摘要
硒是人体必需的营养元素,在体内发挥着重要的生理、生化功能,如抗氧化、增强免疫力、抗衰老等。汞是剧毒元素,能对人体免疫系统、肾脏、肺和神经系统产生毒理效应,且和许多疾病密切相关。贵州省万山特区是汞暴露地区,由于自然和人为的因素,附近居民面临汞带来的健康风险和增加的氧化性损伤。众所周知,硒能对汞产生拮抗作用,并减少汞在体内的积累。由于硒在体内含量较少,且具有各种不同的形态,所以能否精确测量硒成为一个关键问题。本文开展了硒形态的分析方法研究,并将该方法运用于探究贵州汞暴露地区补硒人群血清中硒形态分布情况及补硒后的生物效应。
(1)本文优化了阴离子交换高效液相色谱与电感耦合等离子体质谱联用同时测定不同硒的形态的在线分离检测方法,并将其应用于长期汞暴露人群血清中小分子硒形态的研究。采用不同浓度柠檬酸铵及甲醇作流动相,流动相A为0.5 mM柠檬酸铵溶液+2%(Ⅴ/Ⅴ)甲醇,pH值3.7;流动相B为100 mM柠檬酸铵溶液+2%(Ⅴ/Ⅴ)甲醇,pH值8.0;当利用浓度梯度93% A + 7% B的流动相时,SeCys、Se(Ⅳ)、SeMet、Se(Ⅵ)的保留时间分别为2.48 min,3.28 min,5.19 min,7.02 min,四种硒的形态能完全分离。采用外标法定量,加标回收率在93.0%~117.0%之间,相对标准偏差小于9.0%,硒代半胱氨酸、四价硒离子、硒代蛋氨酸、六价硒离子的检出限分别为0.34μg/L、0.67μg/L、1.38μg/L、0.63μg/L (S/N=3)。本方法简单、快速、重现性好,适合于各种生物样品中含硒小分子的形态分析。本方法已成功应用于研究长期汞暴露地区硒干预人群中血清中硒的化学形态,结果表明血清中小分子硒主要是Se(Ⅳ),补硒后血清中总硒及Se(Ⅳ)含量均增加。
(2)本文提出了一种对生物样品中硒形态进行全定量分析的新方法。通过利用反相和亲合色谱结合电感耦合等离子体质谱联用技术,并结合在线同位素稀释法和碰撞池技术的HPLC-ICP-MS联用技术,成功分离和测定了汞暴露地区硒干预人群血清中硒形态,包括含硒蛋白(SelP,SeAlb, GPx)和小分子硒(SeCys, SeMet,无机硒);与此同时,对连续半年每天服用富硒酵母补硒100ug的汞暴露人群体内血清中硒形态动态分布进行了研究。结果表明:SelP占总硒含量的一半以上,且随着体内总硒含量的增加而增加。连续十次重复测定得出SelP的相对标准偏差为6.0%,SeAlb的相对标准偏差为5.0%。检测限分别为: GPx和其他未保留的硒(0.1μg Se L~(-1) ), SelP (1.0μg Se L~(-1) ), SeAlb (1.2μg Se L~(-1) ),无机硒(1.3μg Se L~(-1)),SeCys (1.2μg Se L~(-1)), SeMet (1.1μg Se L~(-1))。
(3)本研究探索了柱后同位素稀释法高效液相色谱电感耦合等离子体质谱测定含硒蛋白中汞含量的分析方法。SelP中汞的相对标准偏差为5.0%,检测限为1.2μg Hg L~(-1)。结果表明,硒对汞有抑制作用,补硒能减少汞在体内的积累。
Selenium is recognized as the necessary nutritional element for human health, which plays many important physiological and biochemical roles in the body, such as anti-oxygenic property, improvement of immunity, prevention of human aging etc. As a highly hazardous element, mercury can toxicly influence the immune system, kidneys, lungs, and nervous tissues. So it is linked with a number of human health diseases. The town of Wanshang in Guizhou was once the major Hg-mining area in china. Owing to the natural and anthoropogenic factors, people living in the area are suffering from the exposure to elevated Hg and increased oxidative damage. As is well known, Se can antagonized Hg and redcuce the accumulation of Hg in body. Accurate determination of selenium species in bilolgical samples is a critical issue because selenium commonly presents at low levels and in diverse species in body. Therefore, methods for exploring selenium species were established and applied to evaluate the distribution of selenium species in serum and their biological effects after the mercury-exposed volunteers in Guizhou had taken selenium supplement.
(1) A on-line detecting technique, namely, anion exchange (AE)-inductively coupled plasma mass spectrometry (ICP-MS), was optimized to simultaneously determine different selenium species, including Se(Ⅳ), Se(Ⅵ), selenocysteine (SeCys) and selenomethionine (SeMet),. Ammonium citrate and methanol were used as mobile phase. Mobile phase A is composed of 0.5 mmol/L ammonium citrate and 2% (Ⅴ/Ⅴ) methanol. Mobile phase B consists of 100 mmol/L ammonium citrate and 2% (Ⅴ/Ⅴ) methanol. When 93% A + 7% B were used, the four kinds of Se species can be separated within 7.5 min. The detection limits for SeCys, Se(Ⅳ), SeMet and Se(Ⅵ) were 0.34μg/L, 0.67μg/L, 1.38μg/L and 0.63μg/L, respectively, according to 3 times standard deviation of the blank criterion. The recoveries of standard addition of Se(Ⅳ) in serum samples ranged from 93% to 117%, and the relatively standard deviations were less than 9% (n=3).Apparently, this simple, rapid and repeatable method is suitable for analyzing selenium speciations of small selenium-containing moleculars in various biological samples. We successfully applied this method to analyze serum samples from long-term mercury-exposed people who had taken selenium-enriched yeast supplement. The results show that Se of small moleculars in serum mainly presents in the form of Se(Ⅳ) and the concentrations of the total Se and Se(Ⅳ) in serum samples increase when people took Se supplementation.
(2) In this study, a novel method was established for full quantitativly analyzing the species of selenium. The combination of RP, AF chromatography and ICP-MS coupled with on-line isotope dilution analysis (IDA) and a collision cell technique (CCT), can separate and quantify Se-containing proteins (SelP, SeAlb, GPx), and small selenocompounds like SeCys, SeMet, inorganic Se in serum from Hg-exposed people after Se supplementation. Meanwhile, it was used to qualitatively and quantitatively study the dynamic distribution of Se species in serum samples from the Hg--exposed people, who had a daily Se supplementation of 100μg by continuously taking Se-enriched yeast for 180 days. The results show that SelP takes up more than half of the total Se and increases with the increase of the total Se in body. The repeatability in terms of relative standard deviation (R.S.D. %, n=10) is 6.0% for GPx and SelP, and 5.0% for SeAlb. The detection limits are 0.1μg Se L~(-1) for GPx and other non-retained Se compounds, 1.0μg Se L~(-1) for SelP and 1.2μg Se L~(-1) for SeAlb, 1.3μg Se L~(-1) for inorganic Se; 1.2μg Se L~(-1) for SeCys; 1.1μg Se L~(-1) for SeMet, respectively.
(3) Post-column isotope dilution analysis by HPLC-ICP-MS was explored to determine mercury content in selenium-containing proteins. The relative standard deviation is 5.0% for Hg in SelP and the detection limits is 1.2μg Hg L~(-1).It is found that selenium can antagonize the toxicity of mercuy in mammals and selenium supplement can reduce the accumulation of mercury in body.
引文
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