芥子气血浆暴露标志物定量分析方法及其毒物代谢动力学的研究

发布时间：2018-04-04 22:03

本文选题：芥子气　切入点：血浆　出处：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：芥子气（Sulfur Mustard, SM）是强效的糜烂性毒剂，是日本在我国遗留化学武器的主要填充剂，它具有合成简便、作用持久、防护困难等特点，是一类难防难治的化学战剂。芥子气进入生物体后迅速分布于各组织中，主要存在形式为毒剂原型、水解氧化产物、β-裂解产物和大分子加合物等。这些游离或结合的具有芥子气结构特征的化合物，统称为芥子气的生物标志物。按照其来源和体内停留时间，芥子气生物标志物可分为两类：小分子代谢产物和生物大分子的加合物。代谢产物标志物包括芥子气的水解和氧化产物、以及与谷胱甘肽加合后的β-裂解产物；这些小分子标志物在芥子气暴露后很快出现在体液中，取决于染毒途径和剂量，它们在机体内的停留时间为数日至两周；这些代谢产物标志物可以作为芥子气的早期暴露标志物，在接触或染毒的早期用于中毒鉴别和诊断。本研究以芥子气的代谢产物标志物为研究对象，应用现代仪器分析、分析毒理学、定量毒理学和毒物代谢动力学的理论和技术，通过标准品合成、定量检测关键技术的建立、标志物血浆动力学的研究和分析，以及技术方法和研究成果的应用研究，为芥子气的医学防护提供毒剂标志物体内处置过程的新认识，为芥子气的中毒诊断和防治措施研究提供新颖可行的定量检测技术。本研究以芥子气为起始原料，通过优化和控制反应条件，成功合成了9个芥子气代谢产物标准对照品和1个内标标准品，应用NMR、IR、MS等技术对它们的结构进行了确证，获得的标准品纯度均95%。在此基础上，首次建立了芥子气血浆标志物定量检测关键技术，应用LC-MS/MS技术同时检测大鼠血浆中7个芥子气的代谢产物标志物（TDG、TDGO，SMO、SBMTE、MSMTESE、SBMSE和SBSNAE），并对方法学进行了全面的验证。结果表明，本方法的专属性好、灵敏度高，回收率、精密度和准确度均符合生物样品定量分析要求。与已报道的方法相比，本方法的灵敏度与其相当或更高，并具有线性范围宽、样品需用量少、采用一步沉淀蛋白法处理样品、简便快捷、可用于大量样品的快速分析等优点，为芥子气染毒血浆样品的检测提供一个综合解决方案。应用建立的定量检测技术，系统研究了代谢产物标志物在芥子气染毒大鼠的血浆动力学。大鼠皮下和皮肤染毒3个剂量芥子气后，在0.08-72h的时间段内可在血浆中检出芥子气的7个代谢产物(SMO、TDG、TDGO、SBMTE、SBMSE、MSMTESE和SBSNAE)。研究还发现，SMO是芥子气染毒后血循环中的主要产物，也是良好的暴露标志物。动力学分析结果表明，SMO、TDGO、SBMSE、MSMTESE和SBSNAE的血浆暴露与剂量之间有明确并良好的线性相关性，血浆浓度随剂量增大呈线性增高，定量时间窗为染毒后5min至48h的不同时间段，因此，这些代谢产物可以用作芥子气中毒血浆暴露和诊断标志物。比格犬皮下染毒芥子气后的毒物代谢动力学研究表明，芥子气血浆代谢产物的生成及动力学在比格犬和大鼠之间存在种属差异，主要表现为比格犬体内较弱的谷胱甘肽加合-β裂解反应以及SBSNAE生成的缺失。比格犬染毒芥子气后，SMO、TDG和TDGO是更适宜的血浆暴露和诊断标志物。将建立的芥子气标志物定量检测关键技术应用于芥子气皮肤防护膏和洗消剂的效能评价中，以标志物的血浆暴露水平为指标，确证了自行研制的皮肤防护膏和洗消剂能有效地防护芥子气中毒损伤。染毒前正确使用皮肤防护膏，可防止约96%的芥子气通过皮肤吸收；在芥子气染毒后及时（6min）用洗消剂消毒，至少能减少80%的体内毒剂暴露量。此外，应用研究的结果也证明标志物定量检测技术在芥子气防护措施评价中的可行性，为芥子气防治研究提供了快速、准确的定量评价手段。将标志物定量检测技术用于意外化学品中毒伤员生物样品的检测，通过血浆、组织渗出液和尿样的定量检测，确证4位伤员是因接触芥子气中毒，并为伤员的暴露时间及中毒程度判断提供重要的定量化数据。这一应用实例展示了早期暴露标志物及其定量检测技术在芥子气暴露确证和中毒诊断中的良好应用前景。本论文有如下的创新点或新发现： 1)首次建立了芥子气血浆标志物的定量检测关键技术，该技术可同时检测血浆样品中芥子气的7个代谢产物，技术指标与已报道方法相当或达到更高水平；本研究将SMO作为芥子气暴露标志物纳入定量检测方法，以及将LC-MS/MS技术用于生物样品中TDG和TDGO的直接定量分析也未见报道。 2)通过芥子气血浆标志物毒物代谢动力学的系统研究和标志物血浆暴露水平-时间动力学过程的分析，揭示了毒剂染毒剂量与标志物血浆暴露水平之间的相关性，提出了适用于暴露确证和中毒诊断的血浆标志物种类及其检测时间窗，为将血浆标志物检测用于中毒诊断提供了必要的科学依据。 3)本研究发现SMO是芥子气染毒后血循环中的主要产物，也是良好的暴露标志物；比格犬染毒芥子气后体内谷胱甘肽加合-裂解的转化途径显著弱于大鼠，犬体内因缺乏N-乙酰转移酶而导致SBSNAE生成的缺失。 4)本研究首次将血浆暴露标志物的定量检测技术应用于芥子气防护措施的评价，，为芥子气中毒的医学防治研究提供了可靠、准确和快速的定量化评价手段。
