Functional Research of Emulsifiers in Humans
Phase: NA
Conditions: Healthy
Sponsor: University of Pennsylvania

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University of Pennsylvania 
 
 
 
 
 
 
 
Functional Research on Emulsifiers in Humans (FRESH) 
 
 
Regulatory Sponsor: 
James D. Lewis, MD, MSCE 
Department of Medicine and Department of 
Biostatistics and Epidemiology 
720 Blockley Hall 
215-573-5137 
Funding Sponsor: 
National Institutes of Health  
Study Product: 
Carboxymethylcellulose 
Protocol Number: 
828422 
NCT # 
03440229 
IND Number: 
Exempt 
 
 
 
 
 
Initial Date: 
October 19, 2017 
Amended: 
November 21, 2017 
December 7, 2017 
January 19, 2018 
March 2, 2018 
April 6, 2018 
May 4, 2018 
June 2, 2018 
June 14, 2018 
July 23, 2018 
July 31, 2018 
December 4, 2018 
May 14, 2019 
Administrative 
Change: 
 
 
 
CONFIDENTIAL 
 
This document is confidential and the property of the University of Pennsylvania.  
No part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 
 
 

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Table of Contents 
Study Summary .................................................................................................... 7 
1 
Introduction ..................................................................................................... 8 
1.1 
Background .............................................................................................. 8 
1.2 
Investigational Agent ................................................................................ 9 
1.3 
Preclinical Data ........................................................................................ 9 
1.3.1 Risk-Benefit Balance ........................................................................ 10 
2 
Study Objectives ........................................................................................... 10 
3 
Study Design ................................................................................................ 11 
3.1 
General Design ...................................................................................... 11 
3.2 
Primary Study Endpoints ........................................................................ 11 
4 
Participant Selection and Withdrawal ............................................................ 11 
4.1 
Inclusion Criteria ..................................................................................... 11 
4.2 
Exclusion Criteria ................................................................................... 12 
4.3 
Participant Recruitment and Screening .................................................. 13 
4.4 
Early Withdrawal of Participants ............................................................. 14 
4.4.1 When and How to Withdraw Participants ......................................... 14 
5 
Study Intervention ......................................................................................... 14 
5.1 
Randomization procedures .................................................................... 14 
5.2 
Prior and Concomitant Therapy .............................................................. 14 
5.3 
Supervision of access to food outside of the study protocol ................... 15 
5.4 
Activity Level .......................................................................................... 15 
6 
Study Procedures ......................................................................................... 15 
6.1 
Sample collection methods for metabolic and microbial profiling ........... 15 
6.1.1 Urine sample collection and storage ................................................ 15 
6.1.2 Blood sample collection and storage ................................................ 15 
6.1.3 Stool sample collection .................................................................... 16 
6.1.4 Sigmoidoscopy to obtain mucosal biopsy samples .......................... 16 
6.1.5 Food Satiety Questionnaire .............................................................. 17 
6.1.6 Oral Glucose Tolerance Test ........................................................... 17 
6.1.7 PROMIS scale…………………………………………………………......17 
6.2 
Sequence of study activities ................................................................... 18 
6.2.1 Pre-screening and Informed Consent .............................................. 19 
6.2.2 Screening (Day 0/Visit 1).................................................................. 19 
    6.2.2.1 Physical Examination .................................................................. 20 

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    6.2.2.2 Laboratory Studies ...................................................................... 20 
    6.2.2.3 Dietitian Evaluation ...................................................................... 20 
    6.2.2.4 Three Day Dietary Recall ............................................................ 20 
6.2.3 Post Screening (Visit 2) .................................................................... 20 
    6.2.3.1 Fitbit Distribution .......................................................................... 20 
    6.2.3.2 Stool Sample Collection .............................................................. 20 
    6.2.3.3 Emulifier Free Diet ....................................................................... 21 
6.2.4 Day 1 of Inpatient Procedures .......................................................... 21 
    6.2.4.1 History and Physical Examination ............................................... 21 
    6.2.4.2 Adverse Events ........................................................................... 21 
    6.2.4.3 Urine pregnancy Test .................................................................. 21 
    6.2.4.4 Concomitant Medications ............................................................ 21 
    6.2.4.5 Oral Glucose Tolerance Test ....................................................... 21 
    6.2.4.6 Sample Collection ........................................................................ 22 
    6.2.4.7 Flexible Sigmoidoscopy ............................................................... 22 
    6.2.4.8 Food Frequency Questionnaire ................................................... 22 
    6.2.4.9 Three Day Dietary Recall ............................................................ 22 
    6.2.4.10 Recording of Diet ....................................................................... 22 
    6.2.4.11 Timing of meals ......................................................................... 22 
    6.2.4.12 Activity Level Maintenace .......................................................... 23 
    6.2.4.13 PROMIS Scale .......................................................................... 23 
6.2.5 Day 2-10 of Inpatient Procedures ..................................................... 23 
    6.2.5.1 Vital Signs and Weight ................................................................ 23 
    6.2.5.2 Adverse Events ........................................................................... 23 
    6.2.5.3 Concomitant Medications ............................................................ 23 
    6.2.5.4 Sample Collection ........................................................................ 23 
    6.2.5.5 Recording of diet ......................................................................... 23 
    6.2.5.6 Timing of Meals ........................................................................... 23 
    6.2.5.7 Satiety Questionnaire .................................................................. 24 
    6.2.5.8 Activity Level Maintenance .......................................................... 24 
6.2.6 Day 11 of Inpatient Procedures ........................................................ 24 
    6.2.6.1 Vital Signs and Weight ................................................................ 24 
    6.2.6.2 Adverse Events ........................................................................... 24 
    6.2.6.3 Concomitant Medications ............................................................ 24 

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    6.2.6.4 Oral Glucose Tolerance Test ....................................................... 24 
    6.2.6.5 Sample Collection ........................................................................ 24 
    6.2.6.6 Flexible Sigmoidoscopy ............................................................... 25 
    6.2.6.7 Recording of diet ......................................................................... 25 
    6.2.6.8 Timing of Meals ........................................................................... 25 
    6.2.6.9 Activity Level Maintenance .......................................................... 25 
    6.2.6.10 PROMIS Scale .......................................................................... 25 
    6.2.6.11 Discharge .................................................................................. 25 
6.3 
Post-study follow-up ............................................................................... 25 
    6.3.1 Adverse Event Follow Up Phone Call ............................................. 25 
    6.3.1 One Month Follow-Up Sample Collection ....................................... 26 
    6.3.1 Three Month Follow-Up Sample Collection .................................... 26 
6.4 Sample preparation, processing, and storage at the University of 
Pennsylvania ........................................................................................... 27 
6.4.1 Aliquoting and processing of samples for archiving ......................... 27 
6.4.2 Assay for CMC ................................................................................. 27 
6.4.3 Microbiome Characterization ............................................................ 27 
6.4.4 Metabolic profiling based on plasma samples .................................. 28 
6.4.5 Long term sample storage and reuse of samples ............................ 28 
7 
Statistical Plan .............................................................................................. 28 
7.1 
Sample Size Determination .................................................................... 28 
7.2 
Overview of Statistical Methods ............................................................. 29 
7.2.1 Determining the effect of diet on fecal and mucosal microbiome 
composition………….. ...................................................................... 29 
7.2.2 Comparison of metabolites between diets ....................................... 29 
7.2.3 Comparison of metabolites before and after antibiotics ................... 30 
7.2.4 Comparison of SCFAs and triglycerides in the fecal matter between 
the study groups ............................................................................... 30 
8 
Safety and Adverse Events ........................................................................... 30 
8.1 
Recording of Adverse Events ................................................................. 32 
8.2 
Reporting of Serious Adverse Events ..................................................... 32 
8.2.1 Study Sponsor Notification by Investigator ....................................... 32 
8.2.2 Ethics Committee/Internal Review Board Notification by 
Investigator…………….. ................................................................... 33 
9 
Data Handling and Record Keeping .............................................................. 33 

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9.1 
Confidentiality ......................................................................................... 33 
9.2 
Source Documents ................................................................................. 34 
9.3 
Case Report Forms ................................................................................ 34 
9.4 
Records Retention .................................................................................. 35 
10 
Study Monitoring, Auditing, and Inspecting ................................................ 35 
11 
Ethical Considerations ............................................................................... 35 
12 
Study Finances .......................................................................................... 36 
12.1 
Funding Source ................................................................................... 36 
12.2 
Conflict of Interest ............................................................................... 36 
12.3 
Participant Stipends or Payments ....................................................... 36 
13 
Publication and Data Sharing Plan ............................................................ 36 
14 
References ................................................................................................ 36 
15 
Attachments ............................................................................................... 40 
16 
Protocol Signature Page ............................................................................ 41 
 
 
 
 
 

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List of Abbreviations 
 
 
CRF – case report form 
CHPS - Center for Human Phenomic Science (CHPS) 
IRB – Internal Review Board 
EC – Ethics Committee 
CMC – carboxymethylcellulose 
 
 
 

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Study Summary 
Title 
Functional Research on Emulsifiers in Humans 
Short Title 
FRESH Study 
Protocol Number 
828422 
Phase 
Not applicable 
Methodology 
Controlled feeding experiment 
Study Duration 
161 days 
Study Center(s) 
1 
Objectives 
Establish a tractable and physiologic means of administering and 
measuring CMC consumption and its metabolic impact in healthy 
volunteers. 
 
Examine extent to which CMC consumption impacts human gut 
microbiota composition, gene expression, and/or localization. 
 
