Synthesis, secretion, and circulation of bile acids
Bile acids (BAs) are synthesized from cholesterol in the liver; the rate limiting enzyme in the classical pathway of synthesis is 7α-hydroxylase (cytochrome P450 7A1, and CYP7A1). The primary BAs produced in the liver are cholic acid (CA) and chenodeoxycholic acid (CDCA), which are conjugated with taurine and glycine, thereby increasing the solubility of the BA in bile, excreted in the bile, stored in the gallbladder, and delivered into the duodenum with ingestion of meals to emulsify fats and fat-soluble vitamins and to aid in their absorption (1). BAs have detergent properties, and they retain their cholesterol “backbone”; their conjugated state with taurine and glycine is preserved in the small intestine.
In the ileum, BAs that are not involved in micelles are efficiently (∼95%) absorbed via an energy-requiring process involving the apical sodium BA transporter (ASBT). Within the ileal enterocytes (Figure 1), BAs stimulate the nuclear farsenoid X receptor (FXR) to produce fibroblast growth factor 19 (FGF-19), an enteroendocrine hormone that is transported to the liver and enters the hepatocyte through FGF-receptor 4 on interaction with a surface protein (klotho β), leading to the induction of a small heterodimer protein to decrease hepatic BA synthesis by inhibiting the rate limiting enzyme, 7α-hydroxy-4-cholesten-3-one (C4) (Figure 2) (1).
About 5% of the BAs (CA and CDCA) that are unabsorbed in the ileum are deconjugated on reaching the colon by bacterial bile salt hydrolases and by 7α-dehydroxylating bacteria to form secondary BAs (Figure 2), predominantly deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid. Thus, colonic microbiota are integral to the effects of BAs. In the colon, CDCA and DCA stimulate fluid secretion (2), increase mucosal permeability, and induce high amplitude-propagated contractions (3,4). The colon reabsorbs, by diffusion, at least 50% of the mass of BAs reaching the human colon (5).
The FXR is highly expressed in the intestine and liver and is a natural receptor for BAs. CDCA is the most potent FXR agonist, followed by CA (81%), DCA (40%), and LCA (4%) relative to CDCA's potency (6).
The second natural BA receptor is G protein-coupled BA receptor 1 (GPBAR1), also called Takeda G-coupled receptor 5 (TGR5); it is located on cholangiocytes, intestinal cells, the basolateral surface of smooth muscle, neural cells, brown adipose tissue, immune cells including dendritic cells and macrophages, and enteroendocrine cells that produce glucagon-like peptide 1 (7). TGR5 is most potently activated by LCA, among the natural BAs, and it mediates effects of BAs on motility, directly by acting on neurons and indirectly by stimulating serotonin release (1).
Symptoms and signs of bile acid malabsorption
Diarrhea is the hallmark of BA malabsorption (BAM). In an online survey of 100 patients with BAM of 1,300 members of a BAM support group, 85% reported urgency, 54% abdominal pain, 88% occasional incontinence, and 52% felt the need to be close to the bathroom. Among those with abdominal discomfort, 40% reported fatigue and at least 60% “brain fog” which prevented work efficiency. After treatment with BA sequestrants, gastrointestinal and systemic symptoms improved or resolved by at least 50%, and there was a significant improvement in work absences and altered work hours (8). Patients with unexplained diarrhea or irritable bowel syndrome-diarrhea (IBS-D) who have increased fecal BA excretion typically have higher body mass index, increased stool weight and fat, and accelerated colonic transit as compared to patients without increased fecal BA excretion (9).
Disease states resulting in BA malabsorption
BAM is characterized by diarrhea, abdominal discomfort, and bloating (10,11). Four types of BAM (12) are recognized: type 1 BAM includes ileal disease, such as Crohn's disease, resection, and radiation ileitis; type 2 BAM is “idiopathic” and manifests clinically as functional diarrhea or IBS-D; type 3 BAM is malabsorption of BAs secondary to diseases, such as chronic pancreatitis, cholecystectomy, and celiac disease; type 4 BAM results from increased BA synthesis induced by treatment with metformin, which inhibits ileal reabsorption of BAs, thereby increasing fecal excretion of BAs (13).
