Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 10050, Taiwan.
Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei 10050, Taiwan.
Chem Biol Interact. 2015 Jul 25;237:38-46. doi: 10.1016/j.cbi.2015.05.002. Epub 2015 May 15.
Acrylamide (AA) is reported present in high-temperature-processed food and classified as a possible human carcinogen. In vivo metabolic activation of AA by CYP 2E1 to glycidamide (GA) may play an important role on AA carcinogenicity. AA and GA can be detoxified by glutathione-S-transferase to form AA and isomeric GA glutathione conjugates (AA-, GA2- and GA3-GSH, respectively), which can be further metabolized to mercapturic acids (MAs). Although many studies analyzed MAs in urine of rodents and humans, few studies have characterized and analyzed the GSH conjugates. The objectives of this study were to synthesize, purify, and characterize AA-GSH, GA2-GSH, GA3-GSH, ((13)C3)-AA-GSH, ((13)C3)-GA2-GSH, and ((13)C3)-GA3-GSH to develop an isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method to analyze AA- and GA-GSHs in blood of rats treated with AA. After purification and characterization of these conjugates, the LC-MS/MS method was developed and validated. This method reveals a limit of detection (S/N=3) at 0.017 and a limit of quantitation (S/N=10) at 0.05ng/mL of serum for AA-GSH, 0.075 and 0.25ng/mL for GA2-GSH, and 0.15 and 0.5ng/mL for GA3-GSH. Analyzed with this method, AA-GSH, GA2-GSH and GA3-GSH were 1651.1±374.5, 18.4±6.3 and 75.3±31.3ng/mL in blood of male rats at 2h after treatment with 5mg/kgbw of AA by ip injection. These results showed that the LC-MS/MS method was successfully developed to analyze AA-GSH, GA2-GSH and GA3-GSH with satisfying sensitivity of AA and GA which were conjugated by glutathione in vivo.
丙烯酰胺（AA）存在于高温加工食品中，被归类为可能的人类致癌物质。AA 在体内通过 CYP2E1 代谢激活为缩水甘油酰胺（GA），可能在 AA 的致癌性中起重要作用。AA 和 GA 可以被谷胱甘肽-S-转移酶解毒形成 AA 和异构 GA 谷胱甘肽轭合物（分别为 AA-、GA2-和 GA3-GSH），然后可以进一步代谢为硫醚氨酸（MA）。尽管许多研究分析了啮齿动物和人类尿液中的 MA，但很少有研究对 GSH 轭合物进行特征分析和分析。本研究的目的是合成、纯化和表征 AA-GSH、GA2-GSH、GA3-GSH、((13)C3)-AA-GSH、((13)C3)-GA2-GSH 和 ((13)C3)-GA3-GSH，以开发一种同位素稀释液相色谱串联质谱法 (LC-MS/MS) 来分析 AA 处理大鼠血液中的 AA 和 GA-GSH。在对这些轭合物进行纯化和表征后，开发并验证了 LC-MS/MS 方法。该方法显示，对于 AA-GSH，血清的检测限（S/N=3）为 0.017，定量限（S/N=10）为 0.05ng/mL；对于 GA2-GSH，检测限为 0.075，定量限为 0.25ng/mL；对于 GA3-GSH，检测限为 0.15，定量限为 0.5ng/mL。通过该方法分析，雄性大鼠经腹腔注射 5mg/kgbw AA 2 小时后，血液中的 AA-GSH、GA2-GSH 和 GA3-GSH 分别为 1651.1±374.5、18.4±6.3 和 75.3±31.3ng/mL。这些结果表明，成功开发了 LC-MS/MS 方法来分析体内谷胱甘肽结合的 AA 和 GA 的 AA-GSH、GA2-GSH 和 GA3-GSH，具有令人满意的灵敏度。
