Association Between Luminal Bile Salt Content and Duodenal Mucosal Integrity in Functional Dyspepsia

Study Overview
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Brief Summary
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Functional dyspepsia (FD) is an extremely common disorder of gastrointestinal function. Recently, impaired duodenal mucosal integrity was reported as a potential pathophysiological mechanism in FD. However, the factors controlling duodenal mucosal integrity remain unknown. In this study, we evaluated whether the luminal bile salt content could play a role in impaired duodenal permeability in FD. Duodenal biopsies were obtained from 25 healthy volunteers (HV) and 25 FD patients. Biopsies were mounted in Ussing chambers to measure transepithelial resistance (TEER) and paracellular permeability using fluorescein isothiocyanate dextran (FITC-dx4, MW 4kDa). Expression of bile acid-sensing receptors (TGR5, VDR, PXR, FXR and CAR) in duodenal biopsies was measured by western blot and real time RT-PCR. Immunohistochemistry was used to evaluate eosinophil and mastcell infiltration in duodenal biopsies of FD patients and HV. Duodenal fluid aspirates were collected at fixed time points during 1 hour in fasted state and 1.5 hours after a liquid meal (Nutridrink, 200ml). Concentration and composition of the bile salt pool (including glycocholic acid (GC), taurocholic acid (TC), glycochenodeoxycholic acid (GCDC), taurochenodeoxycholic acid (TCDC), glycodeoxycholic acid (GDC), taurodeoxycholic acid (TDC), glycoursodeoxycholic acid (GUDC) and tauroursodeoxycholic acid (TUDC)) in these aspirates was evaluated by liquid chromatography-mass spectrometry-selected ion monitoring analysis (LC-MS/MS).

Detailed Description
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The Rome III criteria defined functional dyspepsia (FD) as the presence of symptoms thought to originate in the gastroduodenal region, in the absence of any organic, systemic or metabolic disease that readily explains the complaints. FD is extremely common, affecting up to 15-20% of the population and is associated with significantly decreased quality of life and substantial healthcare costs. The available treatment options for FD are of limited effectiveness, which reflects the poorly understood pathogenesis. Studies indicate that FD is a heterogeneous disorder, in which different pathophysiological mechanisms underlie specific symptom patterns. Traditionally, gastric abnormalities such as impaired accommodation, delayed emptying and hypersensitivity have been believed to be involved in the pathophysiology of FD. More recent studies have suggested that also a number of duodenal abnormalities can be responsible for the generation of symptoms, like increased sensitivity to duodenal acid, increased sensitivity to duodenal lipids and low-grade mucosal inflammation. The investigators recently showed that FD patients display impaired duodenal mucosal integrity. The trigger of increased permeability is unknown, but it is possible that increased exposure to duodenal bile acids or an altered composition of bile acids leads to impairment of the intestinal barrier. This sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa and lead to local mucosal immune responses that manifest as inflammation and finally result in generation of dyspeptic symptoms. The investigators hypothesized that increased duodenal bile acid exposure or a change in the composition of bile acids lead to impaired duodenal mucosal integrity in FD, allowing luminal substances to pass through the mucosa and result in immune responses and finally in dyspeptic symptom generation. The general aim of this project is to assess if FD patients display increased endogenous duodenal bile acid exposure and a different bile acid composition. In addition, it will be tested whether duodenal mucosal permeability of FD patients with an endogenous duodenal bile acid exposure above the normal range and an altered bile acid composition is higher than in FD patients with a normal endogenous duodenal acid exposure and composition. Participants will be expected on the department endoscopy of the UZ Gasthuisberg after they have fasted overnight. Before the study, they are asked to fill in a bundle of questionnaires concerning physical complaints, depression, anxiety (disturbances), pain/disease, body/interoceptive awareness, trauma/abuse and personality. Gastroduodenoscopy will be performed by an experienced endoscopist (Jan Tack). Hereby, 12 duodenal biopsies (2 biopsies at a time) (Radial Jaw™3 with needle; outside diameter 2.2mm; Boston Scientific, 302 Parkway, Global Park, Heredia, Costa Rica) will be obtained. To measure the in vitro transepithelial resistance, 4 biopsies will be examined using an adapted mini-Ussing chambers system. After equilibration, the mucosal side of the tissue will be exposed to 4kDa FITC-dextran as a measure of paracellular permeability. A sample will be taken from the serosal side during 2h at 30min interval. The concentration of fluorescein will then be measured using a fluorescence plate reader. Also, 2 biopsies will be used for mRNA extraction and subsequent cDNA synthesis. This cDNA will be used to measure the gene expression of cell-to-cell adhesion proteins and acid-sensing receptors by means of real-time PCR. In addition, 2 biopsies will be prepared for immunofluorescence and immunohistochemistry and 2 will be used for western blot to measure changes in distribution/expression of the cell-to-cell adhesion proteins and of bile acid-sensitive receptors. Two biopsies will be obtained to study ultrastructural alterations by transmission electron microscopy. After recovery, a catheter will be introduced in the second duodenum via the nose and the position of the catheter will be checked fluoroscopically. This catheter allows collection of intestinal fluids by means of a syringe to collect duodenal fluid aspirates and characterization of the bile acid composition of those samples (8). After 30 minutes, the participants will be given a specified amount of water (250 mL) (fasted state) and another 30 minutes later a nutritional drink (fed state). Intestinal fluids will be sampled every 15 min for a period of 1 h before the liquid meal intake and until 90 minutes after the liquid meal intake. So, after the total collection period, 7 fractions for the fed state and 4 fractions for the fasted state will be obtained per participant in a time frame of 2 hours and a half. The composition of bile acids of the intestinal samples will be determined by GC-MS-selected ion monitoring analysis.

