Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Central Laboratory, The Second Hospital of Jilin University, Key Laboratory of Zoonosis Research, Ministry of Education, Jilin University, Changchun, China.
Chem Res Toxicol. 2021 Jan 18;34(1):144-153. doi: 10.1021/acs.chemrestox.0c00454. Epub 2021 Jan 7.
Emerging evidence suggests that chronic exposure to aristolochic acids (AAs) is one of the etiological pathways leading to chronic kidney disease (CKD). Due to the traditional practice of herbal medicine and AA-containing plants being used extensively as medicinal herbs, over 100 million East Asians are estimated to be at risk of AA poisoning. Given that the chronic nephrotoxicity of AAs only manifests itself after decades of exposure, early diagnosis of AA exposure could allow for timely intervention and disease risk reduction. However, an early detection method is not yet available, and diagnosis can only be established at the end stage of CKD. The goal of this study was to develop a highly sensitive and selective method to quantitate protein adducts of aristolochic acid I (AAI) as a biomarker of AA exposure. The method entails the release of protein-bound aristolactam I (ALI) by heat-assisted alkaline hydrolysis, extraction of ALI, addition of internal standard, and quantitation by liquid chromatography-tandem mass spectrometric analysis. Accuracy and precision of the method were critically evaluated using a synthetic ALI-containing glutathione adduct. The validated method was subsequently used to detect dose-dependent formation of ALI-protein adducts in human serum albumin exposed to AAI and in proteins isolated from the tissues and sera of AAI-exposed rats. Our time-dependent study showed that ALI-protein adducts remained detectable in rats even at 28 days postdosing. It is anticipated that the developed method will fill the technical gap in diagnosing AA intoxication and facilitate the biomonitoring of human exposures to AAs.
新出现的证据表明，慢性接触马兜铃酸（AAs）是导致慢性肾病（CKD）的病因之一。由于传统的草药医学实践和作为药用植物的 AA 含量植物被广泛使用，估计有超过 1 亿的东亚人面临 AA 中毒的风险。鉴于 AA 的慢性肾毒性只有在暴露几十年后才会表现出来，因此尽早诊断 AA 暴露可以及时进行干预和降低疾病风险。然而，目前还没有早期检测方法，只能在 CKD 晚期才能确诊。本研究的目的是开发一种高灵敏度和选择性的方法来定量测定马兜铃酸 I（AAI）的蛋白加合物作为 AA 暴露的生物标志物。该方法通过热辅助碱性水解释放蛋白结合的马兜铃内酰胺 I（ALI），提取 ALI，加入内标，并用液相色谱-串联质谱分析定量。使用含有合成 ALI 的谷胱甘肽加合物对方法的准确性和精密度进行了严格评估。随后，该验证方法用于检测 AAI 暴露后人血清白蛋白中剂量依赖性形成的 ALI-蛋白加合物，以及从 AAI 暴露大鼠的组织和血清中分离的蛋白质。我们的时间依赖性研究表明，即使在给药后 28 天，大鼠中仍可检测到 ALI-蛋白加合物。预计该方法将填补诊断 AA 中毒的技术空白，并有助于对人类接触 AA 进行生物监测。
