Methods for Diagnosis of Bile Acid Malabsorption in Clinical Practice

      Altered concentrations of bile acid (BA) in the colon can cause diarrhea or constipation. More than 25% of patients with irritable bowel syndrome with diarrhea or chronic diarrhea in Western countries have BA malabsorption (BAM). As BAM is increasingly recognized, proper diagnostic methods are needed to help direct the most effective course of treatment for the chronic bowel dysfunction. We review the methodologies, advantages, and disadvantages of tools that directly measure BAM: the 14C-glycocholate breath and stool test, the 75selenium homotaurocholic acid test (SeHCAT), and measurements of 7 α-hydroxy-4-cholesten-3-one (C4) and fecal BAs. The 14C-glycocholate test is laborious and no longer widely used. The 75SeHCAT has been validated but is not available in the United States. Measurement of serum C4 is a simple and accurate method that can be used for most patients but requires further clinical validation. Assays to quantify fecal BA (total and individual levels) are technically cumbersome and not widely available. Regrettably, none of these tests are routinely available in the United States; assessment of the therapeutic effects of a BA binder is used as a surrogate for diagnosis of BAM. Recent data indicate the advantages to studying fecal excretion of individual BAs and their role in BAM; these could support the use of the fecal BA assay, compared with other tests. Measurement of fecal BA levels could become a routine addition to the measurement of fecal fat in patients with unexplained diarrhea. Availability ultimately determines whether the C4, SeHCAT, or fecal BA test is used; more widespread availability of such tests would enhance clinical management of these patients.

      Keywords

      Abbreviations used in this paper:

