Using targeted LC‐MS/MS‐based metabolomics to measure a broad constellation of bile acids/salts in disorders of human health

Abstract

BackgroundBile is an important liver secretion which aids the digestion of fats in the intestine. Bile acids are the major organic solute component of bile and are synthesized from cholesterol in the liver and stored in the gallbladder. After secretion in the intestine, bile acids are re‐absorbed in the distal small intestine (ileum). Imbalances in the reabsorption of bile acids can lead to bile acid diarrhea when bile acid malabsorption occurs. Conversely, when there is an increase in bile acid absorption hepatic disorders such as nonalcoholic fatty liver disease can arise. A wide array of distinct bile acid structures have been identified in vertebrates. Developing and refining experimental tools to identify the role of bile acids in health and disease will enhance our ability to treat complex disorders impacting multiple organ systems.Methods & ResultsWe have expanded our existing reverse‐phase liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐based targeted metabolomics method to accommodate a simple‐to‐use standards kit that includes reference standards for 16 bile acids/salts and their corresponding deuterated isotopologs as internal standards. LC‐MS/MS method enhancements include scheduled Selected‐Reaction Monitoring (sSRM) to reduce instrument duty cycle, and carefully tailored chromatographic conditions to resolve the following groups of structurally similar or isobaric bile acids/salts: i) Glycocholic Acid (GCA) and Taurocholic Acid (TCA); ii) Lithocholic Acid (LCA), Glycolithocholic Acid (GLCA), and Taurolithocholic Acid (TLCA); iii) isobaric Cholic Acid (CA) and beta‐Muricholic Acid (b‐MCA); iv) isobaric Ursodeoxycholic Acid (UDCA), Chenodeoxycholic Acid (CDCA), and Deoxycholic Acid (DCA); v) isobaric Glycoursodeoxycholic Acid (GUDCA), Glycochenodeoxycholic Acid (GCDCA), and Glycodeoxycholic Acid (GDCA); and vi) isobaric Tauroursodeoxycholic Acid (TUDCA), Taurochenodeoxycholic Acid (TCDCA), and Taurodeoxycholic Acid (TDCA). Our tissue processing and metabolite extraction procedures have been optimized for hepatic and fecal tissues. Individuals with inactivating mutations in the molecular motor, Myosin Vb, have severe diarrhea and often present with liver cholestasis. We used an animal model of loss of Myosin Vb to determine alterations in bile acid composition in the distal small intestine and liver. Whole segments of the ileum and liver were processed in methanol and analyzed by LC‐MS/MS.ConclusionWe have developed a targeted metabolomics method to accurately identify and quantify bile acids by LC‐MS/MS and this technique can be used to investigate changes in bile acid composition in gastrointestinal disorders.