DOI: 10.1177/0004563220966139
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Journal: Annals of Clinical Biochemistry
Manuscript ID ACB-20-234.R1
Manuscript Type: Review Article
Date Submitted by the 20-Jul-2020 Author:
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Complete List of Authors: Hughes, Lauren; New Cross Hospital, Clinical Chemistry Ford, Clare; Royal Wolverhampton Hospitals NHS Trust, Clinical Chemistry Brookes, Matthew; Royal Wolverhampton Hospitals NHS Trust, Department of Gastroenterology; University of Wolverhampton Faculty of Education Health and Wellbeing Gama, Rousseau; Royal Wolverhampton Hospitals NHS Trust, Blood Sciences Keywords: Gastro-intestinal disorders < Clinical studies
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Clinical Sciences Review Committee (CSRC) Commissioned Review
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CSRC Article Number Review Title
Running Title Author(s)
Bile Acid Diarrhoea: Current and Potential methods of Diagnosis
Methods for Diagnosing Bile Acid Diarrhoea Lauren Elizabeth Hughes Clare Ford Matthew James Brookes Rousseau Gama
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Author Affiliations Including email address for each author
Lauren Elizabeth Hughes: [email protected] 1) Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK Clare Ford: [email protected] 1) Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK Matthew James Brookes: [email protected] 1) Department of Gastroenterology, Royal Wolverhampton NHS Trust, Wolverhampton, UK 2) Faculty of Education, Health and Wellbeing, University of Wolverhampton, Wolverhampton, UK
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Rousseau Gama: [email protected] 1. Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK 2) School of Medicine and Clinical Practice, Wolverhampton
University, Wolverhampton, UK
Word Count
Declaration of Interests Funding N/A
Guarantor
Rousseau Gama
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Contributorship Acknowledgements
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Ethical Approval
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This article was prepared at the invitation of the Clinical Sciences Reviews Committee of the Association for Clinical Biochemistry and Laboratory Medicine.
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Key Words
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Abstract Chronic diarrhoea is common and mostly due to diarrhoea predominant irritable bowel syndrome (IBS-D). IBS-D affects about 11% of the population, however up to a third of these patients actually have bile acid diarrhoea (BAD). There are, therefore, more than one
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million sufferers of BAD in the UK. BAD is caused by small bowel malabsorption of bile acids and the increased bile acids in the large intestine cause diarrhoea. Once diagnosed, the
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treatment of BAD is simple and effective. BAD, however, is often not diagnosed because of a lack of easily available and reliable diagnostic methods. In the United Kingdom, the
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radiolabelled 23-seleno-25-homotaurocholic acid test (SeHCAT) is the gold-standard method of diagnosis. SeHCAT, however, is expensive, inconvenient to the patient, involves radiation exposure and has limited availability. As such, a laboratory biomarker is desirable. This review briefly discusses the pathophysiology and management of BAD and critically evaluates methods for its diagnosis, including serum 7α-hydroxy-4-cholesten-3-one, faecal
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bile acid measurement, serum fibroblast growth factor 19, urine-2-propanol, and the 14Cglycocholate breath and stool test.
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Introduction Irritable Bowel Syndrome (IBS) is estimated to affect 11% of the global population [1]. IBS is a chronic, relapsing, often lifelong disorder with typical symptoms of abdominal pain, diarrhoea or constipation associated with bloating. IBS is classified into four subtypes
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depending on the primary stool composition; diarrhoea-predominant IBS (IBS-D), constipation-predominant IBS, mixed IBS and un-subtyped IBS [2]. IBS is not life-
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threatening, but significantly diminishes the quality of life for sufferers [3].
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It, however, is estimated that 15 to 50% of patients with IBS-D have bile acid diarrhoea (BAD) [4-6], but the lack of diagnostic tests makes it difficult to determine the exact prevalence. BAD may be associated with other gastrointestinal disorders, such as Crohn’s Disease (CD) or post-cholecystectomy, but may also be idiopathic (primary BAD). BAD is caused by bile acid malabsorption (BAM) in the small intestine. In healthy individuals,
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approximately 95% of bile acids (BAs) are reabsorbed in the ileum and returned to the liver via the enterohepatic circulation [7]. If bile acid (BA) reabsorption is reduced or small bowel
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BA concentrations overwhelm the resorptive capacity of the ileum, increased
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concentrations of BAs move into the large intestine and, by stimulating colonic motility and secretions, cause diarrhoea. BAM and BAD are often used interchangeably.