      ASBT (Na+-dependent bile salt transporter), BA (bile acid), BAM (bile acid malabsorption), CA (cholic acid), CDCA (chenodeoxycholic acid), CYP7A1 (cholesterol 7α hydroxylase), C4 (7 α-hydroxy-4-cholesten-3-one), DCA (deoxycholic acid), GC-MS (gas chromatography-mass spectrometry), HPLC (high-performance liquid chromatography), IBAT (ileal BA transporter), IBS (irritable bowel syndrome), LCA (lithocholic acid), MS (mass spectrometry), NADH (reduced nicotinamide adenine dinucleotide), 75SeHCAT (75selenium homotaurocholic acid test)
      Podcast interview: www.gastro.org/cghpodcast. Also available on iTunes; See related article by Shin A et al, on page 1270 in this issue of CGH.
      Bile acids (BAs) are detergent molecules
      • Hofmann A.F.
      • Small D.M.
      Detergent properties of bile salts: correlation with physiological function.
      excreted from the liver and are responsible for fat emulsification, aiding lipid absorption and digestion in the small intestine. There are several BA species that are differentiated structurally by their hydroxylation and conjugation status. Chenodeoxycholic acid (CDCA) and cholic acid (CA) are the primary BAs (Figure 1) that are synthesized in the liver from cholesterol.
      • Rao A.S.
      • Wong B.
      • Camilleri M.
      • et al.
      Chenodeoxycholate in females with irritable bowel syndrome-constipation: a pharmacodynamic and pharmacogenetic analysis.
      The majority of BAs excreted from the liver are conjugated to the amino acids, taurine or glycine, and remain ionized in the duodenum, which increases their solubility. This allows a high enough concentration of BAs to reach the critical micellar concentration, allowing for spontaneous formation of micelles whereby the polar BAs surround fat molecules and can present the hydrophobic fat molecules (which are insoluble in the aqueous phase) to the brush border membrane of the small intestine for digestion and absorption. The colonic bacteria avidly deconjugate and dehydroxylate BA; therefore, the major proportion of fecal BAs consists of the deconjugated secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA).
      Figure thumbnail gr1
      Figure 1BA chemistry: CDCA, CA, DCA, LCA, and ursodeoxycholic acid (UDCA).
      Sulfonation is an additional conjugation reaction that can occur in a subset of BAs; the reaction is catalyzed by sulfotransferase 2A1, preferentially transferring a sulfonate group (SO3-) to the 3α hydroxyl group on the BA molecule (Figure 1). Sulfonated BAs maintain a permanent negative charge, which influences their solubility but also decreases their affinity for the apical Na+-dependent bile salt transporter (ASBT) (also called ileal BA transporter [IBAT] or SLC10A2 [solute carrier family 10, member 2]). IBAT is responsible for the active reuptake of BAs in the terminal ileum. Because of reduced affinity to IBAT and extraction by the liver, sulfonated BAs have limited enterohepatic recirculation,
      • Alnouti Y.
      Bile acid sulfation: a pathway of bile acid elimination and detoxification.
      and desulfonation occurs in the colon as a result of bacterial hydrolysis.
      • Robben J.
      • Caenepeel P.
      • Van Eldere J.
      • et al.
      Effects of intestinal microbial bile salt sulfatase activity on bile salt kinetics in gnotobiotic rats.
      Sulfonation has been shown to decrease the secretory effects of CDCA, CA, and DCA in rats.
      • Breuer N.F.
      • Rampton D.S.
      • Tammar A.
      • et al.
      Effect of colonic perfusion with sulfated and nonsulfated bile acids on mucosal structure and function in the rat.
      A functional enterohepatic circulation (Figure 2) reabsorbs ∼95% of BAs in the terminal ileum
      • Hofmann A.F.
      The syndrome of ileal disease and the broken enterohepatic circulation: cholerhetic enteropathy.
      and transports the BAs back to the liver; interruption of the enterohepatic circulation results in cholerheic or BA diarrhea.
      • 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.
      Bile acid malabsorption (BAM) is one of the mechanisms underlying the pathophysiology of diarrhea associated with ileal disease (Crohn's disease, surgical resection, radiation ileitis) and occurs in 32% of irritable bowel syndrome (IBS) diarrhea, 30%-50% of chronic diarrhea, and up to 35% of microscopic colitis.
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
      Perfusion of BAs in the human colon results in colonic secretion of water and electrolytes
      • Mekjian H.S.
      • Phillips S.F.
      • Hofmann A.F.
      Colonic secretion of water and electrolytes induced by bile acids: perfusion studies in man.
      and high amplitude, propagated contractions.
      • Bampton P.A.
      • Dinning P.G.
      • Kennedy M.L.
      • et al.
      The proximal colonic motor response to rectal mechanical and chemical stimulation.
      The presence of two α-hydroxyl groups at the 3, 7, or 12 positions in the BA molecules (CDCA, CA, DCA) is responsible for their secretory effects.
      • Chadwick V.S.
      • Gaginella T.S.
      • Carlson G.L.
      • et al.
      Effect of molecular structure on bile acid-induced alterations in absorptive function, permeability, and morphology in the perfused rabbit colon.
      When the colon is exposed to an increased or decreased amount of those BAs, their presence promotes or decreases fluid and electrolyte secretion, which resembles symptoms of chronic diarrhea or constipation.
      • Rao A.S.
      • Wong B.
      • Camilleri M.
      • et al.
      Chenodeoxycholate in females with irritable bowel syndrome-constipation: a pharmacodynamic and pharmacogenetic analysis.
      Figure thumbnail gr2
      Figure 2Hepatic synthesis of BA from cholesterol involves entry into hepatocytes of low-density lipoprotein (LDL) cholesterol by binding to LDL receptors on the hepatocyte cell surface. Up-regulation of hepatic BA synthesis promotes maintenance of BA pool size and leads to an increase in serum C4, a surrogate for the activity of cholesterol 7 α-hydroxylase, the rate-limiting enzyme in hepatic BA synthesis. LBAT, liver bile acid transporter.
      Adapted with permission from Wong BS, et al. Am J Gastroenterol 2011;106:2154-2164.
      Although BAM is recognized in practice, the most popular current method of diagnosis includes a therapeutic trial of BA binders with symptom improvement; this approach is prevalent and the only resource available in countries like the United States where the noninvasive imaging based on scintigraphic BA retention is unavailable. Unfortunately, in certain disease states, symptoms may only improve with high doses of a BA sequestrant or binder, and the diagnosis of BAM may be missed. Patients report poor palatability and side effects of borborygmi, flatulence, and abdominal pain when using certain BA binders,
      • 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.
      which decrease compliance and ability to diagnose BAM. In addition, resin formulations such as cholestyramine may also bind and inactivate other etiologic agents nonspecifically, including Clostridium difficile toxin.
      • Surowiec D.
      • Kuyumjian A.G.
      • Wynd M.A.
      • et al.
      Past, present, and future therapies for Clostridium difficile-associated disease.
      • Weiss K.
      Toxin-binding treatment for Clostridium difficile: a review including reports of studies with tolevamer.
      Therefore, a definitive diagnosis of BAM is desirable to assess disease severity and direct appropriate treatment modalities.
      We briefly review the rationale, analytical methodology, and advantages/disadvantages of 4 methods that directly measure BAs: 14C-glycocholate breath and stool test, 75selenium homotaurocholic acid test (75SeHCAT), 7 α-hydroxy-4-cholesten-3-one (C4), and fecal BAs. Indirect measurements of BAM have been proposed, such as the serum fibroblast growth factor 19 test,
      • Walters J.R.
      • Tasleem A.M.
      • Omer O.S.
      • et al.
      A new mechanism for bile acid diarrhea: defective feedback inhibition of bile acid biosynthesis.
      but this is not considered because it does not directly measure a BA entity.

