本文選題：芥子氣　切入點(diǎn)：血漿　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：芥子氣（Sulfur Mustard, SM）是強(qiáng)效的糜爛性毒劑，是日本在我國(guó)遺留化學(xué)武器的主要填充劑，它具有合成簡(jiǎn)便、作用持久、防護(hù)困難等特點(diǎn)，是一類(lèi)難防難治的化學(xué)戰(zhàn)劑。芥子氣進(jìn)入生物體后迅速分布于各組織中，主要存在形式為毒劑原型、水解氧化產(chǎn)物、β-裂解產(chǎn)物和大分子加合物等。這些游離或結(jié)合的具有芥子氣結(jié)構(gòu)特征的化合物，統(tǒng)稱(chēng)為芥子氣的生物標(biāo)志物。按照其來(lái)源和體內(nèi)停留時(shí)間，芥子氣生物標(biāo)志物可分為兩類(lèi)：小分子代謝產(chǎn)物和生物大分子的加合物。代謝產(chǎn)物標(biāo)志物包括芥子氣的水解和氧化產(chǎn)物、以及與谷胱甘肽加合后的β-裂解產(chǎn)物；這些小分子標(biāo)志物在芥子氣暴露后很快出現(xiàn)在體液中，取決于染毒途徑和劑量，它們?cè)跈C(jī)體內(nèi)的停留時(shí)間為數(shù)日至兩周；這些代謝產(chǎn)物標(biāo)志物可以作為芥子氣的早期暴露標(biāo)志物，在接觸或染毒的早期用于中毒鑒別和診斷。 本研究以芥子氣的代謝產(chǎn)物標(biāo)志物為研究對(duì)象，應(yīng)用現(xiàn)代儀器分析、分析毒理學(xué)、定量毒理學(xué)和毒物代謝動(dòng)力學(xué)的理論和技術(shù)，通過(guò)標(biāo)準(zhǔn)品合成、定量檢測(cè)關(guān)鍵技術(shù)的建立、標(biāo)志物血漿動(dòng)力學(xué)的研究和分析，以及技術(shù)方法和研究成果的應(yīng)用研究，為芥子氣的醫(yī)學(xué)防護(hù)提供毒劑標(biāo)志物體內(nèi)處置過(guò)程的新認(rèn)識(shí)，為芥子氣的中毒診斷和防治措施研究提供新穎可行的定量檢測(cè)技術(shù)。 本研究以芥子氣為起始原料，通過(guò)優(yōu)化和控制反應(yīng)條件，成功合成了9個(gè)芥子氣代謝產(chǎn)物標(biāo)準(zhǔn)對(duì)照品和1個(gè)內(nèi)標(biāo)標(biāo)準(zhǔn)品，應(yīng)用NMR、IR、MS等技術(shù)對(duì)它們的結(jié)構(gòu)進(jìn)行了確證，獲得的標(biāo)準(zhǔn)品純度均95%。在此基礎(chǔ)上，首次建立了芥子氣血漿標(biāo)志物定量檢測(cè)關(guān)鍵技術(shù)，應(yīng)用LC-MS/MS技術(shù)同時(shí)檢測(cè)大鼠血漿中7個(gè)芥子氣的代謝產(chǎn)物標(biāo)志物（TDG、TDGO，SMO、SBMTE、MSMTESE、SBMSE和SBSNAE），并對(duì)方法學(xué)進(jìn)行了全面的驗(yàn)證。結(jié)果表明，本方法的專(zhuān)屬性好、靈敏度高，回收率、精密度和準(zhǔn)確度均符合生物樣品定量分析要求。與已報(bào)道的方法相比，本方法的靈敏度與其相當(dāng)或更高，并具有線性范圍寬、樣品需用量少、采用一步沉淀蛋白法處理樣品、簡(jiǎn)便快捷、可用于大量樣品的快速分析等優(yōu)點(diǎn)，為芥子氣染毒血漿樣品的檢測(cè)提供一個(gè)綜合解決方案。 應(yīng)用建立的定量檢測(cè)技術(shù)，系統(tǒng)研究了代謝產(chǎn)物標(biāo)志物在芥子氣染毒大鼠的血漿動(dòng)力學(xué)。大鼠皮下和皮膚染毒3個(gè)劑量芥子氣后，在0.08-72h的時(shí)間段內(nèi)可在血漿中檢出芥子氣的7個(gè)代謝產(chǎn)物(SMO、TDG、TDGO、SBMTE、SBMSE、MSMTESE和SBSNAE)。研究還發(fā)現(xiàn)，SMO是芥子氣染毒后血循環(huán)中的主要產(chǎn)物，也是良好的暴露標(biāo)志物。