Synthesized in the liver, bile acids play diverse roles in digestion and metabolism. However, when their processes are disrupted, disease can occur. Scientists seek advanced technologies to better understand these metabolites and their influence on health.

In this Innovation Spotlight, Thomas Horvath, an analytical chemist at Baylor College of Medicine, and Paul Baker, a lipidomics and metabolomics scientist at SCIEX, explore the complexities of bile acid research and its potential effect on human health with Amy Engevik, a cell biologist at the Medical University of South Carolina, Maxim Seferovic, a molecular biologist at Baylor College of Medicine, and Donald Chace, a laboratory director at Capitainer Inc. Here, these experts share their insights into the biochemical intricacies, analytical challenges, and diagnostic potential of bile acids in human disease.

What are bile acids?

Thomas Horvath: Bile acids are cholesterol-derived compounds essential for digesting lipids and regulating metabolic pathways. They traverse a biochemical circuit known as enterohepatic circulation, moving from the liver to the gut, where they undergo transformations through host and microbial interactions, resulting in a complex range of metabolites. This intricate process allows bile acids to function beyond digestion as metabolic regulators. However, disruptions in this circuit can lead to gastrointestinal diseases, making bile acids a critical focus in medical research.

Why is the analysis of bile acids important for understanding human health?

Thomas Horvath: Disturbances in bile acid profiles are linked to gastrointestinal diseases such as inflammatory bowel disease (IBD), necrotizing enterocolitis, primary sclerosing cholangitis, and biliary atresia.¹ My research—along with Amy Engevik’s research on microvillus inclusion disease (MVID) and Maxim Seferovic’s research on intrahepatic cholestasis in pregnancy (IHCP)—seeks to understand these disorders at a molecular level. Advanced tools such as the ZenoTOF 7600 system from SCIEX allow us to analyze bile acids with unprecedented specificity, unlocking new insights into their roles in human health and diseases.

Why are bile acids so difficult to study? What are the technical challenges?

Thomas Horvath: Bile acids are challenging to study for three main reasons. First, their chemical diversity is extensive, with known bile acid structures numbering in the thousands and new types discovered regularly.² Second, analytical techniques face limitations, including high levels of chemical noise in mass spectrometry (MS) methods, particularly for instruments reliant on precursor-to-precursor ion transitions. Third, bile acid molecules share physicochemical similarities, making it difficult to separate and identify isomers accurately.

Is there a way to enable better bile acid analysis? If so, what is it and how does it help researchers gain the insights they need?

Paul Baker: Traditionally, bile acids are analyzed using triple quadrupole mass spectrometry (TQMS). While sensitive, TQMS instruments lack the specificity required for accurate bile acid differentiation, particularly due to the challenges with bile acid fragmentation, isomeric compounds, and chemical noise. Recently, we have developed an improved high-resolution approach using the ZenoTOF 7600 system, which offers higher specificity by capturing unique fragment ions and bypassing the noise challenges typical in traditional MS. This electron-based fragmentation, known as electron activated dissociation (EAD), provides unprecedented specificity and accuracy. Consequently, we are developing an assay that does not solely depend on chromatography for specificity. A sensitive and specific high-throughput assay that can analyze bile acids in under 10 minutes could be a gamechanger.

What excites you the most about recent advancements in bile acid analysis?

Maxim Seferovic: The technological advances in MS open doors to understanding bile acids beyond their digestive roles. They are now recognized as key metabolites influencing gut and systemic health. By identifying new bile acid metabolites, we are piecing together how these compounds interact with various cellular systems, including the gut microbiome, to affect human health and disease. This progress will help decode the communication pathways between microbiota and host, further illuminating the roles bile acids play in biological systems.

Amy Engevik: I am excited by the growing precision in bile acid analysis, which allows us to map the interactions between bile acids, gut bacteria, and the host. With MS, we can differentiate between bile acid types—primary, secondary, and tertiary—which reveals insights into gastrointestinal disorders. Understanding these distinctions could clarify how bile acid imbalances affect inflammation and immune responses in diseases like IBD.³ Technology such as EAD MS enhances our ability to explore these intricate pathways and their impact on health.

What can researchers learn about gastrointestinal disorders such as IBD, MVID, and IHCP by analyzing bile acids using an approach like MS?

Maxim Seferovic: IHCP is a condition where bile acid levels rise, impacting both mother and child, yet its mechanisms remain largely unknown. The ability to differentiate individual bile acid types using MS provides a new layer of understanding for IHCP and other diseases. With this high-resolution data, we can move beyond total bile acid measurements to assess the effect of specific bile acid variants, which may reveal new therapeutic targets and ways to improve fetal health.⁴

Amy Engevik: In diseases such as MVID and IBD, bile acids play crucial roles. MVID, a rare disease that causes severe diarrhea, is linked to bile acid trafficking mutations.⁵ IBD, a chronic inflammatory disease, can involve disrupted bile acid balance. By profiling these diseases, we can track how bile acids contribute to inflammation and explore targeted therapies that may alleviate symptoms. Using MS, we can profile individual bile acids to identify how their imbalance contributes to inflammation and other symptoms in these diseases. This profiling could lead to bile acid-based therapies for improving fat and bile acid absorption, which can benefit patients with nutrient malabsorption issues.

What does the future hold for your work with MS and bile acid analysis?

Maxim Seferovic: My lab is focused on how maternal diet, drugs, and microbiome changes during pregnancy affect fetal development, with bile acids as a key factor. We analyze specific bile acid variants, potentially linking these metabolites to developmental outcomes. This research may clarify how microbiome-derived bile acids influence fetal growth and long-term health, bridging connections between maternal health and future generations.

Amy Engevik: We are integrating bile acid analysis into our animal models for diseases such as MVID and IBD. Using MS, we can trace how microbial communities interact with bile acids to influence host health. This may reveal novel bile acid derivatives with systemic effects beyond the gut, including neurological outcomes. 

What historical context can you draw upon to predict what the future of bile acid analysis might look like?

Donald Chace: The foundation of modern bile acid analysis was laid in the 1960s with Robert Guthrie’s dried blood spot method for newborn screening,⁶ which was further developed in the 1990s for metabolic disorder diagnosis. Advanced MS capabilities can lead the expansion of bile acid research, as we expand into approaches such as dried fecal spots.⁷