      14C-glycocholate Breath and Stool Test

       Clinical Utility

      The 14C-glycocholate breath and stool test is a method to determine bacterial-dependent deconjugation within the gastrointestinal tract, which is due to bacterial overgrowth in the small bowel or BAM.
      • Fromm H.
      • Hoffman A.
      Breath test for altered bile-acid metabolism.
      • Scarpello J.H.B.
      • Sladen G.E.
      Appraisal of the 14C-glycocholate acid test with special reference to the measurement of faecal 14C excretion.
      The 14C-glycocholate solution is orally administered and incorporates into the intraluminal pool of BAs. Bacteria can enzymatically cleave the bond between CA and glycine. 14C-glycine is released, absorbed into the portal circulation, and rapidly metabolized in the liver, and the end product is exhaled as 14CO2. Because bacterial deconjugation is the rate-limiting step, early breath excretion before the expected active absorption of the BA by the ileal BA transporter is highly suggestive of small bowel bacterial overgrowth.
      Conversely, if the 14C-BA is not reabsorbed in the terminal ileum and enters the large intestine, the 14C-BA will be deconjugated by colonic bacteria, and a smaller proportion proceeds intact through the colon to be excreted in stool.
      • Fromm H.
      • Hoffman A.
      Breath test for altered bile-acid metabolism.
      This situation may occur in cases of dysfunction of the IBAT (eg, ileitis due to Crohn's disease or radiation) or ileal resection. Thus, stool collection after the 14C-glycocholate breath test can identify BAM.

       Test Methodology

      14C-glycocholate is ingested with a standard meal. Exhaled air is blown every hour for a minimum of 6 hours into a drying tube and is collected in a vial containing a solution that extracts the 14CO2. Stool is collected for 24 hours and is combusted to create 14CO2. A liquid beta scintillation counter quantifies the amount of 14C and provides estimate of fecal bile acid excretion.
      • Van Tilburg A.J.P.
      • de Rooij F.W.M.
      • van den Berg W.O.
      • et al.
      The selenium-75-homocholic acid taurine test reevaluated: combined measurement of fecal selenium-75 activity and 3α-hydroxy bile acids in 211 patients.

       Advantages and Disadvantages

      Unfortunately, the amount of exhaled 14CO2 during the first 2-4 hours after ingestion does not completely differentiate deconjugation occurring in the small or large intestine (Table 1). The small bowel transit time for liquids in a mixed meal was 181 ± 28 (standard error of the mean) minutes
      • Camilleri M.
      • Brown M.L.
      • Malagelada J.R.
      Impaired transit of chyme in chronic intestinal pseudo obstruction: correction by cisapride.
      ; therefore, detection of 14CO2 in breath from colonic bacterial deconjugation (which would be increased in the presence of BAM as a result of the delivery of 14C-glycocholate to the colon) would occur about 3 hours after meal ingestion. Because the timing of breath excretion does not completely differentiate BAM from bacterial overgrowth, this test necessitates measurement of fecal intact 14C-BA.
      • Fromm H.
      • Hoffman A.
      Breath test for altered bile-acid metabolism.
      Table 1Advantages and Disadvantages of BAM Diagnostic Methods
      BAM diagnostic methodsAdvantagesDisadvantages
      14C glycocholateMay identify small bowel bacterial overgrowthRadiation exposure, β emission, long half-life
      Varying normal values
      Positive breath excretion at 2-4 h does not differentiate BAM from small bowel bacterial overgrowth
      Laborious test method (stool collection)
      75SeHCATGamma emission, short half-life, with decreased radiation to extra-abdominal organsNot available in U.S.
      Well-defined normal values; level of isotope retention predicts response to bile acid sequestrantRadiation exposure
      Simple test method: 2 patient visits
      Serum C4No radiationFasting sample, diurnal variation
      Normal values reported in adultsRequires further validation
      Not dependent on age, gender, or cholesterolFalse positive in liver disease, treatment with statins, and altered circadian rhythm
      Simple blood test: 1 patient visit
      Fecal BANo radiationVariable daily fecal BA excretion, requires at least 48-h sample
      Measures total and individual BAsCumbersome method (stool collection)
      One of the primary disadvantages of the test is the concern over radiation exposure and disposal. Studies have shown that there is a minor concern about radiation exposure to patients,
      • Fromm H.
      • Hoffman A.
      Breath test for altered bile-acid metabolism.
      but the extensive half-life of 5730 years makes disposal of 14C taxing to the environment.
      • Ferguson J.
      • Walker K.
      • Thomson A.B.
      Limitations in the use of 14C-glycocholate breath and stool bile acid determinations in patients with chronic diarrhea.
      In addition, the complexity and laborious nature of 14C-glycocholic breath and stool test reduced its application for diagnosing small bowel bacterial overgrowth or BAM. As other tests were developed to diagnose BAM, increasing the ease and improved accuracy and reliability,
      • Sauter G.H.
      • Munzing W.
      • Von Ritter C.
      • et al.
      Bile acid malabsorption as a cause of chronic diarrhea: diagnostic value of 7[alpha]-hydroxy-4-cholesten-3-one in serum.
      the 14C-glycocholic method was discontinued in many centers.

