Development and validation of a novel 7α-hydroxy-4-cholesten-3-one (C4) liquid chromatography tandem mass spectrometry method and its utility to assess pre-analytical stability.

OBJECTIVES: 7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood. METHODS: Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis. RESULTS: The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4 concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. CONCLUSIONS: We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral.

Food intolerance related to gastrointestinal symptoms amongst adults living with bile acid diarrhoea: A cross-sectional study.

BACKGROUND: The role of food in managing bile acid diarrhoea (BAD) is poorly understood. The present study explored the prevalence of food intolerance amongst adults with BAD. METHODS: The study comprised a cross-sectional survey of adults with BAD determined by the 75 selenium homotaurocholic acid test (SeHCAT) living in the UK. Participants anonymously completed an online questionnaire on 39 food items. Frequency of food in general affecting BAD symptoms, as well as frequencies of diarrhoea, abdominal pain, bloating, flatulence and consequential food avoidance after food item ingestion, were assessed. Food group avoidance was also assessed. RESULTS: There were 434 participants who completed the questionnaire between April and May 2021 of whom 80% reported moderate to severe chronic diarrhoea. Food intolerances were reported by 88.0% (95% confidence interval [CI] = 84.6-90.9) of participants. Diarrhoea was reported most frequently after take-away food, fish and chips, creamy sauces, cream and large quantities of fruit (range 41.0%-33.6%). Lowest frequencies were for potato, avocado, mango, watermelon and pear (range 3.7%-7.4%) for the foods listed in the questionnaire. Similar trends were found for abdominal pain, bloating, flatulence and consequential food avoidance. Symptom-triggering within 30 min of ingestion was more prevalence than after 30 min for almost all foods. Food group avoidance was highest for fatty foods (81.2%; 95% CI = 77.8-85.3) followed by dairy (53.9%; 95% CI = 49.1-58.7). CONCLUSIONS: Perceived food intolerance amongst adults with BAD and persisting diarrhoeal symptoms is high. Important triggers were meals with a higher fat content and higher-fat dairy products. Diets amongst those with persisting diarrhoeal symptoms may be overly restrictive.

The impact of treatment with bile acid sequestrants on quality of life in patients with bile acid diarrhoea.

BACKGROUND: Bile acid diarrhoea (BAD) can be severely debilitating and negatively affect patients' quality of life (QoL). We carried out a multi-centre prospective study exploring QoL outcomes in patients with BAD after treatment with colesevelam. METHODS: Patients with or without a positive 23-seleno-25-homotaurocholic acid (SeHCAT) scan were recruited and categorised into four groups: SeHCAT negative control group (CG), idiopathic BAD, post-cholecystectomy (PC) and post-terminal ileal resection for Crohn's disease (CD). Patients with a positive SeHCAT were treated with colesevelam and dosing was titrated to symptomatic response. Patients were reviewed at 4- and 8-weekly intervals and QoL was evaluated by EQ-5D-3L, SF-36, IBDQ-32 at each visit (where relevant). Patients with a negative SeHCAT (CG cohort) completed one set of questionnaires before being discharged from the study. RESULTS: 47 patients (BAD = 24, PC = 12, CD = 11) completed paired QoL questionnaires before and after treatment and 30 CG patients completed a baseline questionnaire. There was a significant improvement in IBDQ-32 mean scores before and after treatment in CD patients [134.6 (95%CI 112.5-156.6) and 158.4 (136.1-180.6), respectively (p = 0.007). Following treatment, BAD patients had significantly improved mean SF-36 scores in the "Role limitation due to physical health" dimension (p = 0.02) and in the overall mental component summary (p = 0.03). Prior to starting treatment, BAD patients had the lowest scores in the 'activity' dimension of the EQ-5D-3L (p = 0.04), which improved significantly after treatment (p = 0.002). Overall, the BAD and CD cohort showed improved mean scores with treatment in all components of the SF-36 and EQ-5D-3L, while the PC cohort showed a general decline in mean scores after treatment. 55% of patients clinically responded to treatment of which 41.7%, 58.3% and 81.8% responded from the BAD, PC and CD groups respectively. Correlations between those deemed as responders with improvements on the SF-36 and EQ-5D dimensions were not statistically significant. CONCLUSION: Our results demonstrate improved QoL in the BAD and CD cohort with treatment. Further larger studies are recommended specifically investigating the PC cohort and whether patients may improve with newer treatments such as FXR agonists. Trial registration Ethical approval REC Ref: 16/LO/1325.

