A Novel Mass Spectrometric Method For Quantitative Determination Of Acetylcholinesterase Adducts And Its Application On The Efficacy Evaluation Of Reactivators

博士学位论文AbstractA novel mass spectrometric method for quantitativedetermination of acetylcholinesterase adducts and itsapplication on the efficacy evaluation of reactivatorsAbstractNerve agents(NAs), mainly including tabun(GA), sarin(GB), soman(GD), cyclosarin(GF), VX and Russian VX(RVX), are organophosphorus compounds that are extremely potent inhibitors of the enzyme acetylcholinesterase(AChE). The terroristic availability of highly toxic NAs highlights the necessity for a deep understanding of their toxicities and effective medical treatments. Due to the high toxicity of nerve agents, first aid is important for the intoxicated organism. Reactivtors, being able to remove the phosphyl moiety from the active site of AChE, can be expected to act as specific antidotes. However, soman-inhibited AChE undergoes a very quick aging process and resistant to the standard oxime therapy such as pralidoixme and obidoxime. Traditionally, the characterization of nerve agents(NAs) and the evaluation of the effectiveness of antidotes are studied by Ellman assay, which has several limitations due to lack of specificity and easy to be interfered by BChE or many other chemicals such as cholinesterase inhibitors and some drugs etc. Furthermore, the method cannot either precisely or directly demonstrates the exact statuses(uninhibited, inhibited and aged) of AChE before and after NAs intoxication or reactivator administration.With the development of modern instrument analysis,sensitive methods of expose biomarkers such as NAs hydrolytic products and biomacromolecule adducts can achieve the requirement of NAs exposure confirmation and retrospective detect.However,a few of analysis methods from the viewpoint of peptide adduct of target enzyme AChE was reported.In our research,according to the proteomics technology and analytical toxicology strategies,a series of analytical methods for determining effective biomarkers(AChE adducts)of NAs exposure have been developed for the first time applying modern instrument analysis technology to NAs poisoning researches.Particularly,a proteolysis-based liquid chromatography tandem mass博士学位论文Abstract spectrometry(LC-MS/MS)method was developed for quantitatively differentiation of the specific peptides of acetylcholinesterase,i.e.,undecapeptide“GESAGAASVGM”in free,unaged,or aged status after exposure to NAs(GA,GB,GD and VX)and pepsin digestion.A key procedure to make the reaction system“frozen”and precisely“capture”the different real poisoned status is the immediate addition of formic acid to terminate poisoning,aging or/and spontaneous reactivation process of AChE in reaction system.In comparison with conventional Ellman assay,our established method can not only provide the inhibition rate but also characterize the features of NAs exposure by quantatively determining the status of specific adducts.Ellman assay can not provide a convincing evidence for NAs exposure at inhibition levels less than 20%due to its low sensitivity and specificity,while less than 1%inhibition rate of AChE can be precisely determined by this LC-MS/MS method.Based on the established proteolysis-LC-MS/MS method, the poisoning characteristics of four kinds of NAs(GA, GB, GD and VX) were demonstrated by quantitative determination of corresponding adducted biomarkers. The kinetics constant of inhibition rate(ki), aging rate(ka) and half-time of aging(t1/2) were calculated. Subsequently, this analytical platform was applied to evaluate the effectiveness of various of reactivators(HI-6, LuH, 2-PAM, TMB-4 and MMB-4) and some novel antidotes, which developed by our institute, for prevention and treatment of four kinds of NAs(GA, GB, GD and VX) poisoning, and also to disclose the accurate mechanism of these medicine.The established LC-MS/MS analytical platform could determine effective biomarkers of AChE adducts with other kinds of compounds such as cholinesterase inhibitors and organophosphorus pesticides. In our research, LC-MS/MS methods for directly determining the pyridostigmine and chlorpyrifos-inhibited cholinesterase(ChE) adducts have been developed to provide a novel analytical tool for reliable poisoning confirmation and pharmacological and toxicological investigation.There are five chapters compose this dissertation.The first chapter is the preface.The introduction,toxicological mechanism,analytical methods of biomarkers and antidotes research development of nerve agents were systemically described.The evaluation methods of reactivators and some novel antidotes,which developed by our institute,were introduced in detail.Current博士学位论文Abstract research status was pointed out,the purposes and significance,contents and innovations of this study were proposed.Chapter two is the establishment of analytical methods for determining cholinesterase adducts with nerve agents. A LC-MS/MS method for quantatively determining specific peptides of three statuses(free, unaged and aged) as effective biomarkers for the four kinds of NAs(GA, GB, GD and VX) exposed ChE(Electrophorus electricus AChE and human BChE) was established. High resolution mass spectrometric technology(LC-QTOF-MS) was applied to identify the amino acid sequences of specific peptides digested from NAs exposed cholinesterases. Method validation results showed that the method has good selectivity, linearity, precision and accuracy and the limit of detection(LOD) of