       Interpretation

      Interpretive reference range values were not available for normal subjects with different ages and genders, thus making interpretation of results complex and individualized to each study center.

      75Selenium HomotauroCholic Acid Test

       Clinical Utility

      75SeHCAT uses a synthetic 75selenium homotaurocholic BA that is resistant to bacterial degradation
      • Thaysen E.H.
      • Orholm M.
      • Arnfred T.
      • et al.
      Assessment of ileal function by abdominal counting of the retention of a gamma emitting bile acid analogue.
      and passive diffusion.
      • Williams A.J.
      • Merrick M.V.
      • Eastwood M.A.
      Idiopathic bile acid malabsorption: a review of clinical presentation, diagnosis, and response to treatment.
      Thus, 75selenium homotaurocholic BA can be either actively absorbed in the terminal ileum to enter the enterohepatic circulation or excreted into stool, unaltered by its passage through the colon.
      Unlike 14C-glycocholate, 75selenium decays through gamma emission and thus can be measured with an external counter, a gamma camera, which does not require a collimator, therefore reducing the radiation dose necessary to estimate BA retention.

       Test Methodology

      The patient ingests a capsule of 75selenium homotaurocholic acid (gamma radiolabeled BA). The 75selenium is assumed to be appropriately distributed in the gut after approximately 1 hour, at which time a baseline scan is obtained and represents 100% retention. The number of subsequent scans has decreased over the years after introduction of the 75SeHCAT test from daily for a period of 7 days to a single follow-up scan on day 7. The amount of radioactivity from 75selenium on subsequent scans is divided by the baseline scan on day 1, indicating the percentage of 75selenium homotaurocholate remaining in the body and, indirectly, how much was lost in the stool. Sciarretta et al
      • Sciarretta G.
      • Vicini G.
      • Fagioli G.
      • et al.
      Use of 23-selena-25-homocholyltaurine to detect bile acid malabsorption in patients with ileal dysfunction or diarrhea.
      have shown that a single subsequent scan on day 7 has a sensitivity of 89% and specificity of 100% by using whole body retention value. BA may undergo 5 enterohepatic circulations per day, with ∼5% loss in the stool with each circulation. In healthy subjects, 83% of 75selenium homotaurocholic BA is passed into the colon by day 7. Thus, even a minor decrease in BA reabsorption will result in a substantial loss of BA.
      • Notghi A.
      • O'Brien J.
      • Chen S.L.
      • et al.
      Measuring SeHCAT retention: a technical note.
      The test can be conducted by using a single-headed camera, which requires the patient to remain prone and immobile for the duration of the test and then return to that same position for follow-up imaging. Alternatively, use of a dual-headed camera allows the patient to be in a supine position while the anterior and posterior cameras capture the images. There are 3 ways to measure 75selenium: whole body, abdominal, or stool measurements. Currently, abdominal imaging from the chest to the knees is preferred compared with the whole body, because of the lack of customized shielded rooms. However, it is imperative that the patient and camera be in the same position for each image acquisition for abdominal imaging. Stool measurements require 24-hour stool collections for a period of 5 days. After stool collection, a gamma camera is positioned ∼20 cm away from the stool container, and measurements are taken in duplicate to quantitate fecal excretion of the radiolabeled BA. Benefits of the stool measurement include patient convenience and visualization of the stool, which assists in characterizing stool type and form, potentially allowing for additional testing for electrolytes and individual BA measurements if indicated.
      • Notghi A.
      • O'Brien J.
      • Chen S.L.
      • et al.
      Measuring SeHCAT retention: a technical note.
      The most common practice is either abdominal and/or whole body scans.

       Advantages and Disadvantages

      The avoidance of multiple stool collections makes the test less laborious and improves patient compliance. In addition, this practicality allowed for development of normative data in healthy volunteers, as well as a thorough appraisal of the diagnosis and severity of the BAM.
      There are many additional attractive properties about the 75SeHCAT retention test. First, there is little influence of isotope distribution that is due to body build or position of the patient, as well as decreased radiation dose administered to extra-abdominal organs (Table 1). In the presence of BAM, there is increased fecal excretion of the intact radioactive BA, further decreasing the radioactivity exposure to the body, in contrast to 14C, which could be absorbed from the colon by diffusion. Second, 75SeHCAT has a half-life of 118 days, thus remaining in the body for less time as well as simpler disposal in the environment. Third, when directly compared with the 14C-glycocholate test, the 75SeHCAT test resulted in less false negatives and less borderline results.
      • Thaysen E.H.
      • Orholm M.
      • Arnfred T.
      • et al.
      Assessment of ileal function by abdominal counting of the retention of a gamma emitting bile acid analogue.
      The major disadvantage is that unfortunately, this test is approved and used in most European and many other countries, but not in the United States.
      • Ferguson J.
      • Walker K.
      • Thomson A.B.
      Limitations in the use of 14C-glycocholate breath and stool bile acid determinations in patients with chronic diarrhea.
      Second, it is conceivable that false-positive SeHCAT results may occur in patients with accelerated transit. There is no published evidence that experimentally induced diarrhea affects SeHCAT results. However, there are conflicting reports of the nonspecific effects of diarrhea or accelerated transit on BAM; there is abnormal SeHCAT in postvagotomy diarrhea,
      • al-Hadrani A.
      • Lavelle-Jones M.
      • Kennedy N.
      • et al.
      Bile acid malabsorption in patients with post-vagotomy diarrhoea.
      but there was no association between small bowel transit and BAM, analyzed by SeHCAT.
      • Sciarretta G.
      • Fagioli G.
      • Furno A.
      • et al.
      75Se HCAT test in the detection of bile acid malabsorption in functional diarrhoea and its correlation with small bowel transit.

