基于效应标志物质谱定量技术的有机磷毒物中毒与重活化特点研究

发布时间：2018-03-16 09:27

本文选题：有机磷毒物　切入点：效应标志物　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：有机磷毒物属于一类含磷有机物,有机磷毒物依效能可分为神经性毒剂(Nerve agents,NAs)和有机磷农药(Organophosphorus pesticides)。急性有机磷中毒的救治策略主要是利用阿托品进行对症治疗以及利用肟类化合物进行对因治疗。而阿托品仅作用于毒蕈碱型受体,救治效果不佳,增大用药剂量又会引起阿托品中毒;因此肟类化合物作为一种特异性解毒药被越来越广泛地应用于急性有机磷中毒的救治。但肟类重活化剂无法进入中枢神经系统,且目前对其重活化效果存在一定争议:一种观点认为肟类化合物通过重活化神经或肌肉组织内中毒乙酰胆碱酯酶(Acetylcholinesterase,AChE),从而恢复酶活性以及受损的身体机能;另一种观点认为肟类化合物在重活化过程中可与游离毒剂或与离去的磷酸(或膦酸)基团发生反应,形成一种膦酰化肟中间体,该中间体带有季铵正电荷,更易攻击带有负电荷的酶活中心,故具有更强抑酶毒性,或许是造成肟类药物重活化效果不佳甚至出现加深中毒现象的原因。目前对有机磷毒物中毒救治的研究工作多基于经典的Ellman法等酶活测定法开展,然而Ellman法作为酶活测定的金标准,依然存在一定的局限性,例如,AChE的测定易受丁酰胆碱酯酶(Butyrylcholinesterase,BChE)干扰,准确度低;灵敏度低,当中毒浓度低于20%时,无法准确测定;同时测定酶活时,体系中其他胆碱酯酶抑制剂或重活化剂等药物的存在会造成一定干扰,导致假阳性或假阴性结果;更为重要的是该方法特异性较差,无法分辨中毒类型,同时无法准确描述AChE所处的状态(活性态、中毒态、老化态)。本课题研究建立了一种液相色谱-串级质谱联用(Liquid chromatography tandem mass spectrometry,LC-MS/MS)定量测定方法用于研究神经性毒剂中毒特点与救治,该方法通过对毒物特异性加合肽段进行定量检测,提供效应酶中毒程度的数据,同时能够定量获取效应酶中毒后所处的不同状态,尤其是对于低剂量有机磷毒物暴露时,经典Ellman法无法准确获得酶活性抑制率,而该方法对于抑制率小于1%的情况亦可进行准确分析。本课题研究中,针对有机磷毒物中毒救治难题以及现有方法存在的诸多弊端,展开深入研究。本研究论文共分为四章:第一章:前言,对有机磷毒物及其中毒机理、中毒救治研究方法、抗毒药物概况进行了介绍,并提出本论文的研究意义与目的。第二章:有机磷农药与AChE连接的基团多为二甲氧基膦酰基团(Dimethylphosphoryl,DMP)以及二乙氧基膦酰基团(Diethylphosphoryl,DEP),本章针对这两类结构特点制备了二甲基膦酰化十一肽和二乙基膦酰化十一肽,在此基础上利用课题组前期研究建立针对有机磷农药中毒救治研究的LC-MS/MS定量测定方法,对常见有机磷农药敌敌畏、敌百虫、久效磷、对氧磷的中毒特点及重活化特点进行考察,有机磷农药的中毒过程相对神经性毒剂较为缓慢,四种有机磷农药的中毒速率由快至慢依次为对氧磷、敌百虫、敌敌畏、久效磷。对于同一种有机磷农药,不同重活化剂表现出不同的重活化效果:对于敌敌畏,双复磷(Obidoxime,L-1656)的重活化效果更佳;对于敌百虫,酰胺磷定(Asoxime,HI-6)的重活化效果更佳;对于久效磷,氯解磷定(Pralidoxime chloride,2-PAM)表现出显著的重活化效果优势;对于对氧磷,三种重活化剂皆呈现良好的重活化效果,各重活化剂的抗毒效果呈现显著差异,缺乏有效且广谱的抗毒药物;且通过比较发现,二乙氧基膦酰化中毒酶较二甲氧基膦酰化中毒酶更易被重活化剂重活化。该评价工作对有机磷农药的合理使用、有机磷农药中毒的救治具有重要参考意义。第三章:针对有机磷农药救治效果不佳的原因,推测有机磷农药与重活化剂形成的膦酰化肟(Phosphoryl oxime,POX)是重活化效果的重要影响因素。为验证推测,通过对氧磷与双复磷在巴比妥缓冲液中直接反应成功制备得到二者的反应产物——DEP-obidoxime。在体外实验中考察并比较对氧磷以及DEP-obidoxime对AChE的抑制作用差异,利用对氧磷以及DEP-obidoxime对AChE染毒,然后基于酶解-质谱法的分析策略,将中毒酶经胃蛋白酶酶解后产生含有加合位点的特异肽段,利用基于效应标志物的质谱定量分析技术对特异性肽段进行定量检测,实现对氧磷以及DEP-obidoxime中毒特点的考察,证实DEP-obidoxime具有较强的AChE抑制毒性,并进一步完成经典重活化剂(氯解磷定、双复磷、HI-6)抗毒效能评价工作,DEP-obidoxime膦酰化的AChE与对氧磷膦酰化的中毒酶可同样被氯解磷定重活化,且重活化效果无显著差异。同时半数有效浓度(Concentration for 50%of maximal effect,EC50)数据表明,3种经典重活化剂对对氧磷或DEP-obidoxime膦酰化AChE的重活化效果由强至弱依次为:氯解磷定、HI-6、双复磷。上述发现对于有机磷中毒的临床救治用药具有重要的参考价值。第四章:针对本课题组前期研究中发现肟类重活化剂会加深梭曼(Soman,GD)中毒的现象,推测肟类重活化剂作为抗毒药物用于梭曼中毒救治过程中,可能与梭曼发生反应,形成对AChE抑制毒性更大的膦酰化肟,成为中毒加深的根源。基于这一发现,本章开展肟类重活化剂加深梭曼中毒机制的研究。首先,采用光谱法和气相色谱法对神经性毒剂梭曼与重活化剂的相互作用展开研究,研究发现肟类重活化剂双复磷、氯解磷定以及HI-6可与梭曼明显发生反应,而我所自主研发的叔胺类重活化剂L-1978、L-1655不与梭曼发生反应。随后在第三章研究工作的基础上,成功制备得到梭曼与双复磷以及氯解磷定的反应产物PMPA-obidoxime、PMPA-pralidoxime。但由于梭曼中毒及老化十分迅速,无法有效观察梭曼与膦酰化肟中毒及老化速率的差异,且因条件及技术限制,暂无法获得梭曼光学异构体单体,后续工作有待进一步开展。
[Abstract]:Organic phosphorus poison is a kind of organic phosphorous, organic phosphorus poison can be divided into nerve agents (Nerve agents, NAs in efficiency) and organophosphorus pesticides (Organophosphorus pesticides). The therapeutic strategy of acute organophosphate poisoning is the use of atropine for symptomatic treatment and due to treatment. The use of oximes and atropine only on muscarinic receptor, treatment effect, increase the dosage of atropine will cause poisoning; therefore oxime compounds as a specific antidote is more and more widely used in the treatment of acute organophosphorus poisoning. But oxime reactivators cannot enter the central nervous system, and the reactivative effect some controversy: a view of oximes by reactivative nerve or muscle tissue acetylcholinesterase (Acetylcholinesterase, AChE) poisoning, the recovery of enzyme activity As well as impaired physical function; another view is that in the process of