Physiology of bile acids and the enterohepatic circulation
In the classic BA synthesis pathway, the primary BAs, cholic acid and chenodeoxycholic
acid, are synthesised in the liver from cholesterol. The rate limiting step is catalysed by the
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enzyme cholesterol-7-alpha-hydroxylase P4507A1 (CYP7A1), which converts cholesterol to 7-alpha-hydroxy cholesterol [8]. CYP7A1 expression is upregulated by high cholesterol concentrations and down-regulated by high BA concentrations in the intestines. CYP7A1 is under negative feedback from fibroblast growth factor-19 (FGF19) via the ileal Farnesoid X
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Receptor (FXR) [9]. It is important to note that there is an alternative extra-hepatic pathway that may be used if required. This pathway uses oxysterols rather than cholesterol as the
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starting substrate for BA synthesis [10].
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The primary BAs are conjugated at the carboxyl residue with glycine or taurine in the liver [11]. Conjugation has several benefits, including minimising passive absorption and promoting resistance to pancreatic enzyme cleavage. The conjugated BAs are secreted from the liver via a bile salt export pump into bile ducts and then transported, concentrated and stored in the gallbladder [10]. In response to feeding, primary BAs are secreted into
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the duodenum via the common bile duct to enable digestion of fat and fat soluble vitamins by promoting emulsification. This aids the action of lipase and the formation of micelles in
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the intestine [12]. Some reabsorption of BAs occurs passively throughout the length of the
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small intestine, but 95% of bile acids are reabsorbed by active transport in the terminal ileum, and returned to the liver via the enterohepatic circulation for recycling [13].
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There are several transport molecules involved in ileal reabsorption [14]. Within the brush-
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border membranes of the terminal ileum are apical sodium-linked bile salt transporters
(ASBT), which move BAs into the enterocyte in a sodium-dependent manner. The BAs are then intracellularly transported by ileal bile acid binding protein and are extruded into
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portal circulation through the organic solute transporter heterodimer (OSTα and β) [15, 16]. Within the enterocyte, the BAs activate FXR which promotes gene expression and synthesis of FGF19. FGF19 is then transported via the portal circulation to the liver and activates fibroblast growth factor-receptor 4 (FGF-R4) which suppresses expression of
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CYP7A1 [17], thereby inhibiting BA synthesis. If BA concentrations in the intestine and/or terminal ileal cells are low, then FXR is not activated and FGF19 is not synthesised. This
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reduction in FGF19 leads to absence of feedback inhibition, increasing CYP7A1 expression and BA synthesis.
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BAs not reabsorbed in the ileum enter the colon and are excreted in the faeces. In the colon, the primary BAs are converted by bacteria into the secondary BAs, deoxycholic acid and lithocholic acid. About 75% of BAs reaching the colon are passively reabsorbed. In the colon, BAs stimulate secretion of fluids and colonic motility [5-7].
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The BA pool is recycled up to 12 times per day, owing to the high energy cost of new bile
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acid production. This results in approximately 200-600mg of BAs being lost into the faeces
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each day [13]. Every day around 800mg of cholesterol is synthesised and approximately 400mg of this is used for BA synthesis [18].
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Classification of BAD
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BAD is classified into three types, depending on the cause of the BA malabsorption. Type I
BAD is secondary to ileal resection or inflammation, as may occur in CD. Type 2 BAD is also known as primary or idiopathic BAD, and is hypothesised to be caused by increased BA
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synthesis, potentially as a result of defective feedback inhibition or ASBT mutations [4]. Type 3 BAD is secondary to various other gastrointestinal disorders such as cholecystectomy, coeliac disease, small intestinal bacterial overgrowth, post radiation enteritis and chronic pancreatitis.
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Pathophysiology of BAD
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In Type I BAD the loss of the primary location for active and passive reabsorption of bile acids owing to ileal resection or disease results in increased BA delivery to the large
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intestine [15]. Type 2 (primary) BAD is not clearly understood. Mutations in SLC10A2 [19], which codes for the ASBT, leading to failure of BA reabsorption have been reported but are very rare. Rather than malabsorption, it has been suggested that the primary mechanism for type 2 BAD is increased BA production due to defective negative feedback on BA synthesis [15]. The excessive delivery of BAs to the ileum exceeds its capacity for
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reabsorption leading to increased BAs in the colon causing diarrhoea. Although less likely, Type 2 BAD may also result from rapid small bowel transport bypassing the active BA
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reabsorption [7]. As discussed, FGF19 is a regulator of BA synthesis in the liver. A recent
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study reported that patients with Type 2 BAD had significantly lower FGF19 concentrations compared to controls, and this was inversely correlated with serum 7α-hydroxy-4-
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cholesten-3-one (C4) concentrations [20], a marker of BA synthesis. This suggests that defective or reduced FGF19 may lead to increased BA synthesis and subsequently BAD.
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Since the aetiology of Type 3 BAD is varied it is likely that the underlying pathophysiological mechanisms will also be diverse. In small bowel bacterial overgrowth, increased or altered
bacterial deconjugation of BAs reduces the efficiency of BA reabsorption. Other
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gastrointestinal diseases, through increasing intestinal motility, may reduce the time for ileal reabsorption of BAs [15] and reduce the time for bacterial conversion of primary to secondary BAs thereby reducing the efficiency of BA reabsorption. Further research is, therefore, required to fully establish the pathophysiological and molecular mechanisms
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underlying types 2 and 3 BAD.