Explore effects of CMC consumption on a range of inflammatory 
and metabolic parameters that characterize metabolic 
syndrome. 
Number of 
Participants 
20 
Diagnosis and 
Main Inclusion 
Criteria 
Healthy volunteers 
Study Product, 
Dose, Route, 
Regimen 
Participants will be randomly assigned to receive 0 or 15 gm per 
day of carboxymethylcellulose in 6 divided doses baked in 
chocolate brownies and served in sorbet   
Duration of 
administration 
11 days 
Reference therapy 
None 
Statistical 
Methodology 
Determination of the effect of CMC consumption on the fecal 
and mucosal microbiome composition and inflammatory and 
metabolic parameters. Comparison of microbial metabolites 
across diets (with CMC versus without CMC). 
 
 
 

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1 Introduction 
This document is a protocol for a human research study. This study is to be 
conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable 
government regulations and Institutional research policies and procedures.  
1.1 Background 
The human intestine is home to some 100 trillion microorganisms.  The density of 
bacterial cells in the colon has been estimated at 1011 to 1012 per ml, which makes the 
colon one of the most densely populated microbial habitats known on earth.  The 
genome size of this pool of intestinal microbes is estimated to exceed the size of the 
human nuclear genome by two orders of magnitude1. There are far more total cells in 
the gut microbial population than human cells in our bodies--indeed, it has been stated 
that "there are more of them in us than us"2 1, 3.   
The great majority of these gut bacterial species have not been cultured outside 
the human host, so most species are little studied.  Recent advances in scientific 
methods have allowed for new surveys of the intestinal microbiota using DNA 
sequencing of uncultured communities1, 4, 5 or microarray-based methods6.   Many of 
these studies used 16S ribosomal DNA sequences to query the different bacterial taxa, 
since 16S sequences are similar enough to allow amplification of most bacterial species 
with single primer pairs, but diverse enough to allow  "finger printing" of taxa to relatively 
fine taxonomic levels7.  Such studies have revealed that major bacterial phyla in the 
human gut include Bacteroidetes, Firmicutes, Proteobacteria and various minor groups. 
Other recent studies extended this analytical approach by sequencing fragments of bulk 
genomic DNA from intestinal microbiota instead of 16S DNA3, 8-11.   Metagenomic 
analysis allows inferences to be made about the community taxonomy and the 
metabolic pathways in the intestinal microbiota inferred from the genes present.  For 
example, the Gill et al. study10 disclosed that the intestinal microbiota contribute unique 
pathways for metabolism of glycans, amino acids, xenobiotics, methanogenesis, 
vitamins, isoprenoids, and other compounds.   
When maintained in a stable manner, at an appropriately safe distance from gut 
epithelial cells, the microbiota provides benefits to the host, especially in terms of 
energy harvest, pathogen exclusion, and promotion of immune development. In 
contrast, disturbance of the microbiota-host relationship can drive chronic gut 
inflammation that can promote an array of chronic inflammatory diseases including 
metabolic syndrome.  While host genetics are an important determinant of the host-
microbiota relationship, the dramatic increase in incidence of numerous chronic 
inflammatory diseases, including inflammatory bowel disease (IBD) and metabolic 
syndrome over the last 60 years amidst relatively constant genetics, and highlights the 
importance of understanding how non-genetic factors such as diet might alter the 
microbiota-host relationship to promote gut inflammation.  Consequently, we and others 
have focused on how differences in macronutrient consumption influence the 
microbiota. However, dietary patterns can also be characterized by constituents other 
than macro and micronutrients. Post-mid-20th century dietary changes include 
consumption of an array of synthetic compounds that are added to many processed 
foods. The biologic effect of these food additives are not well described, particularly 

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their potential to influence microbiota composition and/or its interaction with the host.  
One particularly disconcerting class of compounds are synthetic dietary emulsifiers 
including polysorbate 80 (P80)and carboxymethylcellulose (CMC). These detergent-like 
compounds, which are incorporated into a variety of processed foods, are not well-
absorbed, thus allowing them to interact with the microbiota and gut mucosa. We have 
recently shown that, in mice, consumption of P80 and CMC, alters microbiota 
composition, have pro-inflammatory potential and promote microbiota encroachment 
into the colonic mucosa, low-grade inflammation, and metabolic syndrome¹. However, it 
is not known whether these compounds have similar effects in humans.  Addressing this 
critically important question is the central goal of this study. Our initial focus will be on 
CMC, which, despite not having been tested in humans and not widely studied in 
animals, has "generally regarded as safe (GRAS)" status from the US FDA, allowing it 
to be widely and unrestrictedly used in a broad variety of processed foods.  
1.2 Investigational Agent 
CMC will be purchased from Modernist Pantry who supplies CMC to small 
bakeries throughout the United States.  
1.3 Preclinical Data 
This proposal seeks to address the post-mid-20th century increased incidence of 
chronic inflammatory diseases of the intestine. Crohn’s disease and ulcerative colitis, 
collectively referred to as the inflammatory bowel diseases (IBD), are debilitating diseases 
that may afflict up to 3 million Americans 12. Like many immune-mediated diseases, the 
incidence of IBD is increasing in the US and elsewhere at rates far in excess of that 
expected by genetic drift. A milder but much more common form of gut inflammation 
referred to as "low-grade" inflammation, is defined by modestly elevated pro-inflammatory 
gene expression amidst a lack of histopathologically-evident inflammation 13. Such low-
grade inflammation associates with, and may promote, metabolic syndrome, which is a 
group of interrelated metabolic disorders including obesity, insulin resistance, 
hyperglycemia, hyperlipidemia, and hepatic steatosis 14. Humanity is facing an epidemic 
of metabolic syndrome, and its downstream consequences, including type 2 diabetes, 
cardiovascular disease, and liver dysfunction that threaten to overwhelm the world's 
healthcare systems and economies thus making it a top public health priority in dire need 
of investigation. Our research suggests that IBD, metabolic syndrome, and other chronic 
inflammatory diseases may share common underlying causes, including a disturbed host-
microbiota relationship.  In accord with this notion, recent studies demonstrate that 
infections in infants increase likelihood of developing both IBD 15 and obesity 16.  
Thus, better understanding of factors that can alter the host-microbiota relationship 
is of paramount importance. Many candidate factors can be reasonably envisaged to 
have impacted the microbiota (e.g. antibiotics, polymers such as BPA, pollutants, 
enhanced sanitation, etc.). Recent studies suggest that some commonly used dietary 
emulsifiers, which are detergent-like molecules that are incorporated into most processed 
foods to improve texture and stability, may be one specific contributor to the increased 
prevalence of chronic inflammatory diseases 17, 18.  We reported that feeding mice CMC 
and P80 results in a microbiota that encroaches upon the epithelium and has inherently 
greater potential to activate host pro-inflammatory signaling 19. Such alterations promote 

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chronic low-grade inflammation and a metabolic syndrome-like phenotype in mice and 
increased incidence of severe colitis in mice that are genetically pre-disposed to this 
disorder.  Investigation of these commonly consumed but poorly studied synthetic 
compounds to determine their impact on human biology, in a manner analogous to our 
mouse studies, is of the utmost significance to human health. 
1.3.1 Risk-Benefit Balance 
The risks associated with participating in this controlled feeding experiment are 
low. The diets will be constructed to meet all nutritional requirements. The CMC will be 
purchased from Modernist Pantry and is the same as that which is sold to small 
bakeries throughout the United States. Thus, consumption of the study diet entails no 
greater risk than consumption of a typical American diet outside of the study 
procedures.  
Participants will undergo phlebotomy and sigmoidoscopy with biopsy. There is a 
small potential risk for complications of sigmoidoscopy, including bleeding and 
perforation. The risks of these complications are estimated to be less than 1% for 
bleeding and less than 0.1% for perforation. Patients with contraindications to biopsy 
will be excluded. The risks of phlebotomy include bleeding, infection, phlebitis, and 
fainting. As with all research, there is a small risk of loss of confidentiality. Participants 
may also experience some discomfort associated with eating a limited diet for 14 days. 
Participants may also feel tired and/or discomfort from staying in the hospital for the 11 
day inpatient stay. 
In summary, the risk of serious or severe adverse events is likely extremely low. 
While there is no direct benefit expected for the participants, the balance of the 
participants’ risk vs. the knowledge to be gained is believed to be favorable. 
2 Study Objectives 
The specific aims for our proposal are:  
 
1. Establish a tractable and physiologic means of administering and measuring 
CMC consumption and its metabolic impact in healthy volunteers. 
 
2. Examine extent to which CMC consumption impacts human gut microbiota 
composition, gene expression, and/or localization. 
 
3. Explore effects of CMC consumption on a range of inflammatory and 
metabolic parameters that characterize metabolic syndrome. 
 
 
 
 
 
 
 

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3 Study Design 
3.1 General Design 
This is a randomized 
double-blind, controlled 
feeding experiment 
examining the effect of CMC 
on the stool microbiota 
composition, gene 
expression, and microbiota 
localization with respect to the epithelium. In addition, we will assess the impact of CMC 
exposure in the diet on markers of metabolic syndrome including response to oral 
glucose tolerance testing and other plasma metabolite measurements.  
Healthy volunteers will be recruited for a 11-day in-patient study. All participants 
will receive 3 full days of freshly prepared emulsifier-free food to begin the study. This 
will be called the “washout period”. Participants will then be randomized into an 
“emulsifier-free” arm and a “CMC” arm.  
Both arms will continue the same diet except that the “CMC” arm’s brownie and 
sorbet will have CMC added to achieve a level of 15 gm per person per day 
consumption, which are the levels of emulsifier consumption that can readily be 
achieved by humans who eat a large amount of processed foods. If the average 
American eats 2kg of processed food per day containing 2% emulsifier (the limit of 
individual emulsifiers), this would amount to 40g of emulsifiers per day. Serial collection 
of fecal, blood, urine and mucosal biopsy samples will define our ability to reliably 
quantitate CMC consumption by assay of biological fluids and preliminarily investigate 
the extent to which CMC consumption by humans impacts the gut microbiota and its 
interaction with the host parameters related to inflammation and metabolism. 
3.2 Primary Study Endpoints 
This is not an efficacy study. There are no pre-specified efficacy or safety 
endpoints for this study. Rather, we will use an unbiased approach to determine the 
composition of the microbiota and metabolome. 
4 Participant Selection and Withdrawal 
4.1 Inclusion Criteria 
1. Participant is capable of giving informed consent  
2. Participant is age 18 to 60 years 
4.2 Exclusion Criteria 
1. Diagnosis with IBD, celiac disease, or other chronic intestinal disorders. Since we 
are interested in assessing the impact of diet on the microbiome in the absence 
of pathologic inflammation, we will exclude participants with chronic intestinal 
abnormalities.  

