大鼠肾脏汞结合金属硫蛋白的联用技术表征研究

通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术，对灌喂HgCl₂大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC-ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征，可确认组织提取液中MT在色谱曲线上的位置。与对照组相比，灌喂HgCl₂大鼠肾中Hg-MT的诱导量显著增加，与此同时，Cu-MT在染毒组的诱导量也相应增加，说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后，用细内径反相色谱柱分离，进行梯度洗脱，并与ESI-MS联用，可获得总离子流色谱图及与色谱峰对应的质谱图，通过对质谱信号的解析及参考脱金属MT构型的有关报道，对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明，在灌喂HgCl₂大鼠的肾组织中存在着一系列汞结合的MT形态。     

同位素示踪SEC-ICP-MS研究大鼠体内镉的形态

通过建立凝胶排阻色谱分离紫外与电感耦合等离子体质谱在线检测的联用技术,研究了灌喂低浓度镉富集同位素后大鼠各器官中镉与蛋白结合的形态.结果发现,镉主要在肝、肾、睾丸中与硫蛋白结合形成金属硫蛋白.与传统方法相比.该法危害性小、具有更接近慢性中毒、能将重金属来源分为天然来源和人为引入等特点.     

小球藻类金属硫蛋白的结构表征研究

金属硫蛋白(metallothioneins,MTs)是一类低分子量、高巯基含量,且能大量结合金属离子的蛋白质,通常由61个氨基酸残基组成,分子量为6～7 ku.MTs具有两个重要特征即金属亲和性和金属诱导性.因此,MTs对多种重金属如Hg、Pb、Zn、Cd等具有螯合作用,且在重金属诱导作用下,MTs可在生物体内诱导合成.近年来小球藻被广泛应用于污水净化,能够去除水体中的多种重金属,其机制是小球藻细胞壁带有负电荷的官能团能吸附金属阳离子,或在细胞内通过诱导生成金属结合蛋白把有毒的金属离子转化为无害的蛋白结合形式.本实验选取分布广泛,易培养的小球藻,经锌诱导后分离、纯化产生的类金属硫蛋白,并对小球藻类金属蛋白进行初步的结构表征.     

高效液相色谱-同位素稀释-电感耦合等离子体质谱法研究大鼠体内的汞结合蛋白

利用高效液相色谱(HPLC)和电感耦合等离子体质谱(ICP-MS)联用技术,对经胚胎早期甲基汞暴露的母、仔鼠脑胞液和肝胞液中汞结合蛋白进行了研究。采用柱后(Post-Column)同位素稀释法(ID)技术定量计算了母、仔鼠组织胞液中与蛋白结合的汞。HPLC-ID-ICP-MS方法的汞回收率与直接ICP-IDMS法的实验结果一致。该方法可以克服生物样品微量元素化合物分析中灵敏度低和标准蛋白不足的缺点,有利于生物体内低丰度金属结合蛋白的分离分析和定量研究。     

Mo/Fe3O4(111)表面对燃煤烟气汞吸附的密度泛函研究

利用CASTEP软件包采用密度泛函理论计算研究了过渡金属Mo掺杂Fe_3O_4(111)Fe_(tet)表面对Hg~0、HgCl和HgCl_2的吸附特征,分析了Mo掺杂前后Fe_3O_4(111)Fe_(tet)表面上不同汞物种的吸附形态。结果表明,Mo掺杂Fe_3O_4(111)Fe_(tet)表面对HgCl和HgCl_2为化学吸附,而对Hg~0的吸附为物理吸附;与纯净表面相比,HgCl在Mo原子掺杂表面上的吸附能提高了40%-66%。HgCl_2在纯净Fe_3O_4(111)Fe_(tet)表面形成"M"形结构;而掺杂Mo原子后,由于Cl原子与Mo原子之间更强的相互作用,使得HgCl_2发生了完全解离,两个Cl原子分别与Mo原子和Fe原子成键吸附在表面,Hg脱附。相关研究结果可为脱除燃煤烟气中的汞提供一定的理论指导。     

高效液相色谱-电感耦合等离子体质谱联用快速测定水样和生物样品中的4种汞形态

建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用快速测定4种汞形态的分析方法。以0.1%L-半胱氨酸、2 mmo L乙酸铵水溶液和甲醇体系(p H 6.8)为流动相,采用梯度洗脱方式,在约3 min内实现了二价汞、甲基汞、乙基汞和苯基汞4种形态在反相色谱C18柱上的完全分离,4种汞形态检出限分别为0.06,0.05,0.08和0.06,在0.5~10μg/L范围内具有良好的线性关系,相关系数均在0.9995以上。用该方法对标准样品人发(GBW07601)、黄鱼(GBW 08573)和金枪鱼(ERM-CE464)中的汞形态和总汞量进行测定,结果与标准参考值一致。方法成功应用于2种水样和3种鱼肉样品中汞形态的测定。两种水样汞形态的加标回收率(加标水平为0.5μg/L和5μg/L)在95%~122%之间,相对标准偏差小于5%。鱼肉样品中无机汞和甲基汞含量进行实验室间比对,结果满意。     

