Alimentary Pharmacology and Therapeutics
Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea-predominant irritable bowel syndrome M. Camilleri*, A. Acosta*, I. Busciglio*, A. Boldingh*, R. B. Dyer†, A. R. Zinsmeister‡, A. Lueke§, A. Gray§ & L. J. Donato§
*Clinical Enteric Neuroscience Translational and Epidemiological Research (C.E.N.T.E.R.), Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. † Immunochemistry Core Laboratory, Center for Clinical and Translational Research, Mayo Clinic, Rochester, MN, USA. ‡ Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA. § Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
Correspondence to: Dr M. Camilleri, Mayo Clinic, Charlton Bldg, Rm 8-110, 200 First Street S.W., Rochester, MN 55905, USA. E-mail: [email protected]
Publication data Submitted 9 October 2014 First decision 3 November 2014 Resubmitted 24 November 2014 Accepted 8 December 2014 EV Pub Online 16 January 2015 This article was accepted for publication after full peer-review.
SUMMARY Background About one-third of patients with IBS-diarrhoea (irritable bowel syndromeD) have evidence of increased bile acid synthesis or excretion. Aims To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam. Methods A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875 mg [3 tablets (625 mg tablets)] orally, twice daily, for 10 days on total 48-h faecal bile acid excretion and fasting serum C4 (7a-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8 days prior and 8 days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4. Results Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48 h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat. Conclusions Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam. Aliment Pharmacol Ther 2015; 41: 438–448
438
ª 2015 John Wiley & Sons Ltd doi:10.1111/apt.13065
Colesevelam and bile acids in IBS-diarrhoea BACKGROUND The pathophysiology of irritable bowel syndrome (IBS) includes abnormal motility and sensation, and mucosal defence, manifested as altered mucosal structure and function or low-grade inflammation. Bile acids (BAs) are synthesised and secreted from the liver, modulate lipid digestion, are reabsorbed mainly in the terminal ileum and, through the enterohepatic circulation, return to the liver. The secretion and circulation of BAs are mainly stimulated by food intake which releases cholecystokinin from the upper small intestine, inducing gall-bladder contraction. Several prior studies in our laboratory have led us to appreciate the role of BAs in diarrhoea-predominant IBS (IBS-D). In a study of 119 patients with IBS,1 32% had abnormal colonic transit measured by scintigraphy at 24 or 48 h, with accelerated transit in 48% of IBS-D patients; the causes of abnormal transit are unclear. A meta-analysis showed bile acid malabsorption (BAM) in up to 50% of patients with chronic functional diarrhoea or IBS-D.2 About one-third of patients with IBS have alterations in faecal BA excretion and hepatic BA synthesis rates compared to healthy controls.3, 4 The effects of BAs on colonic secretion and motility are multifactorial and include stimulation of the G protein-coupled receptor called TGR5 or G protein-coupled bile acid receptor 1 (GPBAR1)5 which mediates colonic secretory and motor effects of BAs.6 The diagnosis of BAM in the United States is based predominantly on a therapeutic trial7 of BA sequestrants such as colestipol or cholestyramine. Recent open-label therapeutic trials have documented the efficacy of cholestyramine in 13 patients with idiopathic bile acid diarrhoea8 and adequate relief of IBS symptoms, with escalating or flexible dosing of colestipol.9 In the cholestyramine trial,8 there was no significant effect on colonic transit, though there were significant prolongations of small bowel transit time and segmental transverse colon transit time. The mechanism of action of these BA sequestrants to ameliorate bowel function is not clearly demonstrated in most studies. In addition, the BA sequestrants may have other effects (e.g. binding of other substances, bacterial toxins) and, therefore, the assumption that BAs are the cause of diarrhoea may be incorrect. Finally, this approach is compromised by poor tolerance of the traditional resin sequestrants like cholestyramine and by inadequate medication compliance.10, 11 The novel BA sequestrant, colesevelam, is administered as a tablet and has had higher patient compliance. In a prior study, we Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
showed that colesevelam slowed colonic transit in patients with unselected IBS-D.12 Our hypothesis in this study was that therapy with the BA sequestrant, colesevelam, sequesters luminal BA, increasing faecal BA excretion and reducing diarrhoea in patients with IBS-D who have documented increased faecal BA excretion. The study aims were to assess the effects of colesevelam on faecal excretion of BAs and on bowel movements IBS-D patients; to study ability of the HPLC assay for faecal BAs to demonstrate responsiveness after treatment with colesevelam and to appraise whether individual or random stool samples accurately reflect 48-h total faecal BA excretion and proportions of the main BAs excreted; and to study the effect of colesevelam on faecal fat excretion.
METHODS Study design We conducted a single-centre, unblinded, single-dose, controlled, 10-day trial of the effects of colesevelam, 1875 mg (3 tablets, 625 mg each), taken orally, twice daily, in 12 patients with IBS-D and prior evidence of increased BA synthesis or faecal excretion (Figure 1). Stool 48-h collections (for BAs) and fasting serum samples (for serum C4, 7a-hydroxy-4-cholesten-3-one) were collected during baseline before treatment, and then during days 9–10 of the 10 days of colesevelam dosing for faecal BAs, and on day 11 (the morning following the last dose) for fasting serum C4. The study was approved by Mayo Clinic Institutional Review Board, and the study was registered with ClinicalTrials.gov. #NCT02111603.
VISIT 2: Fasting blood draw diaries return & study med 100 GRAM FAT DIET & DIARY pickup (Days 7, 8, 9, 10)
Day 1 2 3 4 5 6
First day of bowel diary
7
8
9
10
11
Last day Stool collecting Stool of bowel (Days 9 & 10) sample diary return
Figure 1 | Experimental design of the two phases of the study which were of equal duration: during baseline, no medication was taken; in the treatment phase, patients received colesevelam, 1875 g b.i.d. for 10 days. 439
M. Camilleri et al. Participants From a database of ~950 patients with functional gastrointestinal diseases who reside within ~150 miles of Mayo Clinic, Rochester, MN, we identified potential participants with prior measurements of BA kinetics and invited those with either increased 48-h total faecal BAs or increased serum C4 to participate. Two of the patients had prior cholecystectomy and none were on drugs affecting lipid metabolism. All participants completed a validated bowel disease questionnaire (BDQ, corresponding to Rome criteria)13 and the Hospital Anxiety and Depression Scale (HAD)14 to ensure they had symptoms consistent with IBS-D based on Rome III criteria, and that they had no significant uncontrolled affective disorder based on HAD score. Participants were females or males, 18–65 years of age. Females of child-bearing potential had a negative pregnancy test before initiation of medication. Within the first week prior to the baseline period or during the subsequent 2 weeks during the study period, the following drugs were prohibited: agents that alter gastrointestinal transit (including opioids, narcotics, anticholinergics, tricyclic antidepressants, serotonin and norepinephrine reuptake inhibitors antidepressants), analgesic drugs (including opiates, nonsteroidal anti-inflammatory drugs, COX-2 inhibitors) and medications that could interfere with the interpretation of the study. Birth control pills, oestrogen replacement therapy and thyroxine replacement were permitted. Female subjects who were pregnant or breast-feeding, patients with prior abdominal surgery (except appendectomy) and patients with known chronic liver disease or history of elevated AST/ALT 2.0X upper limit of normal were excluded. Bowel function questionnaires During the study, participants completed a daily diary to record their bowel functions for each bowel movement including consistency (Bristol Stool Form Scale:15 1 – hard lumps, 2 – lumpy sausage, 3 – cracked sausage, 4 – smooth sausage, 5 – soft lumps, 6 – mushy and 7 – watery), ease of passage (1 – manual disimpaction, 2 – enema needed, 3 – straining needed, 4 – normal, 5 – urgent without pain, 6 – urgent with pain, 7 – incontinent) and sense of completeness of evacuation (yes = 1/no = 0). These diaries have been validated for responsiveness.16, 17 Colesevelam Colesevelam was purchased in 625 mg tablet form (Welchol; Daiichi Sankyo, Inc., Parsippany, NJ, USA) through Mayo Clinic Research Pharmacy. Participants took 440
1875 mg (3 tablets, 625 mg each) of the medication orally, twice daily, with lunch and supper for 10 days. Because colesevelam can interfere with absorption of other medications, subjects taking hormonal contraceptives for birth control were instructed to use a double barrier method (such as a condom or diaphragm) with a spermicide.