oxime compounds with free agent or leave phosphoric acid in the heavy (or phosphonic acid) groups react to form a phosphonylation oxime intermediate, the intermediate with positively charged quaternary ammonium, easier to attack with negative charge activity center so, have a stronger inhibitory toxicity, may be caused by oxime drug reactivative effect or even cause poisoning. At present, deepen research work on organic phosphorus poisoning treatment based on Determination of the classical Ellman method to carry out live enzymes, enzyme activity determination method of Ellman as the gold standard, limitations still exist. Some, for example, AChE was susceptible to butyrylcholinesterase (Butyrylcholinesterase, BChE) interference, low sensitivity and low accuracy; when the concentration is below 20%, which can not be measured accurately; simultaneous determination of enzyme activity in the system. He is a cholinesterase inhibitor or reactivators and other drugs exist will cause some interference, leading to false positive or false negative results; more important is this method is not specific, and cannot distinguish the poisoning type, can not accurately describe the state that the AChE (active state, poisoning state, aging state). This research established a liquid chromatography / tandem mass spectrometry (Liquid chromatography tandem mass spectrometry, LC-MS/MS) is used to study the characteristics and treatment of nerve agent poisoning quantitative determination method, this method based on the specific and quantitative detection of toxic synthetic peptides, provide the effect of enzyme poisoning degree data, and to the varying state quantitatively get the effect of enzyme poisoning, especially for the low dose of organophosphorus poison exposure, the classical Ellman method cannot accurately obtain the enzyme activity inhibition rate, and the method for inhibiting rate of less than 1%. Condition can also be accurately analyzed. In this research, aiming at the disadvantages of organophosphorus poisoning and treatment problems of existing methods, in-depth research. This thesis is divided into four chapters: the first chapter: introduction of organic phosphorus poison and its mechanism of poisoning, poisoning and treatment methods, anti drug profiles are introduced. And put forward the significance and purpose of this paper. The second chapter: organophosphorus pesticide is connected with the AChE group for two methoxy phosphonic groups (Dimethylphosphoryl, DMP) and two ethoxylated phosphonic groups (Diethylphosphoryl, DEP), this chapter focuses on these two types of structure were prepared two methyl phosphonic acid eleven peptides and two ethyl phosphonylation eleven peptide, on the basis of previous studies to establish LC-MS/MS quantitative treatment of organophosphorus pesticide poisoning in the determination of common Organophosphorus Pesticide Dichlorvos, trichlorfon, Monocrotophos, inspect the poisoning characteristics and reactivative characteristics of phosphorus, organic phosphorus pesticide poisoning process relative to nerve agents is slow, poisoning rate of four kinds of organophosphorus pesticide from fast to slow in the paraoxon, trichlorfon, dichlorvos, monocrotophos. For the same kinds of organophosphorus pesticides different reactivators showed reactivative effect of different: for dichlorvos, obidoxime (Obidoxime, L-1656) better reactivative effect; for trichlorfon, HI-6 (Asoxime, HI-6) better reactivative effect; for monocrotophos, pralidoxime chloride (Pralidoxime chloride, 2-PAM) showed an advantage reactivative effect; for