大鼠体内铅镉分布的稳定同位素示踪ICP-MS研究

采用稳定同位素示踪技术研究重金属元素铅、镉在大鼠体内的分布,通过对大鼠进行浓缩同位素206Pb、112Cd灌喂,解剖取其肝、肾、睾丸和心脏等脏器,通过相应的化学处理,用电感耦合等离子体质谱(ICP-MS)测量铅、镉元素在各个脏器中的含量及其同位素组成变化.根据同位素比值的变化研究Pb、Cd在各脏器中的分布、蓄积规律和各脏器对引入的浓缩同位素利用率.结果发现:重金属Pb和Cd蓄积的最主要场所是肝和肾,其次是睾丸和心脏,而在脑中的积累最少.在肝和肾中Cd的同位素利用率为0.048 8%、0.015 4%,Pb的同位素利用率为0.010 1%、0.007 8%,远高于其它脏器.与传统的方法相比较,该法具有更接近慢性中毒、危害性小,能将重金属来源分为天然来源和人为引入等特点.     

对氧化还原滴定法测定铁还原Fe(Ⅲ)时用甲基橙代替氯化高汞的正交分析

早在20世纪80年代,文献[1,2]已提出由于HgCl2剧毒,用量大(每个样品消耗20~30 mL的100 g·L-1HgCl2溶液),为避免汞对环境的污染,应采用无汞定铁法。如KIO3-Ag2SO4-K2Cr2O7法[1],由于Ag2SO4加入量的控制不易确定,则严重影响终点观察,同时重金属银的引入又将造成再次的环境污染;再如氯代磺酚S-SnCl2-K2Cr2O7法[2],氯代磺酚S颜色变化不敏锐,易造成SnCl2过量,使结果偏高。因无汞定铁法的不完善,未得到推广。本文就定量分析化学[3,4]中,铁矿石中全铁含量测定的经典方法HgCl2-K2Cr2O7法[1]进行改进。采用甲基橙(MO)-K2Cr2O7无汞定铁…     

高效液相色谱-电感耦合等离子质谱法分析雄黄在大鼠脏器中代谢的砷形态

采用HPLC-ICP-MS联用技术建立了大鼠脏器中AsB,As?,MMA,DMA和As?等砷形态的分析方法。采用超声水浴提取对样品进行前处理,Dionex IonPac AS19阴离子交换柱,20 mmol/L(NH4)2CO3(pH 9.7)为流动相,对雄黄染毒后大鼠肝肾中的砷形态进行分析。实验表明,方法不受40Ar35Cl+干扰,常见的5种砷形态化合物的线性范围为1~300μg/L,相关系数(R2)均大于0.999,检出限为0.3~0.5(μg/L),RSD均小于5%,加标回收率为83.8%~111.7%。通过对肝肾的形态分析,表明经雄黄染毒后大鼠肝脏中的砷形态主要为DMA、As?和未知物2;肾脏中的砷形态主要为DMA、MMA、As?、未知物1、未知物2和未知物3。综合表明,此方法适用于雄黄代谢后的主要砷形态分析。     

气相色谱-电感耦合等离子体质谱联用技术应用于水产品中汞形态分析

对气相色谱(GC)与电感耦合等离子体质谱(ICP-MS)联用的商品化接口进行了改进,并将GC-ICP-MS联用技术应用于水产品中汞的形态分析。在优化条件下,甲基汞(MeHg(Ⅰ))和乙基汞(EtHg(Ⅰ))的检出限(S/N=3)分别为0.5pg和1.0pg,定量限(S/N=8)分别为1.5pg和2.8pg,标准曲线的线性范围为1～1000pg。对三文鱼肉、金枪鱼肉和角鲨鱼肉3种标准物质和市售14种鱼贝类水产品,采用非衍生的样品预处理,进行汞的形态分析。结果表明,采集样品中均检出有甲基汞,未检测出乙基汞,甲基汞的含量在3.7～236.6ng/g(以Hg计)范围,甲基汞的加标回收率为91%～102%。     

Bioaccumulation of metals and induction of metallo‐ thioneins in selected tissues of common carp (Cyprinus carpio L.) co‐exposed to cadmium,mercury and lead