Biological samples Blood samples were collected in the morning after overnight fasting before starting the colesevelam and on the morning after the 10-day dosing with colesevelam. The samples were sent to the Mayo Clinic Immunochemical Core Laboratory (ICL) for storage in a 70 °C freezer, until batch analysis for serum C4 values in the ICL. Stools samples were collected before starting the colesevelam and during days 9–10 of dosing with colesevelam. Samples were collected on both occasions during the last 2 days while patients received a 100-g fat diet each day for 4 days. The samples were sent to a central laboratory to be frozen immediately for subsequent testing for faecal BAs. Participants received standardised instructions for the 100-g fat diet (Mayo Clinic brochure #MC1450) and for the stool specimen collection (Mayo Clinic brochure #MC1335-15) used in clinical practice at Mayo Clinic. Faecal collections over a 48-h period may be considered cumbersome, both for patients and laboratory staff. However, this is presently the only available diagnostic test for BA diarrhoea in the United States.7 Moreover, in this research study with dedicated staff and highly cooperative patients, each stool passed was collected and stored separately to measure BA content. Laboratory measurements Serum 7a-hydroxy-4-cholesten-3-one (C4) is a measurement of hepatic cholesterol synthesis and is closely related to the faecal loss of BAs. Serum C4 is a validated method for detecting BAM. In head-to-head comparisons with the 75SeHCAT retention test, increased serum C4 had sensitivity of 90% and specificity of 79% in diagnosing BAM,18 where shorter retention half-time of 75 SeHCAT is associated with increased level of C4; and it had 98% negative predictive value and 74% positive predictive value for diagnosis of BAM.19 On the basis of the method published from our laboratory, adapted from G€alman et al.,20 we used HPLC/ tandem mass spectrometry to measure serum C4 to evaluate BA synthesis rate.21 Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea Faecal BA excretion. Using HPLC/tandem mass spectrometry, we have adapted a method used with serum samples22 to measure faecal total and individual BAs2 in a 48-h collection of stools collected while ingesting a 100-g fat per day diet. BAs were measured in each individual stool sample and, finally, all samples were mixed to obtain the total faecal BA excretion over 48 h. The stool samples were prepared for assay by HPLC/MS by methanol extraction. Approximately, 100 mg of each stool homogenate was incubated with 2 mL of methanol for 30 min to extract the BAs. Daily faecal fat excretion, while ingesting the 100-g fat diet daily, was measured by nuclear magnetic resonance spectrometry in the Mayo Clinic Department of Laboratory Medicine and Pathology. In vitro studies. To assess the ability of the methanol extraction to separate the BAs from the sequestrant, colesevelam, we mixed BAs with colesevelam and performed several extraction experiments. Ten micromoles of each BA (CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid) were spiked into eight 2-mL aliquots of either water or methanol. Fifty milligram of colesevelam was also added to four of the eight aliquots. Samples were then run on the LC–MS/MS method for detection of free BAs. The mean concentrations for the four replicates with and without colesevelam were compared for both solvents across all analytes. Statistical analysis The primary analysis compared on-treatment total faecal BA excretion with baseline total faecal BA excretion using the signed rank test. The analyses of secondary endpoints were based on the paired t-test or signed rank test as warranted. The concordance between overall mean stool characteristics and randomly selected individual stool samples was based on Lin’s concordance correlation coefficient23 separately for pre- and post-colesevelam periods. Endpoints for analysis. The primary endpoint for analysis was change in total 48-h faecal BAs from baseline in response to treatment with colesevelam. As the faecal BA measurement included an extraction step that freed the BA into the stool water, the effect of colesevelam was measured as an increase in faecal BA excretion. The secondary endpoints addressed other effects of colesevelam in patients with IBS-D and BAM; these endpoints were as follows: Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
(i) Change in faecal BA excretion per gram of stool (ii) Change in fasting serum C4 (iii) Change in faecal fat excretion (iv) Change in stool consistency on colesevelam compared to baseline (v) Change in stool frequency on colesevelam compared to baseline (vi) Relative composition of the main individual BAs (CA, CDCA, DCA, LCA and UDCA) in 48-h stool collection on colesevelam compared to baseline (vii) Comparison of composition of the main individual BAs in individual samples of faeces and total faecal 48-h collection
Sample size assessment. In a prior study of 54 IBS-D patients,24 we identified an IBS-D subgroup (n = 23) with high faecal BA excretion (above the 90th percentile in health of 2337 lM/48 h). The average faecal BA excretion in all IBS-D patients was 2495 382 (S.E.M.) lM/48 h; in patients with IBS-D and evidence of BAM, the mean total faecal BA excretion was 4905 lM/48 h. The estimated standard deviation (s.d.) in this group was 3706 lM/48 h, and it was the basis for estimating the effect size that could be detected with a study of 12 patients, assuming we could recruit about 50% of the patients known to have IBS-D with BAM in the database. The effect size is the difference in means (baseline and on colesevelam) of the total faecal BA excretion over 48 h. Assuming a sample size of 12 subjects, a paired t-test (at two-sided a level of 0.05 and 80% power) would be sufficient to detect a difference in means of 3400 lM/48 h (based on s.d. of 3706 lM/48 h). Thus, if colesevelam inactivated or sequestered luminal BA to effectively reduce faecal 48-h BA to an equivalent of 1500 lM/48 h in the absence of the sequestrants, the drug would have achieved a reduction in luminal BA to bring the latter to be equivalent to the 75th percentile for healthy volunteers. Thus, this effect size would be anticipated to demonstrate a clinically relevant change in total faecal BA and to be associated with improved stool characteristics. All authors had access to the study data and reviewed and approved the final manuscript. RESULTS Demographics The mean age of the 12 participants (11 female, 1 male) was 43.1 3.7 years and mean BMI was 31.1 2.4 kg/m2. 441
M. Camilleri et al. In vitro studies of extraction of bile acids bound to colesevelam To show that our BA extraction and detection method is able to measure all BAs in the methanol extracted
LCA Water BA BA + colesevelam Methanol BA BA + colesevelam
DCA
sample, in vitro studies were performed in different matrices in the presence and absence of colesevelam (Table 1). When BAs are mixed with colesevelam in an aqueous environment, no free BAs are detectable by
UDCA
CDCA
CA
9.4 (1.3) 0.1 (.2)
9.2 (1.5) 0.0 (0.0)
9.5 (1.3) 0.0 (0.0)
9.6 (1.5) 0.0 (0.0)
10.1 (1.6) 0.0 (0.0)
10.0 (1.5) 10.0 (2.4)
9.9 (1.7) 10.0 (2.3)
9.8 (1.2) 9.9 (2.3)
10.1 (1.7) 10.2 (2.5)
9.9 (1.7) 9.7 (2.3)
Table 1 | Bile acids colesevelam in water or methanol (lM s.d.)
BA, bile acid; LCA, lithocholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid.
Total fecal BA (µmoles/48 h) excreted
(a) 10 000
10 000 P = 0.012
8000
8000
6000
6000
4000
4000
2000
2000
0
0
Baseline
(b)
Colesevelam
(c)
16 000 14 000
120
12 000
100
10 000 8000 6000 4000
80 60 40
2000
20
0
0
Baseline
Colesevelam
Colesevelam
140
Serum C4 ng/mL
Total fecal BA (nmoles/g stool) excreted
Baseline
Baseline
Colesevelam
Figure 2 | Comparison of (a) total faecal bile acid excretion over 48 h during baseline and colesevelam treatment in the 12 patients individually (right panel) and as a group (left panel). (b, c) Individual data during baseline and colesevelam treatment for total faecal bile acid excretion per gram of stool, and fasting serum C4 respectively. 442
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea mass spectrometry. Thus, colesevelam does bind all BAs in this environment. In contrast, when colesevelam is mixed with BAs in methanol, all of the BAs are free and detected by our assay. Owing to the extreme solubility differences in methanol between colesevelam (not soluble) and BAs (soluble), BAs do not bind to colesevelam in methanol. Thus, results of methanol-extracted BAs presented in this study include all BAs found in stool, whether they were free or colesevelam-bound in vivo.
Effect of colesevelam on total faecal BA excretion Colesevelam resulted in higher faecal excretion of BAs in stool compared to baseline. The increase in total faecal BAs was observed for the entire group (Figure 2a, left panel) and for 10 of the 12 patients with IBS-D (FigTable 2 | Effects of colesevelam on bowel function endpoints and faecal fat (mean S.E.M.)
ure 2a, right panel). Colesevelam also increased the faecal BA excretion per gram of stool (Figure 2b).