Prevalence of BA malabsorption in diverse conditions
BAM is estimated to affect ∼1% of the population in Western countries (14). The prevalence of BAM in different conditions is generally replicated in numerous studies. For example, type 1 BAM was present in 77/87 patients with Crohn's disease who underwent 75Se-homocholic acid taurine (75SeHCAT) retention (15), in more than 90% of patients with Crohn's disease with ileal resection >100 cm, and in 11%–52% of those without ileal resection (16). This test measures the retention of radiolabeled BAs 7 days after oral ingestion. The lower the retention of radiolabeled BAs, measured by whole body scanning with a gamma camera, indicates that BAs are lost in the feces, and the lowest levels of retention reflect more severe BAM.
The prevalence of type 2 “idiopathic” BAM was assessed in 2 systematic reviews. One involved 15 prospective studies, mostly based on 75SeHCAT retention in patients with functional diarrhea or IBS-D. In this analysis, severe BAM (<5% retention) was present in 10%, moderate BAM (<10% retention) in 32%, and mild BAM (<15% retention) in 26% of patients (17). A second systematic review included other diagnostic methods (discussed below) and concluded that the prevalence was 30% in patients with functional diarrhea or IBS-D (18). The prevalence of types 3 and 4 BAM is unclear because screening for BAM in these patients is limited.
However, the prevalence of BAM in patients postcholecystectomy is lower than generally considered. In a systematic review of 25 studies that included 3,388 patients, only 9.1% of patients developed diarrhea after cholecystectomy, with two-thirds having been diagnosed with BAM (19). Similarly, in 125 consecutive patients who underwent laparoscopic cholecystectomy, 25.2% developed diarrhea at 1 week after surgery, and 5.7% had diarrhea at 3 months (20).
In microscopic colitis, there is considerable evidence of BAM in a subset of patients. In one study, 43% of patients with microscopic colitis had BAM (lymphocytic [60%]; collagenous [27%] colitis), and 86% of patients with BAM responded to cholestyramine (21). Findings in other studies of BAM in microscopic colitis have been conflicting (22–24), and it has also been noted that diarrhea patients without BAM may respond to a BA sequestrant (25). The latter observation suggests that a therapeutic response to BA sequestrant does not prove BAM in such patients. The cause of BAM in microscopic colitis may be related to the villous atrophy, inflammation, and collagen deposition in the ileum that have been reported in patients with microscopic colitis (26,27).
BAs also may play a role in nonalcoholic fatty liver disease, with evidence of higher BA synthesis being associated with higher fibrosis scores (28).
Potential mechanisms underpinning idiopathic BAM
At present, the strongest data suggest that primary BAM results from reduced production of FGF-19 by ileal enterocytes, leading to low fasting serum FGF-19 and reciprocally increased serum 7α-C4, denoting increased hepatic synthesis of BAs (29). The low FGF-19 may result from reduced FGF-19 and ASBT messenger RNA expression in ileal biopsies from patients with BAM (30). The expression data were supported by functional effects. Thus, the in vivo retention of 75SeHCAT was significantly correlated with the basal ileal transcript expression of FGF-19 and ASBT. In addition, in vitro studies showed that CDCA stimulated transcripts of FGF-19 and ileal BA binding protein. Less than 1% of patients with type 2 bile acid diarrhea (BAD) had impairment of BA reabsorption due to a mutation in the gene for the ileal BA transporter (31).
