汞的形态分析、生物有效性及结合蛋白的初步研究
文献类型：学位论文
| 作者 | 贠照军 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 江桂斌，何滨 |
| 关键词 | 汞 含朱砂中成药 生物有效性 在线二维液相色谱 汞结合蛋白 mercury cinnabar-containing Chinese patent medicines bioavailibity on- line 2D HPLC mercury-containing protein |
| 其他题名 | Studies on the speciation, bioavailability of mercury and the mercury-binding protein |
| 学位专业 | 环境科学 |
| 中文摘要 | 汞具有高致毒性、持久性和长距离迁移性等特征，已经成为一种全世界公认的持久性有毒物质（PTS）。汞及其化合物在环境中普遍存在，各形态的汞之间可以相互转化，尤其是无机汞可以通过生物甲基化或者光化学反应等过程形成毒性更大的甲基汞，并可通过食物链最终在人体内富集，严重危害人体健康。虽然现在汞的代谢及毒性受到广泛关注，但是其在生物体内吸收、转化的过程，及其确切的致毒机理尚不十分清楚。本文主要从汞的形态分析、生物有效性及其与生物大分子的相互作用等方面开展了相关研究。 对石油及其产品中汞的形态分析方法进行了优化，对比了多种萃取体系（盐酸氢氧化钾-甲醇、四甲基氢氧化铵和酸性溴化钾/硫酸铜）及多种萃取方法（震荡、超声和微波辅助萃取）对有机汞的萃取效率，萃取后的有机汞用高效液相色谱-冷蒸气发生-原子荧光光谱（HPLC-CV-AFS）联用系统进行分离和定量。结果表明以四甲基氢氧化铵作为萃取试剂，60 W的功率微波辅助萃取 5 min的方法可以得到最优的提取效果。通过对 4个原油样品、3个汽油样品和 1个柴油样品进行检测，充分验证了该方法的可靠性。 中药取材于天然动植物，其中含有的汞等重金属元素使其推广和出口受到了影响，但是仅用汞的总量进行健康风险评价并不全面，还应考虑其生物有效性。对市场上常见的多种中成药中的总汞进行了检测，了解汞的总体含量水平。然后通过模拟消化液提取的方法，对中成药中汞的生物有效性进行分析。结果表明，朱砂是中成药汞超标的主要来源，但是因朱砂的溶解性极差，使得中成药中汞的生物有效性极低，大部分并未被人体吸收。 为了进一步验证服用含朱砂药物对人体健康的影响，将含有朱砂的同仁安神丸暴露于 Wistar模型大鼠，考察其对大鼠健康的影响。结果表明，在各个器官中，肾脏中汞的浓度最高，但是并未影响肾脏的氧化还原状态，肾功能也未受到影响，组织切片的结果进一步表明经同仁安神丸暴露后的大鼠肾脏没有明显的病理损伤。说明含有朱砂的药物虽然总汞的含量高，但是在推荐剂量下并不会对健康造成明显的影响。将暴露后的大鼠用旷场模型进行行为学评价，结果表明同仁安神丸及朱砂确实有显著的镇静作用。 利用二维高效液相色谱技术（2D HPLC）对正常人体血浆中的蛋白进行了分离，同时通过电感耦合等离子体质谱（ICP-MS）对蛋白中汞进行在线监测，发现了血浆中的一种汞结合蛋白，然后利用高效液相色谱-线性离子阱-傅里叶变换离子回旋共振质谱（HPLC-LTQ-FT）对该蛋白进行结构鉴定，经过与蛋白质标准谱库进行对比，表明该蛋白为人血清白蛋白（HSA）。利用等温滴定量热法（ITC）等方法对 Hg2+与HSA的相互作用进行表征，表明 Hg2+主要结合在HSA的 Cys34 上，且结合后会显著影响 HSA的二级结构。这一结果对汞的代谢过程及毒性机制研究具有重要意义。 |
| 英文摘要 | Due to their high toxicity, persistence and long distance transformation, mercury and mercuric compounds are considered as typical persistent toxic substances (PTS)now. As a constituent element of the earth, mercury occurs naturally in the environment and exists in all the matrices. As we all known, the toxicity of mercuric compounds are closely related to their species and concentrations, and the toxicity of organic mercury species, especially methylmercury, are quite higher than the elemental and inorganic mercury. Inorganic mercury could be methylated in some biotic (by microbial metabolism) or abiotic (do not involve living organisms) processes, and then be bio-magnified in the food chain, resulting in high concentrations in fish and mammals and health risk to human beings. So far, the precise mechanisms of metabolism and toxicity of mercury are still unknown. This work mainly focused on the studies on speciation,bioavailability of mercury and the mercury-binding protein. An extraction procedure for extracting organic mercury species including methylmercury (MeHg) and ethylmercury (EtHg) from petroleum samples was developed. Three extraction methods (shaking, ultrasonic and microwave assisted extraction) using different extraction solvents (TMAH, KOH/CH3OH, HCl and acidic CuSO4/KBr) were investigated by comparing the extraction efficiency of the organic mercury species. Microwave assisted extraction at 60 W for 5 min using TMAH (tetramethylammonium hydroxide, 25%, m/v) provided the most satisfactory extraction efficiency for MeHg and EtHg in petroleum at 86.7% ± 3.4% and 70.6% ± 5.9%, respectively. Speciation analysis of mercury was done by on-line coupling of high performance liquid chromatography with cold vapor generation atomic fluorescence spectrometry (HPLC-CV-AFS). The proposed method was successfully applied to analyze several crude oil and light oil samples. The concentrations of MeHg