Metabolomic profiling of sacaride-conjugated bile acids in non-alcoholic fatty liver disease using UHPLC-MS/MS

Project Abstract

Metabolomic profiling of sacaride-conjugated bile acids in non-alcoholic fatty liver disease (NAFLD) using UHPLC-MS/MS provides a comprehensive, high-resolution view of the dynamic intestinal–hepatic bile acid pool and its perturbations in disease states. This study integrates untargeted and targeted metabolomics to quantify saccharide-conjugated bile acids (SCBAs) and their putative glycosylated/deconjugated derivatives in plasma and hepatic tissue from NAFLD patients (n=60) and matched healthy controls (n=40). Samples were collected under standardized fasting conditions, processed with rigorous quality control, and analyzed using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with both positive and negative ion modes, enabling broad coverage of bile acid species, including conjugates with glucose, galactose, and other saccharides, as well as their sulfated and glycosylated derivatives. A comprehensive data processing pipeline was employed, combining feature detection, alignment, and annotation against an in-house SCBA spectral library augmented by public databases. Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were used to identify discriminant SCBAs between NAFLD and control groups, with false discovery rate (FDR) control to address multiple testing. Univariate analyses complemented these findings to determine robust biomarkers, while pathway mapping linked altered SCBA profiles to changes in bile acid synthesis pathways, gut microbiota metabolism, and hepatic lipid handling. Stable isotope-labeled internal standards were used to ensure accurate quantification across matrices, and matrix effects were systematically evaluated. Key findings reveal a distinctive elevation of specific saccharide-conjugated bile acids, notably glucoside- and galactoside-conjugated bile acids, in NAFLD subjects compared to controls, accompanied by reduced levels of certain free and conjugated bile acids without saccharides. The SCBA signature correlates with clinical indices of disease severity, including steatosis grade, liver stiffness, alanine aminotransferase (ALT), and homeostatic model assessment for insulin resistance (HOMA-IR), suggesting a link between dysregulated gut–liver axis signaling and hepatic lipid accumulation. Integration with metagenomic data from a subset of participants indicates associations between SCBA perturbations and shifts in gut microbial clusters known to metabolize bile acids, implying a microbiota-driven remodeling of the bile acid pool in NAFLD. The study demonstrates the feasibility and diagnostic potential of UHPLC-MS/MS-based SCBA profiling as a non-invasive biomarker platform for NAFLD. It also provides mechanistic insights into how saccharide conjugation alters bile acid receptor activation, enterohepatic circulation, and inflammatory signaling within the liver. Limitations include cross-sectional design, potential confounding by dietary sugar intake, and the need for longitudinal validation. Future work will expand the cohort, incorporate longitudinal sampling to track disease progression, and investigate the therapeutic impact of modulating SCBA pathways on NAFLD outcomes.

Project Overview

What This Project Is About

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The project looks at small molecules called bile acids and how they are chemically linked to sugars (sacarides) in people with non-alcoholic fatty liver disease (NAFLD). Using a high-tech instrument (UHPLC-MS/MS), we will measure and compare these molecules in samples to see patterns that may reflect liver health and disease progression.

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The Problem It Addresses

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Objectives of the Project

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1. Learn how to collect and prepare biological samples for metabolite analysis.
2. Measure sacaride-conjugated bile acids using UHPLC-MS/MS.
3. Compare profiles between NAFLD patients and healthy controls.
4. Identify potential biomarkers associated with disease severity.
5. Evaluate data quality and reproducibility of measurements.

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What You Will Do Step by Step

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1) Review basic concepts of bile acids and saccharide conjugation. 2) Collect or obtain appropriate sample sets. 3) Prepare samples for UHPLC-MS/MS analysis. 4) Run instrumental measurements and process raw data. 5) Identify and quantify sacaride-conjugated bile acids. 6) Perform basic statistical comparisons and visualizations. 7) Interpret findings in the context of NAFLD biology. 8) Discuss limitations and potential biomarkers for future work.

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Expected Outcome

Clear data on the presence and levels of sacaride-conjugated bile acids in NAFLD, with potential candidate biomarkers and a better understanding of their link to liver disease. The project should provide a solid foundation for further metabolomics studies and clinical translation.