The concentrations of mercury (Hg), cadmium (Cd) and lead (Pb) at various exposure periods were determined in the gill, kidney, liver and muscle of common carp (Cyprinus carpio L.) co‐exposed to 1.0 µg ml^?1 each of Cd²⁺, Hg²⁺ and Pb²⁺ for up to 10 days. Metallothionein fractions (MTs) in these organs were characterized using the hyphenated technique of size‐exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP‐MS). After 10 days of exposure, maximum toxic metal concentrations of Hg, Cd and Pb were 10.7 (gill), 0.145 (kidney) and 0.112 µg g^?1_dryweight (gill), respectively. The pattern of accumulation of Hg and Pb was in the order gill > kidney > liver > muscle. In the case of Cd, accumulation was in the order kidney > gill > liver > muscle. Cd and Hg binding MTs were significantly induced in the gill, kidney and liver of all the exposure groups in comparison with the control group (p < 0.05), and the amounts of them increased with the longer exposure time. Despite the higher intracellular Hg concentration and the stronger Hg? SH binding affinity, the amount of Cd‐binding MTs was much higher than that of Hg‐binding MTs. The results indicate that MT synthesis in these organs was clearly metal‐specific. MTs in gill may be used as a bio‐marker to detect the metal pollution caused by Hg and Cd. Zinc and copper binding MTs in the organs of the exposed fish were also increased. This may be due to the MTs' important role in the homeostatic regulation of essential metals and their protective role against the acute toxicity of non‐essential metals. Even though there was considerable accumulation of lead in the organs of the exposed fish, Pb‐binding MT synthesis was non‐significant. Copyright © 2006 John Wiley & Sons, Ltd.     

Investigation of metal-binding metallothioneins in the tissues of rats after oral intake of cinnabar

The multi-metal-binding MT fractions in rat tissues after oral intake of cinnabar were characterized by hyphenated size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP–MS). With the increase of both the feeding time and dosage of cinnabar the amounts of Hg-binding MT fractions in rat kidney of groups fed cinnabar increased significantly compared with the control group. Meanwhile, more Cu-binding MT were synthesized in the rat kidney, which confirmed the manipulating effect of MT in the homeostasis of Cu for detoxification of nephritic mercury. Although the Hg-binding MT fractions in rat liver of all cinnabar groups were almost independent of cinnabar dosage (2.5–5.0 g kg^–1 bw) and feeding time (2–4 weeks), the amount was much higher than that of the control group. The amounts of Hg-MT in other rat tissues of the cinnabar groups and the control group were compared and their significance is discussed.     

Investigation of mercury metallothionein complexes in tissues of rat after oral intake of HgCl2

Mercury, a highly toxic metal found widely throughout the environment, is a potent inducer of metallothionein (MT) expression. The role of MTs in the detoxification of mercury after its oral intake in mammals is studied. After feeding rats with mercuric chloride by gastric gavage, the distribution of heavy metals in rat tissues was investigated by inductively coupled plasma mass spectrometry (ICP‐MS). Extensive accumulation of mercury, copper and zinc in kidney and liver is observed. A homemade preparative size‐exclusion chromatography (SEC) column (30 cm × 1.9 cm) packed with Sephadex G‐75 (40–120 µm particle size) gel (Pharmacia) was used for the purification of MT fractions in rat tissues. Preliminary results from SEC indicate that the mercury‐binding MT levels in liver were much lower than in kidney. The MT fractions were collected, desalted, and then separated by reversed‐phase high‐performance liquid chromatography (HPLC) with UV–Vis spectrometry, ICP‐MS and electrospray ionization MS detection. One major and several minor peaks were observed in the HPLC chromatograms of the MT fraction for the kidney sample. UV absorption spectra indicate that MTs were found to bind with mercury. There were no significant mercury‐binding MTs detected in the liver sample using UV detection. ICP‐MS detection showed that mercury‐binding MTs in kidney contained large amounts of mercury and copper but little zinc. Further characterization with ESI‐MS showed that the major peak found in kidney contained Hg₆Cu, Hg₅Cu₂‐MT‐2c and Hg₆‐MT‐2β, Hg₆Cu‐MT‐1γ, Hg₇‐MT‐2α. However, distinction between copper and zinc could not be made based on current mass spectrometric analysis because of instrumental resolution limitations. Copyright © 2005 John Wiley & Sons, Ltd.     

