Effect of SB CD on Chylomicron
Condition: Crohn Disease
MeSH terms: Crohn Disease
Study type: Observational

Summary: 
      It is often suggested that lymphatic vessels are ineffective in transporting cargo in Crohn's
      disease. Our own work on surgically resected tissue supports this concept (1), but the
      concept has not been directly tested. Chylomicrons are packaged lipids from the diet with an
      obligatory absorption route through the lymphatic vasculature to reach host plasma. This
      protocol takes an approach to directly quantify chylomicron secretion using a fatty meal that
      incorporates stable isotopic tracers for trioleate and cholesterol in the meal. We will
      collect baseline plasma and then plasma every 30 minutes for 6 hours to chart the kinetic and
      magnitude of chylomicron secretion and transport in all subjects using mass spectrometry
      analysis. We will characterize a wide variety of parameters on the chylomicrons as well using
      ELISA. Infusions i.v. of stable isotope labeled triglyceride and glycerol will allow us to
      consider whether there are changes in VLDL metabolism that could account for differences in
      chylomicron handling once the chylomicrons are secreted into plasma. Collection and analysis
      of breath samples will also be carried to normalize against possible incomplete lipid
      absorption in some subjects.
    

Detailed description: 
      Background:

      B1 Prior Literature and Studies Crohn's disease (CD) is characterized by spontaneous, chronic
      relapsing and remitting inflammation of the gastrointestinal tract. The underlying etiology
      of the disease is unknown but it likely develops secondary to an environmental stimulus in a
      genetically predisposed person that results in a dysregulated immune response to the
      intestinal microbiome. Considerable effort has been paid to understanding the role of the
      intestinal microbiome, the genetic susceptibilities at play, and the immunology behind the
      condition to tailor treatments aimed at reducing inflammation.

      Prior to the advent of current medical therapies a defining characteristic of CD noted by
      early pathologists evaluating tissue specimen's was a significant alteration of the
      gastrointestinal lymphatic system with lymphocytic thrombi, aggregates of lymphocytes and
      granuloma-obstructed lymphatics consistent with chronic lymphangitis (2). Furthermore, early
      researchers showed that obstructing segments of the regional lymphatics of the small
      intestine resulted in a segmental intestinal disease similar to CD (3). Immunohistochemical
      staining has confirmed the presence of lymphangiectasias, lymphocytic perilymphangitis,
      lymphocyte or granuloma obstructed lymphatics and inflammatory lymphoid follicles in patients
      with CD (4). Upstream of these obstructed lymphatics the vessels remain distended with
      lymphocytes (5). More recent studies have shown that lymphatic density is also significantly
      increased in the ileal and colonic tissue of patients with CD (6). Furthermore, the
      relationship between intestinal health and lymphatics has been highlighted in studies that
      found certain types of bacteria and viruses had resulted in a profoundly remodeled mesenteric
      lymphatic system. Findings included the development of chronic lymphadenopathy and increased
      permeability, similar to what is seen in CD (7). Given the prominent structural changes that
      occur to the mesenteric lymphatic system in CD (1), studies are needed to evaluate if
      lymphatic function is altered as well.

      Chylomicrons are cholesterol and triacylglycerol rich lipoproteins synthesized by the small
      intestine and transported through the lymphatics. Thus, oral uptake of trioleate and
      cholesterol will lead to incorporation of the tracers into chylomicrons.

      Using ELISA and mass spectrometric analysis these labelled chylomicrons can be quantitated in
      the serum, thus serving as a marker of absorption and transit through the lymphatics. Studies
      of healthy subjects have utilized this validated means to assess lymphatic uptake and
      transport. After ingestion, there is a delay of <60 minutes in the appearance of 13C labelled
      chylomicrons in the serum, with a peak occurring between 180 and 240 minutes. Our protocol is
      designed to take advantage of the knowledge that other oral tracer studies at Washington
      University have learned in optimizing such studies, including ongoing studies by Dr. Todd
      Cade that focuses on chylomicron secretion in pre-diabetic patients. Our study will evaluate
      patients with significant small bowel CD and compare them to healthy controls using a similar
      method.

      This pilot study would be the first to evaluate the quantitative function of lymphatics in
      CD. It would provide the basis for further studies evaluating the mesenteric lymphatics and
      their role in the pathogenesis of CD and as a treatment target.

      C Study Objectives

      C1 Primary Aim Follow lymphatic vessel transported cargo in Crohn's disease patients with
      small bowel involvement as a way to assess possible defects in the lymphatic system in
      patients compared with healthy control subjects.