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2. Baseline bowel frequency less than every 2 days or greater than 3 times daily. 
Normal bowel frequency is every 3rd day to 3 times per day.20-23 Although 
unknown, stool frequency could be related to the microbiome composition.24-26 
Furthermore, change in diet could alter baseline stool frequency, potentially 
causing diarrhea, particularly in those with high baseline stool frequency, or 
severe constipation in those with low stool frequency. To avoid the need for use 
of antidiarrheal medications or laxatives, which themselves could alter the 
microbiome composition, these patients will be excluded.  
3. Current smoker. What effect smoking has on the microbiome of the gut is 
unknown. Furthermore, because our hospital is a smoke-free environment, 
volunteers will not be able to smoke. Thus, inclusion of smokers would increase 
the risk of early withdrawal from the study.  
4. Body Mass Index (BMI) <18.5 or >40 at screening. Volunteers with BMI below 
normal27 will be excluded to prevent inclusion of participants with a subclinical 
systemic disease that may influence the gut microbiome. Volunteers with severe 
obesity will be excluded as obesity may be associated with altered gut 
microbiome composition. 
5. More than two of the criteria for metabolic syndrome: 
o A waist circumference greater than 35 inches (89 centimeters) for women 
and 40 inches (102 centimeters) for men at screening. 
o A diagnosis of diabetes mellitus or baseline HbA1c > 6.4% or a fasting 
glucose level of greater than 100mg/dL  
o Systolic blood pressure >130 mmHg or diastolic blood pressure >85 
mmHg or treated with medications for hypertension at screening. 
o Fasting triglycerides >149 mg/dl or treated with medications for 
hypertriglyceridemia 
o Fasting HDL cholesterol <40 mg/dl in men or <50 mg/dl in women or 
treated with medications for hypercholesterolemia 
6. Known substance abuse disorder or consumption of illicit drugs or alcohol in the 
24 hours prior to admission to the Center for Human Phenomic Science (CHPS). 
7. Prior bowel resection surgery other than appendectomy. It is unknown how prior 
bowel resection surgery may influence the microbiome composition, hence we 
will exclude these participants. 
8. Contraindication to flexible sigmoidoscopy and biopsies. Patients with 
suppressed white blood count may be at increased risk of systemic infection 
following sigmoidoscopy with biopsies. As such, participants with a WBC less 
than 3,500 or an absolute neutrophil count of less than 1,000 will be excluded. 
Patients with thrombocytopenia or with a coagulopathy may be at increased risk 
of bleeding complications after colonoscopic biopsies. As such, patients with a 
platelet count of less than 100,000 or an INR greater than 1.2 will be excluded 
from the study. 
9. Estimated GFR<60ml/min/1.73m2 based on measured serum creatinine 
concentration  
10. Pregnant and lactating women. To avoid any risk to an unborn fetus or new born 
baby from changing the mother’s diet, pregnant and lactating women will be 
excluded.  

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11. Use of antibiotics in the 6 months prior to Visit 2. A small proportion of bacteria 
may require 6 months to recover after treatment with antibiotics.28 
12. Use of antacids, NSAIDs, or dietary supplements in the week prior to Visit 2. 
NSAIDs have been associated with C. difficile colitis, although whether this is 
causative and whether this is mediated through changing the fecal microbiota 
composition is unknown.29 Antacids could potentially alter the gut microbiota by 
changing the acid milieu or by altering fecal transit time. For our study purposes, 
multivitamins will not be considered dietary supplements.  
13. Use of laxatives or anti-diarrhea medications in the two weeks prior to Visit 2. 
14. Use of anticholinergics in the week prior Visit 2. 
15. Use of narcotics in the week prior to Visit 2. 
16. HIV infection, AIDS, or other known conditions resulting in immunosuppression -  
we will determine this by direct participant query; no formal testing will be done. 
17. Allergies or intolerance to the components of the study diets. 
18. Participant has experienced diarrhea within the two weeks prior to Visit 2. 
Diarrhea is defined as a change in bowel habits with an increased frequency or 
loose stools such that the stool could not be lifted with a fork. 
19. Refusal to use a medically accepted method of birth control while participating in 
this study, such as a barrier method, hormonal contraceptives, implanted birth 
control devices, permanent methods (such as a vasectomy), and/or abstinence.  
20. Vegans and Vegetarians. 
21. Student or employee of any one of the investigators. 
22. Anyone who cannot receive study payment (ie: visa) 
23. Any condition that the investigator feels may limit the volunteer’s ability to 
complete the study protocol.  
4.3 Participant Recruitment and Screening 
Our goal is to have 16 participants complete this study; however, it is anticipated 
we may need to screen up to 50 participants to meet our target enrollment. 
Participants will be recruited through local and web-based advertising. We may 
also contact participants who have been enrolled in other studies under the same PI in 
the past if they have consented to future contact. Other participants may contact the 
research coordinator by phone or email in response to local paper flyers or web based 
advertisements (i.e.: craigslist). All advertisements will be approved by the local IRB 
before being used.    
Potential participants will be pre-screened via telephone for eligibility prior to the 
screening visit. At the screening visit, potential participants will be screened for eligibility 
before being enrolled in the study. Potential participants will be directly queried 
regarding exclusion criteria. If there are questions regarding eligibility, relevant 
information from the participant’s medical record will be obtained, with appropriate 
permission for release of records as necessary to comply with HIPAA. In the absence of 
any concerns, the participant’s medical records will not be requested or reviewed as 
part of this study.  

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4.4 Early Withdrawal of Participants 
4.4.1 When and How to Withdraw Participants 
Participants may be withdrawn from the study at any time if the investigator or 
the participant believes that the participant’s safety will be jeopardized by continued 
participation, or if the participant does not follow study procedures. Likewise, 
participants will be withdrawn from the study if they withdraw consent.  
5 Study Intervention  
All foods will be prepared within the CHPS’s metabolic kitchen without emulsifiers 
(unless specifically added). All participants will follow the same Western style diet (i.e. 
the only difference being portion size). The macronutrient percentages of calories for 
the study diet will be 55% carbohydrate, 30% fat, and 15% protein. The diet will be 
composed of two menus that will be consumed on alternating days. Water, coffee and 
tea will be provided as desired. Participants will have access to additional food outside 
of the meals provided, however, the entire serving of the previous meal must have been 
consumed.  The additional food provided outside of the scheduled meals will consist of 
the food from the same Western style diet provided for each meal. 
For the three days prior to admission, participants will be eating an emulsifier free 
diet at home.  Food will be provided by the CHPS metabolic kitchen. After admission to 
CHPS, no emulsifiers will be consumed prior to dinner on Day 1. Thus, accounting for 
the 8 hours of nil per os status prior to admission, all participants will have 
approximately 80 hours of emulsifier free washout time prior to administration of the 
food containing CMC.  
Participants will be randomly assigned to receive 0 or 15 gm per day of CMC (8 
participants in each arm of the study). Beginning with the dinner meal on Day 1, all 
participants will consume one serving of brownie or sorbet (15 gm) per day that contain 
0 or 2.5 gm CMC per serving. The brownie and sorbet servings will be provided at all 
three scheduled meals and scheduled snacks. Prior to eating any other food on the 
study menu, participants must consume the brownie and sorbet servings. 
5.1 Randomization procedures 
The double-blind randomization order will be generated by Dr. Hongzhe Li. 
Because of the small sample size, we will utilize block randomization with a block size 
of 4 participants.  
5.2 Prior and Concomitant Therapy 
Participants will be allowed to take their usual outpatient medications. In order to 
facilitate this, participants will bring these medications with them to the CHPS on the 
day of admission and they will be administered under the supervision of the nursing 
staff during the course of their in-patient stay. Participants will be allowed to take 
acetaminophen as needed for minor ailments, such as a headache. Consumption of 
concomitant medications will be recorded in the study records daily.  Participants will 
not be permitted to take any medications listed in the exclusion criteria during their 
participation in the study, prior to discharge from CHPS.   

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5.3 Supervision of access to food outside of the study protocol 
In order to prevent access to food outside of the study protocol, participants will 
be accompanied by a member of the research staff whenever they leave the CHPS. 
When sufficient staff is available to supervise the participant, participants will be allowed 
to walk outside.  
5.4 Activity Level  
Once a participant is determined to be eligible, they will be given a Fitbit Flex. 
Participants will be required to wear this Fitbit Flex for at least 3 days prior to admission 
to CHPS to record the number of steps they take daily.  The study team will calculate 
the average number of daily steps the participant took during the 3 days prior to 
admission. During the inpatient stay, participants will be required to attain within 10% of 
the average number of daily steps that they took in the 3 days prior to admission.  To 
achieve this, participants will be escorted on walks outside of the inpatient unit by a 
coordinator, as needed. 
6 Study Procedures  
The sequence of study procedures is summarized in Section 6.2. The study 
procedures section is divided into two parts, one describing the collection methods of 
samples for metabolic and microbial profiling and a second describing the sequence of 
events.  
6.1 Sample collection methods for metabolic and microbial profiling 
6.1.1 Urine sample collection and storage 
There is increasing evidence that the composition of the gut microbiome 
influences the metabolic profile of blood, urine, and other tissues.30-32 Urine will 
collected at the post-screening visit and during the inpatient stay. During the post-
screening visit, urine will be collected following an overnight fast. During the inpatient 
stay, urine samples will be collected daily prior to 09:00h after an overnight fast. Up to 
15 mL of urine will be obtained for each sample, aliquoted into three 5ml aliquots, and 
frozen at minus 80°C for use in metabolomic studies. 
6.1.2 Blood sample collection and storage 
Blood will be collected after an overnight fast prior to breakfast at the screening 
visit, at the post-screening visit, and on Days 1-4, 8, 10, and 11 of the inpatient study. 
Approximately 16cc of blood will be collected for metabolomics at each collection. 
Plasma will be separated from the blood samples. The plasma will be stored frozen at 
minus 80°C for use in metabolomic studies. Peripheral blood monocytes (PBMCs) will 
also be collected from the same samples and stored at minus 80°C for potential future 
use in cell phenotyping studies.  
Participants may be contacted and asked to return 1 month after discharge for a 
follow-up blood collection. 

