Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16 Nanxiaojie, Dongzhimen Nei Ave, Beijing, 100700, China.
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16 Nanxiaojie, Dongzhimen Nei Ave, Beijing, 100700, China.
J Chromatogr A. 2022 Sep 13;1680:463417. doi: 10.1016/j.chroma.2022.463417. Epub 2022 Aug 11.
Bile acids (BAs) play an important role in pre-diagnosing drug-induced liver injury (DILI). However, in clinical practice, different types of liver injury are characterized by different pathogeneses and pathological manifestations. Therefore, whether BAs can be used as biomarkers across different DILIs remains unclear. In this study, an ultra-performance chromatography-mass spectrometry (MS)/MS-based technique was developed for the simultaneous quantitative analysis of 31 BAs in the serum, liver, feces, urine, and intestinal contents of rats treated with acetaminophen (APAP) and geniposide to induce liver injury. The total extraction recovery for representative analytes ranged between 80.60% and 99.23% in the serum, urine, liver, feces, and intestinal contents. The correlation coefficients for all standard curves of the different matrices were at least 0.99. Validation of the BA analytical method including selectivity, residue, lower limit of quantification, accuracy, precision, matrix effect, and stability conformed with the biospecimen quality control standards of the Chinese Pharmacopoeia (version 2020). Serum biochemical and pathohistological analyses revealed APAP- and geniposide-induced hepatocellular and cholestatic DILI, respectively, with different effects on BA profiles in the enterohepatic circulation. Metabolomics further revealed that the trends in BA changes in the serum, feces, urine, and intestinal tissues were consistent between the geniposide- and APAP-treated groups. However, in the liver, the total BAs (TBA) concentration increased by 1.70 fold in the geniposide group but decreased by 43% in the APAP group compared with the control group. Multivariate analysis revealed differentially expressed BAs, including TCA, CA, and GCA, which are potential biomarkers for DILI, in the serum, liver, and urine following treatment with geniposide. Interestingly, the differentially expressed BAs in the APAP group were similar to those in the control group. Additionally, the magnitude of changes in the TBA in the urine (3.3 fold and 15.5 fold in the APAP and geniposide groups, respectively) was higher than that in the blood (290 fold and 640 fold in the APAP and geniposide groups, respectively). However, given the BA profiles after geniposide- and APAP-induced liver injury, BAs were found to be more suitable as biomarkers for diagnosing cholestatic liver injury. Overall, the BA assay developed in this study is rapid, simple, accurate, validated, sensitive, and suitable for analyzing the levels and distribution of BAs in various parts of the enterohepatic circulation.
胆汁酸（BAs）在药物性肝损伤（DILI）的早期诊断中发挥着重要作用。然而，在临床实践中，不同类型的肝损伤具有不同的发病机制和病理表现。因此，BAs 是否可以作为不同 DILI 的生物标志物尚不清楚。本研究建立了一种超高效液相色谱-串联质谱（MS/MS）技术，用于同时定量分析乙酰氨基酚（APAP）和栀子苷诱导的大鼠血清、肝脏、粪便、尿液和肠道内容物中 31 种胆汁酸。代表性分析物在血清、尿液、肝脏、粪便和肠道内容物中的总提取回收率在 80.60%至 99.23%之间。不同基质所有标准曲线的相关系数均至少为 0.99。BA 分析方法的验证包括选择性、残留、定量下限、准确性、精密度、基质效应和稳定性，均符合《中国药典》（2020 年版）生物样本质量控制标准。血清生化和组织病理学分析表明，APAP 和栀子苷分别诱导肝细胞性和胆汁淤积性 DILI，对肠肝循环中 BA 谱有不同的影响。代谢组学进一步表明，栀子苷和 APAP 处理组的血清、粪便、尿液和肠道组织中 BA 变化趋势一致。然而，在肝脏中，栀子苷组的总胆汁酸（TBA）浓度增加了 1.70 倍，而 APAP 组则降低了 43%，与对照组相比。多变量分析显示，血清、肝脏和尿液中 TCA、CA 和 GCA 等潜在的 DILI 生物标志物的表达差异较大。有趣的是，APAP 组的差异表达 BA 与对照组相似。此外，尿液中 TBA 的变化幅度（APAP 和栀子苷组分别为 3.3 倍和 15.5 倍）高于血液（APAP 和栀子苷组分别为 290 倍和 640 倍）。然而，鉴于栀子苷和 APAP 诱导的肝损伤后的 BA 谱，BA 被认为更适合作为诊断胆汁淤积性肝损伤的生物标志物。总之，本研究建立的 BA 分析方法快速、简单、准确、验证、灵敏，适用于分析肠肝循环中各种部位的 BA 水平和分布。
