Effect of SB CD on Chylomicron
Effect of SB CD on Chylomicron
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.
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.
Inclusion criteria (experimental group):

 1. Age 18-80 year's old.

 2. Diagnosed with Crohn's disease.

 3. Active small bowel Crohn's disease on MR enterography or CT enterography confirmed
 within the prior 30 days.

 4. Inflammatory markers (eg.,. CRP, ESR) elevated above the upper limit of normal.

 5. Ability to provide informed consent.

 Inclusion criteria (control group):

 1. Age 18-80 years old.

 2. Ability to provide informed consent.

 3. No diagnosis of inflammatory bowel disease.

 Inclusion criteria (inflammatory arthritis or skin inflammation group):

 1. Age 18-80 years old.

 2. Ability to provide informed consent.

 3. Taking a biologic targeting TNF (infliximab, adalimumab) for at least 4 weeks.

 Exclusion Criteria

 1. A history of diabetes mellitus.

 2. A history of hyperlipidemia.

 3. A history of thyroid dysfunction.

 4. A history of hepatic, cardiac, renal or endocrine dysfunction.

 5. A history of cancer requiring systemic chemotherapy

 6. Prior radiation therapy to pelvis or abdomen

 7. Taking medications (current or past 30 days) known to alter plasma lipid levels
 including statins, corticosteroids (oral, IV or topical), bile acid sequestrants,
 thyroid drugs, or fibrates.

 8. Currently pregnant or trying to become pregnant.

 9. History of abdominal or pelvic surgery.

 10. Complicated small bowel CD with obstruction, penetrating disease and/or abscess.

 11. Recent surgery of any type within the last 30 days that, in the discretion of the
 principal investigator, might affect the study or pose risk to the subject

 Exclusion for control group: all of the above and no recent participation in other studies
 as a control.