Dietary Impact on Intestinal Sulfate Metabolism
Phase: NA
Conditions: Healthy Nutrition
Sponsor: University of Minnesota

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Dietary Impact on Intestinal Sulfate Metabolism 
 
 
 
Sponsor Investigator:  Alexander Khoruts, MD 
 
 
 
 
PWB, Room 1-214 
 
 
 
 
516 Delaware St SE 
 
 
 
 
Minneapolis, MN 55455 
 
 
 
 
612-625-5956 
 
 
 
 
Khoru001@umn.edu 
 
Protocol Number:  
CTSI# 25182 
Trial Registration:  
pending 
Version / Date:   
Version 10 / 20 January 2021 
 
 
 
 
 
 
 
 
 
CONFIDENTIAL 
 
 
This document is confidential and the property of the University of Minnesota. No part of it may be 
transmitted, reproduced, published, or used by other persons without prior written authorization from the 
study sponsor-investigator. 
 

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VERSION HISTORY 
 
Version # 
Version Date 
Significant Changes from Previous Version 
Version 1 
18 Oct 2016 
Original Protocol Version 
Version 2 
15 May 2018 
Raised age and BMI cutoffs for inclusion criteria; 
removed vegetarianism as exclusion criteria; allowed 
coffee and tea as beverages in High-S diet; removed 
physical exam from baseline study data collection; 
changed titled to align with grant submission process 
Version 3 
21 June 2018 
Added 24-hour urine collection to help characterize dietary 
sulfur intake 
Version 4 
19 July 2018 
Changed status of vital signs and blood collection procedures to 
“per PI discretion” 
 
22 August 2018 
Revised section 4.1 according to reviewer comments: “Revise 
the section 4.1 of the protocol to state the following, to ensure 
the health and safety of all participants entering and exiting the 
study: ‘Of note, Vital signs and collection of blood samples will 
be completed based on PI discretion, except for the Screening 
and Safety Follow-up Visit.’ 
 
Version 5 
20 December 2018 
Decreased diet intervention duration to from 14 days to 7 days 
and overall duration of the study from ~2 months to ~1 
month; Decreased clinic visits from 11 down to 6;  
Modified High-S and Low-S diets to incorporate the emphasis 
on animal-based for high sulfur and plant-based for low sulfur 
described in “Research Design and Methods”; Updated 
FFQ to most recent version (web-based DHQ-III) 
Version 6 
19 February 2019 
1. Added participant compensation of up to $150. Payments will 
be received in the form of gift cards.  
 
2. Added additional fecal sample collections. The fecal sample 
collections are not full stool samples, but are small samples 
collected in collection tubes. These samples will be stored in the 
participant’s freezer and collected at each study visit. These will 
be collected on days (±2 days) -4, -2, 3, 5, 9, 11, 16, 18, 23, 25, 
30, and 32. The additional samples will enhance analysis of 
longitudinal changes and help correct for some of the inherent 
variability associated with stool sampling.  
 
3. We would also like to remove Matthew Hamilton as a study 
team member. 
Version 7 
8 May 2019 
1. Increased population to 15 volunteers 
2. Changed 24-hour urine collection to “per PI discretion” 
 
Version 8 
17 September 2019 
1. Increased population to 20 volunteers 
Version 9 
27 February 2020 
1. Increased population to 30 volunteers 
Version 10 
20 January 2021 
1. Increased “Inclusion Criteria” age from maximum of 70 years 
of age to 80 years of age 
 
 

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TABLE OF CONTENTS 
 
1 
BACKGROUND INFORMATION AND SIGNIFICANCE ................................................ 8 
2 
STUDY HYPOTHESES AND OBJECTIVES ................................................................. 10 
2.1 
Hypotheses ................................................................................................................... 10 
2.2 
Primary Objectives ...................................................................................................... 10 
2.3 
Secondary Objectives: ................................................................................................ 10 
2.4 
Exploratory Objectives ................................................................................................ 10 
2.5 
Study Outcome Measures .......................................................................................... 10 
2.5.1 
Primary Outcome Measures ............................................................................... 10 
2.5.2 
Secondary Outcome Measures ......................................................................... 11 
2.5.3 
Exploratory Outcome Measures ........................................................................ 11 
3 
RESEARCH DESIGN AND METHODS ......................................................................... 11 
3.1 
Study Participants and Recruitment ......................................................................... 11 
3.1.1 
Inclusion Criteria ................................................................................................... 11 
3.1.2 
Exclusion Criteria ................................................................................................. 12 
3.2 
Diet Intervention Design ............................................................................................. 12 
3.3 
Dietary Intake Assessment ........................................................................................ 13 
3.3.1 
Tolerability and Feasibility................................................................................... 14 
3.4 
Sample Collection ........................................................................................................ 14 
3.4.1 
Fecal Samples ...................................................................................................... 14 
3.4.2 
Urine Samples ...................................................................................................... 15 
3.4.3 
Blood Samples ...................................................................................................... 15 
3.5 
Gas Concentration Measurement ............................................................................. 15 
3.6 
16S Sequencing .......................................................................................................... 15 
3.6.1 
DNA Extraction from Fecal Samples................................................................. 15 
3.6.2 
Amplification of 16S rRNA Gene and Sequencing ......................................... 15 
3.7 
Fecal Bile Acid Analysis ............................................................................................. 16 
3.8 
Treatment Assignment ................................................................................................ 16 
3.9 
Withdrawal .................................................................................................................... 16 
3.9.1 
Reasons for Withdrawal ...................................................................................... 16 
3.9.2 
Handling of Withdrawal ....................................................................................... 16 
3.10 
Study Termination .................................................................................................... 17 
4 
STUDY PROCEDURES AND EVALUATION SCHEDULE ......................................... 17 