Effect of colesevelam on fasting serum C4 Colesevelam significantly increased fasting serum C4 measurement [P < 0.01 (signed rank test); Table 2 and individual data shown in Figure 2c]. Effects of colesevelam on proportion of main primary and secondary BAs and faecal fat Colesevelam was associated with increased proportion of deoxycholic acid in the stool (Figure 3) and a numerical reduction in the proportion of cholic acid in stool; there were no significant differences in the proportions of CDCA, lithocholic or ursodeoxycholic acids. There was no significant effect of colesevelam on faecal fat excretion Baseline
# stools per week Stool form (BSFS) Ease of stool passage Total faecal BA excretion (lmoles/48 h) Total faecal BA excretion per gram of stool (nmoles/g stool) Faecal fat (g/day) Serum C4 (ng/mL)
17.6 4.8 4.7 1558 2332
0.4 0.3 0.2 229 344
6.4 1.2 24.7 4.2
Colesevelam 15.1 4.4 4.2 3496 6585
1.9 0.3 0.1 709 1196
6.8 0.9 72.3 12.2
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
100
P = 0.028
DCA 100
80
80
60
60
40
40
20
20
0
0
Baseline
Percent of total fecal BA
Figure 3 | Changes in percentage of deoxycholic acid (DCA) and cholic acid (CA) in stool with colesevelam, suggesting that the medication sequesters the secretory, dehydroxylated secondary bile acid, DCA, which is derived from the primary bile acid, CA. There were no significant differences in lithocholic acid or chenodeoxycholic acid in stool.
Percent of total fecal BA
BSFS, Bristol Stool Form Scale; BA, bile acid. There were significant differences in stool form, faecal total BA excretion, faecal total BA excretion per gram of stool, and serum C4 (shown in figures).
Baseline
Colesevelam
Colesevelam CA 60
60
P = 0.061
50
50
40
40
30
30
20
20
10
10
0
0
Baseline Baseline
Colesevelam
Colesevelam 443
Fecal fat g/day
M. Camilleri et al. (Table 2, Figure 4). There was no major difference in the total faecal BA in the two patients with prior cholecystectomy compared to the other patients. Thus, faecal BA excretions in these two patients were 2916 and 2498 lM/48 h at baseline and 1878 and 2932 lM/48 h on colesevelam treatment.
20 18 16 14 12 10 8 6 4 2 0 Baseline
Colesevelam
Figure 4 | Effects of colesevelam on faecal fat in patients with diarrhoea-predominant irritable bowel syndrome and bile acid malabsorption.
(a) 7.0
7
Stool form BSF scale
6.5
P = 0.043
6.5
6.0
6
5.5
5.5
5.0
5
4.5
4.5
4.0
4
3.5
3.5
3.0
(b)
Effects of colesevelam on stool form and bowel function Colesevelam resulted in a significant decrease in the average score of stool consistency on the Bristol Stool Form Scale (Figure 5a). There were numerical reductions in number of bowel movements per week (Figure 5b) and improvements in ease of passage, but these were not statistically significant (Table 1).
3
Baseline
Colesevelam
Baseline
Colesevelam
Baseline
Colesevelam
40 35
# BM/week
30 25 20 15 10 5 0
Figure 5 | Effects of colesevelam on (a) stool form [Bristol Stool Form Scale (BSF scale)] and (b) stool frequency per week in patients with diarrhoea-predominant irritable bowel syndrome and bile acid malabsorption. 444
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea individual patients, both at baseline and after colesevelam treatment (Figure 7). However, individual stool samples could not be used in lieu of the total 48-h faecal samples to estimate the total or the proportion of the main primary and secondary BAs in stool, as shown by the relatively low concordance correlations for total BAs and moderate coefficients for LCA and DCA, which are the greatest proportions of faecal BAs, and higher coefficients for CDCA, CA and UDCA, which are relatively low proportions of BAs in patients with intact colon (Table 3).
35
BM # per week
30
Rs = –0.621, P = 0.03
25 20 15 10 5 0 0
2000
4000
6000
8000
10 000
Total bile acid (µmoles) in stool with colesevelam treatment
DISCUSSION Our study has shown that, among patients with IBS-D who have prior evidence of BAM, the sequestrant, colesevelam, resulted in increased delivery of BAs to stool. This is accompanied by a significantly more solid stool form, and the stool frequency correlates inversely with the BA sequestered by colesevelam and delivered to the stool. This paradox, that is, the increase in faecal BAs and the more solid stool and reduced number of bowel movements per week, is explained by the binding of the luminal BAs to the sequestrant, colesevelam, and the ability of the preparatory step in the faecal analysis to deliver the BAs from the bound to the free aqueous phase of stool. Specifically, BAs were obtained from stool homogenates using methanol extraction while leaving colesevelam in the insoluble precipitate. Therefore, the extraction contained both free and sequestrant-bound BAs, even though most of the BAs are likely sequestrant-bound in vivo. Colesevelam also resulted in a significant increase in serum C4, suggesting that the greater loss of BA into
Figure 6 | Significant inverse correlation between the faecal excretion of total bile acids and number of bowel movements (BM) per week, suggesting that sequestration of the bile acids by colesevelam reduced the diarrhoea.
Correlation of faecal BA excretion and number of stools per week There was a significant inverse correlation between the faecal excretion of total BAs and number of bowel movements per week, suggesting that sequestration of the BAs by colesevelam reduced the diarrhoea (Figure 6). Comparison of proportions of primary and secondary BAs in individual and total stool samples in each participant The faecal BA excretion in a random sample of stool was closely correlated (concordance correlation coefficient 0.947) with the mean excretion in all samples from
Baseline
Colesevelam
15 000
Figure 7 | Concordance between mean total faecal bile acid (BA) excretion in all samples from an individual patient, and the total faecal BA content in a randomly selected individual sample. Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Mean total fecal BA, µmoles
Overall concordance correlation coefficient 0.947
10 000
5000
0 0
2500 5000 7500 10 000 12 500 0
2500 5000 7500 10 00012 500
Random sample total fecal BA, µmoles 445
M. Camilleri et al. Random sample vs. total 48 h collection
Baseline
Total BAs Cholic acid Deoxycholic acid Ursodeoxycholic acid Chenodeoxycholic acid Lithocholic acid
0.53 0.84 0.68 0.96 0.88 0.44
Colesevelam 0.13 0.08 0.14 0.20 0.05 0.21
stool stimulated hepatic synthesis rate. This increased synthesis of BAs was functionally important in avoiding the development of steatorrhoea. However, longer term trials are required to ensure that there are no steatorrhoea or malabsorption of fat-soluble vitamins with prolonged treatment. We had previously observed that colesevelam treatment in unselected patients with IBS-D was associated with slower emptying of the ascending colon (average 4 h longer) compared with placebo.12 In these unselected patients, we noted that the treatment effect was associated with baseline serum C4 levels (P = 0.0025) suggesting that patients with IBS-D and increased BA synthesis were more likely to respond to colesevelam. Overall, colesevelam was associated in the prior study with greater ease of stool passage (P = 0.048) and somewhat firmer stool consistency (P = 0.12). The current study extends the previous findings and demonstrates that the significantly improved stool consistency is associated with sequestration of the BAs in the colon, preventing their diarrhoeagenic effects. This is also supported by the inverse correlation between number of stools per week and the total faecal BAs measured in stool after extraction of the BAs from their bound state. The diagnosis of BAM has been a clinical challenge in the United States as the 75SeHCAT retention test is not approved. A definitive diagnosis is desirable before committing patients to ingesting the relatively unpalatable resins in powder form or the relatively expensive tablet forms such as colesevelam. With the availability of tests such as serum C4, serum fibroblast growth factor-19 (FGF-19)25 and faecal BA excretion at reference laboratories, we anticipate that there will be greater use of these diagnostic tests after their fuller validation. One of the steps of validation of the diagnostic is demonstration of responsiveness to treatment and concordance with clinical benefit. The current study has shown that the HPLC/MS method used by Mayo Clinic Department of Laboratory Medicine and Pathology to measure total and individual BAs is responsive to treatment with the BA sequestrant, colesevelam, and that this 446
0.54 0.83 0.85 0.54 0.90 0.88
0.15 0.06 0.07 0.20 0.06 0.07
Table 3 | Concordance correlation coefficients (mean S.E.M.) of stool total and individual bile acids (BAs) for the 48-h collection and a randomly selected stool sample
biochemical effect is associated with clinical improvement in the diarrhoea. We have set up the method to be run together with a measurement of faecal fats, which is commonly investigated in patients with chronic diarrhoea. This combination would also be useful in patients with diarrhoea due to ileal resection (type 1 BA diarrhoea) and cholecystectomy or pancreatic exocrine insufficiency (type 3 BA diarrhoea). Thus, in such patients, knowing both the faecal fat and faecal BA excretion would help clinicians determine the degree of dietary fat restriction in addition to the sequestration of BAs to relieve the diarrhoea. Our study also addressed the clinically relevant question: Does a random measurement of stool BA (total content and proportion of main primary and secondary BAs) provide sufficient information to avoid the need for a 48-h collection? Unfortunately, we came to the same conclusion as with random faecal fat estimates relative to 48-h fat excretion that the random sample provides insufficient information on BA excretion. Variation in BA excretion per gram faecal weight in each bowel movement, and in the faecal pellet and supernatant were reported in three patients with ileal resection diarrhoea and suggested that individual stool samples were not representative of the total faecal BA excretion.26 The main limitations of our current study are the absence of a placebo arm and the small sample size; these limitations reduce our confidence in the clinical benefit of colesevelam in patients with IBS-D and BAM. However, the major objective of our study was to demonstrate responsiveness of faecal BAs with the HPLC/MS assay, and future, appropriately powered studies will be designed to address the clinical efficacy of this therapeutic approach. A recent systematic review also endorsed the potential use of colesevelam in the management of chronic diarrhoea due to BAM.27 In conclusion, the current study demonstrates the utility of measurement of faecal BAs and serum C4 to characterise either increased synthesis or excretion of BAs as targets for treatment with a BA sequestrant. We have Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea demonstrated the responsiveness to colesevelam at the biochemical level and in the bowel dysfunction of such patients, particularly stool consistency and number of bowel movements. We have also shown that, as with faecal fat estimation, the 48-h stool collection is necessary to measure faecal BA excretion and prove BAM in 12 such patients with IBS-D, confirming the experience published over 40 years ago of Mitchell et al. in studies performed on three patients with prior ileal resection.26 These studies also add to the plethora of data suggesting that there is an opportunity to diagnose and specifically treat the cause of symptoms in patients who carry a label of IBS-D.
