Analysis of Bile Acids

Bile acids are present in numerous biological materials including bile (stored in the gallbladder), liver tissue, cecal contents, and stool. Bile acids are synthesized in the liver from cholesterol, stored in the gallbladder, and secreted into the intestine to aid in digesting fats. Most bile acids are re-absorbed from the intestine and return to the liver via portal circulation. A small percentage remains in the intestine and is excreted in stool.

The primary bile acids synthesized in the liver include cholic acid (CA) and chenodeoxycholic acid (CDCA). Mice generate a primary bile acid called muricholic acid (MCA), which comes in enantiomers designated alpha-MCA and beta-MCA. The last step of synthesis is conjugation to a charged molecule, such as sulfate or an amino acid. The amino acid is typically glycine or taurine, though there is some species dependence; e.g., in mice, bile acids are conjugated to taurine almost exclusively. Examples of conjugated primary bile acids are taurocholic acid (TCA) or glycocholic acid (GCA)

Upon reaching the intestine, bile acids may be metabolized by gut microbes. There are two metabolic transformations that bacteria can perform: deconjugation, or removal of the charged moiety; and dehydroxylation, which produces a more hydrophobic secondary bile acid. The dehydroxylation product of cholic acid is deoxycholic acid (DCA), while that of CDCA is lithocholic acid (LCA). Like primary bile acids, secondary bile acids can also be reabsorbed and returned to the liver, where they may be conjugated to form, e.g., taurodeoxycholic acid (TDCA). A table of bile acid species is provided below (Table 1).

LC-MS/MS can be a powerful technique for measuring bile acid content in biological samples. Because bile acids differ based on conjugation state, number of hydroxyl groups, and isomerism, it is easy to measure individual bile acids based on a combination of retention time in reverse-phase chromatography and m/z value. A tandem mass spectrometer, such as a QQQ, provides further information based on fragmentation of the target molecule, although not all bile acids fragment equally well (hence the need for LC to help make an identification).