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.

中文翻译：

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通过液相色谱-串联质谱法定量马兜铃酸 I 的蛋白质加合物：一种生物监测马兜铃酸暴露的新方法

新出现的证据表明，长期接触马兜铃酸 (AAs) 是导致慢性肾病 (CKD) 的病因途径之一。由于传统的草药做法和含 AA 的植物被广泛用作药材，估计有超过 1 亿东亚人面临 AA 中毒的风险。鉴于 AA 的慢性肾毒性仅在暴露数十年后才显现出来，AA 暴露的早期诊断可以允许及时干预和降低疾病风险。然而，目前尚无早期检测方法，只能在 CKD 末期确立诊断。本研究的目标是开发一种高度灵敏和选择性的方法来定量马兜铃酸 I (AAI) 的蛋白质加合物作为 AA 暴露的生物标志物。该方法需要通过热辅助碱性水解、ALI 提取、添加内标和液相色谱-串联质谱分析定量来释放蛋白质结合的马兜铃内酰胺 I (ALI)。使用合成的含有 ALI 的谷胱甘肽加合物对该方法的准确度和精密度进行了严格评估。随后使用经过验证的方法检测暴露于 AAI 的人血清白蛋白和从暴露于 AAI 的大鼠的组织和血清中分离的蛋白质中 ALI 蛋白质加合物的剂量依赖性形成。我们的时间依赖性研究表明，即使在给药后 28 天，大鼠体内仍可检测到 ALI 蛋白加合物。预计所开发的方法将填补诊断 AA 中毒的技术空白，并促进人体暴露于 AA 的生物监测。