synthetic undecapeptide and nonapeptide is 0.2 n M. The limit of quantitation(LOQ) of synthetic peptides is 0.5 nM. The matrix effect and stability fulfill the requirement for method application. In comparison with conventional Ellman assay, our proteolysis-based LC-MS/MS method could not only determine enzyme activity or inhibition rate, but also directly provid the precise status(free, unaged and aged) of NAs exposed AChE poisoning process especially under extremely low NAs exposure. Because of accurate determination of enzyme statuses, our method is more sensitive and specific than conventional Ellman assay.Chapter three is the characterization of nerve agents. Applying the proteolysis-based LC-MS/MS method, the poisoning and aging characteristics of four kinds of NAs(GA, GB, GD and VX) were demonstrated by quantitative determination of corresponding specific peptides of three statuses(free, unaged and aged) digested from intoxicated ChE(AChE and BChE).The results showed under physiological condition,GD exposed AChE would complete its poisoning and aging during 3 minutes.For GB exposure,the poisoning was completed during 4 min and the aged peptide could simultaneously be detected after 10 min.For VX exposure,the poisoning was completed within about 10 min,aged peptide could be continuously detected after 1 hour.GA exposed AChE aging contained two processes.Under 0°C condition,poisoning and aging process of GD exposure is slow down and the unaged peptide could be continuously detected.With the increase of pH value,poisoning and aging process of GD exposure would slow down further.When BChE exposed to four kinds of NAs(GA,GB,GD and VX),the博士学位论文Abstract inhibition rate were basically similar.Comparing with AChE,unaged peptide of GD exposed BChE could be simultaneously detected.For GB exposure,aged peptide slowly and continuously increased in 1 hour.For VX exposure,aged peptide could be continuously detected after 20 min.Furthermore,by quantitative determining specific peptides of intoxicated ChE,the calculating method of kinetics constant of inhibition rate(ki),aging rate(ka)and half-time of aging(t1/2)was promoted for the first time based on our established mass spectrometric method.Chapter four is the evaluation the effectiveness of various of medicines for prevention and treatment of NAs poisoning. Based on the established proteolysisLC-MS/MS platform, a novel in vitro method for evaluating the efficacy of reactivators was promoted. In this method, reactivation rate and relative ChE activity were calculated by quantitively determining free peptide of three experimental groups(unintoxicated, intoxicated, reacivated) of ChE. Additionally, the changing tendencies of unaged and/or aged peptides were contribute to confirm the reactivation mechanism. The calculating method of reactivation rate and concentration for 50% of maximal effect(EC50) was promoted for the first time based on our established mass spectrometric method. The evaluation results for classic reactivators(HI-6, LuH, 2-PAM) were consistent with the previous studies. This method could avoid the disadvantages of conventional Ellman assay, such as easy to be interfered by other chemicals, lack of sensitivity and specificity. The big advantage of our method is that, the specific free peptides and peptide adducts with NAs were directly and separately measured, giving the most direct evidence of reactivation effect of reactivators. The method is a proper substitute of Ellman assay in evaluation of reactivator efficacy.The evaluation results of classic oxime reactivators and novel compounds or antidotes developed by our institute showed that these various reactivators and antidotes are effective for GB and VX exposed AChE.TMB-4,LuH and“85”first aid injection are shown to be therapeutic effective for AChE exposed to GA.For GD exposed AChE in physiological conditions,only L-1655,L-1978 and“85”prophylaxis tablet have prophylaxis effect.The reason why GD exposed AChE couldn’t be treated is that the aging rate of GD-inhibited AChE is extremely quick.It is noteworthy that LuH,TMB-4 and some other reactivators could aggravate GD intoxication.Based on the thought of analytical toxicology,reactivation mechanism of various reactivators and other antidotes were discussed according to changing博士学位论文Abstract tendency of effective biomarkers.Chapter five is the determination of specific peptide adducts of other kinds of compounds inhibited ChE. The established proteolysis-LC-MS/MS analytical platform was applied to determine effective biomarkers of other kinds of compounds. In this chapter, novel proteolysis-based quantitative LC-MS/MS methods have been developed for directly determining the effective biomarkers of ChE inhibitor pyridostigmine and organophosphorus pesticide chlorpyrifos-inhibited AChE and BChE adducts. We confirmed that NAs prophylaxis antidote pyridostigmine could form a covalent bond with active serine site of ChE. As for chlorpyrifos incubated with ChE in vitro, the compound added with ChE is actually the oxide of chlorpyrifos, i.e. chlorpyrifos oxon. The established method for determining CPO-AChE adduct is help to toxicological research of chlorpyrifos, while the established method for determining CPO-BCh E adduct provide a new tool for clinical intoxication conformation and health care. These cases indicate that the proteolysis-based LC-MS/MS platform for ChE inhibitors established in this dissertation could be widely applied to poisoning confirmation and pharmacological and toxicological investigation.