       Interpretation

      Retention at 7 days of ≥15% BA is consistent with a normal result. Mild BAM is considered 10%-15%, moderate 5%-10%, and severe <5% retention. Wedlake et al
      • 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.
      analyzed 18 studies and found response to cholestyramine, a BA binder, in 96% of patients with <5% retention, 80% with <10%, and 70% with <15%, indicating that the more severe the BAM, the greater the response to BA binders. Response was assessed differently in the 15 studies that provided the information and is detailed elsewhere. The studies did not evaluate the response to cholestyramine in non-BAM patients.
      • 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.

      Serum 7 α-hydroxy-4-cholesten-3-one

       Clinical Utility

      C4 was initially developed to measure BA synthesis and the associated removal of circulating low-density lipoprotein cholesterol in clinical trials investigating modalities to reduce cardiovascular risk.
      • Axelson M.
      • Aly A.
      • Sjövall J.
      Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man.
      BA synthesis occurs via neutral and acidic pathways. In humans, 90% of BA synthesis occurs through the neutral pathway, which is regulated by the rate-limiting enzyme cholesterol 7α hydroxylase (CYP7A1). C4 is a downstream product of CYP7A1 (Figure 2). The correlation of C4 with BA synthesis has been validated in multiple studies, even in the presence of rapid CYP7A1 activity.
      • Galman C.
      • Arvidsson I.
      • Angelin B.
      • et al.
      Monitoring hepatic cholesterol 7α hydroxylase activity by assay of the stable bile acid intermediate 7α-hydroxy-4-cholesten-3-one in peripheral blood.
      Unlike the 14C and SeHCAT tests, which require multiple visits and testing with special equipment, serum C4 is a simple blood test that requires only a standardized specimen collection time. Galman et al
      • Galman C.
      • Angelin B.
      • Rudling M.
      Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis.
      reported diurnal variability in C4, with peak concentrations lasting for ∼1.5-3 hours postprandially at 1:00 and 9:00 pm. These C4 peaks reflect a true diurnal pattern as they were observed in patients who fasted, and they were not related to postprandial excretion of bile. C4 levels in the gallbladder were minimal, and the diurnal peak was also seen in patients who underwent cholecystectomy. Similarly, Ma et al
      • Ma K.
      • Xiao R.
      • Tseng H.
      • et al.
      Circadian dysregulation disrupts bile acid homeostasis.
      demonstrated alteration of the circadian rhythm or a severed connection between the central and peripheral "clock" changes in the diurnal rhythm for BA synthesis in mice. Thus, it is hypothesized that C4 is not released directly into the bile but reaches the bloodstream directly. Although peaks are found during regular meal hours, C4 release is not dependent on meal ingestion but reflects the circadian rhythm.

       Test Methodology

      Quantitation of C4 requires a single blood draw in the morning after an overnight fast. C4 is isolated by using liquid chromatography-tandem mass spectrometry. Although there are many methods and reagents that are used, the most recent method has an average of 99% recovery and is described elsewhere.
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
      Briefly, lipids and proteins are precipitated out with high-performance liquid chromatography (HPLC) grade water, acetonitrile, and saturated ammonium sulfate, and the specimen is vortexed and centrifuged. The supernatant is dried, reconstituted with 100% methanol, and injected onto a liquid chromatography-tandem mass spectrometry system (AB Sciex API 5000 MS/MS; AB Sciex, Framingham, MA) coupled with an electrospray ionization interface on a Cohesive HPLC System (Thermo Fisher Scientific, Franklin, MA) with a Phenomenex MAX-RP column (150 × 2.0 mm, 4μm; Phenomenex, Torrance, CA).
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.