ranged from under detection limit to 0.515 ng g-1 , whereas EtHg was not detected in the samples. This method can be a very useful tool in evaluating the risk of mercury emission from petroleum. Mercury, mainly in cinnabar species, has been used in medicine for the treatment of diseases for thousands of years in China. The bioavailability of mercury in Chinese patent medicines (CPMs) was assessed by using a simulated digestion extraction test. The levels of total mercury in sixteen CPMs ranged from not detected to 11.9 mg g-1 with a mean value of 1.1 mg g-1, while the extractable mercury ranged from not detected . Mercury bioavailability variedto 4373.6 ng g -1 , with a mean value of 417.2 ng g-1 depending on the type of medicine analyzed. Mercury solubility in the gastrointestinal supernatants was quite lower in cinnabar-containing medicines (0.037%) than in ordinary medicines without cinnabar (2.5% - 31%). Human health risk by taking Chinese patent medicine was accessed by the results of simulated digestion extraction. In order to further investigate the potential health risk by taking the cinnabar containing Chinese patent medicines, Tong Ren An Shen Wan (TRASW) was exposed to Wistar mice, and then the effects about the medicine was studied. The data showed that the amount of mercury in the mice tissues increased after intake of TRASW, and the highest concentration of mercury was found in the kidney. But the redox state of the kidney was well balanced, and the kidney function was not damaged compared with the negative control. Obvious injury was not found in the HE slide of kindey exposed to TRASW. All the results of the in-vivo experiments showed that the recommended dose of TRASW would not injure kidney. Open-field test indicated that the TRASW and cinnabar had obvious sedation effects. Characterization of mercury binding protein in human body is very important to understand the metabolism and the mechanism of toxication of the ingested mercuric compounds. In this study, the mercury-containing protein in human plasma was separated by on-line heart-cutting two-dimensional high performance liquid chromatography (2D-HPLC) system. This 2D separation system used size exclusion liquid chromatography (SEC) followed by weak anion exchange liquid chromatography (WAX) and the two LC parts were coupled by a six-port valve equipped with a storage loop and controlled by the computer. The WAX effluent was determined by both a UV detector and inductively coupled plasma-mass spectrometry (ICP-MS) to locate the mercury-containing protein. Unique mercury-containing protein fraction was obtained from 2D-HPLC separation and subsequently identified by HPLC coupled with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-LTQ-FT). The database search confirmed that the mercury-containing protein in the human plasma is human serum albumin (HSA). The stoichiometry and thermodynamics interaction of inorganic mercury (Hg2+ ) with HSA was studied by isothermal titration calorimetry (ITC) and two binding types were observed. Mercury-containing protein in the human plasma was separated and identified in the present study and it is important to understand the metabolism of mercury in the human body. |
| 公开日期 | 2015-07-07 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/15628]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 贠照军. 汞的形态分析、生物有效性及结合蛋白的初步研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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