Characterization of Metabolic Correlations of Ursodeoxycholic Acid with Other Bile Acid Species through In Vitro Sequential Metabolism and Isomer-Focused Identification

As a first-line agent for cholestasis treatment in a clinic, ursodeoxycholic acid rectifies the perturbed bile acids (BAs) submetabolome in a holistic manner. Considering the endogenous distribution of ursodeoxycholic acid and extensive occurrences of isomeric metabolites, it is challenging to point out whether a given bile acid species is impacted by ursodeoxycholic acid in a direct or indirect manner, thus hindering the therapeutic mechanism clarification. Here, an in-depth exploration of the metabolism pattern of ursodeoxycholic acid was attempted. Sequential metabolism in vitro with enzyme-enriched liver microsomes was implemented to simulate the step-wise metabolism and to capture the metabolically labile intermediates in the absence of endogenous BAs. Squared energy-resolved mass spectrometry (ER2-MS) was utilized to achieve isomeric identification of the conjugated metabolites. As a result, 20 metabolites (M1-M20) in total were observed and confirmatively identified. Of those, eight metabolites were generated by hydroxylation, oxidation, and epimerization, which were further metabolized to nine glucuronides and three sulfates by uridine diphosphate-glycosyltransferases and sulfotransferases, respectively. Regarding a given phase II metabolite, the conjugation sites were correlated with first-generation breakdown graphs corresponding to the linkage fission mediated by collision-induced dissociation, and the structural nuclei were identified by matching second-generation breakdown graphs with the known structures. Together, except for intestinal-bacteria-involved biotransformation, the current study characterized BA species directly influenced by ursodeoxycholic acid administration. Moreover, sequential metabolism in vitro should be a meaningful way of characterizing the metabolic pathways of endogenous substances, and squared energy-resolved mass spectrometry is a legitimate tool for structurally identifying phase II metabolites.

Automated summary

As a primary treatment for cholestasis, ursodeoxycholic acid corrects the imbalances in bile acid metabolism in a comprehensive way. The complex metabolism of ursodeoxycholic acid makes it difficult to determine whether a specific bile acid species is directly or indirectly impacted by the drug, which hinders the understanding of its therapeutic mechanism. In this study, the metabolism pattern of ursodeoxycholic acid was explored using liver microsomes. Through sequential metabolism in vitro, 20 metabolites were identified, including hydroxylated, oxidized, and epimerized products that were further metabolized to glucuronides and sulfates. The conjugation sites and structural nuclei of the metabolites were determined using mass spectrometry. This study provides insight into the direct effects of ursodeoxycholic acid administration on bile acid species and highlights the utility of sequential metabolism in vitro and squared energy-resolved mass spectrometry in characterizing metabolic pathways and identifying metabolites.