Effectiveness of diet, psychological, and exercise therapies for the management of bile acid diarrhoea in adults: A systematic review.

BACKGROUND: Bile acid diarrhoea (BAD) causes chronic diarrhoea and is primarily treated pharmacologically. This systematic review aimed to evaluate the effectiveness of non-pharmacological therapies for evidence-based management of BAD in adults. METHODS: A systematic review of the medical literature was performed from 1975 to 13 July 2021 to identify studies on diet, psychological, and exercise therapies that met diagnostic criteria for BAD in adults with diarrhoea. Effectiveness was judged by responder or improvement in diarrhoea at study endpoint according to each study's definition of diarrhoea. Therapeutic effect on abdominal pain and flatulence was also measured. Risk of bias was assessed using the Risk Of Bias In Non-Randomised Studies of Interventions tool. A narrative review was conducted using 'Synthesis Without Meta-analysis' guidance. Certainty of the evidence was assessed using Grading of Recommendations Assessment, Development, and Evaluation. RESULTS: Eight prospective cohort studies were identified on diet therapies from 2 weeks to over 2 years involving 192 patients. No psychological or exercise therapies were found. Carbohydrate modification (one study, n = 2) in primary BAD, and dietary fat intake reductions (five studies, n = 181) and an exclusive elemental diet therapy (two studies, n = 9) in secondary BAD, showed beneficial directions of effect on diarrhoea, abdominal pain, and flatulence. Risks of bias for each study and across studies for each therapy type were serious. Certainty of the evidence was very low for all outcomes. CONCLUSIONS: No conclusions could be drawn on the effectiveness of diet, psychological, or exercise therapies on diarrhoea, abdominal pain, and flatulence for the management of BAD in adults. High-quality randomised controlled trials are needed.

Prevalence of biliary acid malabsorption in patients with chronic diarrhoea of functional characteristics: a prospective study.

BACKGROUND: Bile acid malabsorption occurs in up to one third of patients with chronic diarrhoea of functional characteristics. The gold standard test for its diagnosis is the 75Selenium homocholic acid taurine (75SeHCAT) test. The aim of this work is to confirm previous data suggesting that bile acid malabsorption, diagnosed by 75Se-HCAT test, is the underlying cause of diarrhoea in a significant proportion of patients previously diagnosed with a functional disorder. In addition, we have analysed the clinical response of bile acid sequestrants in those patients with a bile acid diarrhoea diagnosis. METHODS: This is a prospective, single-centre study including consecutive adult patients diagnosed with chronic diarrhoea of unknown origin and with functional characteristics; systematic rule out of common causes of chronic diarrhoea was performed before bile acid malabsorption evaluation by 75SeHCAT scanning. A retention percentage less than 10% was considered positive. Clinical response to cholestyramine was further evaluated in those patients with a positive diagnosis of bile acid diarrhoea RESULTS: 38 patients (20 male, mean age 37.5 years) were finally included. Twenty (52.6%) patients included had a positive 75SeHCAT test. Median body mass index was significantly higher in those patients. We did not find significant differences in other clinical or biochemical variables 75SeHCAT-positive and 75SeHCAT-negative groups. Only 6 of 17 (35.3%) patients responded to cholestyramine treatment; 10 patients did not have response or withdraw the drug due to adverse events. Logistic regression analysis showed that none of the included variables was a predictor of clinical response to cholestyramine. CONCLUSIONS: Bile acid malabsorption occurs in a high proportion of patients suffering from chronic diarrhoea with functional characteristics. Systematic investigation of bile acid malabsorption should be included in the diagnostic algorithms of patients with chronic watery diarrhoea in the routine clinical practice. Absence of response to cholestyramine does not rule out bile acid diarrhoea.

Incidence and predictive factors for positive 75SeHCAT test: improving the diagnosis of bile acid diarrhoea.

