carotid artery were as poisoned blood samples,and stored at-80℃.2.Sample pretreatment and detection methodsThe blood samples were derivatized,the cyanide was detected by gas chromatography electron capture detector,after protein precipitation,the SCN^-was tested by ion chromatography conductivity detector,and the ATCA was tested by liquid liquid extraction and LC-MS/MS.Blood samples were extracted by liquid-liquid extraction,dried by cryo-rotary concentrator,redissolved with methanol,and detected by LC-QTOF.After liquid-liquid extraction,the blood samples were blown with nitrogen,redissolved with methanol,and ATCA content was detected by LC-MS/MS.3.Data processing methodpharmacokinetic software（DAS3.0）,statistical software（SPSS20）,Profinder B.08.00 and Mass Profiler Professional.Results:1.Cyanide entry time inferenceThe ratio of the concentrations of ATCA,CN^-and SCN^-was taken as the independent variable,and the time as the dependent variable Inferring the time of potassium cyanide into the body,ATCA/SCN^-y=283.67e^-81.36x,R²=0.9608ATCA/CN^-y=7.9994x³-23.365x²+23.603x-5.0549 R²=0.9851SCN^-/CN^-y=0.0003x³-0.0232x²+0.6276x-1.9197 R²=0.98262.Cyanide metabolomicsUsing Profinder B.08.00,Mass Proprofiler Professional software and Metlin database,combined with the conditions of FC>2.0 and P<0.05,a total of 18differential metabolites were obtained in the control group and the model group:Lysope（0:0/18:2（9Z,12Z））,Threonine,PIP2（16:0/20:1（11Z））,Lysine,Cis-enic acid,11-beta-hydroxyandrosterone-3-glucuronide;N-acetylglutamic acid,Glutamine Histidine,Tryptophan,Phytosphingosine,Vignatic acid A,Dihydrourac,MG（0:0/16:0/0:0）,Lysope（0:0/20:3（11Z,14Z,17Z））and Arginine,Oleic Acid,In the KEGG database,according to-log（P）>0.5 and Pathway impact value greater than0.05,6 metabolic pathways were screened out:Arginine biosynthesis pathway,beta-Alanine metabolism,Tryptophan metabolism,Histidine metabolism,Alanine metabolism,aspartate,metabolism,glutamate metabolism,Arginine metabolism and proline metabolism.3.The conversion conditions from ITCA to ATCAThe same rabbit blood samples were statistically significant differences in the concentration of ATCA at each time point within 48 hours in the blank blood group at room temperature（17℃）,the group(2LD₅₀)at room temperature（17℃）and the group(2LD₅₀,p H=10)at room temperature（17℃）（S-N-K multiple test and the test levelα=0.05）.At 0 h,the concentration of ATCA in the blank blood group was62.35±2.51ng/m L,the concentration of ATCA in the group(2LD₅₀)was629.88±8.06ng/m L,and the concentration of ATCA in the group(2LD₅₀,p H=10)was 669.41±14.96ng/m L.At 3 h,the concentrations of ATCA in the three groups were 61.09±7.50m L,512.27±1.56ng/m L and 971.15±4.55ng/m L,respectively.At 12h,the concentrations of ATCA in the three groups were 72.20±3.42ng/m L,440.41±8.88ng/m L and 814.60±3.97ng/m L,respectively.At 16 h,the concentrations of ATCA in the three groups were 79.80±3.90ng/m L,444.53±5.51ng/m L and613.39±4.75ng/m L,respectively.Conclusion:1.The metabolic rules of cyanide and its metabolites in vivo were preliminarily investigated,and the time inference equation within 24 hours after potassium cyanide was fitted according to the concentrations of ATCA,CN^-and SCN^-.2.Based on the technique and principle of metabonomics,the different metabolites affected by potassium cyanide and determined the main metabolic pathways were preliminarily inferred.2.3.The study showed that the external temperature was 17℃and p H=10,the conversion of ITCA to ATCA could be promoted,and the external conditions of the infected blood were changed,the increase of ATCA was much higher than that of the blank blood under the same conditions.The above results could distinguish the infected blood samples from the blank blood samples,so it might be used as determining cyanide poisoning...