Official Title
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Association Between Luminal Bile Salt Content and Duodenal Mucosal Integrity in Functional Dyspepsia

Conditions
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Functional Dyspepsia

Intervention / Treatment
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* Procedure: Duodenogastroscopy
* Device: Duodenal aspiration catheter
* Dietary Supplement: Nutri drink


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: FD patients have to fulfill the Rome III criteria for functional dyspepsia. Exclusion Criteria: Symptoms or history of gastrointestinal disease (for healthy volunteers) First degree relatives with celiac disease Diabetes mellitus Allergy/atopy (eczema, asthma, allergic rhinoconjunctivitis) Coagulation disorders/anticoagulant therapy First degree relatives with Crohn's disease or type I diabetes mellitus Intake of antihistamines, ketotifen, cromoglycate, acetylsalicylates, NSAIDs, anticholinergics, theophylline, β2-agonists, codeine or opioid derivatives for at least 2 weeks prior to the study. Steroid or immunosuppressive drug intake any time in the last 6 months

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 60 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Duodenal fluid aspiration<br> | Procedure: Duodenogastroscopy<br>* Catheter goes through the mouth in the duodenum to take duodenal biopsies.<br>Device: Duodenal aspiration catheter<br>* Catheter goes through the nose in the duodenum to aspirate duodenal fluid in fasted and fed state.<br>Dietary Supplement: Nutri drink<br>* Liquid meal of 200 ml.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Duodenal mucosal transepithelial electrical resistance (Ohm*cm^2) | Measurement for mucosal integrity | 2 hours | 
| Duodenal paracellular passage of Fitc Dextran 4 kDa (pmol) | Measurement for mucosal integrity | 2 hours | 
| Glycocholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Taurocholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Glycochenodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Taurochenodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Glycodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Taurodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Glycoursodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 
| Tauroursodeoxycholic acid concentration (mM) | Bile salt | 2.5 hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Duodenal mucosal mastcell count (number of mastcells/mm^2 lamina propria) |  | 2 years | 
| Duodenal mucosal eosinophil count (number of eosinophils/mm^2 lamina propria) |  | 2 years | 
| RNA expression of Vitamin D receptor (VDR) | Real-time RT-PCR | 2 years | 
| RNA expression of bile acid receptor FXR | Real-time RT-PCR | 2 years | 
| RNA expression of bile acid receptor PXR | Real-time RT-PCR | 2 years | 
| RNA expression of bile acid receptor TGR5 | Real-time RT-PCR | 2 years | 
| RNA expression of bile acid receptor CAR | Real-time RT-PCR | 2 years | 
| Protein expression of bile acid receptor VDR | Western blot | 2 years | 
| Protein expression of bile acid receptor FXR | Western blot | 2 years | 
| Protein expression of bile acid receptor PXR | Western blot | 2 years | 
| Protein expression of bile acid receptor TGR5 | Western blot | 2 years | 
| Protein expression of bile acid receptor CAR | Western blot | 2 years | 
| Stomach complaints questionnaire | Questions about stomach complaints | 2 years | 
| ReQuest Questionnaire | Reflux evaluation | 2 years | 
| Council of Nutrition appetite questionnaire | Questions about appetite | 2 years | 
| Bowel complaints questionnaire | Questions about bowel complaints | 2 years | 
| Patient health questionnaire | Questions about health | 2 years | 
| Anxiety sensitivity index | Questions about anxiety sensitivity | 2 years | 
| Visceral sensitivity index | Questions about visceral sensitivity | 2 years |