AUTHORSHIP Guarantor of the article: Dr Michael Camilleri. Author contributions: Michael Camilleri: principal investigator; author of manuscript, takes responsibility for the integrity of the work as a whole, from inception to published article. Andres Acosta: fellow investigator, screening of patients; co-author. Irene Busciglio: recruitment of participants, conduct of study. Amy Boldingh: recruitment of participants. Roy B. Dyer: measurement of serum C4. Alan R. Zinsmeister: biostatistician. Alan Lueke: measurement of faecal fat and bile acids. Amber Gray: measurement of faecal fat and bile acids. Leslie J. Donato: supervision of measurement of faecal fat and
bile acids, co-author. All authors approved the final version of the manuscript.
ACKNOWLEDGEMENTS The authors thank the Mayo Clinic Department of Laboratory Medicine and Pathology for waiving charges for measurements of faecal bile acids and fats, and we thank Mrs Cindy Stanislav for excellent secretarial assistance. Declaration of personal interests: Dr Camilleri has served as a consultant for NPS Pharmaceuticlas, AstraZeneca, Navartis, Targacept, NGM Biopharmaceuticals, Altos Therapeutics, and Purdue Pharma with personal remuneration. He has served as an advisory board member for AstraZeneca and BioKier, also with personal remuneration. With remuneration to his employer, Mayo Clinic, he has served as a consultant for Entera Health, Rhythm, Theravance, GlaxoSmithKline, Takeda, and Shire. He has received research funding from Rhythm, Salix, SK Life Sciences, NPS Pharmaceuticals, Second Genome, and Entera Health. Declaration of funding interests: Dr Camilleri’s research on bile acid diarrhoea is supported by NIH R01 DK92179 and by Mayo Foundation (Atherton and Winifred W. Bean Endowed Professorship). The study was conducted with the help of the Nursing Core of Mayo Clinic CCaTS (grant #UL1-TR000135 from National Institutes of Health).
REFERENCES 1. Camilleri M, McKinzie S, Busciglio I, et al. Prospective study of motor, sensory, psychological and autonomic functions in 119 patients with irritable bowel syndrome. Clin Gastroenterol Hepatol 2008; 6: 772–81. 2. Wedlake L, A’Hern R, Russell D, et al. Systematic review: the prevalence of idiopathic bile acid malabsorption as diagnosed by SeHCAT scanning in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther 2009; 30: 707–17. 3. Wong BS, Camilleri M, Carlson P, et al. Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea. Clin Gastroenterol Hepatol 2012; 10: 1009–15. 4. Shin A, Camilleri M, Vijayvargiya P, et al. Bowel functions, fecal unconjugated primary and secondary bile acids, and colonic transit in patients with irritable bowel syndrome.
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
5.
6.
7.
8.
Clin Gastroenterol Hepatol 2013; 11: 1270–5. Poole DP, Godfrey C, Cattaruzza F, et al. Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system. Neurogastroenterol Motil 2010; 22: 814– 25. Alemi F, Poole DP, Chiu J, et al. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology 2013; 144: 145–54. Vijayvargiya P, Camilleri M, Shin A, et al. Methods for diagnosis of bile acid malabsorption in clinical practice. Clin Gastroenterol Hepatol 2013; 11: 1232–9. Stotzer P-O, Abrahamsson H, Bajor A, Sadik R. Effect of cholestyramine on gastrointestinal transit in patients with idiopathic bile acid diarrhea: a prospective, open-label study.
9.
10.
11.
12.
Neuroenterology 2013; 2: 5. doi:10.4303/ ne/235657 Article ID 235657. Bajor A, T€ ornblom H, Rudling M, et al. Increased colonic bile acid exposure: a relevant factor for symptoms and treatment in IBS. Gut 2015; 64: 84–92. adell K, Holme I, et al. Eriksson M, H Compliance with and efficacy of treatment with pravastatin and cholestyramine: a randomized study on lipid-lowering in primary care. J Intern Med 1998; 243: 373–80. Bays HE, Maki KC, Schmitz K. Colesevelam hydrochloride powder for oral suspension versus cholestyramine powder for oral suspension: comparison of acceptability and tolerability. Endocr Pract 2011; 17: 218–25. Odunsi-Shiyanbade ST, Camilleri M, McKinzie S, et al. Effects of chenodeoxycholate and a bile acid sequestrant, colesevelam, on intestinal transit and bowel function. Clin
447
M. Camilleri et al.
13.
14.
15.
16.
17.
18.
448
Gastroenterol Hepatol 2010; 8: 159–65. Talley NJ, Phillips SF, Wiltgen CM, et al. Assessment of functional gastrointestinal disease: the bowel disease questionnaire. Mayo Clin Proc 1990; 65: 1456–79. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983; 67: 361–70. Heaton KW, Radvan J, Cripps H, et al. Defecation frequency and timing, and stool form in the general population: a prospective study. Gut 1992; 33: 818–24. Andresen V, Camilleri M, Busciglio I, et al. Effect of 5 days linaclotide on transit and bowel function in females with constipation-predominant irritable bowel syndrome. Gastroenterology 2007; 133: 761–8. Manini ML, Camilleri M, Goldberg M, et al. Effects of velusetrag (TD-5108) on gastrointestinal transit and bowel function in health and pharmacokinetics in health and constipation. Neurogastroenterol Motil 2010; 22: e7–8. Sauter GH, M€ unzing W, von Ritter C, et al. Bile acid malabsorption as a cause of chronic diarrhea: diagnostic value of
19.
20.
21.
22.
7alpha-hydroxy-4-cholesten-3-one in serum. Dig Dis Sci 1999; 44: 14–9. Brydon WG, Nyhlin H, Eastwood MA, et al. Serum 7 alpha-hydroxy-4cholesten-3-one and selenohomocholyltaurine (SeHCAT) whole body retention in the assessment of bile acid induced diarrhoea. Eur J Gastroenterol Hepatol 1996; 8: 117–23. G€alman C, Arvidsson I, Angelin B, et al. Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alphahydroxy-4-cholesten-3-one in peripheral blood. J Lipid Res 2003; 44: 859–66. Camilleri M, Nadeau A, Tremaine WJ, et al. Measurement of serum 7alphahydroxy-4-cholesten-3-one (or 7alphaC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry. Neurogastroenterol Motil 2009; 21: 734–e43. Tagliacozzi D, Mozzi AF, Casetta B, et al. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem
23.
24.
25.
26.
27.
mass spectrometry: a simple and rapid one-step method. Clin Chem Lab Med 2003; 41: 1633–41. Lin LI-K. A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989; 45: 255–68. Camilleri M, Busciglio I, Acosta A, et al. Effect of increased bile acid synthesis or fecal excretion in irritable bowel syndrome-diarrhea. Am J Gastroenterol 2014; 109: 1621–30. Pattni SS, Brydon WG, Dew T, et al. Fibroblast growth factor 19 in patients with bile acid diarrhoea: a prospective comparison of FGF19 serum assay and SeHCAT retention. Aliment Pharmacol Ther 2013; 38: 967–76. Mitchell WD, Findlay JM, Prescott RJ, et al. Bile acids in the diarrhoea of ileal resection. Gut 1973; 14: 348–53. Wilcox C, Turner J, Green J. Systematic review: the management of chronic diarrhoea due to bile acid malabsorption. Aliment Pharmacol Ther 2014; 39: 923–39.
