**2. Classification of bile acids and their metabolites**

Bile acids are synthesized from cholesterol in the liver; in humans, cholic acid (CA) and chenodeoxycholic acid (CDCA) are typical primary bile acids [5]. In the rodent liver, chenodeoxycholic acid is further converted to muricolic acid (MCA). After biosynthesis, CA and CDCA undergo further glycine conjugation or taurine conjugation in the liver. Among human conjugated bile acids, taurine conjugates and glycine conjugates accumulate in the gallbladder at a concentration ratio of approximately 1:3 and are secreted from the bile duct into the duodenum in response to food intake. On secretion into the duodenum, taurine conjugates (tauro-CA, tauro-CDCA) and glycine conjugates (glyco-CA, glyco-CDCA) are deconjugated by the bile salt hydrolase (BSH) of the gut microbiota; these deconjugated bile acids are involved in the formation of micelles and the absorption of dietary fat (**Figure 1**).

*Lactobacillus plantarum*, *Lactobacillus johnsonii*, *Clostridium perfringens*, and *Bifidobacterium longum* have been reported to be specific bacteria carrying the BSH gene [6]. Because the BSH gene is expressed in many bacteria and the presence of BSH is favorable to the host, the presence of these bacteria can be interpreted to be a result of selection by the host. BSH has been suggested to play an important role in the colonization and survival of bacteria in the gut [7]. Jarocki et al. [8] first analyzed the occurrence of BSH in 14 strains belonging to the *Bifidobacterium* genus and purified and analyzed two BSHs from *B. pseudocatenulatum* and *B. longum* subsp. *suis* for their selected biochemical and molecular features. Deconjugation by BSH seems to be meaningful in human physiology and is involved in the lowering of cholesterol levels, maintenance of intestinal homeostasis, maintenance of the intestinal circadian rhythm, and supply of glycine and taurine to the surrounding bacteria [6]. In particular, free bile acids (CA, CDCA) have been reported to be directly involved in the expression of clock genes in intestinal epithelial cells that can control peripheral circadian rhythms in the intestinal tract and liver. Govindarajan et al. [9] demonstrated that unconjugated bile acids are potential chronobiological regulators of host circadian gene expression, especially in the intestine and liver. These data may indicate the potential role of microbiota-generated bile acids as chronological regulators of the peripheral circadian clock and

**Figure 1.** *Synthesis, conjugation, and metabolism of bile acids by gut microbiota.*

#### *Role of Gut Microbiota in Bile-Acid Metabolism DOI: http://dx.doi.org/10.5772/intechopen.100440*

suggest that intervention strategies that alter gut bile-acid profiles could influence the circadian clock. Joyce et al. [10] investigated the role bacterial BSH in the host physiology and have demonstrated that bacterial BSH activity significantly impacts the systemic metabolic processes and adiposity in the host and represents a key mechanistic target for the control of obesity and metabolic syndrome.

Primary bile acids are actively reabsorbed by the ileal bile-acid transporter (IBAT) present in the terminal ileum in addition to being passively absorbed. Consequently, bile acids secreted into the intestinal tract could return to the liver via the portal vein, 95% of which is reused. Bile acids are reported to be reused by enterohepatic circulation and circulated in the human body 4–12 times a day [11]. The details of the mechanism regulating IBAT expression have not yet been elucidated; however, IBAT expression appears to be affected by gut microbiota and is markedly enhanced in germ-free mice [12].

Bile acids that flow from the small intestine to the large intestine are further metabolized by abundant gut bacteria. First, multi-step reactions of specific bacteria result in the hydroxyl group at the C-7a position of the deconjugated bile acids (CA and CDCA) being dehydroxylated (7α-dehydroxylation) to form secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA). Specifically, CA is metabolized to deoxycholic acid (DCA), which in turn is metabolized to lithocholic acid (LCA). Specific *Eubacterium* and *Clostridium* cluster XIVa species, belonging to the Firmicutes phylum, are involved in this conversion in a complex manner, but the entire pathway has not yet been clarified. More than 90% of bile acids in feces are secondary bile acids, and in humans, DCA and LCA are the most abundant [13]. These bile acids affect host physiological function via the TGR5 receptor, which is a bile-acid receptor, and are involved in water secretion to the lumen of the large intestine and peristaltic movement of the tract. Therefore, secondary bile acids produced by the gut microbiota are essential for the physiological function of the host, and a decrease in their concentration could lead to a corresponding decrease in intestinal peristalsis. Thus, a decrease in bile-acid concentrations may not only induce constipation symptoms, but also adversely affect the gut-brain axis.

Furthermore, some amount of CDCA is further metabolized to UDCA by gut bacteria carrying the 7α- and 7β-hydroxylsteroid dehydrogenase (HSDH) genes. Bacteria such as *Ruminococcus gravus*, *Ruminococcus productus*, *Collinsella aerofaciens*, and *Clostridium absonum* have the HSDH gene but have not been studied in detail. Although the luminal concentrations of these bile-acid metabolites in the large intestine are low, recent reports have reported anti-inflammatory, anti-bacterial, and wound healing promoting effects of these metabolites [14]; thus, further studies are required.

As described above, primary bile acids secreted into the duodenum in a conjugated form are metabolized by various intestinal bacterial genes. This interaction is complex because bile acids also have a more direct effect on the survival of the gut microbiota. Islam et al. [15] have demonstrated that bile acid is a host factor that regulates the composition of the cecal microbiota in rats, and that CA feeding simplifies the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Furthermore, importantly, several bile-acid receptors have been discovered and each bile acid has differing binding ability to these receptors; these aspects should be considered to understand the bile acid-mediated host response. Metabolic disorders have an impact on longevity and the recent findings showed the relationship between bile acid metabolism and metabolic disorders [16–18]. Broeders et al. [16] have showed that CDCA promotes mitochondrial uncoupling via bile-acid receptor (TGR5) in human brown adipocytes and increases brown fat activity and energy expenditure in women.