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6.1.3 Stool sample collection 
Stool will be collected from the inpatient participants daily.  Only the first sample 
of each day will be aliquoted and frozen.  Subsequent samples (if available) will be 
weighed and the weight will be recorded.  Each stool specimen that is collected will be 
processed  in the same manner. The participant will be provided with a collection device 
to be placed in the commode that allows for separation of urine and feces, and keeps 
the feces separated from the toilet water. After they have finished voiding their first stool 
of the day, the specimen will be immediately processed according to the following 
protocol by the CHPS nurses.  Using vials with small spoons attached to the lid, a small 
amount of stool will be scooped on to the spoon from the surface of the stool sample 
and leveled off with a tongue depressor.  The weight of the sample will be recorded.  A 
minimum of seven samples will be obtained from one stool collection. All samples will 
be labeled with the subject ID #, and the date of collection from the volunteer. The core 
sample will be labeled the same way.  The aliquoted samples and the core sample will 
be immediately frozen at minus 80°C.  The nurses will rate the sample based on the 
Bristol Stool Chart.  Residual stool samples will be stored into up to five 15ml coring 
vials. Subsequent stools will be weighed in the collection device, rated on the Bristol 
Stool Chart, and then discarded. 
In order to have a baseline stool sample prior to study diet,  participants will be 
required to collect a stool sample prior to the post-screening visit.  Participants will also 
collect a stool sample on the day prior to admission to CHPS.  Study staff will provide 
the participant with collection materials and instructions for collecting the stool at home 
and bringing it in to study staff.  Immediately after voiding the sample, the participant will 
put a small amount of stool into a spoon-top vial containing ethanol.  They will label this 
vial and the collection device containing the remainder of the stool sample with the date 
of collection, and will rate the stool on the Bristol Stool Chart.  They will then arrange a 
drop-off time with the study coordinator within 48 hours of sample collection.  The study 
coordinator will bring this stool sample to the study lab on the 9th floor Biomedical 
Research Building (BRB). Lab personnel will aliquot the stool sample into a minimum of 
7 spoon-top vials.   
Participants may be asked to provide a stool sample 1 month after discharge. In 
addition, follow-up stool samples (which may be collected every 3 months for up to 5 
years after the participant’s last study day) may be collected and will be processed in 
the same manner as the inpatient samples. The collection kit may be shipped to the 
participant’s home and the participant will either return the stool sample to study staff or 
ship it to Dr. Gary Wu’s lab at the University of Pennsylvania.  
6.1.4 Sigmoidoscopy to obtain mucosal biopsy samples 
On days 1 and 11, each participant will undergo a sigmoidoscopy to obtain 
biopsies from the area of approximately 15 cm from the anal verge, which correlates 
with approximately the rectosigmoid junction. No bowel preparation will be utilized prior 
to the sigmoidoscopy since it is unknown what affect the bowel preparation may have 
on the fecal microbiome or the mucosally adherent microbiome. No sedation 
medications will be administered.  
16 biopsies obtained from the distal 20 cm of the colon will be collected; 4 placed 
in Carnoy solution for non-denaturing confocal microscopy, 4 flash frozen in liquid 

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nitrogen, 4 placed in RNAlater, and 4 will be kept fresh for immediate cell isolation 
studies. Additional biopsies may be taken (up to a total of 20), to replace 
lost/dropped/unusable biopsies (i.e., if the biopsy is too small), for quality control and/or 
for methods testing.  
6.1.5 Rectal Swabs  
On Day 1, prior to the flexible sigmoidoscopy, participants will undergo a rectal 
swab for assessment of the mucosally-associated gut microbiota.   
Rectal swabs will be obtained during the physical examination by the CHPS 
nurse practitioner or study investigator. Four total swabs will be collected per 
participant. Three (3) sterile swabs will be inserted 3 cm into the rectum, turned 360 
degrees, removed, placed into a sterile tube, and frozen at -80 degrees celcius until 
analysis. One of the swabs will be delivered immediately to Dr. Wu’s laboratory for 
anaerobic culture. Finally, one (1) swab will be waved in the air and collected as a 
control. 
6.1.6 Food Satiety questionnaire 
On days 2, 3, 5, 6, 9, and 10, following lunch, participants will complete a 
standard food satiety questionnaire. A standardized 150mm VAS approach to 
measuring satiety and hunger as described by Hill and Blundell and modified by Doucet 
will be employed. 81, 82 The following questions will be asked at 12:15h before lunch is 
served.  Participants will be given their meal at 12:30h and will finish eating their meal 
by 13:00h.  They will complete the questionnaire again at 13:00h. The questions should 
be asked in the participant’s room with all lights on and with no food in the room.  
 
Questions to be asked are as follows. 
1) How strong is your desire to eat (very weak to very strong)?  
2) How hungry do you feel (not hungry at all to as hungry as I have ever felt)? 
3) How full do you feel (not full at all to very full)?  
4) How much food do you think you could eat (nothing at all to a large amount)? 
5) How preoccupied are you with thoughts of food (no thoughts to very 
preoccupied? 
6) What is your urge to eat (no urge to strong urge)? 
6.1.7 Oral glucose tolerance test 
On day 1 and 11, participants will complete a 2.5 hour oral glucose tolerance 
test. This test will be completed prior to breakfast following a 12 hour fast. At 08:00h, a 
catheter will be placed in an antecubital or forearm vein for blood sampling, with the arm 
placed in a heating pad to promote arterialization of venous blood.  After 20 min of 
acclimatization to the catheter placement, baseline blood samples will be taken at -5 
and -1 min prior to the ingestion of 75 grams anhydrous oral glucose over a 5 min 
period starting at t = 0 at ~08:30h.  Subsequent blood samples will be collected at t = 
15, 30, 60, 90, and 120 min post-ingestion.  All samples will be collected on ice into 
chilled EDTA tubes with protease inhibitor cocktail containing DPP4-inhibitor added 
immediately, centrifuged at 4 degrees C, aliquoted in ice, and stored at minus 80 
degrees C until biochemical analysis. A total of 4 ml of blood will be collected at each 

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test. Glucose, free fatty acids, insulin, glucagon, and GLP-1/GIP will be the substances 
tested.  
6.1.8 PROMIS scale 
On days 1 and 11, participants will complete the PROMIS scale for abdominal 
pain and gas/bloating. This should be completed prior to eating study food that day. On 
Days 1 and 11, this can be completed during the oral glucose tolerance test.  

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6.2 Sequence of study activities 
Visit Number  
Visit 
1 
 
Visit 
1.1 
Visit 
2 
 
Visit 
2.1 
Visit 3 
 
Visit 4 
 
Visit 
5 
 
Visit 
6 
Study Day 
-60 to 
-9 
-7 
-4 
-3 to 
-1 
1 
2 
3  
4 
5 
6 
7 
8 
9 
10 
11 
 
24 
 
48* 
 
107* 
Food pick up 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Emulsifier free diet 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomized diet 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Satiety Questionnaire 
 
 
 
 
 
X 
X 
 
X 
X 
 
 
X 
X 
 
 
 
 
Record Calories 
Consumed 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
DHQII 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fitbit Data Collection  
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Walks (as needed) 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Weight 
X 
 
X 
 
 
   X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Waist Circumference 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC 
X 
 
 
 
 
 
CMP 
X 
 
 
X 
 
 
 
PT and INR 
X 
 
 
 
 
 
HbA1c 
X 
 
 
 
 
 
Fasting Lipid Panel 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Physical Exam 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24 hour recall x3 
X 
 
 
 
 
Urine Collection 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Stool Collection 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Stool Kit Pick Up 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
Stool Kit Return 
 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Blood Collection 
 
 
X 
 
X 
X 
X 
X 
 
 
 
X 
 
X 
X 
 
X 
 
PROMIS scale 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Oral glucose tolerance 
test 
 
 
X 
X 
 
 
 
Rectal swabs 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sigmoidoscopy with 
biopsy 
 
 
X 
X 
 
 
 
Monitoring adverse 
events 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
Concomitant 
medications 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
AE Follow Up Phone Call 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Follow Up 
Questionnaire 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
*Fitbit will be distributed at Visit 2 (4 days prior to admission to inpatient CHPS). 
*Urine pregnancy test will be conducted for females at screening and Day 1 of inpatient procedures.

