Bile Acid, Bile Metabolism Research Strategy

1. Introduction to bile acids

Bile acids are exclusively synthesized in the liver, the only organ harboring the enzymes required for bile acid synthesis. They can be categorized into two groups based on their structure: free and conjugated bile acids. The former include cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithophanic acid, while the latter are produced by conjugating free bile acids with glycine or taurine, consisting mainly of glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid. Alternatively, bile acids can also be classified as primary or secondary bile acids based on their origin. Primary bile acids, including cholic acid and chenodeoxycholic acid, and their conjugation products with glycine or taurine are directly synthesized from cholesterol in hepatocytes. Secondary bile acids are produced by intestinal bacteria via 7α-hydroxydeoxygenation of primary bile acids, which primarily includes deoxycholic acid, lithotrizoic acid, and their conjugation products with glycine or taurine.

 Figure from Luo, W., Guo, S. et al. "Hepatocellular carcinoma: Novel understandings and therapeutic strategies based on bile acids (review)". International Journal of Oncology vol. 61,4 (2022). doi.org/10.3892/ijo.2022.5407 

2. The synthesis and physiological functions of bile acids

The human understanding of bile acids can be traced back nearly 3,000 years when animal bile was widely used in traditional Chinese medicine. Since the 19th century, scientists have conducted extensive research on bile acid synthesis, which occurs through two pathways: the classical pathway initiated by the microsomal cytochrome P450 cholesterol 7α-hydroxylase (CYP7A1) and the alternative pathway initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In humans, the classical pathway accounts for at least 75% of bile acid production and is considered the predominant pathway. Bile acids possess a variety of physiological functions, namely:

(1) Facilitate the digestion and absorption of lipids

Bile acids are amphiphilic molecules containing hydrophilic hydroxyl and carboxyl groups, as well as hydrophobic alkyl groups. This unique structure gives them strong interfacial activity, allowing them to reduce the interfacial tension between oil and water phases and promote lipid emulsification. Additionally, bile acids increase the contact surface between lipids and lipases, speeding up lipid digestion.

(2) Regulate metabolism as signaling molecules

Bile acids mediate downstream signaling by recognizing multiple receptors, including the farnesol X receptor (FXR), pregnane X receptor, and vitamin D receptor. FXR, the first identified bile acid receptor, is essentially a transcription factor that triggers transcriptional changes upon binding to bile acids. Research indicates that FXR signaling has various physiological roles, including regulating bile acid synthesis, transport, energy metabolism, and immune responses in a feedback loop. Among cell membrane receptors, TGR5 (also known as GPBAR1) is the primary receptor recognized by bile acids. In addition to TGR5, bile acids can activate sphingosine-1-phosphate receptor 2, which is also a G protein-coupled receptor.

(3) Protect the liver, promote bile secretion and excretion, and enhance the body's immune system

Bile acids prevent the formation of cholesterol stones by inhibiting the precipitation of cholesterol in the bile. The liver is a vital metabolic organ responsible for digestion, nutrient absorption, and detoxification. Liver impairments or diseases can cause metabolic abnormalities that lead to digestive and absorption disturbances. Incorporating bile acids into the daily diet can boost the body's immunity. Specifically, deoxycholic acid, when consumed in appropriate quantities, binds with endotoxins and promotes their degradation, ultimately protecting the liver's health. Moreover, deoxycholic acid and ursodeoxycholic acid can help clear the biliary tract, promote bile secretion by liver cells, and eliminate bile stagnation.

(4) Regulate intestinal flora

Bile acids exhibit potent antimicrobial properties that enable them to modulate the body's intestinal microbiota and impede the growth of harmful intestinal bacteria like Escherichia coli, Salmonella, and Streptococcus coli. Research indicates that bile acids can reduce the number of harmful bacteria and decrease plasma endotoxin levels in the terminal ileum of cirrhotic mice, thereby promoting their well-being. Furthermore, grass carp bile acids have been found to be highly effective in inhibiting the growth of Gram-positive bacteria, highlighting their potential antimicrobial properties.