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Copyright of Alimentary Pharmacology & Therapeutics is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea-predominant irritable bowel syndrome M. Camilleri*, A. Acosta*, I. Busciglio*, A. Boldingh*, R. B. Dyer†, A. R. Zinsmeister‡, A. Lueke§, A. Gray§ & L. J. Donato§
*Clinical Enteric Neuroscience Translational and Epidemiological Research (C.E.N.T.E.R.), Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. † Immunochemistry Core Laboratory, Center for Clinical and Translational Research, Mayo Clinic, Rochester, MN, USA. ‡ Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA. § Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
Correspondence to: Dr M. Camilleri, Mayo Clinic, Charlton Bldg, Rm 8-110, 200 First Street S.W., Rochester, MN 55905, USA. E-mail: [email protected]
Publication data Submitted 9 October 2014 First decision 3 November 2014 Resubmitted 24 November 2014 Accepted 8 December 2014 EV Pub Online 16 January 2015 This article was accepted for publication after full peer-review.
SUMMARY Background About one-third of patients with IBS-diarrhoea (irritable bowel syndromeD) have evidence of increased bile acid synthesis or excretion. Aims To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam. Methods A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875 mg [3 tablets (625 mg tablets)] orally, twice daily, for 10 days on total 48-h faecal bile acid excretion and fasting serum C4 (7a-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8 days prior and 8 days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4. Results Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48 h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat. Conclusions Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam. Aliment Pharmacol Ther 2015; 41: 438–448
438
ª 2015 John Wiley & Sons Ltd doi:10.1111/apt.13065
Colesevelam and bile acids in IBS-diarrhoea BACKGROUND The pathophysiology of irritable bowel syndrome (IBS) includes abnormal motility and sensation, and mucosal defence, manifested as altered mucosal structure and function or low-grade inflammation. Bile acids (BAs) are synthesised and secreted from the liver, modulate lipid digestion, are reabsorbed mainly in the terminal ileum and, through the enterohepatic circulation, return to the liver. The secretion and circulation of BAs are mainly stimulated by food intake which releases cholecystokinin from the upper small intestine, inducing gall-bladder contraction. Several prior studies in our laboratory have led us to appreciate the role of BAs in diarrhoea-predominant IBS (IBS-D). In a study of 119 patients with IBS,1 32% had abnormal colonic transit measured by scintigraphy at 24 or 48 h, with accelerated transit in 48% of IBS-D patients; the causes of abnormal transit are unclear. A meta-analysis showed bile acid malabsorption (BAM) in up to 50% of patients with chronic functional diarrhoea or IBS-D.2 About one-third of patients with IBS have alterations in faecal BA excretion and hepatic BA synthesis rates compared to healthy controls.3, 4 The effects of BAs on colonic secretion and motility are multifactorial and include stimulation of the G protein-coupled receptor called TGR5 or G protein-coupled bile acid receptor 1 (GPBAR1)5 which mediates colonic secretory and motor effects of BAs.6 The diagnosis of BAM in the United States is based predominantly on a therapeutic trial7 of BA sequestrants such as colestipol or cholestyramine. Recent open-label therapeutic trials have documented the efficacy of cholestyramine in 13 patients with idiopathic bile acid diarrhoea8 and adequate relief of IBS symptoms, with escalating or flexible dosing of colestipol.9 In the cholestyramine trial,8 there was no significant effect on colonic transit, though there were significant prolongations of small bowel transit time and segmental transverse colon transit time. The mechanism of action of these BA sequestrants to ameliorate bowel function is not clearly demonstrated in most studies. In addition, the BA sequestrants may have other effects (e.g. binding of other substances, bacterial toxins) and, therefore, the assumption that BAs are the cause of diarrhoea may be incorrect. Finally, this approach is compromised by poor tolerance of the traditional resin sequestrants like cholestyramine and by inadequate medication compliance.10, 11 The novel BA sequestrant, colesevelam, is administered as a tablet and has had higher patient compliance. In a prior study, we Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
showed that colesevelam slowed colonic transit in patients with unselected IBS-D.12 Our hypothesis in this study was that therapy with the BA sequestrant, colesevelam, sequesters luminal BA, increasing faecal BA excretion and reducing diarrhoea in patients with IBS-D who have documented increased faecal BA excretion. The study aims were to assess the effects of colesevelam on faecal excretion of BAs and on bowel movements IBS-D patients; to study ability of the HPLC assay for faecal BAs to demonstrate responsiveness after treatment with colesevelam and to appraise whether individual or random stool samples accurately reflect 48-h total faecal BA excretion and proportions of the main BAs excreted; and to study the effect of colesevelam on faecal fat excretion.
METHODS Study design We conducted a single-centre, unblinded, single-dose, controlled, 10-day trial of the effects of colesevelam, 1875 mg (3 tablets, 625 mg each), taken orally, twice daily, in 12 patients with IBS-D and prior evidence of increased BA synthesis or faecal excretion (Figure 1). Stool 48-h collections (for BAs) and fasting serum samples (for serum C4, 7a-hydroxy-4-cholesten-3-one) were collected during baseline before treatment, and then during days 9–10 of the 10 days of colesevelam dosing for faecal BAs, and on day 11 (the morning following the last dose) for fasting serum C4. The study was approved by Mayo Clinic Institutional Review Board, and the study was registered with ClinicalTrials.gov. #NCT02111603.
VISIT 2: Fasting blood draw diaries return & study med 100 GRAM FAT DIET & DIARY pickup (Days 7, 8, 9, 10)
Day 1 2 3 4 5 6
First day of bowel diary
7
8
9
10
11
Last day Stool collecting Stool of bowel (Days 9 & 10) sample diary return
Figure 1 | Experimental design of the two phases of the study which were of equal duration: during baseline, no medication was taken; in the treatment phase, patients received colesevelam, 1875 g b.i.d. for 10 days. 439
M. Camilleri et al. Participants From a database of ~950 patients with functional gastrointestinal diseases who reside within ~150 miles of Mayo Clinic, Rochester, MN, we identified potential participants with prior measurements of BA kinetics and invited those with either increased 48-h total faecal BAs or increased serum C4 to participate. Two of the patients had prior cholecystectomy and none were on drugs affecting lipid metabolism. All participants completed a validated bowel disease questionnaire (BDQ, corresponding to Rome criteria)13 and the Hospital Anxiety and Depression Scale (HAD)14 to ensure they had symptoms consistent with IBS-D based on Rome III criteria, and that they had no significant uncontrolled affective disorder based on HAD score. Participants were females or males, 18–65 years of age. Females of child-bearing potential had a negative pregnancy test before initiation of medication. Within the first week prior to the baseline period or during the subsequent 2 weeks during the study period, the following drugs were prohibited: agents that alter gastrointestinal transit (including opioids, narcotics, anticholinergics, tricyclic antidepressants, serotonin and norepinephrine reuptake inhibitors antidepressants), analgesic drugs (including opiates, nonsteroidal anti-inflammatory drugs, COX-2 inhibitors) and medications that could interfere with the interpretation of the study. Birth control pills, oestrogen replacement therapy and thyroxine replacement were permitted. Female subjects who were pregnant or breast-feeding, patients with prior abdominal surgery (except appendectomy) and patients with known chronic liver disease or history of elevated AST/ALT 2.0X upper limit of normal were excluded. Bowel function questionnaires During the study, participants completed a daily diary to record their bowel functions for each bowel movement including consistency (Bristol Stool Form Scale:15 1 – hard lumps, 2 – lumpy sausage, 3 – cracked sausage, 4 – smooth sausage, 5 – soft lumps, 6 – mushy and 7 – watery), ease of passage (1 – manual disimpaction, 2 – enema needed, 3 – straining needed, 4 – normal, 5 – urgent without pain, 6 – urgent with pain, 7 – incontinent) and sense of completeness of evacuation (yes = 1/no = 0). These diaries have been validated for responsiveness.16, 17 Colesevelam Colesevelam was purchased in 625 mg tablet form (Welchol; Daiichi Sankyo, Inc., Parsippany, NJ, USA) through Mayo Clinic Research Pharmacy. Participants took 440
1875 mg (3 tablets, 625 mg each) of the medication orally, twice daily, with lunch and supper for 10 days. Because colesevelam can interfere with absorption of other medications, subjects taking hormonal contraceptives for birth control were instructed to use a double barrier method (such as a condom or diaphragm) with a spermicide.