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6.2.1 Pre-screening and Informed Consent 
Each potential participant will be pre-screened for eligibility over the phone.  
Additionally, inclusion and exclusion criteria may be reviewed with the potential 
participant during the pre-screening visit. Those who appear to meet all eligibility criteria 
will then be introduced to the study.  Full explanation of the study and the general 
procedures will be explained thoroughly to the participant.  Potential participants will be 
given an opportunity to ask questions and have them answered by study staff.   
Potential participants will sign and date written consent to participate prior to 
screening visits. No steps for screening or beyond will occur prior to informed consent. 
The consent process will be completed in English.  
A copy of the signed consent form will be given to the participant. The original 
signed consent form should be placed in the participant study file. A second copy 
should be placed in a confidential study folder containing copies of consents for all 
study participants. A third copy will be provided to the CHPS. 
6.2.2 Screening (Visit 1) 
Screening evaluation should be completed no more than 60 days prior to 
admission to the CHPS. After providing informed consent, the participant’s medical 
history will be collected to assess for exclusion criteria, including vital signs and 
demographic data. Demographic data collected will include age, sex, race, ethnicity, 
lifetime weight history, current and recent medication use, and past medical and 
surgical history. A physical exam will also be conducted.  
In addition, blood will be drawn for CBC, comprehensive metabolic panel, lipid 
panel, Hba1c, coagulation parameters following an overnight fast. Women will complete 
a urine pregnancy test. 
During the screening process, participants will be presented with the study diets 
to assure that they are able to consume this for a 14 day period. Screening will not 
proceed further unless participants agree that they can follow the study diet, regardless 
to which they are assigned.  
 
Demographics 
      Birth date 
      Sex 
      Race and ethnicity 
      PMH & concurrent medical problems 
Current medications and dose 
Tobacco use 
Alcohol use 
Exclusion criteria lab work 
     CBC 
     Comprehensive metabolic panel (CMP) 
     PT and INR 
     Fasting lipid panel 
     Urine pregnancy test 
     Hba1c 
Physical exam 

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6.2.2.1 Physical examination 
The physical exam will document any baseline abnormalities as well as the 
baseline BMI, waist circumference and a seated blood pressure measurement.  
6.2.2.2 Three Day Dietary Recall (Visit 1.1) 
Data on recent dietary patterns will be collected for 7 days immediately prior to 
Visit 2. The participant will complete a diet diary during the week prior to Visit 2 that will 
be used to facilitate the dietary recall. Note that the last 24 hour recall for the day 
immediately prior to Visit 2 can be completed on the same day as Visit 2. 
Three day dietary recalls will be performed by the CHPS bionutritionists. Dietary 
assessments are to be performed interactively using the Nutrition Data System program 
(University of Minnesota) that includes prompts for questions depending on the food 
item entered as part of the interview. Using this software, the diet information will be 
summarized according to the macronutrient composition (daily consumption of calories, 
protein, fat, carbohydrates and dietary fiber). Additionally, the data on fat and other 
macronutrients can be further subcategorized, such as according percent of calories 
from saturated, monounsaturated, and polyunsaturated fats.  
6.2.2.3 Dietitian Evaluation 
Caloric requirements will be estimated by the dietitian in the CHPS prior Visit 2 
using standard methods to estimate metabolic requirements.33 This will be completed 
prior to Visit 2 so that the CHPS metabolic kitchen can be prepared for the dietary 
needs of the participant prior to providing 3 days of outpatient emulsifier free food.. 
Participants who gain or lose weight will not have their food quantity adjusted to prevent 
further weight loss or gain as the effect of emulsifiers on body weight is one of the 
measures that the study will evaluate. 
 
6.2.3 Post Screening Visit (Visit 2) 
Post screening should be completed 4 days prior to admission to the CHPS.  
6.2.3.1 Fitbit Distribution 
Once participants are determined to be eligible, they will be given a Fitbit Flex to 
measure their activity level each day in the 4 days prior to admission. A study 
coordinator will set up the Fitbit Flex for the participant. The participants will be sent 
home with the Fitbit Flex and instructions on how to use, charge, and wear their Fitbit 
every day and through their return on the day of admission. 
6.2.3.2 Sample Collection 
Eligible participants will have blood, urine, and stool samples will be collected 
prior to beginning the emulsifier free diet. Blood and urine will be collected following an 
overnight fast (see section 6.1.1 and 6.1.2) at Visit 2.  A stool sample will be collected 
by the participant at home either the day prior to Visit 2 or the morning of Visit 2. 
Participants will bring the stool sample to the visit. Study staff will provide the participant 
with collection materials and instructions for collecting the stool at home and returning it 
to study staff (See section 6.1.3).  

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6.2.3.3 Emulsifier Free Diet 
Participants will be given food prepared by the CHPS kitchen. All participants will 
receive 3 full days’ worth of freshly prepared emulsifier-free food at Visit 2. Participants 
will consume the emulsifier-free food exclusively for the three days prior to admission to 
CHPS. Other than the prepared study meals, participants may have water, coffee and 
tea with no added sugar or dairy as desired.  No other food or drink is allowed. 
6.2.3.4 Vital signs and Weight 
Vital signs and weight will be recorded. Weight should be recorded with the 
participant wearing undergarments and a hospital gown prior to the first meal of the day. 
The patient should urinate prior to being weighed. Weight will be measured in triplicate. 
The participant should not be able to see the readout on the scale and should not be 
told their weight. 
6.2.4  
Day 1 of Inpatient Procedures (Visit 3.1) 
All participants will arrive at the CHPS in the morning, having not eaten since 
11:59PM the evening prior.   All of the below procedures for day 1 will be the same for 
both study arms. 
6.2.4.1 History and physical exam 
Vital signs (blood pressure should be done sitting) and weight will be recorded.  
Weight should be recorded with the participant wearing undergarments and a hospital 
gown prior to the first meal of the day. The patient should urinate prior to be weighed. 
Weight will be measured in triplicate. The participant should not be able to see the 
readout on the scale and should not be told their weight.  
A complete physical exam and medical history will be completed to assess for 
abnormalities present at baseline and to confirm absence of intercurrent illnesses that 
would make the participant ineligible. Because the participants may be anxious on the 
first day of the inpatient stay, we will not use blood pressure on Day 1 as an exclusion 
criteria as long as the blood pressure at screening met the inclusion criteria (section 
4.2).  
6.2.4.2 Adverse Events 
Patients will be queried for adverse events. 
6.2.4.3 Urine pregnancy Test 
A urine pregnancy test will be repeated for all women.  
6.2.4.4 Concomitant medications 
Consumption of concomitant medications will be recorded in the study records. 
Participants will not be permitted to take any medications that are listed in the exclusion 
criteria during the study.  
6.2.4.5 Oral Glucose Tolerance Test 
Participants will complete a 2.5 hour oral glucose tolerance test. This test will be 
completed prior to breakfast following a 12 hour fast.  

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6.2.4.6 Sample collection 
Blood, urine, and feces samples will be obtained. Up to 15 ml of urine samples 
will be collected prior to 09:00h after an overnight fast starting at midnight.  
Approximately 16cc of blood will be collected for metabolomics at each collection 
following an overnight fast prior to breakfast. Participants will be provided a stool 
collection kit to collect a stool sample on the day prior to admission or on the morning of 
admission.  The first stool sample will be weighed, aliquoted and frozen.  Participants 
will collect stool from all subsequent bowel movements.  This stool will be weighed, the 
weight recorded and discarded. In addition, a stool sample will be collected by the 
participant at home either the day prior to Visit 3 or the morning of Visit 3. Participants 
will bring the stool sample to the visit. Study staff will provide the participant with 
collection materials and instructions for collecting the stool at home and returning it to 
study staff (See section 6.1.3). 
 
6.2.4.7 Rectal Swabs 
Prior to the flexible sigmoidoscopy, each participant will undergo a rectal swab. 
(See section 6.1.5). 
6.2.4.8 Flexible Sigmoidoscopy 
Following the oral glucose tolerance test, each participant will undergo a 
sigmoidoscopy to obtain biopsies from the area of approximately 15 cm from the anal 
verge. 
6.2.4.9 Food Frequency Questionnaire 
We will collect information on the participant’s usual diet utilizing the Diet History 
Questionnaire II (DHQ II), a food frequency questionnaire (FFQ) developed by the Risk 
Factor Monitoring and Methods Branch (RFMMB) of the National Cancer Institute (NCI). 
The original DHQ I was designed based on cognitive research findings, to be easy to 
use. It had 124 food items and included both portion size and dietary supplement 
questions. The DHQ II has a food list that has been updated based on more recent 
dietary data and consists of 134 food items and 8 dietary supplement questions. There 
are four different versions of DHQ II. These four versions differ by time frame (the past 
year versus the past month) and whether or not questions are included about portion 
size. We will utilize the questionnaire that asks about the past month and includes 
questions about portion sizes for this study since we are interested in most recent 
intake.  
6.2.4.10 
Recording of diet 
Participants will begin their randomized diets following the oral glucose tolerance 
test and flexible sigmoidoscopy at dinner. Participants will be instructed to consume 
only the food provided and no other food. Participants must eat all of the brownie and 
servings of sorbet provided. Participants will have access to additional food outside of 
the meals provided, however, the entire serving of the previous meal must have been 
consumed.  The amount of food consumed will be recorded on the case report forms.  