AIMS: To determine the value of 75SeHCAT retention in determining bile acid diarrhoea (BAD), treatment response and predictors of a positive result. METHODS: Retrospective casenote review of consecutive patients undergoing 75SeHCAT from 2008 to 2014, including gender, age, history, clinical, and laboratory parameters. This included diseases associated with Type 1 BAD (ileal resection, Crohn's disease) and Type 3 BAD. Chi-squared test and logistic regression determined factors predictive of BAD. Subjective response to treatment with bile acid sequestrants (BAS) was analysed with respect to the 75SeHCAT result. RESULTS: Of 387 patients, 154 (39.7%) were male and average age was 50 years. Ninety-five patients (24.5%) were investigated for Type 1 BAD, 86 (22.2%) for Type 3, and 206 patients (53.2%) for Type 2 or idiopathic BAD. There was a large increase in the number performed with time but no difference in percentage positive tests. One hundred and seventy-nine patients (46.2%) had BAD. Positive result was commonest in possible Type 1 and they had most severe BAD. Ninety-nine patients had severe BAD (<5% 75SeHCAT retention), 47 moderate BAD (5% to <10% retention), and 33 mild BAD (10% to <15% retention). Predictors of a positive 75SeHCAT were right hemicolectomy (OR 4.88), cholecystectomy (OR 2.44), and Crohn's (OR 1.86). A positive 75SeHCAT predicted a good or partial response to BAS of 66.7% (mild), 78.6% (moderate), or 75.9% (severe BAD). CONCLUSION: 75SeHCAT test use increased in 2008-2014, with high positive results throughout. Ileal resection, Crohn's, and cholecystectomy independently predict BAD. 75SeHCAT predicted response to BAS.

The Effect of Colesevelam on the Microbiome in Postoperative Crohn's Disease.

BACKGROUND: While surgery plays a pivotal role in the management of ileal Crohn's disease, the risk of endoscopic recurrence following an ileocaecal resection can be greater than 65% within 12 months of surgery. More than 90% of patients with Crohn's disease have a concomitant diagnosis of bile acid diarrhea following an ileal resection. This pilot study aimed to assess whether the use of bile acid sequestrants in patients with Crohn's disease who have undergone a primary terminal ileal resection with concomitant bile acid diarrhea can alter the microbiome and prevent disease recurrence. METHODS: Patients with Crohn's disease who underwent a primary terminal ileal resection and had symptoms of diarrhea within 1-3 months of surgery underwent 75SeHCAT testing for bile acid diarrhea. If positive (75SeHCAT ≤ 15%), patients were treated with colesevelam and stool samples were collected at 4 weeks, 8 weeks, and 6-12 months posttreatment. If negative (75SeHCAT > 15%), treatment was not given and were reviewed in the clinic as per local guidelines. All patients underwent a 6-12 month postoperative colonoscopy where further stool samples and mucosal biopsies were taken. Disease activity was established using the endoscopic Rutgeert's score, with disease remission defined as Rutgeert's score

This pilot study demonstrated that patients with Crohn's disease who underwent a primary terminal ileal resection and were given colesevelam were more likely to be in disease remission at their 6-12 month postoperative colonoscopy review compared with those not treated; there was a notable change in abundance in certain bacteria following treatment compared to their pretreatment microbiome.

The analysis of gut microbiota in patients with bile acid diarrhoea treated with colesevelam.

Introduction: Bile acid diarrhoea (BAD) is a common disorder that results from an increased loss of primary bile acids and can result in a change in microbiome. The aims of this study were to characterise the microbiome in different cohorts of patients with BAD and to determine if treatment with a bile acid sequestrant, colesevelam, can alter the microbiome and improve microbial diversity. Materials and methods: Patients with symptoms of diarrhoea underwent 75-selenium homocholic acid (75SeHCAT) testing and were categorised into four cohorts: idiopathic BAD, post-cholecystectomy BAD, post-operative Crohn's disease BAD and 75SeHCAT negative control group. Patients with a positive 75SeHCAT (<15%) were given a trial of treatment with colesevelam. Stool samples were collected pre-treatment, 4-weeks, 8-weeks and 6-12 months post-treatment. Faecal 16S ribosomal RNA gene analysis was undertaken. Results: A total of 257 samples were analysed from 134 patients. α-diversity was significantly reduced in patients with BAD and more specifically, in the idiopathic BAD cohort and in patients with severe disease (SeHCAT <5%); p < 0.05. Colesevelam did not alter bacterial α/ß-diversity but patients who clinically responded to treatment had a significantly greater abundance of Fusobacteria and Ruminococcus, both of which aid in the conversion of primary to secondary bile acids. Conclusion: This is the first study to examine treatment effects on the microbiome in BAD, which demonstrated a possible association with colesevelam on the microbiome through bile acid modulation in clinical responders. Larger studies are now needed to establish a causal relationship with colesevelam and the inter-crosstalk between bile acids and the microbiome.

Diagnosis and treatment of post-cholecystectomy diarrhoea.