Biological samples Blood samples were collected in the morning after overnight fasting before starting the colesevelam and on the morning after the 10-day dosing with colesevelam. The samples were sent to the Mayo Clinic Immunochemical Core Laboratory (ICL) for storage in a 70 °C freezer, until batch analysis for serum C4 values in the ICL. Stools samples were collected before starting the colesevelam and during days 9–10 of dosing with colesevelam. Samples were collected on both occasions during the last 2 days while patients received a 100-g fat diet each day for 4 days. The samples were sent to a central laboratory to be frozen immediately for subsequent testing for faecal BAs. Participants received standardised instructions for the 100-g fat diet (Mayo Clinic brochure #MC1450) and for the stool specimen collection (Mayo Clinic brochure #MC1335-15) used in clinical practice at Mayo Clinic. Faecal collections over a 48-h period may be considered cumbersome, both for patients and laboratory staff. However, this is presently the only available diagnostic test for BA diarrhoea in the United States.7 Moreover, in this research study with dedicated staff and highly cooperative patients, each stool passed was collected and stored separately to measure BA content. Laboratory measurements Serum 7a-hydroxy-4-cholesten-3-one (C4) is a measurement of hepatic cholesterol synthesis and is closely related to the faecal loss of BAs. Serum C4 is a validated method for detecting BAM. In head-to-head comparisons with the 75SeHCAT retention test, increased serum C4 had sensitivity of 90% and specificity of 79% in diagnosing BAM,18 where shorter retention half-time of 75 SeHCAT is associated with increased level of C4; and it had 98% negative predictive value and 74% positive predictive value for diagnosis of BAM.19 On the basis of the method published from our laboratory, adapted from G€alman et al.,20 we used HPLC/ tandem mass spectrometry to measure serum C4 to evaluate BA synthesis rate.21 Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea Faecal BA excretion. Using HPLC/tandem mass spectrometry, we have adapted a method used with serum samples22 to measure faecal total and individual BAs2 in a 48-h collection of stools collected while ingesting a 100-g fat per day diet. BAs were measured in each individual stool sample and, finally, all samples were mixed to obtain the total faecal BA excretion over 48 h. The stool samples were prepared for assay by HPLC/MS by methanol extraction. Approximately, 100 mg of each stool homogenate was incubated with 2 mL of methanol for 30 min to extract the BAs. Daily faecal fat excretion, while ingesting the 100-g fat diet daily, was measured by nuclear magnetic resonance spectrometry in the Mayo Clinic Department of Laboratory Medicine and Pathology. In vitro studies. To assess the ability of the methanol extraction to separate the BAs from the sequestrant, colesevelam, we mixed BAs with colesevelam and performed several extraction experiments. Ten micromoles of each BA (CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid) were spiked into eight 2-mL aliquots of either water or methanol. Fifty milligram of colesevelam was also added to four of the eight aliquots. Samples were then run on the LC–MS/MS method for detection of free BAs. The mean concentrations for the four replicates with and without colesevelam were compared for both solvents across all analytes. Statistical analysis The primary analysis compared on-treatment total faecal BA excretion with baseline total faecal BA excretion using the signed rank test. The analyses of secondary endpoints were based on the paired t-test or signed rank test as warranted. The concordance between overall mean stool characteristics and randomly selected individual stool samples was based on Lin’s concordance correlation coefficient23 separately for pre- and post-colesevelam periods. Endpoints for analysis. The primary endpoint for analysis was change in total 48-h faecal BAs from baseline in response to treatment with colesevelam. As the faecal BA measurement included an extraction step that freed the BA into the stool water, the effect of colesevelam was measured as an increase in faecal BA excretion. The secondary endpoints addressed other effects of colesevelam in patients with IBS-D and BAM; these endpoints were as follows: Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
(i) Change in faecal BA excretion per gram of stool (ii) Change in fasting serum C4 (iii) Change in faecal fat excretion (iv) Change in stool consistency on colesevelam compared to baseline (v) Change in stool frequency on colesevelam compared to baseline (vi) Relative composition of the main individual BAs (CA, CDCA, DCA, LCA and UDCA) in 48-h stool collection on colesevelam compared to baseline (vii) Comparison of composition of the main individual BAs in individual samples of faeces and total faecal 48-h collection
Sample size assessment. In a prior study of 54 IBS-D patients,24 we identified an IBS-D subgroup (n = 23) with high faecal BA excretion (above the 90th percentile in health of 2337 lM/48 h). The average faecal BA excretion in all IBS-D patients was 2495 382 (S.E.M.) lM/48 h; in patients with IBS-D and evidence of BAM, the mean total faecal BA excretion was 4905 lM/48 h. The estimated standard deviation (s.d.) in this group was 3706 lM/48 h, and it was the basis for estimating the effect size that could be detected with a study of 12 patients, assuming we could recruit about 50% of the patients known to have IBS-D with BAM in the database. The effect size is the difference in means (baseline and on colesevelam) of the total faecal BA excretion over 48 h. Assuming a sample size of 12 subjects, a paired t-test (at two-sided a level of 0.05 and 80% power) would be sufficient to detect a difference in means of 3400 lM/48 h (based on s.d. of 3706 lM/48 h). Thus, if colesevelam inactivated or sequestered luminal BA to effectively reduce faecal 48-h BA to an equivalent of 1500 lM/48 h in the absence of the sequestrants, the drug would have achieved a reduction in luminal BA to bring the latter to be equivalent to the 75th percentile for healthy volunteers. Thus, this effect size would be anticipated to demonstrate a clinically relevant change in total faecal BA and to be associated with improved stool characteristics. All authors had access to the study data and reviewed and approved the final manuscript. RESULTS Demographics The mean age of the 12 participants (11 female, 1 male) was 43.1 3.7 years and mean BMI was 31.1 2.4 kg/m2. 441
M. Camilleri et al. In vitro studies of extraction of bile acids bound to colesevelam To show that our BA extraction and detection method is able to measure all BAs in the methanol extracted
LCA Water BA BA + colesevelam Methanol BA BA + colesevelam
DCA
sample, in vitro studies were performed in different matrices in the presence and absence of colesevelam (Table 1). When BAs are mixed with colesevelam in an aqueous environment, no free BAs are detectable by
UDCA
CDCA
CA
9.4 (1.3) 0.1 (.2)
9.2 (1.5) 0.0 (0.0)
9.5 (1.3) 0.0 (0.0)
9.6 (1.5) 0.0 (0.0)
10.1 (1.6) 0.0 (0.0)
10.0 (1.5) 10.0 (2.4)
9.9 (1.7) 10.0 (2.3)
9.8 (1.2) 9.9 (2.3)
10.1 (1.7) 10.2 (2.5)
9.9 (1.7) 9.7 (2.3)
Table 1 | Bile acids colesevelam in water or methanol (lM s.d.)
BA, bile acid; LCA, lithocholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid.
Total fecal BA (µmoles/48 h) excreted
(a) 10 000
10 000 P = 0.012
8000
8000
6000
6000
4000
4000
2000
2000
0
0
Baseline
(b)
Colesevelam
(c)
16 000 14 000
120
12 000
100
10 000 8000 6000 4000
80 60 40
2000
20
0
0
Baseline
Colesevelam
Colesevelam
140
Serum C4 ng/mL
Total fecal BA (nmoles/g stool) excreted
Baseline
Baseline
Colesevelam
Figure 2 | Comparison of (a) total faecal bile acid excretion over 48 h during baseline and colesevelam treatment in the 12 patients individually (right panel) and as a group (left panel). (b, c) Individual data during baseline and colesevelam treatment for total faecal bile acid excretion per gram of stool, and fasting serum C4 respectively. 442
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea mass spectrometry. Thus, colesevelam does bind all BAs in this environment. In contrast, when colesevelam is mixed with BAs in methanol, all of the BAs are free and detected by our assay. Owing to the extreme solubility differences in methanol between colesevelam (not soluble) and BAs (soluble), BAs do not bind to colesevelam in methanol. Thus, results of methanol-extracted BAs presented in this study include all BAs found in stool, whether they were free or colesevelam-bound in vivo.
Effect of colesevelam on total faecal BA excretion Colesevelam resulted in higher faecal excretion of BAs in stool compared to baseline. The increase in total faecal BAs was observed for the entire group (Figure 2a, left panel) and for 10 of the 12 patients with IBS-D (FigTable 2 | Effects of colesevelam on bowel function endpoints and faecal fat (mean S.E.M.)
ure 2a, right panel). Colesevelam also increased the faecal BA excretion per gram of stool (Figure 2b).