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6.2.4.11 
Timing of meals 
The first meal will be served following the oral glucose tolerance test or after the 
flexible sigmoidoscopy. All scheduled snacks and meals will be provided following the 
oral glucose tolerance test except during the flexible sigmoidoscopy. Lunch will be 
served at approximately 12:30h and dinner at approximately 18:00h. A snack will be 
provided at 15:00h and 21:00h. Beginning at the dinner meal, all participants will 
consume one serving of brownie or sorbet (15 gm) per day that contain 0 or 2.5 gm 
CMC per serving. Participants will be expected to finish each meal by the time of their 
next meal. There will be no limitation on the time to consume snacks.  All food must be 
consumed by midnight.  
6.2.4.12 
Activity Level Maintenance 
Participants will be required to attain within 10% of the average number of daily 
steps that they took in the 3 days prior to admission.  To achieve this, participants may 
be escorted on walks outside of the inpatient unit by a coordinator, as needed. 
6.2.4.13 
PROMIS Scale 
Participants will complete the PROMIS scale prior to eating their first meal.  
6.2.5 Day 2-10 of Inpatient Procedures (Visit 3.2-3.10) 
All of the below procedures for day 2-10 will be the same for both study arms. 
6.2.5.1 Vital Signs and Weight 
Vital signs and weight will be recorded. Weight should be recorded with the 
participant wearing undergarments and a hospital gown prior to the first meal of the day. 
The patient should urinate prior to being weighed. Weight will be measured in triplicate. 
The participant should not be able to see the readout on the scale and should not be 
told their weight. The number of bowel movements during the prior day will also be 
recorded.  
6.2.5.2 Adverse Events 
Participants will be queried for adverse events. 
6.2.5.3 Concomitant medications 
Consumption of concomitant medications will be recorded in the study records. 
Participants will not be permitted to take any medications that are listed in the exclusion 
criteria during the study.  
6.2.5.4 Sample collection 
On days 2, 3, 4, 8, and 10 blood will be obtained. Approximately 16cc of blood 
will be collected for metabolomics after an overnight fast prior to breakfast. Urine and 
feces samples will also be obtained. Up to 15 ml of urine samples will be collected prior 
to 09:00h after an overnight fast starting at midnight. The first stool sample will be 
aliquoted and frozen.   

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6.2.5.5 Recording of diet 
Participants will continue on their randomized diets. Participants will be instructed 
to consume only the food provided and no other food. Participants must eat all of the 
brownie and servings of sorbet provided. Participants will have access to additional food 
outside of the meals provided, however, the entire serving of the previous meal must 
have been consumed.  The amount of food consumed will be recorded on the case 
report forms.  
6.2.5.6  Timing of meals 
Breakfast will be served at 09:00h. Lunch will be served at approximately 12:30h 
and dinner at approximately 18:00h. A snack will be provided at 10:30h, 15:00h and 
21:00h on Days 2-9. Participants will be expected to consume snacks by the next meal 
with the last snack consumed by midnight on Days 2-9. On Day 10, the 21:00h 
intervention snack will be provided in addition to the intervention and meal at 18:00h. 
Participants will be expected to finish each meal by the time of their next meal or snack. 
Lunch should be finished within 30 minutes. On Day 10, all food must be consumed by 
19:30h.  No food or beverage other than water is to be consumed between midnight and 
breakfast on Days 2-9 and between 19:30h and breakfast on Day 10.   
6.2.5.7 Satiety Questionnaire 
On days 2, 3, 5, 6, 9 and 10, participants will complete a standard food satiety 
questionnaire at 12:15h before lunch and at 13:00h following the completion of lunch. 
6.2.5.8 Activity Level Maintenance 
See Day 1 of inpatient procedures. 
6.2.6 Day 11 of Inpatient Procedures (Visit 3.11) 
All of the below procedures for day 11 will be the same for both study arms. 
6.2.6.1 Vital Signs and Weight 
See Day 2-3 of inpatient procedures. 
6.2.6.2 Adverse Events 
Participants will be queried for adverse events. 
6.2.6.3 Concomitant medications 
Consumption of concomitant medications will be recorded in the study records. 
Participants will not be permitted to take any medications that are listed in the exclusion 
criteria during the study.  
6.2.6.4  Oral Glucose Tolerance Test 
Participants will complete a 2.5 hour oral glucose tolerance test. This test will be 
completed prior to breakfast following a 12 hour fast.  

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6.2.6.5 Sample collection 
Blood, urine, and feces samples will be obtained as described above. Up to 15 
ml of urine samples will be collected prior to 09:00h after an overnight fast starting at 
midnight.  Approximately 16cc of blood will be collected for metabolomics after an 
overnight fast prior to breakfast. The first stool sample will be aliquoted and frozen.   
6.2.6.6  Flexible Sigmoidoscopy 
Following the oral glucose tolerance test, each participant will undergo a 
sigmoidoscopy to obtain biopsies from the area of approximately 15 cm from the anal 
verge. 
6.2.6.7 Recording of diet 
Participants will be given their scheduled, randomized diets following the oral 
glucose tolerance test except during the flexible sigmoidoscopy. Participants will be 
instructed to consume only the food provided and no other food. Participants must eat 
all of the brownie and servings of sorbet provided. Participants will have access to 
additional food outside of the meals provided, however, the entire serving of the 
previous meal must have been consumed.  The amount of food consumed will be 
recorded on the case report forms.  
6.2.6.8  Timing of meals 
The first meal will be served following the oral glucose tolerance test. All 
scheduled snacks and meals will be provided following the oral glucose tolerance test 
except during the flexible sigmoidoscopy. If the flexible sigmoidoscopy directly follows 
the oral glucose tolerance test, participants will be given their final meal before 
discharge. 
6.2.6.9  Activity Level Maintenance 
Participants will not be walked to maintain activity level.  
6.2.6.10 
PROMIS Scale 
 
Participants will complete the PROMIS scale prior to eating their first meal. 
6.2.6.11 
 Discharge 
 
Participants will be discharged following the completion of the oral glucose 
tolerance test and the flexible sigmoidoscopy, and the collection of blood, urine, and 
stool or by 5pm. They will be given post-flexible sigmoidoscopy instructions. An AE log 
and a participant payment form will be completed. Participants will then be escorted out 
of the hospital. 
6.3 Post-study follow-up 
There will be three additional visits following the completion of inpatient visits.  

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6.3.1 Adverse Event Follow Up Phone Call (Visit 4) 
The research team will contact the participant by phone once during the week 
following the completion of the inpatient procedures to assess for any new or 
unresolved adverse events.  
6.3.2 One Month Follow-Up Sample Collection (Visit 5) 
Participants will provide a stool sample 1 month (+/- 1 week) after discharge. 
Participants will return for a blood collection 1 month after discharge. Participants will 
complete a post study follow up questionnaire at this visit. 
6.3.3 Three Month Follow-Up Sample Collection (Visit 6) 
In addition, follow-up stool samples (which may be collected every 3 months for 
up to 5 years after the participant’s last study day) may be collected and will be 
processed in the same manner as the full study. Participants may provide a stool 
sample every three months (+/- 2 weeks). Participants will complete a post study follow 
up questionnaire at this visit. 
6.4 Sample preparation, processing, and storage at the University of 
Pennsylvania 
6.4.1 Aliquoting and processing of 
samples for archiving 
Stool samples are aliquoted into spoon-
capped vials labeled with a barcode that is a 
unique identifier, the date and a protocol 
number, usually 6 per sample, for archiving.  
Any residual stool is aliquoted for specific 
immediate demands by investigators and/or 
removed from the collection vessel into up to 
five 15 ml coring vials, which is also labeled 
with a similar barcode.  Each sample is then 
scanned, and placed in an -80oC freezer in an 
appropriate freezer box with its own separate 
barcode.  The freezer box barcode is scanned 
at the same time.  This provides a 
computerized list of where each sample has 
been stored which can then be matched with 
other information that is collected on this 
sample in the LabVantage lab management 
system.  Blood samples are processed for either serum or plasma, depending on the 
preservatives added at the time of sampling.  Once in the lab, the blood is centrifuged to 
clear the plasma/serum and the supernatant is measured out in 300-500 ul aliquots into 
polypropylene cryotubes.  Each aliquot is labeled in the same manner as the stool 
aliquots and immediately placed in a barcoded box in an -80oC freezer to be archived 
for later use.  Finally, when retrieving a stored sample the box and aliquot barcodes are 
Figure 6. Assessment of microbiota 
encroachment. Carnoy-fixed biopsies are stained 
with probes to label bacteria (red), mucus (green) , 
nuclei (blue) or actin (purple). The location of the 5 
closest bacteria are measured. 5 fields per sample 
are analyzed.  
Figure 2. Assessment of microbiota 
encroachment. Carnoy-fixed biopsies are stained 
with probes to label bacteria (red), mucus (green), 
nuclei (blue) or actin (purple). The location of the 5 
closest bacteria are measured. 5 fields per sample 
are analyzed. 

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scanned and compared to a list of samples needed to ensure the correct material is 
being removed for use.                                
 
6.4.2 Assay for CMC 
Levels of CMC in feces and urine will be quantitated by co-investigator Andrew 
Patterson at The Penn State University  via NMR-based approaches developed by 
Tajima and colleagues34, 35.  
6.4.3 Microbiome characterization 
Microbiota localization. For each subject, 4 distal biopsies, collected pre/post 
CMC and immediately preserved in Carnoy fixative, will be assayed by fluorescence 
microscopy to determine bacterial localization relative to the epithelium.  Specifically, for 
each specimen, we will examine 5 high-powered fields, selected arbitrarily while blinded 
to the accompanying clinical data. We will use staining protocols developed by Gunnar 
Hansson and colleagues 36, 37 that we have ourselves used extensively in our recent 
publications 19, 38. For each field, we will measure the distance of the 5 closest bacteria 
per field as illustrated in Figure 2. The 25 distances (5 per field x 5 fields) will then be 
averaged to produce the "average closest bacteria" and compared between groups as 
reported in our mouse-based studies 19, 38. 2) Microbiota composition. Each fecal 
sample will be subjected to 16S, metagenomics, and meta-transcriptomic sequence-
based analysis as previously described.  Sequencing via the Illumina platform (Miseq for 
16S, NextSeq500 for metagenomics/transcriptomic). Data will be processed and 
analyzed via the Qiime platform and R software, in a manner similar to our previous 
publications 19, 38-40. 3) Microbiota pro-inflammatory potential. For each fecal sample 
collected, we will measure fecal levels of bioactive flagellin and LPS as reported in our 
previous publications 19, 38, 40.  Briefly, for each sample, we will prepare a soluble surface 
extract to be generated by bead-beating followed by centrifugation. Such samples will be 
diluted and then applied to cells engineered to express receptors for TLRs 4 or 5 and 
produce peroxidase under the control of a NF-B responsive promoter - our initial 
publication using this technique describe an extensive set of control studies we have 
utilized to validate this approach 41. These assays, standardized with E. coli flagellin and 
LPS will report the levels of bioactive LPS and flagellin, which we have shown are broadly 
reflective of a fecal specimens ability to drive pro-inflammatory gene expression upon 
injection into mice 41. Comparison between groups will use t-test, repeated measures 
ANOVA or analogous non-parametric tests.  
6.4.4 Microbial DNA Sequencing 
DNA will be prepared in the PennCHOP Microbiome Center. Samples will be 
sent to the center de-identified and coded with a study number. We will perform 16S 
rRNA and ITS gene sequencing to evaluate the bacterial and fungal microbiota, 
respectively, in rectal swab samples. Isolated DNA will be quantified using the 
Picogreen system and 50 ng of DNA will be amplified.  Pyrosequencing will be carried 
out using barcoded primers as previously described42.  For pyrosequencing of bacteria, 
primers annealing to the V1V2 region of the 16S bacterial gene will be used.  The 
development of the ITS1 fungal primers is described in42.  For pyrosequencing, we will 