Other data support an association of BAM with proteins involved in the enterohepatic circulation, based on associations of genetic variants in GPBAR1 rs11554825, klotho β rs17618244, and FGFR4 (fibroblast growth factor receptor 4) rs351855, with acceleration of colonic transit (as a surrogate of BAM) in patients with IBS-D or functional diarrhea (Figure 1) (32–34). In mice, mutations within the Diet1 gene, exclusively expressed in the epithelial cells lining the small intestinal villi and kidney proximal tubules, have resulted in decreased FGF-15, the mice counterpart for the human FGF-19 (35). The relevance to humans with idiopathic BAM is unclear.
Mechanisms leading to gastrointestinal symptoms in BA malabsorption
BAs cause diarrhea by increasing colonic motility and secretion, and they affect inflammation and the microbiome. Water and electrolyte secretion in response to BAs is based on several mechanisms (Table 1).
Stimulation of motility results from a series of processes. First, BAs are passively absorbed by diffusion to activate TGR5 receptors on enteric neurons to release serotonin, thereby inducing colonic contractions (1), chloride secretion (36), and stimulation of defecation in mice (37). The greatest potency for stimulation of TGR5 is with LCA, followed by DCA, CDCA, and CA (38). Stimulatory effects of BAs on colonic motility are induction of high amplitude propagating contractions in the colon (e.g., with rectal infusion of 1 mM CDCA (4)) and acceleration of colonic transit with Na CDC (39). When CDC undergoes colonic dehydroxylation to LCA, the latter stimulates colonic motility through TGR5 receptors.
An additional effect on BA-induced colonic dysfunction may result from changes in the microbiome. Patients with BAM have a higher proportion of fecal primary BAs, particularly CDCA, a secretory BA (10,40), suggesting either insufficient time for dehydroxylation due to rapid colonic transit or due to changes in the microbiome, resulting in alteration of BA-transforming bacteria in the feces. Indeed, although patients with IBS-D have a higher proportion of Escherichia coli and decreased Clostridium leptum and Bifidobacterium (40), a recent report showed that 24.5% of patients with IBS-D exhibited excessive excretion of total BAs and increase in Clostridia bacteria (e.g., Clostridium scindens), which was positively associated with the levels of fecal BAs and serum 7α-hydroxy-4-cholesten-3-one (C4) (41). The latter finding suggested that Clostridia bacteria have potential as a biomarker for BAD and as a target for therapy (42), although this still requires formal testing in humans.
Diagnosis of BA malabsorption
Current BAM diagnostic methods are based on the documentation of impaired ileal BA absorption, decreased hepatic feedback inhibition, and increased hepatic BA synthesis. Table 2 summarizes the characteristics of the different diagnostic tests (43).
75Selenium HomotauroCholic acid test.
75SeHCAT measures the retention of radiolabeled BAs 7 days after ingestion. This is the gold standard diagnostic method. Low retention of radiolabeled BAs indicates the loss of BAs in the feces; the current cutoffs for mild, moderate, and severe BAM are <15%, <10%, and <5% retentions, respectively, at 7 days. 75SeHCAT is simple and noninvasive, but it requires a gamma camera, exposes patients to radiation, and is not available in many countries including the United States. A potential confounder in the measured retention of BAs in the whole body is the number of times the BA pool undergoes enterohepatic recycling, which can vary from 4 to 16 per day (44). For example, a small deficit in ileal BA absorption may result in high loss of isotope if the individual has 16 cycles per day, whereas, a more substantial deficit in ileal BA absorption may result in greater overall retention if there are fewer cycles of the BA pool per day. This confounder should be resolved by estimating retention over a longer period of time, such as the recommended 7 days for the test.
48-hour fecal BA test.
In places without access to 75SeHCAT, a 48-hour fecal BA test is the best current option. Patients consume a high fat diet (100 g/d) for 4 days and collect stool for the past 48 hours. Total and primary fecal BA levels have demonstrated a significant positive association with 75SeHCAT retention (45,46). Total and primary fecal BAs and fecal fat were significant predictors of increased stool weight, frequency, and consistency, with area under the curve >0.71 (sensitivity >55%, specificity >74%) (47). In addition, primary fecal BA excretion was associated with fecal weight (>400 g/48 h) and colonic transit average location of isotope at 24 hours > 3.34, corresponding to sigmoid colon (48).
