Ultra-performance chromatography-electrospray tandem mass spectrometry analysis of bile acid profiles in the enterohepatic circulation following geniposide and acetaminophen-induced liver injury

Abstract

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.