[Abstract]:Mustard (Sulfur Mustard SM) is a potent vesicant agent in Japan, is China's main filler chemical weapons left, it has simple synthesis, long lasting protection is so difficult, is a kind of chemical warfare agents are difficult to prevent and cure. Mustard gas enters the organism rapidly distributed in various tissues, mainly exist as agent prototype, hydrolysis oxidation products, beta pyrolysis products and macromolecular adducts. These free or combined with the structure characteristics of mustard compounds, collectively known as mustard gas biomarkers. According to their sources and in vivo residence time, mustard gas biomarkers can be divided into two categories: small molecule metabolites and biological macromolecular adducts. Metabolite markers including mustard gas hydrolysis and oxidation products, and glutathione adduct after beta lysate; these small molecular markers in mustard gas exposure soon after In body fluids, depending on the exposure routes and doses, their in vivo residence time for a few days to two weeks; these metabolites can be used as early markers of mustard gas exposure markers for identification and diagnosis of poisoning in the early contact or exposure.
In this study, metabolites of mustard gas markers as the research object, analysis of toxicological analysis, application of modern instrument, quantitative toxicology and toxicokinetics of theory and technology, through the standard synthesis, key techniques for establishing quantitative detection, symbol study and analysis of plasma dynamics, applied research and technical methods and research results. Provide a new understanding of the objects within the agent sign the disposal process for medical protection of mustard gas, providing a novel and feasible quantitative detection technology for poisoning diagnosis and prevention measures of mustard gas.