paraoxon, three kinds of reactivators are showing reactivative effect is good, showing significant difference in each reactivators antitoxic effect, lack of effective and broad-spectrum antiviral drugs; and it was found through comparison that two ethoxy phosphonylation poisoning with enzyme Two methoxy phosphonylation poisoning were more susceptible to the enzyme reactivators reactivative. Reasonable use of the evaluation work of organophosphorus pesticides, has important significance in the treatment of organophosphorus pesticide poisoning. The third chapter: the reasons for the organophosphorus pesticide treatment effect, that of organic phosphorus pesticide and phosphonylation oxime reactivative the formation of agent (Phosphoryl oxime POX) is an important factor influencing the reactivative effect. In order to verify that by paraoxon and obidoxime in barbital buffer was prepared by direct reaction of the success of the two reaction product DEP-obidoxime. in vitro experiments to investigate and compare the inhibitory effect of DEP-obidoxime on phosphorus and oxygen the differences in AChE, using paraoxon and DEP-obidoxime to AChE exposure, then enzyme analysis strategy based on mass spectrometry, poisoning the enzyme by pepsin produced containing specific peptide binding site, using the medium To mark the effect of mass spectrometry quantitative analysis technique for quantitative detection of specific peptide for the investigation of oxygen phosphorus and DEP-obidoxime poisoning, confirmed that DEP-obidoxime has strong inhibition of AChE toxicity, and further complete the classic reactivators (pralidoxime chloride, obidoxime, HI-6) antitoxic effectiveness evaluation, enzyme poisoning DEP-obidoxime phosphonylation AChE and paraoxon phosphonylation can also be pam-ci reactivative, and the activation effect was no significant difference. At the same time the median effective concentration (Concentration for 50%of maximal effect, EC50) data show that 3 kinds of classic reactivators reactivative effect of paraoxon or DEP-obidoxime phosphine acylated AChE were from strong to weak: pralidoxime chloride, HI-6, obidoxime. These findings have important reference value for the clinical treatment of drug organophosphate poisoning. The fourth chapter: according to the previous study Discovery of oxime reactivators will deepen Soman (Soman, GD) poisoning, presumably oxime reactivators as antidotes for Soman poisoning treatment process, may react with Soman, the formation of phosphonylation oxime on AChE inhibited the more toxic, poisoning become a source deepening. Based on this discovery in this chapter, the research development of oxime reactivators deepen Soman poisoning mechanism. Firstly, research the interaction by spectroscopy and gas chromatography of nerve agents Soman and reactivators, the study found that oxime reactivators cmo4, pralidoxime chloride, HI-6 and Soman obvious reaction I work and tertiary amine self-developed agent L-1978, L-1655 does not react with Soman. Then in the third chapter based on the research work, successfully prepared by Soman and obidoxime and pralidoxime chloride reaction products PMPA-obidoxime, PMPA-pralido Xime. but due to soman intoxication and aging rapidly, unable to effectively observe the differences of soman and phosphonylation oxime poisoning and aging rate, and because the conditions and restrictions, temporarily unable to obtain soman stereoisomers of monomer, the follow-up work need to be done.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R595.4

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