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use the Roche/454 Genome Sequencer Junior.  Sequence data will be processed using 
QIIME43. If warranted by our preliminary data, we may analyze samples further using a 
metagenomic approach, in which DNA samples are nebulized, ligated to linkers, and 
subjected to pyrosequencing (IlluminaHiSeq).  This allows enumeration of the types of 
genes present in a sample. To determine bacterial and fungal load, respectively, we will 
determine 16S and 18S gene copy number from the rectal swab samples. The qPCR 
methods including the details of the primers as well as PCR cycling conditions have 
been previously described44. 
6.4.5 Metabolic profiling based on plasma samples 
Fasting glucose and oral glucose tolerance (on day 1 and 11), day 11 sera will be 
assayed for a standard comprehensive metabolic panel (includes albumin, urea, 
calcium, creatinine, glucose, and liver enzymes AST/ALT) performed at Penn’s clinical 
lab. Additionally, sera will be assayed for a range of hormones and cytokines using a 
customized bead-based assay designed to include immune inflammatory and metabolic 
markers of obesity and type 2 diabetes (includes IL-8, IL-6, TNF , lipocalin-2, MCP1, 
insulin, leptin, ghrelin, ESR, CRP) all of which are known to be elevated in metabolic 
syndrome (performed at GSU).  Additionally, fecal samples will be assayed for lipocalin-
2 by ELISA. 
6.4.6 Long term sample storage and reuse of samples 
All specimens remaining after the study is complete will be retained for possible 
future use unless specifically requested by the participant that the specimen be 
destroyed or it is deemed by the investigators that the specimens are no longer needed. 
Future studies, including genetic testing, may be conducted with residual samples. 
7 Statistical Plan 
7.1 Sample Size Determination 
 We will recruit the minimum number of subjects to provide adequate statistical 
power for the aims of the project. For aim 1, statistical power will depend on the 
prevalence of detectable CMC in the feces of participants at the end of follow-up. We 
anticipate that in the CMC treated group, the prevalence will be 100% and in the 
untreated group the prevalence will be 0%. However, with 8 subjects per group, we will 
have 89% power to detect a difference if the true prevalence of detectable CMC after 2 
weeks of a high dose CMC and CMC free diet are 90% and 10%, respectively. For aim 
2, power calculation were based on preliminary data in which the difference in mean 
distance of the nearest bacteria to the epithelium between patients with and without 
diabetes was 19.13 um. The within group standard deviation (SD) for patients without 
diabetes was 7.17. The within group SD for those with diabetes was even smaller. With 
a sample size of 8 subjects per group and assuming a within group SD of 7.17, we will 
have 90% and 80% power to detect a difference in the distance of the nearest bacteria 
to the epithelium between the treatment groups (CMC vs. no CMC) that is 35% and 
44% smaller than the difference observed between patients with and without diabetes, 
respectively. In Aim 3, the study will have 80% power to detect a difference between 

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groups equal to 1.51 units of SD. For paired analyses within treatment groups, there will 
be 80% power to detect a difference of 1.15 units of SD. 
7.2 Overview of Statistical Methods 
Descriptive statistics (mean, SD, median, range, counts, and percentage) will be 
used to describe and compare (t-test or rank sum test for continuous variables and 
Fishers exact test for categorical variables) the baseline characteristics, usual diet, and 
immediate pre-enrollment diet between the treatment arms.  
7.2.1 Determining the effect of CMC on fecal and mucosal microbiome 
composition 
We will first compare the bacteria taxa in each fecal stool communities sampled 
at baseline, after the 3 day emulsifier free period and during the intervention period(total 
of 96 stool samples and 16 biopsy samples per treatment arm – 8 participants on each 
diet) to determine within-diet and between-diet variation prior to the administration of 
antibiotics. The analyses will be conducted separately for microbiomes in fecal samples 
and also in colonic mucosa samples. We will employ both of our methods (PCoA and 
proportion of the community represented by different taxa) to summarize the data at 
each time point. Besides comparing the microbiome among the  treatment groups at 
day 11, we will also perform repeated measurement analysis of variance (RM-ANOVA) 
to study the time effects on microbiome and potential time and diet interaction effects for 
our data on fecal samples. The outcomes of such RM-ANOVA are the significant 
principal components summarizing the bacterial taxa.  
7.2.2 Comparison of metabolites between diets with and without CMC 
The first analysis will compare the abundance of fecal and plasma metabolites 
between the two dietary groups. As assignment to the diet will be based on 
randomization, the primary analysis will be an unadjusted t-test to compare the 
concentration of the various metabolites measured in the plasma and feces. As there 
will be multiple comparisons, we will report both nominal (p values) and q values 
accounting for the multiple testing.   
7.2.3 Effects of CMC consumption on a range of inflammatory and 
metabolic parameters that characterize metabolic syndrome 
Weight change will be compared between the treatment groups from day 1 to 
day 11 using a t-test. If there is evidence of confounding despite randomization, 
additional analyses will use linear regression adjusted for change in weight from day 1 
to day 11 and BMI at day 0.  
We will also assess changes to each subject regarding satiety (from the 
questionnaire re food adequacy/sufficiency). These data are on visual analogue scale 
so will be compared using unpaired t-test for between group comparisons or paired t-
tests for within group comparisons. 
 
The area under the glucose and insulin concentration curves during the oral 
glucose tolerance test at day 1, day 11 and the change from day 1 to day 11 will be 
compared between groups using a t-test. The ΔI30/ΔG30 will be used as a measure of 

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beta cell function45 while homeostasis model assessment of insulin resistance (HOMA-
IR) will be used to estimate insulin resistance46 and compared between the groups with 
a t-test at the same time points.  
  
Sera will be assayed for a range of hormones and cytokines using a customized 
bead-based assay designed to include immune inflammatory and metabolic markers of 
obesity and type 2 diabetes (includes IL-8, IL-6, TNFα, lipocalin-2, MCP1, insulin, leptin, 
ghrelin, ESR, CRP) all of which are known to be elevated in metabolic syndrome 
(performed at GSU).  Additionally, fecal samples will be assayed for lipocalin-2 by 
ELISA.  These data will be compared using unpaired t-test for between group 
comparisons or paired t-tests for within group comparisons. 
 
8 Safety and Adverse Events 
Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that 
develops or worsens in severity during the course of the study.  Intercurrent illnesses 
or injuries should be regarded as adverse events.  Abnormal results of diagnostic 
procedures are considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
Serious Adverse Event (SAE) 
Adverse events are classified as serious or non-serious.  A serious adverse 
event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
 
Important medical events are those that may not be immediately life 
threatening, but are clearly of major clinical significance.   They may jeopardize the 
participant, and may require intervention to prevent one of the other serious 
outcomes noted above.  For example, drug overdose or abuse, a seizure that did 
not result in in-patient hospitalization or intensive treatment of bronchospasm in an 
emergency department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious will be 
regarded as non-serious adverse events. 
Adverse Event Reporting Period 

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The study period during which AEs must be reported is normally defined as 
the period from the initiation of any study procedures to the end of the study 
treatment follow-up.  For this study, the study treatment follow-up is defined as 7 
days following the last administration of study diet.  
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A 
preexisting condition should be recorded as an adverse event if the frequency, 
intensity, or the character of the condition worsens during the study period. 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a 
preexisting condition.  At the end of the study, any new clinically significant 
findings/abnormalities that meet the definition of an AE must also be recorded and 
documented as an AE.  
Post-study Adverse Event 
All unresolved AEs that are categorized by the investigator as definitely or 
probably related to participation in the study should be followed by the investigator 
until the events are resolved, the participant is lost to follow-up, or the AE is 
otherwise explained.  At the last inpatient evaluation, the investigator will instruct 
each participant to report any subsequent event(s) that the participant, or the 
participant’s personal physician, believes might reasonably be related to 
participation in this study.  The investigator will notify the study sponsor of any 
death or AE occurring at any time after a participant has discontinued or 
terminated study participation that may reasonably be related to this study.  The 
sponsor should also be notified if the investigator should become aware of the 
development of cancer or of a congenital anomaly in a subsequently conceived 
offspring of a participant that has participated in this study.  
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse 
event if any one of the following conditions is met:  
• The laboratory abnormality is not otherwise refuted by a repeat test to 
confirm the abnormality 
• The abnormality suggests a disease and/or organ toxicity 
• The abnormality is of a degree that requires active management; e.g. 
change of dose, discontinuation of the drug, more frequent follow-up 
assessments, further diagnostic investigation, etc. 
Hospitalization, Prolonged Hospitalization or Surgery 
Any AE that results in hospitalization or prolonged hospitalization should be 
documented and reported as an SAE unless specifically instructed otherwise in 
this protocol.  Any condition responsible for surgery should be documented as an 
AE if the condition meets the criteria for an AE.  
Neither the condition, hospitalization, prolonged hospitalization, nor surgery 
are reported as an AE in the following circumstances: 