The incidence of cholecystitis is relatively high in developed countries and may usually be attributed to gallstones, the treatment for which involves complete surgical removal of the gallbladder (cholecystectomy). Bile acids produced following cholecystectomy continue to flow into the duodenum but are poorly absorbed by the colon. Excessive bile acids in the colon stimulate mucosal secretion of water and electrolytes leading, in severe cases, to diarrhoea. Bile acid diarrhoea (BAD) is difficult to diagnose, requiring a comprehensive medical history and physical examination in combination with laboratory evaluation. The current work reviews the diagnosis and treatment of BAD following cholecystectomy.

Farnesoid X receptor agonist tropifexor attenuates cholestasis in a randomised trial in patients with primary biliary cholangitis.

Background & Aims: The safety, tolerability, and efficacy of the non-bile acid farnesoid X receptor agonist tropifexor were evaluated in a phase II, double-blind, placebo-controlled study as potential second-line therapy for patients with primary biliary cholangitis (PBC) with an inadequate ursodeoxycholic acid response. Methods: Patients were randomised (2:1) to receive tropifexor (30, 60, 90, or 150 µg) or matched placebo orally once daily for 28 days, with follow-up on Days 56 and 84. Primary endpoints were safety and tolerability of tropifexor and reduction in levels of γ-glutamyl transferase (GGT) and other liver biomarkers. Other objectives included patient-reported outcome measures using the PBC-40 quality-of-life (QoL) and visual analogue scale scores and tropifexor pharmacokinetics. Results: Of 61 enrolled patients, 11, 9, 12, and 8 received 30-, 60-, 90-, and 150-µg tropifexor, respectively, and 21 received placebo; 3 patients discontinued treatment because of adverse events (AEs) in the 150-µg tropifexor group. Pruritus was the most frequent AE in the study (52.5% [tropifexor] vs. 28.6% [placebo]), with most events of mild to moderate severity. Decreases seen in LDL-, HDL-, and total-cholesterol levels at 60-, 90-, and 150 µg doses stabilised after treatment discontinuation. By Day 28, tropifexor caused 26-72% reduction in GGT from baseline at 30- to 150-µg doses (p <0.001 at 60-, 90-, and 150-µg tropifexor vs. placebo). Day 28 QoL scores were comparable between the placebo and tropifexor groups. A dose-dependent increase in plasma tropifexor concentration was observed, with 5- to 5.55-fold increases in AUC0-8h and Cmax between 30- and 150-µg doses. Conclusions: Tropifexor showed improvement in cholestatic markers relative to placebo, predictable pharmacokinetics, and an acceptable safety-tolerability profile, thereby supporting its potential further clinical development for PBC. Lay summary: The bile acid ursodeoxycholic acid (UDCA) is the standard-of-care therapy for primary biliary cholangitis (PBC), but approximately 40% of patients have an inadequate response to this therapy. Tropifexor is a highly potent non-bile acid agonist of the farnesoid X receptor that is under clinical development for various chronic liver diseases. In the current study, in patients with an inadequate response to UDCA, tropifexor was found to be safe and well tolerated, with improved levels of markers of bile duct injury at very low (microgram) doses. Itch of mild to moderate severity was observed in all groups including placebo but was more frequent at the highest tropifexor dose. Clinical Trials Registration: This study is registered at ClinicalTrials.gov (NCT02516605).

A practical approach to the patient with chronic diarrhoea.

Chronic diarrhoea is common, occurring as the first presentation of several diagnoses, or as a prolonged disorder where stool frequency, urgency and incontinence have major impacts on quality of life. Good history taking is necessary, with different causes to be considered: onset and duration of symptoms, previous treatments, co-existing conditions, travel and drug use may all be relevant. Tests include blood and faecal screening. Exclusion of inflammatory bowel disease and colorectal neoplasia is important and may require colonoscopy. Coeliac disease, microscopic colitis and bile acid diarrhoea are all common conditions which should not be missed, as specific therapy is available for each of these. Functional bowel disorders with diarrhoea are prevalent, overlapping with other more treatable conditions. Dietetic assessment and advice are helpful. Awareness of high FODMAP foods, with identification of individual sensitivities, is often beneficial.

What Are the Pearls and Pitfalls of the Dietary Management for Chronic Diarrhoea?

Chronic diarrhoea affects up to 14% of adults, it impacts on quality of life and its cause can be variable. Patients with chronic diarrhoea are presented with a plethora of dietary recommendations, often sought from the internet or provided by those who are untrained or inexperienced. In this review, we summarise the possible causes of chronic diarrhoea that can be managed by diet, the symptom improvement and quality of life benefits but also the potential risks of such dietary treatments. Clinicians need to consider both the benefits and risks of dietary treatments before making dietary recommendations to manage chronic diarrhoea. The pivotal role that dietitians have in ensuring optimal symptom improvement without jeopardising nutritional and overall health is discussed.