In this study, mustard gas as the starting material, by optimizing and controlling the reaction conditions, successfully synthesized 9 metabolites of mustard gas standard control sample and 1 internal standard standard, application of NMR, IR, MS and other techniques of their structure were confirmed, the standard purity 95%. on this basis, for the first time established a blood plasma marks the key technology of mustard quantitative detection, and detection of mustard gas 7 metabolites in rat plasma markers using LC-MS/MS Technology (TDG, TDGO, SMO, SBMTE, MSMTESE, SBMSE and SBSNAE), and the other law to conduct a comprehensive verification. The results show that this method ofspecificity well, high sensitivity, recovery, precision and accuracy were consistent with the quantitative analysis of biological samples. Compared with the reported methods, the sensitivity method of equivalent or higher, and has wide linear range, little amount of sample required, by one-step protein precipitation Sample, convenient, can be used for a large number of samples for rapid analysis of the advantages of providing a comprehensive solution for the detection of mustard in plasma samples.
Quantitative detection technology based on the system of marker metabolites in the plasma dynamics of mustard in rats. The subcutaneous and skin of rats exposed to 3 doses of mustard gas, 7 metabolites were detected in the plasma of mustard gas in the 0.08-72h time period (SMO, TDG, TDGO, SBMTE. SBMSE, MSMTESE and SBSNAE). The study also found that SMO is the main product in the circulation of mustard gas poisoning, is a good biomarker of exposure. Kinetics analysis results show that SMO, TDGO, SBMSE, MSMTESE and SBSNAE plasma exposure there is a linear correlation between clear and good dose, plasma concentration was linear. With the increase of dose, quantitative time window for different time periods, from 5min to 48h after exposure so that these metabolites can be used as a mustard gas exposure and diagnostic markers.
Toxicokinetics of canine subcutaneous exposure after mustard gas shows that the formation and dynamics of blood plasma metabolites in mustard between beagle dog and rat species difference mainly in beagle dogs weaker glutathione adduct - loss of beta pyrolysis reaction and the generation of SBSNAE. After exposure to mustard gas in beagle dogs. SMO, TDG and TDGO are more suitable for plasma exposure and diagnostic markers.
Mustard gas will establish a marker for quantitative detection of key technologies applied in the mustard skin protection cream and decontamination efficiency evaluation agent, with markers of plasma exposure level as the index, confirmed the skin protective cream developed and decontamination agent can effectively protect the mustard gas poisoning injury. After the correct use of skin protection cream, mustard gas can prevent about 96% absorption through the skin; in the mustard in time (6min) with detergent and disinfection, at least 80% agents can reduce the in vivo exposure. In addition, the result of the study also proved feasibility of quantitative detection technology in the sign of mustard gas protective measures in the evaluation of gas. Mustard control provides a quick and accurate quantitative evaluation methods.
The marker for quantitative detection technology for detecting chemical poisoning accident, the biological sample by plasma, quantitative measurement of tissue exudate and urine, were 4 of the wounded is poisoning due to exposure to mustard gas, and the exposure time and the degree of poisoning can provide important quantitative data. The application example shows early exposure markers and quantitative detection of exposure confirmation and good application prospect in the diagnosis of mustard gas poisoning.
This thesis has the following innovations or new discoveries:
1) was established for the first time the blood plasma marker mustard quantitative detection of the key technology, the technology of 7 metabolites in plasma samples and detection of mustard gas, technical indicators with the reported method or to a higher level; this study will SMO as a marker of mustard gas into the quantitative detection method of exposure, and LC-MS/MS technology for direct quantitative analysis of TDG and TDGO in biological samples is reported.
2) exposure analysis level - time kinetics process by blood plasma markers of mustard toxicokinetics study and marking system of plasma, reveals the contamination dose and markers of plasma exposure correlation between levels, put forward for the identification and diagnosis of poisoning plasma marker types and detection time window for the exposure. A plasma marker provides necessary scientific basis for the detection of poisoning diagnosis.
3) this study found that SMO is the main product in the circulation of mustard gas poisoning, is also a good biomarker of exposure; exposure to mustard gas in beagle dogs after glutathione adduct - conversion pathway cracking was significantly weaker than in rats, dogs due to lack of N- acetyltransferase and lead to loss of SBSNAE formation.
4) this is the first study will evaluate the application of quantitative detection of plasma exposure markers in mustard gas protection measures, prevention and control of medical research for mustard gas poisoning and provide a reliable, accurate and rapid quantitative evaluation methods.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

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