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4.1 
Clinical/Laboratory Evaluations and Study Schedule ............................................ 17 
4.2 
Initial Screening Visit ................................................................................................... 17 
4.3 
Visit 1 - Baseline (Day 0) ............................................................................................ 17 
4.4 
Visit 2 .............................................................................. Error! Bookmark not defined. 
4.5 
Visit 3 ............................................................................................................................. 18 
4.6 
Visit 4 (Day 14 ± 2 days; Final day of first diet intervention)................................. 18 
4.7 
Visit 5 .............................................................................. Error! Bookmark not defined. 
4.8 
Visit 6 ............................................................................................................................. 18 
4.9 
Visit 7 (Day 28 ± 2 days; First day of second diet intervention) ........................... 18 
4.10 
Visit 8 .......................................................................... Error! Bookmark not defined. 
4.11 
Visit 9 .......................................................................... Error! Bookmark not defined. 
4.12 
Visit 10 (Day 42 ± 2 days; Final day of second intervention) .. Error! Bookmark 
not defined. 
4.13 
Visit 11 (Day 49 ± 2 days; Follow-up visit) ............ Error! Bookmark not defined. 
4.14 
Unscheduled Visits .................................................................................................. 19 
4.15 
Participant Compensation ...................................................................................... 19 
4.16 
Potential Risks .......................................................................................................... 19 
4.16.1 Risk of gastrointestinal symptoms ..................................................................... 19 
4.17 
Known Potential Benefits ........................................................................................ 19 
4.18 
Risk/Benefit Ratio .................................................................................................... 19 
5 
DATA SAFETY AND MONITORING PLAN ................................................................... 19 
5.1 
Definitions ..................................................................................................................... 19 
5.2 
Recording of Adverse Events .................................................................................... 22 
5.3 
Adverse Event Management ..................................................................................... 23 
5.4 
Reporting of Serious Adverse Events ...................................................................... 23 
5.4.1 
IRB Notification by Sponsor Investigator .......................................................... 23 
5.4.2 
UPIRTSO Events ................................................................................................. 23 
5.5 
Temporary Interruption of Intervention in an Individual Subject .......................... 23 
6 
STATISTICAL CONSIDERATIONS ................................................................................ 23 
6.1 
Sample Size Considerations ...................................................................................... 23 
6.2 
Randomization ............................................................................................................. 24 
6.3 
Blinding .......................................................................................................................... 24 
6.4 
Planned Interim Analyses .......................................................................................... 24 
6.5 
Safety Review .............................................................................................................. 24 

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7 
DATA AND RECORD KEEPING ..................................................................................... 24 
7.1 
Data Capture Methods ................................................................................................ 24 
7.2 
Source Documents ...................................................................................................... 25 
7.3 
Study Records Retention ........................................................................................... 25 
7.4 
Protocol Deviations ..................................................................................................... 25 
8 
QUALITY CONTROL AND QUALITY ASSURANCER ................................................ 25 
8.1 
Dietary Intake ............................................................................................................... 25 
9 
ETHICS/PROTECTION OF HUMAN SUBJECTS ........................................................ 25 
9.1 
Ethical Standard .......................................................................................................... 25 
9.2 
Institutional Review Board .......................................................................................... 26 
10 INFORMED CONSENT PROCESS ................................................................................ 26 
10.1 
Participant Confidentiality ....................................................................................... 26 
10.2 
Principal Investigator Responsibility when Participant Withdraws or is 
Discontinued ........................................................................................................................... 26 
10.3 
Future Use of Stored Specimens .......................................................................... 26 
11 PUBLICATION POLICY .................................................................................................... 27 
12 REFERENCES ................................................................................................................... 28 
13 Appendix 1. Schedule of Events ...................................................................................... 31 
 
 
 

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LIST OF ABBREVIATIONS 
 
AE 
 
 
 
Adverse Event 
CH4 
 
 
 