Metallothionein as a biomarker for mercury in tissues of rat fed orally with cinnabar

Cinnabar, as one of the most widely used mineral drugs in traditional Chinese medicines, has been proven to have prominent curative effects in clinical use for more than 2000 years. But the safety and toxicity of the drug has been under constant debate in clinic usage. Metallothionein (MT) contains about 30% of cysteine in the molecule, and plays an important detoxification role against heavy metals. In this study, it was used as a biomarker to assess mercurial accumulation in rats fed orally with cinnabar. After feeding rats with cinnabar by gastric gavage at different dosages and at different times, the distribution of heavy metals (including mercury, copper and zinc) and MT was investigated among rat tissues, including liver, kidney, heart, brain, testis and blood. Metals and MT determinations were carried out using inductively coupled plasma mass spectrometry (ICP‐MS) and a modified mercury saturation assay technique respectively. The results indicated that mercury was easily accumulated in the tissues of rats exposed to cinnabar, especially in kidney. For example: at a feeding dosage of 5 g kg^?1 (bw) for 4 weeks, the mercury concentrations in kidney were 13, 8.7, 21.6 and 26 times those in liver, testis, brain and heart respectively; and at 2.5 g kg^?1 (bw) for 2 weeks, the mercury concentrations in kidney were 21, 2.1, 3 and 21 times those in liver, testis, brain and heart respectively. In addition, mercury in kidney and liver of all cinnabar groups was significantly higher than that of the control group (P < 0.01). A high positive correlation observed between MT concentrations and mercury levels in both liver and kidney (R² = 0.9299, P < 0.02 for liver; R² = 0.9923, P < 0.0008 for kidney) indicated that MT could be used as a biomarker for mercury in tissues. Copyright © 2004 John Wiley & Sons, Ltd.     

Application of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to Multi-Element Analysis of Human Organs

Abstract

Elemental concentrations of internal organs (cerebrum, cerebellum, heart, spleen, liver and kidney) of Japanese were measured with inductively coupled plasma mass spectrometry (ICP-MS). Sixty-one elements were determined simultaneously, and concentrations of some trace elements in human organs, on which information are scarcely available in the literature, were obtained. The analytical values obtained by ICP-MS and those by other methods were compared and fairly good agreement was found for Cd, Zn and Fe. Moderate agreement was found for Se and Hg, but discrepancy was observed for Mg and Na. Unacceptable values were obtained for some elements, such as Ti, Sc or Ge, probably due to polyatomic molecular interference.     

土壤及蔬菜中微量汞的同位素稀释电感耦合等离子体质谱测定

建立了微波消解电感耦合等离子体质谱同位素稀释(ID/ICP-MS)测定微量汞的方法。考察了仪器参数及测量条件对汞同位素比值RHg(202Hg/200Hg)测量的影响,根据同位素比值测量误差的传递因子优化了富集同位素稀释剂(202Hg98%)的加入量,并以铊同位素比值(205Tl/203Tl)作为RHg(202Hg/200Hg)测量时发生质量歧视效应的校正因子;通过反同位素稀释法标定了富集汞同位素稀释剂的浓度。利用所建立的ID/ICP-MS方法测定了杨树叶(GBW07604)和湖积物(GBW07423)2种标准参考物中汞的含量,回收率分别为112%和100%。该方法具有准确度高、精密度好等优点,且样品前处理简便,适用于土壤及蔬菜等样品中微量及痕量汞的准确测定。     

氯化汞和亚硒酸钠对大鼠的毒性相互作用

用Hom法测定了大鼠经口染毒的LD50,并在此基础上进行90天亚慢性经口毒性试验。测量染毒过程中的体重、食物利用率的变化,测定肝、肾、脾等五种器官脏器系数,并进行血液生化检测。氯化汞、亚硒酸钠及二者等摩尔比联合作用对大鼠LD50分别为127．5、39．55和252mg/kg。汞组和硒组大鼠体重呈进行性下降。汞组肝、肾、胸腺、脾系数增大,睾丸系数减小,联合作用显著降低了汞对各脏器系数的影响,并且随着剂量组中硒剂量的增加,汞对脏器系数的影响减弱。血液学、血清酶化学检测结果无显著性差异。实验结果表明氯化汞和亚硒酸钠相互作用表现为明显的毒性拮抗作用,雌鼠对氯化汞和亚硒酸钠联合毒性有更高的耐受性。联合作用能够减弱单独作用对大鼠生长的影响,减弱汞对五种脏器的毒性。     

电感耦合等离子体质谱(ICP-MS)法测定谷类产品中8种重金属元素

为建立大批量谷类产品中重金属元素的快速检测方法,采用快速消解法,样品于聚丙烯刻度离心管中经硝酸消解,用电感耦合等离子体质谱仪同时检测Pb、Cd、Cr、Cu、Zn、Ni、As、Hg等8种重金属元素含量.研究了消解酸用量及消解时间对结果的影响,并优化了仪器工作参数.研究结果表明,样品在120℃消解1 h后直接定容测定,各元...