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Diagnosis of BAD
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Diarrhoea is defined as the abnormal passage of liquid/loose stools, either greater than three times per day, or generating more than 200g of stool per day [21]. Chronic diarrhoea is defined by the National Institute for Health and Care Excellence (NICE) as diarrhoea lasting more than four weeks [22]. Diagnosis of BAD is, however, often missed owing to difficulties in accessing a suitable diagnostic test, and subsequently patients are diagnosed
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with IBS. The four most explored options for diagnosis of BAD are radiolabelled 23-seleno25-homotaurocholic acid (SeHCAT) test, measurement of faecal bile acids (FBA), serum-C4
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measurement, and a trial of bile acid sequestrants. Other proposed options include serum
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FGF19 measurement, 14C-glycocholate breath and stool test, and urine-2-proponol. These are discussed below.
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SeHCAT Scan
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The current gold standard for the diagnosis of BAD is the SeHCAT test, which assesses BA
retention in the body [21]. After an overnight fast, the patient takes a capsule containing radiolabelled SeHCAT, a synthetic BA. This is followed by two full-body gamma camera
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scans, the first one to three hours after taking the capsule, and the second seven days later. The gamma count from the second scan is then divided by the count from the first scan, and expressed as percentage retention of BAs. In health, the majority of BAs are reabsorbed and recirculated but retention of BAs is reduced in BAD. Retention values of
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greater than 15% are generally classed as normal [21], however different centres may have different cut off points, including an indeterminate range between 12 to 19% retention.
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It has been suggested that the test may not detect some patients with idiopathic BAD, as
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the causative mechanisms of idiopathic BAD are diverse and not well understood. Some patients may reabsorb relatively normal amounts of BAs in the ileum, but overproduction of BAs results in saturation of the retention capacity of the ileum [23]. These patients may demonstrate a relatively normal SeHCAT retention, but increased production means that excess BA still move into the large intestine.
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Whilst the SeHCAT test is considered the gold standard test in the UK, it is time consuming,
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expensive, and results in exposure to radiation. Furthermore, it is not widely available, so
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patients often have to travel to have the test performed. The SeHCAT test is not currently licenced for use in the USA, which complicates comparison of diagnostic methods.
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Faecal Bile Acid measurement
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Several laboratories, particularly in the USA, have developed methods for measuring Faecal
Bile Acid (FBA) excretion. FBA are expected to be higher in patients with BAD, as decreased
reabsorption in the ileum results in increased faecal excretion. There are several published
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enzymatic and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measurement of FBA [24, 26, 27].
Enzymatic methods generally utilise an NAD-dependent steroid dehydrogenase to oxidise
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deconjugated BAs and produce NADH, which is measured [24]. An extraction process is required to remove the BAs from the stool. Owing to the variety of conjugation processes
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that BAs undergo, this method may underestimate total FBA, particularly if hydrolysis time is not sufficient, as different conjugates undergo hydrolysis at different rates [24].
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Furthermore, stool samples may contain significant amounts of 3β-hydroxy and 3-oxo bile acids, which would not be oxidised by the commonly used 3α-hydroxysteroid dehydrogenase (3α-HSD) and thus would not be measured [25]. More recently, Immunodiagnostik (IDK) have developed a kit marketed to measure FBA on a random stool sample, in contrast to other methods which generally require at least a 48 hour stool collection.
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The processes used to extract BAs from faeces for LC-MS/MS analysis are complex due to
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the requirement for a very clean sample. Amplatz et al. described a method that takes several hours, and uses a small amount of freeze-dried stool which is incubated in sodium
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hydroxide (NaOH), followed by addition of distilled water, homogenisation, and addition of acetonitrile. Samples are vortexed, centrifuged and the supernatant dried down and re-
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suspended, prior to a solid phase extraction (SPE) stage with washing and elution [26].
Wong et al. proposed incubating a homogenised stool sample with NaOH and sodium
chloride, followed by centrifugation. The subsequent supernatant is collected and the
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extraction process twice repeated, and the pellet is then re-suspended in methanol and centrifuged. The extract is purified using SPE with several washing steps [27]. These lengthy and complex processes make them impractical for use in a routine laboratory.