動(dòng)力學(xué)分析結(jié)果表明，SMO、TDGO、SBMSE、MSMTESE和SBSNAE的血漿暴露與劑量之間有明確并良好的線性相關(guān)性，血漿濃度隨劑量增大呈線性增高，定量時(shí)間窗為染毒后5min至48h的不同時(shí)間段，因此，這些代謝產(chǎn)物可以用作芥子氣中毒血漿暴露和診斷標(biāo)志物。 比格犬皮下染毒芥子氣后的毒物代謝動(dòng)力學(xué)研究表明，芥子氣血漿代謝產(chǎn)物的生成及動(dòng)力學(xué)在比格犬和大鼠之間存在種屬差異，主要表現(xiàn)為比格犬體內(nèi)較弱的谷胱甘肽加合-β裂解反應(yīng)以及SBSNAE生成的缺失。比格犬染毒芥子氣后，SMO、TDG和TDGO是更適宜的血漿暴露和診斷標(biāo)志物。 將建立的芥子氣標(biāo)志物定量檢測(cè)關(guān)鍵技術(shù)應(yīng)用于芥子氣皮膚防護(hù)膏和洗消劑的效能評(píng)價(jià)中，以標(biāo)志物的血漿暴露水平為指標(biāo)，確證了自行研制的皮膚防護(hù)膏和洗消劑能有效地防護(hù)芥子氣中毒損傷。染毒前正確使用皮膚防護(hù)膏，可防止約96%的芥子氣通過(guò)皮膚吸收；在芥子氣染毒后及時(shí)（6min）用洗消劑消毒，至少能減少80%的體內(nèi)毒劑暴露量。此外，應(yīng)用研究的結(jié)果也證明標(biāo)志物定量檢測(cè)技術(shù)在芥子氣防護(hù)措施評(píng)價(jià)中的可行性，為芥子氣防治研究提供了快速、準(zhǔn)確的定量評(píng)價(jià)手段。 將標(biāo)志物定量檢測(cè)技術(shù)用于意外化學(xué)品中毒傷員生物樣品的檢測(cè)，通過(guò)血漿、組織滲出液和尿樣的定量檢測(cè)，確證4位傷員是因接觸芥子氣中毒，并為傷員的暴露時(shí)間及中毒程度判斷提供重要的定量化數(shù)據(jù)。這一應(yīng)用實(shí)例展示了早期暴露標(biāo)志物及其定量檢測(cè)技術(shù)在芥子氣暴露確證和中毒診斷中的良好應(yīng)用前景。 本論文有如下的創(chuàng)新點(diǎn)或新發(fā)現(xiàn)： 1)首次建立了芥子氣血漿標(biāo)志物的定量檢測(cè)關(guān)鍵技術(shù)，該技術(shù)可同時(shí)檢測(cè)血漿樣品中芥子氣的7個(gè)代謝產(chǎn)物，技術(shù)指標(biāo)與已報(bào)道方法相當(dāng)或達(dá)到更高水平；本研究將SMO作為芥子氣暴露標(biāo)志物納入定量檢測(cè)方法，以及將LC-MS/MS技術(shù)用于生物樣品中TDG和TDGO的直接定量分析也未見(jiàn)報(bào)道。 2)通過(guò)芥子氣血漿標(biāo)志物毒物代謝動(dòng)力學(xué)的系統(tǒng)研究和標(biāo)志物血漿暴露水平-時(shí)間動(dòng)力學(xué)過(guò)程的分析，揭示了毒劑染毒劑量與標(biāo)志物血漿暴露水平之間的相關(guān)性，提出了適用于暴露確證和中毒診斷的血漿標(biāo)志物種類(lèi)及其檢測(cè)時(shí)間窗，為將血漿標(biāo)志物檢測(cè)用于中毒診斷提供了必要的科學(xué)依據(jù)。 3)本研究發(fā)現(xiàn)SMO是芥子氣染毒后血循環(huán)中的主要產(chǎn)物，也是良好的暴露標(biāo)志物；比格犬染毒芥子氣后體內(nèi)谷胱甘肽加合-裂解的轉(zhuǎn)化途徑顯著弱于大鼠，犬體內(nèi)因缺乏N-乙酰轉(zhuǎn)移酶而導(dǎo)致SBSNAE生成的缺失。 4)本研究首次將血漿暴露標(biāo)志物的定量檢測(cè)技術(shù)應(yīng)用于芥子氣防護(hù)措施的評(píng)價(jià)，，為芥子氣中毒的醫(yī)學(xué)防治研究提供了可靠、準(zhǔn)確和快速的定量化評(píng)價(jià)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R114

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相關(guān)期刊論文 前2條

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