High prevalence of primary bile acid diarrhoea in patients with functional diarrhoea and irritable bowel syndrome-diarrhoea, based on Rome III and Rome IV criteria.

BACKGROUND: A high prevalence of primary bile acid diarrhoea (BAD) has been reported for Rome III defined irritable bowel syndrome (IBS)-diarrhoea and functional diarrhoea. We determined whether this still applies under the contemporaneous Rome IV criteria, given that the latter characterises IBS-diarrhoea as having more frequent abdominal pain compared with previous iterations, whilst no longer recognising abdominal discomfort. METHODS: Patients referred for a 75SeHCAT test completed a baseline questionnaire comprising, i) demographic data, ii) risk factors for BAD (inflammatory bowel disease, bowel resection, cholecystectomy, microscopic colitis, celiac disease, abdominal-pelvic radiotherapy), iii) the Rome III and IV bowel disorder questionnaire, and iv) mood and somatisation scores. A diagnosis of BAD constituted a 75SeHCAT of ≤15%, with moderate to severe disease being defined as ≤10% and ≤5%, respectively. FINDINGS: Of 300 patients with complete dataset, 184 had no risk factors for BAD and fulfilled criteria for either IBS-diarrhoea or functional diarrhoea. The prevalence of primary BAD was 38% (n = 70/184), with almost half having moderate (n = 16) to severe (n = 17) disease. Using the Rome III criteria, the prevalence of primary BAD was 36% in IBS-diarrhoea (n = 63/173) and 64% (n = 7/11) in functional diarrhoea; p = 0.11. Using the Rome IV criteria, the prevalence of primary BAD was 38% (n = 53/139) in IBS-diarrhoea and 38% (n = 17/45) in functional diarrhoea; p = 0.97. Patients with primary BAD experienced more frequent loose stools (p = 0.01) and had a higher body mass index (p<0.0001) compared to those without BAD, but otherwise no significant differences were seen in age, gender, mood, somatisation, or abdominal pain. The presence of primary BAD in patients classified as overweight or obese was approximately 40% and 60%, respectively. INTERPRETATION: Over a third of patients with Rome IV IBS-diarrhoea or functional diarrhoea have primary BAD, similar to Rome III. We therefore recommend that, in secondary care settings, generic testing for primary BAD should be considered in patients presenting with chronic diarrhoea of presumed functional origin regardless of concomitant abdominal pain. Centres that lack tests for primary BAD, and who empirically treat instead, may consider targeting patients who are overweight or obese.

Effects of conventional and a novel colonic-release bile acid sequestrant, A3384, on fibroblast growth factor 19 and bile acid metabolism in healthy volunteers and patients with bile acid diarrhoea.

BACKGROUND: Primary bile acid diarrhoea (BAD) is associated with increased bile acid synthesis and low fibroblast growth factor 19 (FGF19). Bile acid sequestrants are used as therapy, but are poorly tolerated and may exacerbate FGF19 deficiency. AIM: The purpose of this study was to evaluate the pharmacological effects of conventional sequestrants and a colonic-release formulation preparation of colestyramine (A3384) on bile acid metabolism and bowel function in patients with BAD. METHODS: Patients with seven-day 75selenium-homocholic acid taurine (SeHCAT) scan retention <10% were randomised in a double-blind protocol to two weeks treatment with twice-daily A3384 250 mg (n = 6), 1 g (n = 7) or placebo (n = 6). Thirteen patients were taking conventional sequestrants at the start of the study. Symptoms were recorded and serum FGF19 and 7α-hydroxy-4-cholesten-3-one (C4) measured. RESULTS: Median serum FGF19 on conventional sequestrant treatment was 28% lower than baseline values in BAD (p < 0.05). C4 on conventional sequestrant treatment was 58% higher in BAD (p < 0.001). No changes were seen on starting or withdrawing A3384. A3384 improved diarrhoeal symptoms, with a median reduction of 2.2 points on a 0-10 Likert scale compared to placebo, p < 0.05. CONCLUSIONS: Serum FGF19 was suppressed and bile acid production up-regulated on conventional bile acid sequestrants, but not with A3384. This colonic-release formulation of colestyramine produced symptomatic benefit in patients with BAD.