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There are advantages and disadvantages for both enzymatic methods and LC-MS/MS
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methods. The LC-MS/MS method used by the Mayo Clinic in the US requires a 48 hour stool sample collection following a four day fat controlled diet, making it inconvenient and
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unpleasant for the patient. The enzymatic assay developed by IDK requires a random stool sample, which is more convenient and less unpleasant for the patient and the laboratory. Mitchell et al., suggested that there is variable excretion in BAs throughout the day in different stool samples [28], therefore measurement of FBA in a random stool sample may result in missed diagnosis. This will need further clarification. The extraction methods prior
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to LC-MS/MS analysis are cumbersome and time consuming for the laboratory. Enzymatic methods are generally quicker and easier, but have the potential to underestimate total
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BAs within the stool [24]. One benefit of LC-MS/MS analysis is that it allows quantitation
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of individual BAs within the stool, as opposed to the total BA measurement obtained in enzymatic methods [26].
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FBA measurement as a direct biomarker of BA excretion offers an attractive proposition
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for the diagnosis of BAD. The diagnostic accuracy of measurement of FBA, however, in
comparison to SeHCAT testing has not yet been fully assessed due to unavailability of the former in the United Kingdom (UK) and the latter in the United States (US).
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Bile acid sequestrant trial A therapeutic trial of bile acid sequestrants in suspected BAD leading to an improvement
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in patient symptoms would support a diagnosis of BAD. BA sequestrants, however, are often poorly tolerated, making them a less attractive diagnostic option. Furthermore,
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patient non-compliance may result in misdiagnosis. A trial of BA sequestrants is, therefore, not recommended by the British Society of Gastroenterology for the diagnosis of BAD [29].
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Serum 7α-hydroxy-4-cholesten-3-one measurement Cholesterol is converted by CYP7A1 to 7α-hydroxycholesterol and is the rate-limiting step in the classic BA synthesis pathway. The enzyme 3β-hydroxy-D5-C27-steroid dehydroxylase converts 7 α-hydroxycholesterol to 7α-hydroxy-4-cholesten-3-one (C4), which is the
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common precursor for the primary BAs, cholic acid and chenodeoxycholic acid. Serum C4 is, therefore, utilised as a biomarker of BA synthesis. Serum C4 would be expected to be
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higher in patients with BAD, as BA synthesis increases to compensate for the increased
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faecal BA loss. Several studies have looked at the utility of serum C4 as a biomarker of BAD [7, 30-32]; however, its adoption as a routine test has been limited. Reported methods use
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LC-MS/MS or high performance liquid chromatography (HPLC); with extraction protocols varying between studies. Donato et al. and Kent et al. used acetonitrile precipitation in
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conjunction with a deuterated internal standard. The supernatant is then injected directly
onto the LC-MS/MS system for quantification [33, 34]. This method is rapid and efficient and does not require specialist equipment for solvent evaporation unlike other published
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methods, making it an attractive option for a routine laboratory. This method may, however, be unsuitable with less sensitive mass spectrometers. Camilleri et al. used acetonitrile and ammonium sulphate precipitation followed by incubation and separation, before evaporation under nitrogen and reconstitution [31]. Gothe et al. utilised a HPLC
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method, however this method required several washing steps on octadecylsilane-bonded silica columns, using hexane-chloroform for elution [30] making it unsuitable for routine use.
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The major benefit of C4 measurement is that it requires a single blood sample, rather than a long stool collection or two visits to hospital for the SeHCAT test, making it more convenient for patients. BA synthesis and therefore C4 levels, have diurnal variation and increase post-prandially, thus a fasting morning sample is preferred [34]. Gothe’s study demonstrated that children with CD and persistent diarrhoea had significantly higher
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serum C4 concentrations compared to those with formed stools. Furthermore, C4 concentrations were increased in patients with ileal resection compared to those with
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intact ileum [30]. Serum C4 concentration had 90% sensitivity and 79% specificity
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respectively, in the diagnosis of BAD when SeHCAT testing was used as the gold standard [31]. C4 has also been shown to be inversely related to SeHCAT retention [35], and several
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studies have shown its correlation with FBA loss [5, 7]. Owing to its high negative predictive value, C4 has been proposed as a rule-out test for BAD, reducing the number of patients
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requiring referral for SeHCAT testing [36]. However research is still limited owing to the small number of centres that measure the analyte.
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Serum fibroblast growth factor 19 measurement FGF19 inhibits BA synthesis. In BAD, loss of FBA and reduced ileal BA concentrations decrease FGF19 which leads to increased hepatic BA synthesis. Several commercial enzyme-linked immunosorbent assay (ELISA) kits are available for measurement of FGF19
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in serum and plasma. The kits generally use several steps, including multiple washing and incubation steps, and thus the assay may be time consuming in a routine laboratory
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environment.