Although the stool collection is cumbersome, a 48-hour fecal BA test allows direct measurements of total and individual BAs without radiation exposure, with 3 criteria diagnostic of BAM: total fecal BAs ≥2,337 μmol/48 h (5), primary BAs (CDCA and CA) > 10%, or total fecal BAs ≥1,000 μmol/48 h plus primary BAs >4% compared with healthy controls (<5%) (Figure 3) (48,49).
Fasting serum 7α-hydroxy-4-cholesten-3-one (C4).
Fasting serum C4 (before 9:00 am because of diurnal variation) is measured by C18 liquid chromatography–tandem mass spectrometry (50). Serum C4 is a direct measure of BA synthesis; higher values indicate BAM. C4 has been validated in comparison with 75SeHCAT (C4 cutoff value > 48.4 ng/mL: sensitivity of 90% and specificity of 79% (51)) and in comparison with the fecal 48-hour BA test (C4 cut-off >52.5 ng/mL: sensitivity of 29% and specificity of 83% (52)).
Fasting serum C4 is an efficient and convenient method to rule out BAM (52), and it is available via commercial testing in the United States (Mayo Medical Laboratories and PROMETHEUS IBcause).
In patients with Crohn's disease, high C4 was associated with ileal disease or resection and nonbloody diarrhea. By contrast, C4 levels in ulcerative colitis were similar to healthy controls (53). Thus, fasting serum C4 >48.3 ng/mL identifies Crohn's patients with diarrhea likely attributable to BAM (90.9% sensitivity, 84.4% specificity) (54).
Serum fasting FGF-19.
Serum FGF-19 is inversely correlated with serum C4 (55), and the fifth percentile in healthy volunteers is ≤ 61.7 pg/mL. However, fasting FGF-19 is not sensitive (29%) or specific enough (78%) relative to fecal BA excretion (52) to screen for BAD. Therefore, fasting serum FGF-19 would seem to be most helpful to rule out BAM, if and when it becomes available for use in clinical practice. Further research is required to assess a modification on the test, which is CDCA-stimulated FGF-19 (acting as a “stress test”) in the diagnosis of BAD (56). Recent guideline did not recommend the test as a first line diagnostic test for BAD (57).
Diagnostic test vs empiric trial of BA sequestrants
When diagnostic tests are not available, empiric treatment with BA sequestrants is advocated for patients with suspected BAM. However, compliance with a therapeutic trial may be suboptimal (58), compromising interpretation of a negative response. Because severity of BAM can predict response to treatment, there is strong rationale to measure BAM rather than just empiric treatment (17,59). British and Canadian gastroenterology organization guidelines also support diagnosis over empiric trial for suspected BAM in patients presenting with chronic diarrhea (57,60). In addition, a positive test for BAM was associated with reduced healthcare utilization in a referral center in the United Kingdom (61), and a retrospective study of almost 1,000 patients evaluated for chronic unexplained diarrhea in a US tertiary center showed high healthcare utilization in referred patients that could have been avoided by earlier implementation of a diagnostic test for BAM (62).
Treatment of BAM
A low fat diet with <20% of total daily caloric intake complements efficacy of BA sequestrant treatment in the relief of abdominal discomfort, distension, urgency, and stool consistency and frequency (63).
Three BA sequestrants are available in either powder or tablet formulations: cholestyramine, colestipol, and colesevelam. Patients should take these medications with meals to bind free BAs and prevent the colonic effects of increased colonic motility and secretion.
The only randomized trial of cholestyramine efficacy in BAD showed response rates of 40% and 53.8% in patients with 75SeHCAT retention <10% or 20%, respectively. Less than 15% retention is usually the cutoff for abnormal BA loss (10%–15% retention mild, 5%–10% moderate, and <5% severe BA loss).