       Advantages and Disadvantages

      The clinical performance of the C4 assay demonstrated a sensitivity of 90%, specificity of 79%, negative predictive value of 98%, and positive predictive value of 74% when compared with the SeHCAT test, identifying BAM when the half-life remaining in the body was ≤1.2 days. The high negative predictive value makes the assay attractive as a screening test to rule out BAM. C4 was unrelated to age, gender, or serum cholesterol when analyzed against potential covariates (Table 1).
      • Ferguson J.
      • Walker K.
      • Thomson A.B.
      Limitations in the use of 14C-glycocholate breath and stool bile acid determinations in patients with chronic diarrhea.
      Unlike other diagnostic tests, measurement of C4 allows for a dynamic understanding of the CYP7A1 enzyme.
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      The availability of a test like C4 would be particularly advantageous in the pediatric population because of the lack of radiation exposure, and it is relatively simple to obtain normal values in children.
      Quantitation of C4 requires specialized equipment and personnel, a disadvantage to the assay. There may be false-positive or false-negative C4 results in patients with liver disease (cholestatic disease with hypertriglyceridemia, aspartate aminotransferase or alanine aminotransferase >2× upper limit of normal), in patients taking medications that alter BA production (statins),
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
      or in individuals with an altered circadian rhythm. The utility of the test is still relatively limited, and it is unclear whether age, emotional conditions, or environmental factors such as shift work or jet lag may alter the circadian rhythm and thus the synthesis of BAs. The 9:00 am fasting blood draw is a routine practice in most laboratories.
      Patients who have slow colonic transit constipation have C4 levels that are often difficult to interpret. Although recent studies demonstrated lower C4
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      and lower fecal BAs in a subset of patients with constipation phenotypes,
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      • Shin A.
      • Vijayvargiya P.
      • Busciglio I.
      • et al.
      Quantitative assessment of fecal primary and secondary bile acids in health and irritable bowel syndrome (IBS) with diarrhea or constipation.
      slow colonic transit constipation was associated with an elevated C4 in the morning (9:00 am) and decreased peak values at noon.
      • Abrahamsson H.
      • Ostlund-Lindqvist A.
      • Nilsson R.
      • et al.
      Altered bile acid metabolism in patients with constipation-predominant irritable bowel syndrome and functional constipation.
      This change in pattern was hypothesized to depict the body's attempt to increase production, but no conclusive evidence has been reported.
      • Abrahamsson H.
      • Ostlund-Lindqvist A.
      • Nilsson R.
      • et al.
      Altered bile acid metabolism in patients with constipation-predominant irritable bowel syndrome and functional constipation.

       Interpretation

      C4 is reported as a serum concentration (ng/mL). Two studies have reported the median (5th, 95th percentiles) as 14.3 ng/mL (6, 60.7)
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
      and 16.9 ng/mL (5, 50.5)
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      in 111 and 23 healthy volunteers, respectively.
      • Camilleri M.
      • Nadeau A.
      • Tremaine W.J.
      • et al.
      Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      In our laboratory, an elevated serum C4 concentration is >60.7 ng/mL. Unfortunately, there are no data on the response to BA binders in patients with an elevated C4.

      Fecal Bile Acid

       Clinical Utility

      Apart from the fecal measurement of 14C-glycocholate, the tests discussed above indirectly assist in the diagnosis of BAM by measuring BA synthesis or retention. In contrast, it is now possible to quantify fecal total and individual BA. Increased total fecal BAs is reported in patients with chronic functional diarrhea
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      ; moreover, recent data show that functional diarrhea and diarrhea-predominant IBS are associated with higher fecal levels of secretory BAs (CA,CDCA, DCA), whereas functional constipation and constipation-predominant IBS are associated with higher fecal LCA levels.
      • Shin A.
      • Vijayvargiya P.
      • Busciglio I.
      • et al.
      Quantitative assessment of fecal primary and secondary bile acids in health and irritable bowel syndrome (IBS) with diarrhea or constipation.
      Measurement of fecal BAs is technically challenging and requires skilled technicians to perform the test. Although in time fecal BAs may become the gold standard assay, it is currently a test that is only available at a few clinical laboratories.

       Test Methodology

      Stool is collected during the last 48 hours of the high fat intake diet. There are 2 predominant approaches to measure fecal BAs, enzymatic and chromatographic.

       Enzymatic assays

      The assays use an oxidized nicotinamide adenine dinucleotide-dependent steroid dehydrogenase enzyme to oxidize deconjugated BAs and produce reduced nicotinamide adenine dinucleotide (NADH). Because the sensitivity of detecting NADH is low, another reaction is coupled that uses the reducing potential of NADH to create a product that is detected with greater sensitivity. With a variety of BA conjugations (sulfonation, glucuronidation) and hydroxyl groups, the quantified total BAs are underestimated because this method requires proper stereotactic alignment of enzyme and substrate. When hydrolysis time and BA concentration were increased, there was a significant correlation between enzymatic assay and gas chromatography-mass spectrometry (GC-MS) when measuring 3α-hydroxyl BAs.
      • Porter J.L.
      • Fordtran J.S.
      • Santa Ana C.A.
      • et al.
      Accurate enzymatic measurement of fecal bile acids in patients with malabsorption.