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Vijayvargiya et al. reported FGF19 to have a negative predictive value of 78% and specificity of 78% for BAD when using 48 hour FBA measurement as the gold standard [36]. Pattni et al. demonstrated a negative predictive value and positive predictive value of 82% and 61% respectively, when utilising an FGF19 cut off of ≤145 pg/mL, for predicting a SeHCAT of
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t Au Bile Acid Diarrhoea: Current and Potential methods of Diagnosis
rP A
ie eevp r Rc ee c
Journal: Annals of Clinical Biochemistry
Manuscript ID ACB-20-234.R1
Manuscript Type: Review Article
Date Submitted by the 20-Jul-2020 Author:
u ann Mrsio dVe
twe
Complete List of Authors: Hughes, Lauren; New Cross Hospital, Clinical Chemistry Ford, Clare; Royal Wolverhampton Hospitals NHS Trust, Clinical Chemistry Brookes, Matthew; Royal Wolverhampton Hospitals NHS Trust, Department of Gastroenterology; University of Wolverhampton Faculty of Education Health and Wellbeing Gama, Rousseau; Royal Wolverhampton Hospitals NHS Trust, Blood Sciences Keywords: Gastro-intestinal disorders < Clinical studies
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Clinical Sciences Review Committee (CSRC) Commissioned Review
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CSRC Article Number Review Title
Running Title Author(s)
Bile Acid Diarrhoea: Current and Potential methods of Diagnosis
Methods for Diagnosing Bile Acid Diarrhoea Lauren Elizabeth Hughes Clare Ford Matthew James Brookes Rousseau Gama
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Author Affiliations Including email address for each author
Lauren Elizabeth Hughes: [email protected] 1) Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK Clare Ford: [email protected] 1) Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK Matthew James Brookes: [email protected] 1) Department of Gastroenterology, Royal Wolverhampton NHS Trust, Wolverhampton, UK 2) Faculty of Education, Health and Wellbeing, University of Wolverhampton, Wolverhampton, UK
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Rousseau Gama: [email protected] 1. Clinical Chemistry, Royal Wolverhampton NHS Trust, Wolverhampton, UK 2) School of Medicine and Clinical Practice, Wolverhampton
University, Wolverhampton, UK
Word Count
Declaration of Interests Funding N/A
Guarantor
Rousseau Gama
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Contributorship Acknowledgements
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Ethical Approval
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This article was prepared at the invitation of the Clinical Sciences Reviews Committee of the Association for Clinical Biochemistry and Laboratory Medicine.
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Key Words
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Abstract Chronic diarrhoea is common and mostly due to diarrhoea predominant irritable bowel syndrome (IBS-D). IBS-D affects about 11% of the population, however up to a third of these patients actually have bile acid diarrhoea (BAD). There are, therefore, more than one
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million sufferers of BAD in the UK. BAD is caused by small bowel malabsorption of bile acids and the increased bile acids in the large intestine cause diarrhoea. Once diagnosed, the
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treatment of BAD is simple and effective. BAD, however, is often not diagnosed because of a lack of easily available and reliable diagnostic methods. In the United Kingdom, the
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radiolabelled 23-seleno-25-homotaurocholic acid test (SeHCAT) is the gold-standard method of diagnosis. SeHCAT, however, is expensive, inconvenient to the patient, involves radiation exposure and has limited availability. As such, a laboratory biomarker is desirable. This review briefly discusses the pathophysiology and management of BAD and critically evaluates methods for its diagnosis, including serum 7α-hydroxy-4-cholesten-3-one, faecal
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bile acid measurement, serum fibroblast growth factor 19, urine-2-propanol, and the 14Cglycocholate breath and stool test.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Introduction Irritable Bowel Syndrome (IBS) is estimated to affect 11% of the global population [1]. IBS is a chronic, relapsing, often lifelong disorder with typical symptoms of abdominal pain, diarrhoea or constipation associated with bloating. IBS is classified into four subtypes
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depending on the primary stool composition; diarrhoea-predominant IBS (IBS-D), constipation-predominant IBS, mixed IBS and un-subtyped IBS [2]. IBS is not life-
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threatening, but significantly diminishes the quality of life for sufferers [3].
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It, however, is estimated that 15 to 50% of patients with IBS-D have bile acid diarrhoea (BAD) [4-6], but the lack of diagnostic tests makes it difficult to determine the exact prevalence. BAD may be associated with other gastrointestinal disorders, such as Crohn’s Disease (CD) or post-cholecystectomy, but may also be idiopathic (primary BAD). BAD is caused by bile acid malabsorption (BAM) in the small intestine. In healthy individuals,
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approximately 95% of bile acids (BAs) are reabsorbed in the ileum and returned to the liver via the enterohepatic circulation [7]. If bile acid (BA) reabsorption is reduced or small bowel
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BA concentrations overwhelm the resorptive capacity of the ileum, increased
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concentrations of BAs move into the large intestine and, by stimulating colonic motility and secretions, cause diarrhoea. BAM and BAD are often used interchangeably.
