Using Multidimensional Separations to Distinguish Isomeric Amino Acid–Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems

Abstract

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid–bile acid (AA–BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC–IMS–MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Offline Two-Dimensional Liquid Chromatography–Mass Spectrometry for Deep Annotation of the Fecal Metabolome Following Fecal Microbiota Transplantation

“…The differential compounds include bile acids, amino acids, lipids, and several previously unreported metabolites. Our work complements a recent manuscript by Stewart et al . that studied FMT samples from the same clinical trial, and includes data from four additional subjects.…”

“…MCBAs were first identified in samples collected from the small intestine and are expected to have higher concentration in that location within the gut. Furthermore, MCBAs are relatively low-abundance compounds, reported as generating approximately three times lower signal than canonical bile acids in human fecal extracts . Nevertheless, we confirmed the identification of 16 of the 22 MCBAs reported by Shalon et.…”

“…All patients were enrolled under IRB #16–2283 at the University of North Carolina Hospital . Fecal samples were collected pre-FMT and 2 weeks post-FMT for eight patients with prior unsuccessful antibiotic treatment for rCDI.…”

Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrentClostridioides difficileinfection

“…Specifically, the library included 35 unique BA standards purchased from Cambridge Isotope Laboratories (CIL, Tewksbury, MA) and Cayman Chemical (Ann Arbor, MI), and an additional 173 MCBAs. 45 The synthesis and evaluation of MCBA standards were conducted by Quinn et al 61 , employing the methodologies established by Ezawa et al 73 The BAs and MCBAs as listed in Table S8 and the full library can be found at https://panoramaweb.org/rT2GQI.url in Panorama Public 74,75 (MacCoss lab, Seattle, WA).…”

“…However, recent studies suggest that BSHs have the ability to reverse this reaction and reconjugate bile acids with a variety of other amino acids thereby creating microbially conjugated bile acids (MCBAs), also referred to as bacterial bile acid amidates (BBAAs) 43,44 . MCBAs have previously been identified in a small sample set of rCDI patients treated with FMT by our group 45 . In a recent study, a BSH cocktail given to mice increased MCBAs and was able to restrict C. difficile growth in vivo 46 .…”