       Chromatographic assays

      The stool sample is weighed and frozen at -25°C until analysis. The chemical measurement of fecal BA involves separate steps of extraction, deconjugation, derivatization, and quantification. The specifics of each chromatography process are described in detail elsewhere
      • Griffiths W.J.
      • Sjövall J.
      Bile acids: analysis in biological fluids and tissues.
      • Story J.A.
      • Furumoto E.J.
      Bile acid analysis: methods and problems.
      and are briefly described below. The stool specimen is homogenized with water, deconjugated, and separated from proteins, lipids, and salts. Unfortunately, these steps may be complicated by the formation of molecules that may interfere with subsequent steps and result in loss of BAs.
      Quantitation can be accomplished by using 3 methods: GC-MS, liquid chromatography-tandem mass spectrometry, or HPLC-mass spectrometry (HPLC-MS).
      GC-MS requires additional steps to deconjugate all the fecal BAs and derivatize the BAs to allow for improved chromatographic separation.
      • Griffiths W.J.
      • Sjövall J.
      Bile acids: analysis in biological fluids and tissues.
      • Story J.A.
      • Furumoto E.J.
      Bile acid analysis: methods and problems.
      Derivatization provides the best analysis of complex mixtures of BAs. GC-MS also measures sulfonated BAs. Hofmann et al
      • Hofmann A.F.
      • Loening-Baucke V.
      • Lavine J.E.
      • et al.
      Altered bile acid metabolism in childhood functional constipation: inactivation of secretory bile acids by sulfation in a subset of patients.
      studied pediatric functional constipation and found that in a small subset of children, 3-sulfonate (sulfate) CDCA was the predominant fecal BA. This was accomplished by use of electrospray ionization-single ion monitoring-mass spectrometry, and individual BAs were measured by GC-MS.
      Compared with liquid chromatography-tandem mass spectrometry, GC-MS provides more structural information and allows proper quantification of individual BAs. Because GC and LC focus on 2 different aspects of the analysis, it is plausible that they can be performed sequentially.
      HPLC-MS requires limited sample preparation (protein extraction) but focuses more on the number of observable features and less on the structure or character of the BAs. HPLC-MS allows simultaneous analysis of free and conjugated BAs.
      • Griffiths W.J.
      • Sjövall J.
      Bile acids: analysis in biological fluids and tissues.

       Advantages and Disadvantages

      An unresolved aspect of fecal BA measurement includes variation in daily BA excretion (Table 1).
      • Griffiths W.J.
      • Sjövall J.
      Bile acids: analysis in biological fluids and tissues.
      Serum C4 studies suggest that there are diurnal variations in BA synthesis.
      • Galman C.
      • Angelin B.
      • Rudling M.
      Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis.
      Mitchell et al
      • Mitchell W.D.
      • Findlay J.M.
      • Prescott R.J.
      • et al.
      Bile acids in the diarrhoea of ileal resection.
      measured fecal BAs in individual bowel movements of 3 patients (total of 20 bowel movements) with ileal resections and noted substantial variations between samples from the same individual (Figure 3). Patient G reported in their study had 11 stool samples that were homogenized and centrifuged to separate the supernatant and pellet. These were separately analyzed for CDCA and CA. There was a wide range of BA concentrations in the pellet for each BA (Figure 3). Because of the variability, a single stool sample to quantify fecal BA is insufficient to assess BA excretion or achieve an accurate diagnosis of BAM. Although there is no documented evidence in the literature, multiple sources recommend a stool collection of about 3-5 days to accurately diagnose BAM from fecal BA measurements.
      • Griffiths W.J.
      • Sjövall J.
      Bile acids: analysis in biological fluids and tissues.
      • Setchell K.D.
      • Ives J.A.
      • Cashmore G.C.
      • et al.
      On the homogeneity of stools with respect to bile acid composition and normal day-to-day variations: a detailed qualitative and quantitative study using capillary column gas chromatography-mass spectrometry.
      Currently at our institution, we collect a 48-hour stool sample, allowing for measurement of both fecal fat and BAs. This increases the practicality of the test and appears to be a reasonable approach for patients with suspected BAM who have diarrhea, because scintigraphic transit measurements show that the majority of isotope is excreted in stool at 48 hours in patients with diarrhea.
      • Manabe N.
      • Wong B.S.
      • Camilleri M.
      • et al.
      Lower functional gastrointestinal disorders: evidence of abnormal colonic transit in a 287 patient cohort.
      However, more studies must be conducted to evaluate how many days of stool collection are required to produce precise, accurate, and clinically representative fecal BA measurements, particularly in patients with constipation.
      Figure thumbnail gr3
      Figure 3Variation in BA excretion per gram fecal weight in each bowel movement in a single patient. Data show median, interquartile range, 5th and 95th percentiles, and each individual value.
      Data from Mitchell WD, et al. Gut 1973;14:348-353.
      Another methodological question pertains to the measurement of BAs in stool supernatant, compared with the solid phase. From Mitchell et al
      • Mitchell W.D.
      • Findlay J.M.
      • Prescott R.J.
      • et al.
      Bile acids in the diarrhoea of ileal resection.
      (Figure 3), the fecal BAs present in the supernatant are minimal and always less than the minimum values in the pellet or solid phase of the stool. These observations argue for homogenization of the solid and liquid phase of the stool in water during the extraction step of the assay and for collection of stool during 48 hours rather than a random sample for fecal BA measurement.