In an open-label trial in patients with BAD with 75SeHCAT retention <20%, colestipol reduced stool frequency and IBS severity score (64).
In another open-label study in patients with high 48-hour stool BA excretion, colesevelam, 1875 mg twice daily for 10 days, decreased stool consistency and increased stool excretion of sequestered BAs (65). Because of the loss of BAs in the stool with BA sequestrant treatment, hepatic BA synthesis and, thus, serum C4 were increased (65). Colesevelam also slowed emptying of the ascending colon compared with placebo in IBS-D; the treatment effect was associated with baseline serum C4, which reflects the hepatic BA synthesis rate (66).
Further controlled trials are necessary to assess the effects of BA sequestrants for diarrhea, and patients will likely need long-term therapy with BA sequestrants for symptom relief. In a long-term follow-up study of patients with a median time from diagnosis of BAD of 6.8 years, 38% were still on BA sequestrants, with adequate relief of their symptoms, whereas 24% discontinued therapy, most commonly because of poor tolerability (67).
Two double-blinded, placebo-controlled, randomized trials with the BA sequestrant, colesevelam, in patients with BAM, based on elevated serum C4 in Crohn's disease, showed a higher number of patients with >30% reduction of liquid stool and reduction of median number of liquid stools from 5 to 2 per week, compared with placebo (68). In a comparison of cholestyramine and hydroxypropyl cellulose in patients with chronic water diarrhea (some with 75SeHCAT <15%), there was higher decrease in watery stools in the cholestyramine group, although there was no difference in the primary endpoint of proportion with mean ≤3 liquid bowel movements per week (69). However, the equipoise between these treatments may be confounded by the fact that hydroxypropyl cellulose actually binds BAs without affecting hepatic BA synthesis, and it was shown in a separate study to improve stool frequency, consistency, urgency, and incontinence after 6 weeks' treatment in patients with idiopathic BAM and Crohn's disease with ileal resection (70).
Although BA sequestrants are effective in improving abdominal symptoms and stool characteristics, this treatment option does not target the underlying pathophysiology. This is addressed more directly by FXR agonists.
FXR agonists were initially developed for cholestatic liver diseases. However, efficacy of FXR agonists in BAM has been shown in vitro and in 2 in vivo studies. FXR agonists attenuated calcium and cyclic adenosine monophosphate dependent chloride secretion on colonic epithelium (71). In 2 clinical trials in patients with BAM, a 2-week trial of obeticholic acid (6-ethyl CDCA) daily in patients with primary and secondary BAM and chronic diarrhea showed improvement in stool frequency, form, and total diarrhea index, with corresponding increase in FGF-19 and decrease in C4 and fecal BAs. However, obeticholic acid (which is chemically 6-ethyl CDCA) is associated with pruritus (72). A preliminary report showed the non-BA molecule, tropifexor, retarded ascending colon emptying in patients with BAD, although the clinical endpoints were not significantly altered in that small clinical trial (73).
The BA field has expanded in relevance, particularly in clinical diagnosis of unexplained diarrhea in patients with IBS-D, microscopic colitis, and inflammatory bowel disease without ileal inflammation or resection. This has been facilitated by the validation of screening serum tests and fecal BA excretion. Novel therapeutic approaches targeting FXR receptors might open new avenues for treatment of intestinal diseases.
CONFLICTS OF INTEREST
Guarantor of the article: Michael Camilleri, MD.
Specific author contributions: M.C.: Primary author, principal investigator in many relevant mechanistic and diagnostic aspects of bile acid diarrhea P.V.: Co-author and fellow who anchored most of the original studies in the past 4 years.
Financial support: Michael Camilleri is supported by grant RO1-DK115950 from National Institutes of Health.
Potential competing interests: M.C. has conducted sponsored research on elobixibat and tropifexor. P.V. has no conflicts of interest.
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