Physiology of bile acids and the enterohepatic circulation
In the classic BA synthesis pathway, the primary BAs, cholic acid and chenodeoxycholic
acid, are synthesised in the liver from cholesterol. The rate limiting step is catalysed by the
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enzyme cholesterol-7-alpha-hydroxylase P4507A1 (CYP7A1), which converts cholesterol to 7-alpha-hydroxy cholesterol [8]. CYP7A1 expression is upregulated by high cholesterol concentrations and down-regulated by high BA concentrations in the intestines. CYP7A1 is under negative feedback from fibroblast growth factor-19 (FGF19) via the ileal Farnesoid X
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Receptor (FXR) [9]. It is important to note that there is an alternative extra-hepatic pathway that may be used if required. This pathway uses oxysterols rather than cholesterol as the
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starting substrate for BA synthesis [10].
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The primary BAs are conjugated at the carboxyl residue with glycine or taurine in the liver [11]. Conjugation has several benefits, including minimising passive absorption and promoting resistance to pancreatic enzyme cleavage. The conjugated BAs are secreted from the liver via a bile salt export pump into bile ducts and then transported, concentrated and stored in the gallbladder [10]. In response to feeding, primary BAs are secreted into
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the duodenum via the common bile duct to enable digestion of fat and fat soluble vitamins by promoting emulsification. This aids the action of lipase and the formation of micelles in
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the intestine [12]. Some reabsorption of BAs occurs passively throughout the length of the
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small intestine, but 95% of bile acids are reabsorbed by active transport in the terminal ileum, and returned to the liver via the enterohepatic circulation for recycling [13].
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There are several transport molecules involved in ileal reabsorption [14]. Within the brush-
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border membranes of the terminal ileum are apical sodium-linked bile salt transporters
(ASBT), which move BAs into the enterocyte in a sodium-dependent manner. The BAs are then intracellularly transported by ileal bile acid binding protein and are extruded into
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portal circulation through the organic solute transporter heterodimer (OSTα and β) [15, 16]. Within the enterocyte, the BAs activate FXR which promotes gene expression and synthesis of FGF19. FGF19 is then transported via the portal circulation to the liver and activates fibroblast growth factor-receptor 4 (FGF-R4) which suppresses expression of
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CYP7A1 [17], thereby inhibiting BA synthesis. If BA concentrations in the intestine and/or terminal ileal cells are low, then FXR is not activated and FGF19 is not synthesised. This
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reduction in FGF19 leads to absence of feedback inhibition, increasing CYP7A1 expression and BA synthesis.
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BAs not reabsorbed in the ileum enter the colon and are excreted in the faeces. In the colon, the primary BAs are converted by bacteria into the secondary BAs, deoxycholic acid and lithocholic acid. About 75% of BAs reaching the colon are passively reabsorbed. In the colon, BAs stimulate secretion of fluids and colonic motility [5-7].
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The BA pool is recycled up to 12 times per day, owing to the high energy cost of new bile
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acid production. This results in approximately 200-600mg of BAs being lost into the faeces
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each day [13]. Every day around 800mg of cholesterol is synthesised and approximately 400mg of this is used for BA synthesis [18].
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Classification of BAD
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BAD is classified into three types, depending on the cause of the BA malabsorption. Type I
BAD is secondary to ileal resection or inflammation, as may occur in CD. Type 2 BAD is also known as primary or idiopathic BAD, and is hypothesised to be caused by increased BA
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synthesis, potentially as a result of defective feedback inhibition or ASBT mutations [4]. Type 3 BAD is secondary to various other gastrointestinal disorders such as cholecystectomy, coeliac disease, small intestinal bacterial overgrowth, post radiation enteritis and chronic pancreatitis.
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Pathophysiology of BAD
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In Type I BAD the loss of the primary location for active and passive reabsorption of bile acids owing to ileal resection or disease results in increased BA delivery to the large
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intestine [15]. Type 2 (primary) BAD is not clearly understood. Mutations in SLC10A2 [19], which codes for the ASBT, leading to failure of BA reabsorption have been reported but are very rare. Rather than malabsorption, it has been suggested that the primary mechanism for type 2 BAD is increased BA production due to defective negative feedback on BA synthesis [15]. The excessive delivery of BAs to the ileum exceeds its capacity for
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reabsorption leading to increased BAs in the colon causing diarrhoea. Although less likely, Type 2 BAD may also result from rapid small bowel transport bypassing the active BA
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reabsorption [7]. As discussed, FGF19 is a regulator of BA synthesis in the liver. A recent
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study reported that patients with Type 2 BAD had significantly lower FGF19 concentrations compared to controls, and this was inversely correlated with serum 7α-hydroxy-4-
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cholesten-3-one (C4) concentrations [20], a marker of BA synthesis. This suggests that defective or reduced FGF19 may lead to increased BA synthesis and subsequently BAD.