       Interpretation

      Fecal BAs are reported as a concentration in stool. Wong et al
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.
      showed a median (5th, 95th percentiles) of 363 μmol/24 hours (83, 2008) in 20 healthy volunteers, suggesting an elevated fecal BA measurement is >2.0 mmol/24 hours.

      Cross-validation of Methods to Detect Bile Acid Malabsorption

      A number of studies published in the literature describe cross-validation between the various methods described; the comparative data are shown in Figures 4 and 5. Figure 4A compares 75SeHCAT retention at 3 days with total fecal BAs. The decrease in 75SeHCAT retention at day 3 is significantly associated with an increase in fecal BAs. After these initial validation studies in which the cutoff at 3 days was 34% retention, subsequent studies focused on the 75SeHCAT retention at 7 days and described different degrees of BAM that were based on the proportion retained, with 5% or lower being associated with severe BAM. Figure 4B demonstrates the inverse relationship between serum C4 (a reflection of BA synthesis) and 75SeHCAT half-life in days.
      • Ferguson J.
      • Walker K.
      • Thomson A.B.
      Limitations in the use of 14C-glycocholate breath and stool bile acid determinations in patients with chronic diarrhea.
      A shorter half-life reflects lower retention of the radioisotope in the body.
      Figure thumbnail gr4
      Figure 4(A) 75SeHCAT values and total fecal BAs of 9 patients. Vertical dotted line marks the lower normal limit for total fecal BAs (250 mg/day). Reproduced from Sciaretta G, et al. Gut 1987;28:970-975. (B) Relationship between half-life of 75SeHCAT and HCO (7 α-hydroxy-4-cholesten-3-one) serum concentrations in patients with diarrhea of unknown origin. The response to treatment is indicated by different symbols (○ = response to treatment, ▪= no response to treatment, ●= response not evaluated). The dark gray shaded area denotes pathologic values for both tests; the light gray shaded area denotes normal values for both tests.
      Modified with permission from Sauter GH, et al. Dig Dis Sci 1999;44:14-19.
      Figure thumbnail gr5
      Figure 5(A and B) Quantification of serum C4 and total stool BAs in IBS-constipation, IBS-diarrhea, and healthy controls. Data show median and interquartile ranges, 5th and 95th percentiles. Note the higher serum C4 and fecal total BAs excreted during 48 hours in patients with IBS-diarrhea. (C) Relationship between fasting serum C4 and total 48-hour stool BA excretion.
      Adapted from Wong BS, et al. Clin Gastroenterol Hepatol 2012;10:1009-1015.
      Figure 5A and B shows the results of serum C4 and fecal total BAs in patients with symptoms of chronic diarrhea or constipation, compared with healthy controls. Note that both measurements identify subgroups of patients with abnormal fecal excretion, especially in patients with IBS-diarrhea. There is also a cross-validation comparing the fecal 48-hour total BA excretion and serum C4, as illustrated in Figure 5C, plotted from the results in the same study.
      • Wong B.S.
      • Camilleri M.
      • Carlson P.
      • et al.
      Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.

      Conclusions

      Currently, 75SeHCAT retention, serum C4, and fecal BA measurements are the 3 viable tests available to diagnose BAM. Unfortunately, 75SeHCAT is not available in several countries, including the United States. Serum C4 is a simple and accurate method for patients who do not have liver disease or take statins and maintain a normal circadian rhythm; however, it has been studied in relatively small numbers of patients, and further validation, including response to therapy in patients selected for BA binders on the basis of serum C4 test, is desirable. Fecal BA measurement by enzymatic assay provides an estimate of total fecal BAs, but its accuracy is considered suboptimal. The chromatographic assays can quantify total and individual fecal BAs, but the method is cumbersome, not widely available, and requires further validation of the optimal collection time of stool samples for patients with constipation. Recent data suggest that there is an advantage to studying the individual BAs and their role in BAM or constipation. Regrettably, for the U.S. physician, the only current option is to continue with trials of BA binders in patients suspected of having BAM. Hopefully, increased availability of serum C4 and fecal BAs through samples mailed to centralized clinical laboratories will enhance the opportunity to optimize treatment directed at disorders of BA homeostasis that are responsible for either chronic diarrhea or constipation.

      Acknowledgments

      The authors thank Mrs Cindy Stanislav for excellent secretarial assistance.

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