Alava Pradeep, Tack Filip, Laing Gijs Du, de Wiele Tom Van
Laboratory of Analytical Chemistry and Applied Ecochemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
Biomed Chromatogr. 2012 Apr;26(4):524-33. doi: 10.1002/bmc.1700. Epub 2011 Sep 8.
Inorganic arsenic (iAs) has been classified as a type 1 carcinogen and has also been linked to several noncancerous health effects. Prior to 1995, the As(V) methylation pathway was generally considered to be a detoxification pathway, but cellular and animal studies involving MMA(III) (mono metyl arsonous acid) and DMA(III) (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs, suggesting an activation process. In addition, thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies, for which the toxicological profile was not yet fully characterized in human cells. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. This presystemic metabolism necessitates the development of protocols that enable the detection of not only inorganic arsenic species, but also pentavalent and trivalent methylated, thiolated arsenicals in a gastrointestinal environment. We aim to study the biotransformation of arsenic (As) using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME). To be able to analyze the arsenicals resulting from biotransformation reactions occurring in this system, a method using liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer (HPLC-ICP-MS) was developed. A Hamilton PRP-X100 anion exchange column was used. The method allowed separation, identification and quantification of As(III) (arsenite), As(V) (arsenate), DMA(V) (dimethylarsinicacid), MMA(V) (monomethylarsonicacid) and MMMTA (monomethylmonothioarsenate). Attempts to optimize the same method for also separating MMA(III) and DMA(III) did not succeed. These compounds could be successfully separated using a method based on the use of a Zorbax C₁₈ column. The properties of the column, buffer strength, pH and polar nature of mobile phase were monitored and changed to optimize the developed methods. Linearity, sensitivity, precision, accuracy and resolution of both methods were checked. The combination of the two methods allowed successful quantification of arsenic species in suspensions sampled in vitro from the SHIME reactor or in vivo from the human colon and feces.
无机砷（iAs）已被列为1类致癌物，还与多种非癌性健康影响有关。1995年之前，As（V）甲基化途径通常被认为是一种解毒途径，但涉及MMA（III）（一甲基胂酸）和DMA（III）（二甲基胂酸）的细胞和动物研究表明，它们的毒性等于或超过无机砷，这表明存在一个活化过程。此外，在一些研究中，口服无机砷后在尿液中观察到了硫醇化砷代谢物，其毒理学特征在人体细胞中尚未完全明确。研究表明，肠道环境中的微生物是砷形态变化的重要促成因素。这种系统前代谢需要开发能够检测胃肠道环境中不仅有无机砷物种，还能检测五价和三价甲基化、硫醇化砷化合物的方案。我们旨在使用人体肠道微生物生态系统模拟器（SHIME）研究砷（As）的生物转化。为了能够分析该系统中发生的生物转化反应产生的砷化合物，开发了一种将液相色谱与电感耦合等离子体质谱联用（HPLC-ICP-MS）的方法。使用了汉密尔顿PRP-X100阴离子交换柱。该方法能够分离、鉴定和定量As（III）（亚砷酸盐）、As（V）（砷酸盐）、DMA（V）（二甲基胂酸）、MMA（V）（一甲基胂酸）和MMMTA（一甲基单硫代胂酸盐）。尝试优化同一方法以分离MMA（III）和DMA（III）未成功。使用基于Zorbax C₁₈柱的方法可以成功分离这些化合物。监测并改变柱的性质、缓冲强度、pH值和流动相的极性以优化所开发的方法。检查了两种方法的线性、灵敏度、精密度、准确度和分辨率。这两种方法的结合使得能够成功定量从SHIME反应器体外采样的悬浮液或从人体结肠和粪便体内采样的悬浮液中的砷化合物。