      C2 Secondary Aim Characterize changes in pre- and post-prandial lipid metabolism resulting
      from Crohn's disease affecting the small bowel.

      C3 Tertiary Aim Carry out lipidomic analyses to determine if chylomicrons absorbed from the
      small bowel carry distinct microbial lipid signatures compared with healthy control subjects
      (eg., LPS, commendamide (8)).

      C4 Quaternary Aim Measure lipid absorption from the intestine using 13CO2 breath analysis.

      D Study Design

      D1 Overview or Design Summary This study will utilize a 2-group trial design in which 40
      participants (20 CD patients and 20 controls) will visit once for this feeding (oral tracer)
      and infusion study, following an overnight fast.

      Participants will be recruited from the Washington University School of Medicine IBD Center,
      Washington University's Volunteers for Health, the Center for Community Based Research, and
      IBD clinics affiliated with Washington University in the surrounding St. Louis community.

      The principal investigator, collaborating physicians, and study coordinator(s) will be
      responsible for identifying participants through the use of posters, email, and existing
      participant databases.

      E Study Procedures

      E1 Screening for Eligibility IBD Center staff will identify eligible subjects from the
      patient arm. By phone, each participant will be asked a series of preliminary screening
      questions to determine immediate expulsion from the study based on exclusion and inclusion
      criteria. The purpose of the study, and a brief overview of the procedures, time-commitment,
      and compensation will be discussed over the phone prior to the individual agreeing to
      participate.

      E2 Study Visit

        1. 7:00 AM: Subject reports to the Clinical Research Unit at Washington University School
           of Medicine.

             1. The subject will be asked to skip breakfast and report to the Clinical Research
                Unit after an overnight fast (> 8 hours) with nothing taken by mouth except water.
                Subjects will be asked to avoid caffeine and alcohol for at least 24 h before
                admission for the study visit. The day prior to the study visit, subjects will be
                provided instructions for a standardized meal for breakfast, lunch, and dinner
                containing a total of 2,165 calories (46% of total energy from carbohydrates, 40%
                from fat, and 14% from protein). A liquid formula (Ensure; Ross Laboratories,
                Columbus, OH) containing 250 kcal (40 g carbohydrates, 6.1 g fat, and 8.8 g
                protein) to ensure complete filling of hepatic glycogen stores as a snack before
                bedtime. This snack is included in the total calories stated above.

             2. The participant will have his/her height and weight measured using the electronic
                weight scale and stadiometer within the Clinical Research Unit. BMI will be
                calculated from height and weight using the formula: [mass(kg)/[height(meters)2].
                Blood pressure will be measured and recorded.

             3. The participant will complete a medical history questionnaire, which includes
                obtaining a list of current medications. Completion of the questionnaire may occur
                following the baseline blood draw and meal administration

        2. A catheter will be inserted into the other antecubital vein to obtain blood samples.

        3. 8 AM: Metabolism Study: Baseline Period: Baseline blood and breath samples to determine
           background enrichment of isotopes. Then the subject will then consume a liquid test meal
           prepared by CRU nutrition staff consisting of 27 g Sol Carb, 1032 g Boost Plus, 3.2 g
           canola oil, 0.2 g lecithin, 5 mg/kg of [1,2, 3, 7, 8-13C8] glyceryl trioleate, and 40 g
           of water. Participants will be asked to consume the liquid meal within 20 minutes for
           consistency, with the sides of the container scraped to ensure maximal ingestion.
           Additionally, 30 minutes after consuming the meal a 75 micromol/kg bolus infusion of
           (1,1,2,3,3-2H5)glycerol will be initiated to track VLDL catabolism. Blood and breath
           samples will be collected at 0, 15. 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360
           minutes post-meal ingestion. Indirect calorimetry will also be administered using the
           ventilated hood technique for 10 minutes prior to each breath sample collection to
           determine resting metabolic rate for tracer calculations and measurement of total fatty
           acid oxidation. Blood samples will be analyzed for tracer enrichment and total
           concentration of free fatty acids, triglycerides, and cholesterol.

        4. 2 pm: Lunch will be served by CRU, prepared by CRU kitchen. End of Study Visit.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Participants will be recruited from the Washington University School of Medicine IBD
        Center, Washington University's Volunteers for Health, the Center for Community Based
        Research, and IBD clinics affiliated with Washington University in the surrounding St.
        Louis community.
      
Biospecimen description: 
      Stool, Blood