Methane 
CH3SH 
 
 
Methanethiol 
CRF 
 
 
 
Case Report Form 
CTCAE 
 
 
Common Terminology Criteria for Adverse Events 
DHQ  
 
 
Dietary History Questionnaire 
DNA  
 
 
Deoxyribonucleic Acid 
GC 
 
 
 
Gas Chromatography 
H2S 
 
 
 
Hydrogen sulfide 
H2 
 
 
 
Hydrogen Gas 
High-S  
 
 
High-Sulfur Diet 
HIPAA 
 
 
Health Insurance Portability and Accountability Act 
IBD 
 
 
 
Inflammatory Bowel Disease 
IRB 
 
 
 
Institutional Review Board 
Low-S  
 
 
Low-Sulfur Diet 
LC-MS 
 
 
Liquid Chromatography-Mass Spectrometry 
NCI 
 
 
 
National Cancer Institute 
NDSR  
 
 
Nutrient Data System for Research 
SAA  
 
 
Sulfur-containing Amino Acid 
SAE 
 
 
 
Serious Adverse Event 
SCFA  
 
 
Short-Chain Fatty Acid 
SRB 
 
 
 
Sulfur-Reducing Bacteria 
TMAO 
 
 
Trimethylamine N-oxide 
UC 
 
 
 
Ulcerative Colitis 
UPIRTSO 
 
 
Unanticipated Problems Involving Risks to Subjects or Others 
 
 
 
 

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PROTOCOL SUMMARY 
 
 
Title:  
Dietary Impact on Intestinal Sulfate Metabolism 
 
Phase:  
 
 
1 
 
Population:  
30 healthy male and female volunteers. Subjects will be recruited 
from the Twin Cities metropolitan area and will participate as 
outpatients in this study. 
 
Number of Sites:  
 
1 (University of Minnesota) 
 
Study Duration:  
 
12 months 
 
Subject Participation  
Approximately 1 month 
Duration:  
 
Agent or Intervention:  
Dietary intervention will consist of following diets high in sulfur-
containing amino acids (SAAs) and low in SAAs for 7 days each, 
with a 14-day washout period between interventions 
 
Objectives:  
The primary objectives of this proposal are to: 1) Compare low 
sulfur and high sulfur diets with respect to fecal production of 
hydrogen sulfide (H2S) and other end products of microbial 
fermentation; and 2) Determine whether the diet alters the fecal 
microbiota and the relative proportion of fecal sulfate reducing 
bacteria. 
 
Endpoints:  
The primary endpoint is the change in H2S concentration measured 
by gas chromatography. Comparisons will be made between the 
baseline measurement (Day 0) and end of intervention (Day 7) for 
each diet (within-group comparison) and between high- and low-
sulfur diet H2S concentration on Day 7 (between group 
comparison). 
 
 

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1 
BACKGROUND INFORMATION AND SIGNIFICANCE 
Ulcerative colitis (UC) is one of the two major inflammatory bowel disease (IBD) subtypes and 
is characterized by chronic, relapsing inflammation in the colon causing diarrhea and rectal 
bleeding. UC currently affects 0.25% of the population in North America (~ 1 million people in 
the United States), having risen dramatically over recent decades, and is now emerging as a 
global disease.1 UC is responsible for significant personal, professional, and economic 
consequences - annual costs associated with medical management approach $5 billion.2 The 
causes for flares and remissions in UC activity are largely unknown, although epidemiologic 
studies suggest a significant role of the environment in the emergence of IBD. Thus, there is 
rising incidence in Europe and North America,3,4 higher incidence in developed countries as 
compared to non-industrialized countries,5,6 and increasing incidence rates in developing  
countries as they have Westernized.7 
 
Patients with UC instinctively understand that diet is an important variable in diseases involving 
the intestinal tract, and they have a strong interest in dietary modifications that may aid in 
maintaining remission of symptoms and improving their disease. When first diagnosed, virtually 
all patients with IBD ask what they should eat to help control their disease. To a large extent, this 
reasonable and intuitively logical question remains unanswered, as there is a scarcity of well-
designed, prospective interventional studies that can guide dietary recommendations for this 
patient population. Current allopathic therapeutic strategies rely exclusively on drugs and surgery 
to control intestinal inflammation and deal with its complications. However, these available 
therapies are commonly not sufficient to control the symptoms, risk serious side effects, and 
come at a high financial cost. Therefore, patients and doctors alike seek effective complementary 
therapies. 
 