Effect of colesevelam on fasting serum C4 Colesevelam significantly increased fasting serum C4 measurement [P < 0.01 (signed rank test); Table 2 and individual data shown in Figure 2c]. Effects of colesevelam on proportion of main primary and secondary BAs and faecal fat Colesevelam was associated with increased proportion of deoxycholic acid in the stool (Figure 3) and a numerical reduction in the proportion of cholic acid in stool; there were no significant differences in the proportions of CDCA, lithocholic or ursodeoxycholic acids. There was no significant effect of colesevelam on faecal fat excretion Baseline
# stools per week Stool form (BSFS) Ease of stool passage Total faecal BA excretion (lmoles/48 h) Total faecal BA excretion per gram of stool (nmoles/g stool) Faecal fat (g/day) Serum C4 (ng/mL)
17.6 4.8 4.7 1558 2332
0.4 0.3 0.2 229 344
6.4 1.2 24.7 4.2
Colesevelam 15.1 4.4 4.2 3496 6585
1.9 0.3 0.1 709 1196
6.8 0.9 72.3 12.2
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
100
P = 0.028
DCA 100
80
80
60
60
40
40
20
20
0
0
Baseline
Percent of total fecal BA
Figure 3 | Changes in percentage of deoxycholic acid (DCA) and cholic acid (CA) in stool with colesevelam, suggesting that the medication sequesters the secretory, dehydroxylated secondary bile acid, DCA, which is derived from the primary bile acid, CA. There were no significant differences in lithocholic acid or chenodeoxycholic acid in stool.
Percent of total fecal BA
BSFS, Bristol Stool Form Scale; BA, bile acid. There were significant differences in stool form, faecal total BA excretion, faecal total BA excretion per gram of stool, and serum C4 (shown in figures).
Baseline
Colesevelam
Colesevelam CA 60
60
P = 0.061
50
50
40
40
30
30
20
20
10
10
0
0
Baseline Baseline
Colesevelam
Colesevelam 443
Fecal fat g/day
M. Camilleri et al. (Table 2, Figure 4). There was no major difference in the total faecal BA in the two patients with prior cholecystectomy compared to the other patients. Thus, faecal BA excretions in these two patients were 2916 and 2498 lM/48 h at baseline and 1878 and 2932 lM/48 h on colesevelam treatment.
20 18 16 14 12 10 8 6 4 2 0 Baseline
Colesevelam
Figure 4 | Effects of colesevelam on faecal fat in patients with diarrhoea-predominant irritable bowel syndrome and bile acid malabsorption.
(a) 7.0
7
Stool form BSF scale
6.5
P = 0.043
6.5
6.0
6
5.5
5.5
5.0
5
4.5
4.5
4.0
4
3.5
3.5
3.0
(b)
Effects of colesevelam on stool form and bowel function Colesevelam resulted in a significant decrease in the average score of stool consistency on the Bristol Stool Form Scale (Figure 5a). There were numerical reductions in number of bowel movements per week (Figure 5b) and improvements in ease of passage, but these were not statistically significant (Table 1).
3
Baseline
Colesevelam
Baseline
Colesevelam
Baseline
Colesevelam
40 35
# BM/week
30 25 20 15 10 5 0
Figure 5 | Effects of colesevelam on (a) stool form [Bristol Stool Form Scale (BSF scale)] and (b) stool frequency per week in patients with diarrhoea-predominant irritable bowel syndrome and bile acid malabsorption. 444
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea individual patients, both at baseline and after colesevelam treatment (Figure 7). However, individual stool samples could not be used in lieu of the total 48-h faecal samples to estimate the total or the proportion of the main primary and secondary BAs in stool, as shown by the relatively low concordance correlations for total BAs and moderate coefficients for LCA and DCA, which are the greatest proportions of faecal BAs, and higher coefficients for CDCA, CA and UDCA, which are relatively low proportions of BAs in patients with intact colon (Table 3).
35
BM # per week
30
Rs = –0.621, P = 0.03
25 20 15 10 5 0 0
2000
4000
6000
8000
10 000
Total bile acid (µmoles) in stool with colesevelam treatment
DISCUSSION Our study has shown that, among patients with IBS-D who have prior evidence of BAM, the sequestrant, colesevelam, resulted in increased delivery of BAs to stool. This is accompanied by a significantly more solid stool form, and the stool frequency correlates inversely with the BA sequestered by colesevelam and delivered to the stool. This paradox, that is, the increase in faecal BAs and the more solid stool and reduced number of bowel movements per week, is explained by the binding of the luminal BAs to the sequestrant, colesevelam, and the ability of the preparatory step in the faecal analysis to deliver the BAs from the bound to the free aqueous phase of stool. Specifically, BAs were obtained from stool homogenates using methanol extraction while leaving colesevelam in the insoluble precipitate. Therefore, the extraction contained both free and sequestrant-bound BAs, even though most of the BAs are likely sequestrant-bound in vivo. Colesevelam also resulted in a significant increase in serum C4, suggesting that the greater loss of BA into
Figure 6 | Significant inverse correlation between the faecal excretion of total bile acids and number of bowel movements (BM) per week, suggesting that sequestration of the bile acids by colesevelam reduced the diarrhoea.
Correlation of faecal BA excretion and number of stools per week There was a significant inverse correlation between the faecal excretion of total BAs and number of bowel movements per week, suggesting that sequestration of the BAs by colesevelam reduced the diarrhoea (Figure 6). Comparison of proportions of primary and secondary BAs in individual and total stool samples in each participant The faecal BA excretion in a random sample of stool was closely correlated (concordance correlation coefficient 0.947) with the mean excretion in all samples from
Baseline
Colesevelam
15 000
Figure 7 | Concordance between mean total faecal bile acid (BA) excretion in all samples from an individual patient, and the total faecal BA content in a randomly selected individual sample. Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Mean total fecal BA, µmoles
Overall concordance correlation coefficient 0.947
10 000
5000
0 0
2500 5000 7500 10 000 12 500 0
2500 5000 7500 10 00012 500
Random sample total fecal BA, µmoles 445
M. Camilleri et al. Random sample vs. total 48 h collection
Baseline
Total BAs Cholic acid Deoxycholic acid Ursodeoxycholic acid Chenodeoxycholic acid Lithocholic acid
0.53 0.84 0.68 0.96 0.88 0.44
Colesevelam 0.13 0.08 0.14 0.20 0.05 0.21
stool stimulated hepatic synthesis rate. This increased synthesis of BAs was functionally important in avoiding the development of steatorrhoea. However, longer term trials are required to ensure that there are no steatorrhoea or malabsorption of fat-soluble vitamins with prolonged treatment. We had previously observed that colesevelam treatment in unselected patients with IBS-D was associated with slower emptying of the ascending colon (average 4 h longer) compared with placebo.12 In these unselected patients, we noted that the treatment effect was associated with baseline serum C4 levels (P = 0.0025) suggesting that patients with IBS-D and increased BA synthesis were more likely to respond to colesevelam. Overall, colesevelam was associated in the prior study with greater ease of stool passage (P = 0.048) and somewhat firmer stool consistency (P = 0.12). The current study extends the previous findings and demonstrates that the significantly improved stool consistency is associated with sequestration of the BAs in the colon, preventing their diarrhoeagenic effects. This is also supported by the inverse correlation between number of stools per week and the total faecal BAs measured in stool after extraction of the BAs from their bound state. The diagnosis of BAM has been a clinical challenge in the United States as the 75SeHCAT retention test is not approved. A definitive diagnosis is desirable before committing patients to ingesting the relatively unpalatable resins in powder form or the relatively expensive tablet forms such as colesevelam. With the availability of tests such as serum C4, serum fibroblast growth factor-19 (FGF-19)25 and faecal BA excretion at reference laboratories, we anticipate that there will be greater use of these diagnostic tests after their fuller validation. One of the steps of validation of the diagnostic is demonstration of responsiveness to treatment and concordance with clinical benefit. The current study has shown that the HPLC/MS method used by Mayo Clinic Department of Laboratory Medicine and Pathology to measure total and individual BAs is responsive to treatment with the BA sequestrant, colesevelam, and that this 446
0.54 0.83 0.85 0.54 0.90 0.88
0.15 0.06 0.07 0.20 0.06 0.07
Table 3 | Concordance correlation coefficients (mean S.E.M.) of stool total and individual bile acids (BAs) for the 48-h collection and a randomly selected stool sample
biochemical effect is associated with clinical improvement in the diarrhoea. We have set up the method to be run together with a measurement of faecal fats, which is commonly investigated in patients with chronic diarrhoea. This combination would also be useful in patients with diarrhoea due to ileal resection (type 1 BA diarrhoea) and cholecystectomy or pancreatic exocrine insufficiency (type 3 BA diarrhoea). Thus, in such patients, knowing both the faecal fat and faecal BA excretion would help clinicians determine the degree of dietary fat restriction in addition to the sequestration of BAs to relieve the diarrhoea. Our study also addressed the clinically relevant question: Does a random measurement of stool BA (total content and proportion of main primary and secondary BAs) provide sufficient information to avoid the need for a 48-h collection? Unfortunately, we came to the same conclusion as with random faecal fat estimates relative to 48-h fat excretion that the random sample provides insufficient information on BA excretion. Variation in BA excretion per gram faecal weight in each bowel movement, and in the faecal pellet and supernatant were reported in three patients with ileal resection diarrhoea and suggested that individual stool samples were not representative of the total faecal BA excretion.26 The main limitations of our current study are the absence of a placebo arm and the small sample size; these limitations reduce our confidence in the clinical benefit of colesevelam in patients with IBS-D and BAM. However, the major objective of our study was to demonstrate responsiveness of faecal BAs with the HPLC/MS assay, and future, appropriately powered studies will be designed to address the clinical efficacy of this therapeutic approach. A recent systematic review also endorsed the potential use of colesevelam in the management of chronic diarrhoea due to BAM.27 In conclusion, the current study demonstrates the utility of measurement of faecal BAs and serum C4 to characterise either increased synthesis or excretion of BAs as targets for treatment with a BA sequestrant. We have Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Colesevelam and bile acids in IBS-diarrhoea demonstrated the responsiveness to colesevelam at the biochemical level and in the bowel dysfunction of such patients, particularly stool consistency and number of bowel movements. We have also shown that, as with faecal fat estimation, the 48-h stool collection is necessary to measure faecal BA excretion and prove BAM in 12 such patients with IBS-D, confirming the experience published over 40 years ago of Mitchell et al. in studies performed on three patients with prior ileal resection.26 These studies also add to the plethora of data suggesting that there is an opportunity to diagnose and specifically treat the cause of symptoms in patients who carry a label of IBS-D.
