Spotlight on: bile acids

What are bile acids?

Bile acids are cholesterol-derived molecules synthesized in the liver that function as amphipathic surfactants and systemic endocrine hormones. Once considered mere dietary surfactants, they have recently been identified as critical modulators of macronutrient metabolism as well as systemic pro-inflammatory/anti-inflammatory balance⁽¹⁾. We know now bile acids play multifaceted roles in human physiology, health and disease. The wider impact of these diverse molecules in cancer research, drug development, cardiovascular health, nutrition, obesity and exposomics makes them a focus of ongoing research.

There are two primary bile acids found in humans: cholic acid (CA) and chenodeoxycholic acid (CCDA), which get conjugated by the liver with either glycine or taurine to form a total of eight possible conjugated bile acids. These conjugated bile acids are commonly referred to as bile salts and are water-soluble, thereby used to emulsify fats. Bile salts can then be dehydroxylated by gut bacteria, giving rise to secondary bile acids, deoxycholic acid and lithocholic acid. Remarkably, all four of these bile acids get recycled!

What are the challenges with analysis by LC-MS?

Bile acids are a complex class of molecules with diverse structures and properties, making their analysis intricate. Because they exist in so many various forms, including free bile acids, conjugated bile acids, and sulfated or glucuronidated bile acids, this structural diversity requires careful method development to ensure comprehensive detection and quantitation.

Some of the challenges of analyzing bile acids by LC-MS are because many bile acids are structural isomers, differing only in the position of hydroxyl groups on the steroid nucleus. Differentiating these isomers can be technically quite demanding. Not only this, but they are often present at very low concentrations, which requires very sensitive detection techniques. Additionally, biological samples are often very complex, containing numerous other compounds that can interfere with the detection of bile acids, giving rise to matrix effects.

Why are bile acids so interesting?

Until recently, the biochemistry of these molecules was relatively poorly understood. But thanks to the rising visibility of the microbiome's importance in the gut-brain axis, renewed interest has prompted many new studies.

This broad interest in bile acids stems from their central role in various biological processes and their potential as therapeutic targets and biomarkers. The interdisciplinary nature of bile acid research underscores their physiological importance and has been driving collaborative efforts across multiple scientific domains.

Understanding bile metabolism through accurate quantitation of bile acid isomers is paving the way for unprecedented metabolomics insights, with implications spanning from metabolic disorders to new therapeutic development.

Using LC-MS for testing bile acids

Liquid chromatography-mass spectrometry (LC-MS/MS) is the gold standard for analyzing bile acid profiles in biological samples. Two main strategies are used to achieve comprehensive, targeted detection and quantitation of bile acids in human plasma.

The first uses a triple-quadrupole MS-based approach that provides highly sensitive quantitation of bile acids using highly resolved chromatographic separation and an internal standard strategy. The second strategy relies on high-resolution mass spectrometry (HR-MS) to provide high-resolution full product ion spectra for each targeted bile acid, and to help reduce background chemical interferences and improve the signal-to-noise (S/N) of the assay.

Recently commercialized by SCIEX, electron activated dissociation (EAD) is a novel fragmentation technology only available on the ZenoTOF 7600 and ZenoTOF 7600+ systems that can help distinguish bile acid isomers in human plasma, removing the need for extensive chromatographic separations and laborious method development.

Some facts about bile acids

Amphipathic: these fascinating molecules have both hydrophilic (water-loving) and hydrophobic (fat-loving) properties, allowing them to act as effective, natural detergents in the body
Regulators: Bile acids are responsible for over 50% of the daily turnover of cholesterol. If they aren’t functioning properly, your cholesterol levels can rise – particularly the “bad” cholesterol known as LDL (low-density lipoprotein)
Activators: After synthesis, bile acids are secreted into bile and concentrated for storage in the gallbladder. Eating stimulates cholecystokinin release, causing gallbladder contraction and the release of bile acids
Complex: Circulating bile acid composition is quite complicated due to their having two very different mechanisms of formation. One input comes from bile acids formed from cholesterol in the hepatocyte, and the other is by formation via bacteria in the colon
Duality: Bile acids promote the elimination of cholesterol by inducing biliary lipid secretion and solubilizing the cholesterol in bile. Whereas in the small intestine, bile acids solubilize dietary lipids promoting their absorption
Cytotoxic: When present in abnormally high concentrations, bile acids can be cytotoxic. This can happen both intracellularly, as occurs in the haptocyte in cholestasis (disrupted bile flow to the intestines), or extracellularly, as occurs in the colon with patients that have malabsorption of bile acids

SCIEX, there where it counts

Dynamic range: bile acids can be present at very low concentrations with a very broad dynamic range. This requires very highly sensitive detection methods
Sensitivity: The flexibility to run electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) on the same high-sensitivity LCMS can address bile acid's wide variety of ionization efficiencies
Quantitation: accurate quantitation requires suitable calibration and internal standards. Isotopically labeled
Flexibility: accurate mass spectrometry can aid in differentiating structural isomers with high-resolution