Because it has been shown that TGR5 localizes in many cells and tissues, including enteroendocrine cells, neurons, macrophages, muscle and endothelial cells, the bile acid-mediated host response should be carefully analyzed.

#### **3. Serum bile-acid profile as a biomarker**

The profiles of various bile acids can be selectively measured using quantitative systematic liquid chromatography–tandem mass spectrometry (LC–MS/MS) for samples such as blood and stool. Furthermore, quantitative measurement using internal standard substances has made it possible to rapidly conduct research using a large number of clinical samples [19, 20]. Features of serum bile-acid profiles have been reported in patients with inflammatory bowel disease [19], colon cancer [21], irritable bowel syndrome [22], chronic constipation [23], liver diseases such as fatty liver and fatty hepatitis [24], and neurodegenerative diseases such as Alzheimer's disease and cognitive dysfunction [25, 26].

The relationship between colorectal cancer and the gut microbiota is a hot topic of research. *Fusobacterium nucreatum*, detected relatively specifically in colorectal cancer tissues, is a sulfate-reducing bacterium that can produce hydrogen sulfide, which is a gene mutagen. Taurine released from taurine-conjugated bile acids has been suggested to be a substrate for hydrogen sulfide production [27]; therefore, the carcinogenic-promoting effect of a high-fat diet may be partially explained by the increase in levels of taurine-conjugated bile acids caused by the diet. Bile-acid profile information for patients with colorectal cancer has also been reported. Uchiyama et al. [21] measured serum bile-acid profiles of healthy individuals, colorectal adenomatous polyps, and colorectal cancer at each clinical stage and analyzed the principal components of 30 types of bile acids. Free CA, 3epi-DCA, CDCA, 3-dehydro CA, glyco-CA, and tauro-CA were extracted as principal components (PC) 1 and free 3-dehydroDCA was extracted as PC 2 by canonical discriminant function coefficients. They concluded that the verification of discriminability using the cross-validation method revealed that the correct classification rate was 66.3% for the original data and 52.6% for the cross-validation data. Kuhn et al. [28] also demonstrated the association between serum concentrations of individual bile acids and colon cancer risk and was the first to show the importance of conjugated bile acids compared to that of unconjugated bile acids. They observed statistically significant positive associations between most conjugated primary bile acids (glyco-CA, tauro-CA, glyco-tauro-CDCA, tauro-CDCA, and glyco-hyocholic acid [GHCA]) and colon cancer risk.

The clinical usefulness of serum bile-acid profiles has also been analyzed in several diseases other than colorectal cancer. Recent reports have demonstrated that the concentration of conjugated secondary bile acids (glycol-DCA, glycol-LCA, tauro-DCA, and tauro-LCA), but not of the unconjugated forms, increases parallelly with the progression of the disease, especially in patients with Alzheimer's disease and dementia. Nho et al. [26] were the first to show that altered bile-acid profiles and increased ratios of glyco-DCA:CA, tauro-DCA:CA, and glyco-LCA:CDCA were significantly associated with structural and functional changes in the brain, as indicated by greater atrophy and reduced glucose metabolism. Higher levels of secondary conjugated bile acids (glyco-DCA, glycol-LCA, and tauro-LCA) were significantly associated with worse cognitive function in 1,464 subjects, including 370 cognitively normal older adults, 284 individuals with early mild cognitive impairment (MCI), 505 individuals with late MCI, and 305 patients with Alzheimer's disease [25]. The increase in the concentration of conjugated bile acids in the blood should be considered to be caused by the high-fat diet-stimulated

#### *Role of Gut Microbiota in Bile-Acid Metabolism DOI: http://dx.doi.org/10.5772/intechopen.100440*

increase in the bile-acid biosynthetic reactions in the liver and the decrease in the bile-acid deconjugation reactions in the intestinal tract, indicating a decrease in the gut microbiota carrying the deconjugation gene for BSH, as has been mentioned above.

We previously performed a correlation analysis between the bile-acid profile and gut microbiota in a fatty liver model and a colon cancer model in mice fed a high-fat diet and indicated that this correlation analysis is useful for assessing the functionality of dietary factors [29, 30]. High-fat dietary load increased serum levels of conjugated bile acids (tauro-CA, tauro-DCA) and decreased levels of unconjugated bile acids (such as CA), but the administration of epigallocatechin gallate (EGCG), a functional ingredient in tea, normalized bile-acid profiles. Furthermore, an analysis of gut microbiota revealed a positive correlation between an increase in the abundance of *Akkermansia* and their effects on bile acids [29]. Similarly, in the colon cancer model, we found that agarooligosaccharide suppressed the increase in levels of taurine-conjugated bile acids and suppressed the increase of bacteria classified into *Clostridium* subcluster XIVa that are involved in secondary bile-acid metabolism.

As described above, quantitative bile-acid profile measurement using the LC– MS/MS method has made it possible to obtain integrated bile-acid information that distinguishes between conjugated and unconjugated types; consequently, the roles of bile acids in physiology and pathophysiology are being elucidated. It is important to note that such analysis should be conducted with the understanding that the bile-acid profiles are influenced by dietary factors, including high-fat diet, liver function, lipid metabolism, cholesterol level, and gut microbiota.