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• Hospitalization or prolonged hospitalization for diagnostic or elective 
surgical procedures for a preexisting condition.  Surgery should not be 
reported as an outcome of an AE if the purpose of the surgery was elective 
or diagnostic and the outcome was uneventful. 
8.1 Recording of Adverse Events 
At each contact with the participant, the investigator must seek information on 
AEs by specific questioning and, as appropriate, by examination.  Information on all AEs 
should be recorded immediately in the source document, and also in the appropriate 
adverse event case report form (CRF).  All clearly related signs, symptoms, and 
abnormal diagnostic procedures results should be recorded in the source document, 
though should be grouped under one diagnosis. 
All AEs occurring during the study period must be recorded.  The clinical course 
of each event should be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause.  SAEs that are still 
ongoing at the end of the study period must be followed up to determine the final 
outcome.  Any SAE that occurs after the study period and is considered to be possibly 
related to the study treatment or study participation should be recorded and reported 
immediately. 
8.2 Reporting of Serious Adverse Events 
8.2.1 Study Sponsor Notification by Investigator 
An SAE must be reported to the study sponsor by telephone within 24 
hours of the event.  An FDA Medwatch 3500A SAE Form must be completed by 
the investigator.  The investigator will keep a copy of this form on file at the study 
site.  Report SAEs by phone to: 
 
James D. Lewis, MD 
Phone - (215) 573-5137 or (856) 906-3173 
 
In the event that Dr. Lewis cannot be reached report SAEs to  
Gary Wu, MD 
Phone - (215) 898-0158 
or 
Lisa Nessel 
Phone – (215) 573-6003 
Or  
Brittaney Bonhomme 
Phone – (215) 898-2625 
 
At the time of the initial report, the following information should be 
provided: 

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• Study Name 
• Participant number 
• A description of the event 
• Date of onset 
• Current status 
• Whether study treatment was 
discontinued 
• The reason why the event is 
classified as serious 
• Investigator assessment of the 
association between the event 
and study treatment 
 
Within the following 48 hours, the investigator must provide further 
information on the SAE in the form of a written narrative.  This should include a 
copy of the completed Medwatch 3500 A SAE Form and any other diagnostic 
information that will assist the understanding of the event.  Significant new 
information on ongoing SAEs should be provided promptly to the study 
investigator. 
8.2.2 Ethics Committee/Internal Review Board Notification by Investigator 
All reports of SAES (including follow-up information)  must be submitted to the 
Ethics Committee (EC)/Internal Review Board (IRB) within 10 working days.  Copies of 
each report and documentation of EC /IRB notification and receipt will be kept in the 
Clinical Investigator’s binder.  
9 Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study participants will be kept confidential and managed according to 
the requirements of the Health Insurance Portability and Accountability Act of 1996 
(HIPAA).  Those regulations require a signed participant authorization informing the 
participant of the following:  
• What protected health information (PHI) will be collected from participants in this 
study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research participant to revoke their authorization for use of their 
PHI.  
In the event that a participant revokes authorization to collect or use PHI, the 
investigator, by regulation, retains the ability to use all information collected prior to the 
revocation of participant authorization.   
 
Analytical data may be handled at Georgia State University and/or University of 
Pennsylvania. All data related to this trial will be recorded using the patients’ assigned 
unique study number. Data will be reported only in a confidential manner such that the 
personal identity of any subject will not be identifiable. All personal identifiers will be 
stored on REDCap (Research Electronic Data Capture). REDCap is a secure, web-
based application designed to support data capture for research studies and secure 

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sensitive data providing physical security of machines by the Penn Medicine Academic 
Computing Services, saving all data on to both the database and webservers located on 
private networks, as well as only providing access to data within the application. Only 
limited data sets will be shared outside of REDCap. Limited data sets will include dates, 
but not direct identifiers.  All study data will be maintained under a double locked 
system, such as a locked closet within a locked office or on a password protected 
computer in a locked office.  At the end of the study these data will be electronically 
archived on a password protected computer or other electronic storage device.  
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, 
or other activities in a clinical trial necessary for the reconstruction and evaluation of the 
trial.  Source data are contained in source documents.  Examples of these original 
documents, and data records include: hospital records, clinical and office charts, 
laboratory notes, memoranda, participants’ diaries or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions 
certified after verification as being accurate and complete, microfiches, photographic 
negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the 
pharmacy, at the laboratories, and at medico-technical departments involved in the 
clinical trial. 
9.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the 
study.  All data requested on the CRF must be recorded.  All missing data must be 
explained.  If a space on the CRF is left blank because the procedure was not done or 
the question was not asked, write “N/D”.  If the item is not applicable to the individual 
case, write “N/A”.  All entries should be printed legibly in black ink.  If any entry error has 
been made, to correct such an error, draw a single straight line through the incorrect 
entry and enter the correct data above it.  All such changes must be initialed and dated.  
DO NOT ERASE OR WHITE OUT ERRORS.  For clarification of illegible or uncertain 
entries, print the clarification above the item, then initial and date it. 
9.4 Records Retention 
Study documents and records will be retained for at least 2 years after the last 
participant has completed the study. 
10 Study Monitoring, Auditing, and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by 
the EC/IRB, the sponsor, government regulatory bodies, and University compliance and 
quality assurance groups of all study related documents (e.g. source documents, 
regulatory documents, data collection instruments, study data etc.).  The investigator 
will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.).  
An independent safety officer has been assigned the role of monitoring adverse 
events. Dr. Yu-Xiao Yang will serve in this role. The safety officer will receive quarterly 

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reports of all adverse events. In addition, the officer will receive reports of serious 
adverse events within 72 hours of the investigators becoming aware of the event. If a 
serious adverse event occurs, the safety officer may request to be unblinded to further 
investigate, if necessary. A biostatistician would generate a list of AEs by arm which 
only be viewed by the safety officer, if necessary, to ensure all study staff remain 
blinded. Additionally, the safety officer will have the authority to review the study 
documents at any time and for any reason to assure the safety of the participants.  
Participation as an investigator in this study implies acceptance of potential 
inspection by government regulatory authorities and applicable University compliance 
and quality assurance offices. 
11 Ethical Considerations 
This study is to be conducted according to US and international standards of 
Good Clinical Practice (FDA Title 21 part 312 and International Conference on 
Harmonization guidelines), applicable government regulations and Institutional research 
policies and procedures. 
This protocol and any amendments will be submitted to a properly constituted 
independent EC/ IRB, in agreement with local legal prescriptions, for formal approval of 
the study conduct.  The decision of the EC/IRB concerning the conduct of the study will 
be made in writing to the investigator and a copy of this decision will be provided to the 
sponsor before commencement of this study.   
All participants for this study will be provided a consent form describing this study 
and providing sufficient information for participants to make an informed decision about 
their participation in this study.  See Attachment A for a copy of the Informed Consent 
Form.  This consent form will be submitted with the protocol for review and approval by 
the EC/IRB for the study.  The formal consent of a participant, using the EC/IRB-
approved consent form, must be obtained before that participant is submitted to any 
study procedure.  This consent form must be signed by the participant or legally 
acceptable surrogate, and the investigator-designated research professional obtaining 
the consent.  
12 Study Finances 
12.1 Funding Source 
This study is financed through a grant from the National Institutes of Health. 
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) 
must have the conflict reviewed by a properly constituted Conflict of Interest Committee 
with a Committee-sanctioned conflict management plan that has been reviewed and 
approved by the study sponsor prior to participation in this study.  All University of 
Pennsylvania investigators will follow the University conflict of interest policy. 

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12.3 Participant Stipends or Payments 
Participants will be compensated $3100 for completion of the entire inpatient stay. 
Participants who do not complete the full inpatient stay will receive only $100 for each 
24 hours of inpatient stay completed. Participants who return to CHPS upon admission 
with their Fitbit Flex will be given the option to keep the Fitbit Flex. Participants who 
complete visit 5 (blood and stool collection) will be compensated an additional $150.  
Participants who complete visit 6 (3 month follow-up stool collection) will be 
compensated an additional $50. Thus, participants who complete visits 1-6 will be 
compensated a total of $3300. 
 
13 Publication and Data Sharing Plan 
Any publications emanating from these data must acknowledge the funding 
source. 
Neither the complete nor any part of the results of the study carried out under this 
protocol, nor any of the information provided by the sponsor for the purposes of 
performing the study, will be published or passed on to any third party without the 
consent of the study regulatory sponsor.  Any investigator involved with this study is 
obligated to provide the regulatory sponsor with complete test results and all data 
derived from the study.  
 
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15 Attachments 
 
 

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16 Protocol Signature Page 
I will provide copies of the protocol, any subsequent protocol amendments and 
access to all information furnished by the sponsor to study personnel under my 
supervision.  I will discuss this material with them to ensure that they are fully informed 
about the investigational drug and study protocol. 
 
I agree to conduct this clinical trial according to the protocol described herein.  I 
also agree to conduct this study in compliance with applicable federal, state and local 
regulations, Guidelines for Good Clinical Practice (GCP), and with the requirements of 
my Institutional Review Board.  I understand that I may not implement this protocol 
without first receiving written IRB approval. 
 
Furthermore, I understand that I may not make changes to this protocol.  (The 
only exception being action needed to remove a subject from immediate harm, with 
subsequent notification to the study PI and IRB). 
 
Investigator’s Signature: __________________________________       
 
Date: ____________ 
 
Investigator’s Name: ______________________________________________ 
 
 
 
 
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