Integrated Multi-Matrix Bile Acid Metabolic Metrics (BAMMs): A Methodological Framework for Functional Metabolic Phenotyping in Human Subjects

Traditional single-matrix bile acid (BA) analysis, which typically relies on a limited set of analytes in plasma or feces, often fails to capture the compartmentalized complexity of the microbiome-gut-liver axis. To address this, we developed a high-coverage, 61-analyte metabolomics platform integrated via a multifaceted Bile Acid Metabolic Metrics (BAMMs) framework across plasma, urine, and feces. In a 12-week clinical intervention study (n=13), conventional single-matrix concentration profiling exhibited high inter-individual variance and failed to reflect significant metabolic shifts. In contrast, the BAMMs successfully identified multifaceted metabolic shifts—including carbon-position-specific transformations—that remained obscured in concentration-based analyses. Notable findings include significant decreases in fecal C-3 oxidation (dehydroLCA/LCA) and epimerization (isoLCA/LCA) metrics, alongside a significant increase in urinary C-7 oxidation (7-ketoCA/CA). These results demonstrate that standardized functional ratios are superior to concentrations for resolving the physiological "uncoupling" between the gut lumen and the systemic circulation. A key advantage of our approach is its high extensibility; by disclosing our full calculation methodologies and metabolic ratios, we provide a transparent and adaptable template that researchers can tailor for additional metabolites, such as muricholic acids, or diverse research models. This validated "Three-Matrix Workflow" provides a comprehensive lens for dissecting the compartmentalized dynamics of BA metabolism, offering a scalable and robust tool for future clinical and microbiome-based investigations.