AUTHORSHIP Guarantor of the article: Dr Michael Camilleri. Author contributions: Michael Camilleri: principal investigator; author of manuscript, takes responsibility for the integrity of the work as a whole, from inception to published article. Andres Acosta: fellow investigator, screening of patients; co-author. Irene Busciglio: recruitment of participants, conduct of study. Amy Boldingh: recruitment of participants. Roy B. Dyer: measurement of serum C4. Alan R. Zinsmeister: biostatistician. Alan Lueke: measurement of faecal fat and bile acids. Amber Gray: measurement of faecal fat and bile acids. Leslie J. Donato: supervision of measurement of faecal fat and
bile acids, co-author. All authors approved the final version of the manuscript.
ACKNOWLEDGEMENTS The authors thank the Mayo Clinic Department of Laboratory Medicine and Pathology for waiving charges for measurements of faecal bile acids and fats, and we thank Mrs Cindy Stanislav for excellent secretarial assistance. Declaration of personal interests: Dr Camilleri has served as a consultant for NPS Pharmaceuticlas, AstraZeneca, Navartis, Targacept, NGM Biopharmaceuticals, Altos Therapeutics, and Purdue Pharma with personal remuneration. He has served as an advisory board member for AstraZeneca and BioKier, also with personal remuneration. With remuneration to his employer, Mayo Clinic, he has served as a consultant for Entera Health, Rhythm, Theravance, GlaxoSmithKline, Takeda, and Shire. He has received research funding from Rhythm, Salix, SK Life Sciences, NPS Pharmaceuticals, Second Genome, and Entera Health. Declaration of funding interests: Dr Camilleri’s research on bile acid diarrhoea is supported by NIH R01 DK92179 and by Mayo Foundation (Atherton and Winifred W. Bean Endowed Professorship). The study was conducted with the help of the Nursing Core of Mayo Clinic CCaTS (grant #UL1-TR000135 from National Institutes of Health).
REFERENCES 1. Camilleri M, McKinzie S, Busciglio I, et al. Prospective study of motor, sensory, psychological and autonomic functions in 119 patients with irritable bowel syndrome. Clin Gastroenterol Hepatol 2008; 6: 772–81. 2. Wedlake L, A’Hern R, Russell D, et al. Systematic review: the prevalence of idiopathic bile acid malabsorption as diagnosed by SeHCAT scanning in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther 2009; 30: 707–17. 3. Wong BS, Camilleri M, Carlson P, et al. Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea. Clin Gastroenterol Hepatol 2012; 10: 1009–15. 4. Shin A, Camilleri M, Vijayvargiya P, et al. Bowel functions, fecal unconjugated primary and secondary bile acids, and colonic transit in patients with irritable bowel syndrome.
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
5.
6.
7.
8.
Clin Gastroenterol Hepatol 2013; 11: 1270–5. Poole DP, Godfrey C, Cattaruzza F, et al. Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system. Neurogastroenterol Motil 2010; 22: 814– 25. Alemi F, Poole DP, Chiu J, et al. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology 2013; 144: 145–54. Vijayvargiya P, Camilleri M, Shin A, et al. Methods for diagnosis of bile acid malabsorption in clinical practice. Clin Gastroenterol Hepatol 2013; 11: 1232–9. Stotzer P-O, Abrahamsson H, Bajor A, Sadik R. Effect of cholestyramine on gastrointestinal transit in patients with idiopathic bile acid diarrhea: a prospective, open-label study.
9.
10.
11.
12.
Neuroenterology 2013; 2: 5. doi:10.4303/ ne/235657 Article ID 235657. Bajor A, T€ ornblom H, Rudling M, et al. Increased colonic bile acid exposure: a relevant factor for symptoms and treatment in IBS. Gut 2015; 64: 84–92. adell K, Holme I, et al. Eriksson M, H Compliance with and efficacy of treatment with pravastatin and cholestyramine: a randomized study on lipid-lowering in primary care. J Intern Med 1998; 243: 373–80. Bays HE, Maki KC, Schmitz K. Colesevelam hydrochloride powder for oral suspension versus cholestyramine powder for oral suspension: comparison of acceptability and tolerability. Endocr Pract 2011; 17: 218–25. Odunsi-Shiyanbade ST, Camilleri M, McKinzie S, et al. Effects of chenodeoxycholate and a bile acid sequestrant, colesevelam, on intestinal transit and bowel function. Clin
447
M. Camilleri et al.
13.
14.
15.
16.
17.
18.
448
Gastroenterol Hepatol 2010; 8: 159–65. Talley NJ, Phillips SF, Wiltgen CM, et al. Assessment of functional gastrointestinal disease: the bowel disease questionnaire. Mayo Clin Proc 1990; 65: 1456–79. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983; 67: 361–70. Heaton KW, Radvan J, Cripps H, et al. Defecation frequency and timing, and stool form in the general population: a prospective study. Gut 1992; 33: 818–24. Andresen V, Camilleri M, Busciglio I, et al. Effect of 5 days linaclotide on transit and bowel function in females with constipation-predominant irritable bowel syndrome. Gastroenterology 2007; 133: 761–8. Manini ML, Camilleri M, Goldberg M, et al. Effects of velusetrag (TD-5108) on gastrointestinal transit and bowel function in health and pharmacokinetics in health and constipation. Neurogastroenterol Motil 2010; 22: e7–8. Sauter GH, M€ unzing W, von Ritter C, et al. Bile acid malabsorption as a cause of chronic diarrhea: diagnostic value of
19.
20.
21.
22.
7alpha-hydroxy-4-cholesten-3-one in serum. Dig Dis Sci 1999; 44: 14–9. Brydon WG, Nyhlin H, Eastwood MA, et al. Serum 7 alpha-hydroxy-4cholesten-3-one and selenohomocholyltaurine (SeHCAT) whole body retention in the assessment of bile acid induced diarrhoea. Eur J Gastroenterol Hepatol 1996; 8: 117–23. G€alman C, Arvidsson I, Angelin B, et al. Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alphahydroxy-4-cholesten-3-one in peripheral blood. J Lipid Res 2003; 44: 859–66. Camilleri M, Nadeau A, Tremaine WJ, et al. Measurement of serum 7alphahydroxy-4-cholesten-3-one (or 7alphaC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry. Neurogastroenterol Motil 2009; 21: 734–e43. Tagliacozzi D, Mozzi AF, Casetta B, et al. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem
23.
24.
25.
26.
27.
mass spectrometry: a simple and rapid one-step method. Clin Chem Lab Med 2003; 41: 1633–41. Lin LI-K. A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989; 45: 255–68. Camilleri M, Busciglio I, Acosta A, et al. Effect of increased bile acid synthesis or fecal excretion in irritable bowel syndrome-diarrhea. Am J Gastroenterol 2014; 109: 1621–30. Pattni SS, Brydon WG, Dew T, et al. Fibroblast growth factor 19 in patients with bile acid diarrhoea: a prospective comparison of FGF19 serum assay and SeHCAT retention. Aliment Pharmacol Ther 2013; 38: 967–76. Mitchell WD, Findlay JM, Prescott RJ, et al. Bile acids in the diarrhoea of ileal resection. Gut 1973; 14: 348–53. Wilcox C, Turner J, Green J. Systematic review: the management of chronic diarrhoea due to bile acid malabsorption. Aliment Pharmacol Ther 2014; 39: 923–39.
Aliment Pharmacol Ther 2015; 41: 438-448 ª 2015 John Wiley & Sons Ltd
Copyright of Alimentary Pharmacology & Therapeutics is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