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Since the aetiology of Type 3 BAD is varied it is likely that the underlying pathophysiological mechanisms will also be diverse. In small bowel bacterial overgrowth, increased or altered
bacterial deconjugation of BAs reduces the efficiency of BA reabsorption. Other
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gastrointestinal diseases, through increasing intestinal motility, may reduce the time for ileal reabsorption of BAs [15] and reduce the time for bacterial conversion of primary to secondary BAs thereby reducing the efficiency of BA reabsorption. Further research is, therefore, required to fully establish the pathophysiological and molecular mechanisms
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underlying types 2 and 3 BAD.
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Diagnosis of BAD
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Diarrhoea is defined as the abnormal passage of liquid/loose stools, either greater than three times per day, or generating more than 200g of stool per day [21]. Chronic diarrhoea is defined by the National Institute for Health and Care Excellence (NICE) as diarrhoea lasting more than four weeks [22]. Diagnosis of BAD is, however, often missed owing to difficulties in accessing a suitable diagnostic test, and subsequently patients are diagnosed
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with IBS. The four most explored options for diagnosis of BAD are radiolabelled 23-seleno25-homotaurocholic acid (SeHCAT) test, measurement of faecal bile acids (FBA), serum-C4
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measurement, and a trial of bile acid sequestrants. Other proposed options include serum
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FGF19 measurement, 14C-glycocholate breath and stool test, and urine-2-proponol. These are discussed below.
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SeHCAT Scan
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The current gold standard for the diagnosis of BAD is the SeHCAT test, which assesses BA
retention in the body [21]. After an overnight fast, the patient takes a capsule containing radiolabelled SeHCAT, a synthetic BA. This is followed by two full-body gamma camera
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scans, the first one to three hours after taking the capsule, and the second seven days later. The gamma count from the second scan is then divided by the count from the first scan, and expressed as percentage retention of BAs. In health, the majority of BAs are reabsorbed and recirculated but retention of BAs is reduced in BAD. Retention values of
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greater than 15% are generally classed as normal [21], however different centres may have different cut off points, including an indeterminate range between 12 to 19% retention.
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It has been suggested that the test may not detect some patients with idiopathic BAD, as
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the causative mechanisms of idiopathic BAD are diverse and not well understood. Some patients may reabsorb relatively normal amounts of BAs in the ileum, but overproduction of BAs results in saturation of the retention capacity of the ileum [23]. These patients may demonstrate a relatively normal SeHCAT retention, but increased production means that excess BA still move into the large intestine.
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Whilst the SeHCAT test is considered the gold standard test in the UK, it is time consuming,
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expensive, and results in exposure to radiation. Furthermore, it is not widely available, so
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patients often have to travel to have the test performed. The SeHCAT test is not currently licenced for use in the USA, which complicates comparison of diagnostic methods.
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Faecal Bile Acid measurement
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Several laboratories, particularly in the USA, have developed methods for measuring Faecal
Bile Acid (FBA) excretion. FBA are expected to be higher in patients with BAD, as decreased
reabsorption in the ileum results in increased faecal excretion. There are several published
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enzymatic and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measurement of FBA [24, 26, 27].
Enzymatic methods generally utilise an NAD-dependent steroid dehydrogenase to oxidise
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deconjugated BAs and produce NADH, which is measured [24]. An extraction process is required to remove the BAs from the stool. Owing to the variety of conjugation processes
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that BAs undergo, this method may underestimate total FBA, particularly if hydrolysis time is not sufficient, as different conjugates undergo hydrolysis at different rates [24].
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Furthermore, stool samples may contain significant amounts of 3β-hydroxy and 3-oxo bile acids, which would not be oxidised by the commonly used 3α-hydroxysteroid dehydrogenase (3α-HSD) and thus would not be measured [25]. More recently, Immunodiagnostik (IDK) have developed a kit marketed to measure FBA on a random stool sample, in contrast to other methods which generally require at least a 48 hour stool collection.
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The processes used to extract BAs from faeces for LC-MS/MS analysis are complex due to
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the requirement for a very clean sample. Amplatz et al. described a method that takes several hours, and uses a small amount of freeze-dried stool which is incubated in sodium
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hydroxide (NaOH), followed by addition of distilled water, homogenisation, and addition of acetonitrile. Samples are vortexed, centrifuged and the supernatant dried down and re-
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suspended, prior to a solid phase extraction (SPE) stage with washing and elution [26].
Wong et al. proposed incubating a homogenised stool sample with NaOH and sodium
chloride, followed by centrifugation. The subsequent supernatant is collected and the
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extraction process twice repeated, and the pellet is then re-suspended in methanol and centrifuged. The extract is purified using SPE with several washing steps [27]. These lengthy and complex processes make them impractical for use in a routine laboratory.