3. Bile acids and intestinal flora in various diseases

(1) Disorders

Metabolic imbalances in the gut microbiota-bile acid axis can lead to heightened levels of secondary bile acids, which activate signaling pathways in the intestinal epithelium, resulting in visceral hypersensitivity (VH), compromising the intestinal mucosal barrier function, and triggering the development of IBS-D. Decreased expression of FXRs not only amplifies NGF/TRPV1 signaling, resulting in VH, but also downregulates FGF19/15, hampering intestinal barrier function and promoting autophagy. Additionally, increased secondary bile acids stimulate TGR5 on endothelial cells, elevating 5-hydroxytryptamine (5-HT) and CGRP levels and augmenting colonic motility. The increased 5-HT in turn upregulates the release of 5-HT3R, leading to VH and transmitting the stimulus to the spinal cord via a process that may involve brain-intestinal interactions.

 Figure from Zhan, K., Zheng, H. et al. "Gut microbiota-bile acid crosstalk in diarrhea-irritable bowel syndrome". BioMed Research International vol. 1–16 (2020). doi.org/10.1155/2020/3828249

(2) Liver Diseases

Liver diseases, including hepatitis, fatty liver, cirrhosis, and other ailments, are associated with the gut microbiota's ability to metabolize bile acids. This metabolic process is crucial in regulating the bile acid pool, which can affect FXR signaling. Studies have shown that inhibiting intestinal FXRs can alleviate high-fat diet-induced hepatic steatosis in mice. According to a study published in Nature Metabolism in 2021, the breakdown of microbiota-mediated negative feedback regulation of bile acid synthesis can cause fatal liver injury due to increased hepatic bile acid levels and the disruption of bile duct barrier function. These changes are driven by reduced bile acid signaling to FXRs, which regulate the activity of CYP7A1, the rate-limiting enzyme in bile acid synthesis.

 Figure from Aron-Wisnewsky, J.et al. "Gut Microbiota and human Nafld: Disentangling microbial signatures from metabolic disorders". Nature Reviews Gastroenterology & Hepatology vol. 17,5 (2020): 279–297. doi.org/10.1038/s41575-020-0269-9 

(3) Metabolic Diseases

Metabolic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD) are characterized by steatoxicity and glucotoxicity. Bile acids, synthesized from cholesterol and stored in the gallbladder, play a crucial role in the absorption and processing of lipids, as well as regulating inflammatory responses and gut microbiota (GM) composition. Dysregulated intestinal flora can induce the secretion of a large amount of 5-HT from intestinal epithelial cells by stimulating them with secondary bile acids. This increase in 5-HT can, in turn, elevate blood glucose levels, resulting in persistent hyperglycemia and the development of diabetes.

4. Intestinal flora + metabolome research protocol

In gastrointestinal and metabolic diseases, the intestinal flora and metabolites interact to mutually regulate and collaborate with each other. The "intestinal flora + metabolome" multi-omics research solution allows for a comprehensive analysis of disease development and drug targets at the species, gene, and metabolic pathway levels. In a classical multi-omics study protocol described in the NatureReviews Gastroenterology &Hepatology journal in 2020, disease cohorts are first recruited for longitudinal sampling and matched to controls; metabolomics can be generated from host samples (serum and urine) and fecal samples, with parallel microbiome sequencing applied to fecal samples; additional clinical data, including disease episodes, diet, activity, and anthropometric measurements, can also be collected; metabolites and microorganisms associated with the disease are identified through data analysis; and specific metabolites and microorganisms are tested in vitro and in vivo and validated using sterile and other methods.

Figure from Lavelle, A. et al. "Gut microbiota-derived metabolites as key actors in inflammatory bowel disease". Nature Reviews Gastroenterology & Hepatology, vol. 17,4 (2020): 223–237. doi.org/10.1038/s41575-019-0258-z 

5. MetwareBio Bile Acid Assay

MetwareBio offers 65 absolute quantification tests for bile acids; we use a validated absolute quantification method of internal and external standards that offers a wide coverage and very accurate results. It is suitable for all kinds of bile acid-related diagnostics and gut microbial-host interaction studies.