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There are advantages and disadvantages for both enzymatic methods and LC-MS/MS
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methods. The LC-MS/MS method used by the Mayo Clinic in the US requires a 48 hour stool sample collection following a four day fat controlled diet, making it inconvenient and
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unpleasant for the patient. The enzymatic assay developed by IDK requires a random stool sample, which is more convenient and less unpleasant for the patient and the laboratory. Mitchell et al., suggested that there is variable excretion in BAs throughout the day in different stool samples [28], therefore measurement of FBA in a random stool sample may result in missed diagnosis. This will need further clarification. The extraction methods prior
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to LC-MS/MS analysis are cumbersome and time consuming for the laboratory. Enzymatic methods are generally quicker and easier, but have the potential to underestimate total
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BAs within the stool [24]. One benefit of LC-MS/MS analysis is that it allows quantitation
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of individual BAs within the stool, as opposed to the total BA measurement obtained in enzymatic methods [26].
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FBA measurement as a direct biomarker of BA excretion offers an attractive proposition
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for the diagnosis of BAD. The diagnostic accuracy of measurement of FBA, however, in
comparison to SeHCAT testing has not yet been fully assessed due to unavailability of the former in the United Kingdom (UK) and the latter in the United States (US).
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Bile acid sequestrant trial A therapeutic trial of bile acid sequestrants in suspected BAD leading to an improvement
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in patient symptoms would support a diagnosis of BAD. BA sequestrants, however, are often poorly tolerated, making them a less attractive diagnostic option. Furthermore,
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patient non-compliance may result in misdiagnosis. A trial of BA sequestrants is, therefore, not recommended by the British Society of Gastroenterology for the diagnosis of BAD [29].
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Serum 7α-hydroxy-4-cholesten-3-one measurement Cholesterol is converted by CYP7A1 to 7α-hydroxycholesterol and is the rate-limiting step in the classic BA synthesis pathway. The enzyme 3β-hydroxy-D5-C27-steroid dehydroxylase converts 7 α-hydroxycholesterol to 7α-hydroxy-4-cholesten-3-one (C4), which is the
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common precursor for the primary BAs, cholic acid and chenodeoxycholic acid. Serum C4 is, therefore, utilised as a biomarker of BA synthesis. Serum C4 would be expected to be
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higher in patients with BAD, as BA synthesis increases to compensate for the increased
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faecal BA loss. Several studies have looked at the utility of serum C4 as a biomarker of BAD [7, 30-32]; however, its adoption as a routine test has been limited. Reported methods use
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LC-MS/MS or high performance liquid chromatography (HPLC); with extraction protocols varying between studies. Donato et al. and Kent et al. used acetonitrile precipitation in
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conjunction with a deuterated internal standard. The supernatant is then injected directly
onto the LC-MS/MS system for quantification [33, 34]. This method is rapid and efficient and does not require specialist equipment for solvent evaporation unlike other published
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methods, making it an attractive option for a routine laboratory. This method may, however, be unsuitable with less sensitive mass spectrometers. Camilleri et al. used acetonitrile and ammonium sulphate precipitation followed by incubation and separation, before evaporation under nitrogen and reconstitution [31]. Gothe et al. utilised a HPLC
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method, however this method required several washing steps on octadecylsilane-bonded silica columns, using hexane-chloroform for elution [30] making it unsuitable for routine use.
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The major benefit of C4 measurement is that it requires a single blood sample, rather than a long stool collection or two visits to hospital for the SeHCAT test, making it more convenient for patients. BA synthesis and therefore C4 levels, have diurnal variation and increase post-prandially, thus a fasting morning sample is preferred [34]. Gothe’s study demonstrated that children with CD and persistent diarrhoea had significantly higher
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serum C4 concentrations compared to those with formed stools. Furthermore, C4 concentrations were increased in patients with ileal resection compared to those with
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intact ileum [30]. Serum C4 concentration had 90% sensitivity and 79% specificity
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respectively, in the diagnosis of BAD when SeHCAT testing was used as the gold standard [31]. C4 has also been shown to be inversely related to SeHCAT retention [35], and several
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studies have shown its correlation with FBA loss [5, 7]. Owing to its high negative predictive value, C4 has been proposed as a rule-out test for BAD, reducing the number of patients
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requiring referral for SeHCAT testing [36]. However research is still limited owing to the small number of centres that measure the analyte.
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Serum fibroblast growth factor 19 measurement FGF19 inhibits BA synthesis. In BAD, loss of FBA and reduced ileal BA concentrations decrease FGF19 which leads to increased hepatic BA synthesis. Several commercial enzyme-linked immunosorbent assay (ELISA) kits are available for measurement of FGF19
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in serum and plasma. The kits generally use several steps, including multiple washing and incubation steps, and thus the assay may be time consuming in a routine laboratory
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environment.
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Vijayvargiya et al. reported FGF19 to have a negative predictive value of 78% and specificity of 78% for BAD when using 48 hour FBA measurement as the gold standard [36]. Pattni et al. demonstrated a negative predictive value and positive predictive value of 82% and 61% respectively, when utilising an FGF19 cut off of ≤145 pg/mL, for predicting a SeHCAT of
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