The intestinal tract contains complex microbial communities that participate in the final steps of 
the digestive process. The structure and composition of these communities is highly responsive to 
dietary input.8 Some of the end products of microbial metabolism in the gut are highly toxic (e.g., 
H2S) and may be linked to the pathogenesis of UC.9,10 These potentially toxic compounds can 
damage the epithelial lining of colon, cause breakdown of the gut barrier, and lead to 
inflammation. Fecal H2S concentration is increased by increasing either the sulfur amino acid11 or 
sulfate content (as additives) of the diet.12 Sulfur-containing amino acids (SAA) are converted to 
sulfides by sulfate reducing bacteria (SRB)13,14, which may expand in response to increased 
availability of SAA and inhibition of alternative end routes of fermentative pathways, e.g., 
production of methane or short-chain fatty acids (SCFAs).15,16 
 
The scientific foundation for the sulfur-containing toxin hypothesis as a driver of UC rests on 
provocative and compelling evidence from clinical observations, animal models, and dietary 
association studies. The characteristic distribution of UC in the colon (always involving the 
rectum and continuously extending proximally to varying extent of involvement) is suggestive of 
a toxin being concentrated along the path of intestinal transit. Sulfated dextrans, but not free 
dextrans, can induce experimental colitis in rodent models.17 H2S, a potent toxin in its own right 
and one of the suspected sulfur-containing toxins in UC, decreases butyrate utilization by 
colonocytes and causes loss of gut barrier function.9,18 UC patients have been found to have 
increased luminal concentrations of H2S and disease activity correlates with sulfide production 

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rates.10,19 Furthermore, some medications that have beneficial effects on UC activity, e.g., 
mesalamine and aminoglycoside antibiotics, can inhibit SRB.20,21 The major dietary sources of 
sulfur are high-protein foods (containing the SAAs methionine and cysteine), cruciferous 
vegetables from the Brassicaceae family, and processed food preservatives.22 Dietary sources of 
sulfur exceed endogenous sources in the colon,23 and dietary intake studies have found a greater 
risk of UC flares in individuals consuming diets with higher sulfur content.24,25 When taken 
together, these mechanisms suggest that minimizing intake of dietary SAAs may improve 
symptoms and reduce risk for disease exacerbation in individuals with UC. 
 
Diet-derived sulfur is not the only determinant for the availability of sulfur to microbiota in the 
colon. Saturated fats (mainly animal-derived), as opposed to unsaturated fats (mainly plant-
derived) promote taurine conjugation of hepatic bile acids. Taurine provides a source of organic 
sulfur to SRB such as Bilophila wadsworthia, which blooms in mice fed a high-milk fat diet, but 
not a high-safflower fat diet.26 Furthermore, the high-milk fat diet induces B. wadsworthia-
specific Th1 cells in the gut-associated lymph nodes and greater incidence of colitis in 
genetically susceptible (IL-10-/-) mice. Different bile acids themselves can have potent effects 
on microbiota, both inhibitory and stimulatory, on different members of microbiota. B. 
wadsworthia is clearly favored by taurocholate, and this is also true of Clostridium difficile, 
which is a common trigger for flares of UC activity.27,28  
 
The notion of a “low sulfur diet” for treatment of gastrointestinal conditions has existed for 
decades. Restricting high-sulfur foods, in combination with milk fat restriction, has historically 
been recommended to UC patients by some practitioners of integrative medicine. However, and 
despite these claims, the methodology of this diet was ill-defined and it is not known whether it 
results in specific changes in gut microbiota, decreases production of toxic sulfur-containing end 
products, or is even feasible in terms of adherence. The medical literature contains only a single 
small clinical case series of 4 patients with UC from approximately 20 years ago that 
demonstrated symptomatic improvement on a low sulfur diet that restricted meat, milk fat, 
cruciferous vegetables, and inorganic sulfates used as food preservatives.29 This work did not 
include objective measurements of disease activity, such as laboratory or endoscopic criteria, 
which would be standard today. One prospective cohort study24 published in 2004 measured 
dietary intake via food frequency questionnaire and found that a high intake of meat and meat 
products (particularly red and processed meats), eggs, protein, alcohol, energy, fat, sulfur, and 
sulfate (a common food and beverage additive) predicted an increased likelihood of relapse. 
However, data on sulfur content of foods was only listed for 29% of foods consumed by 
participants in the study, indicating incompleteness of the full total of sulfur intake. Notably, 
sulfur content was not available for red and processed meats, which are known to be high in 
dietary sulfur. Therefore, more complete food tables for sulfur would be expected to increase the 
differences in sulfur consumption between participants who did or did not relapse.  
 
The scarcity of rigorous, systematic investigations of dietary interventions in UC patients is a 
consequence of several factors: (1) controlling diet in human subjects is difficult, (2) dietary 
input is complex, and (3) clinical end points alone provide few opportunities for mechanistic 
dissections. However, the rise of high throughput DNA sequencing and metabolomics, combined 
with novel computational techniques over the past decade, is opening an entirely new era in 

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nutrition research. The gut microbiota, unlike human individuals, can serve as very accurate 
reporters of environmental changes.  
 
The long-term goal of our research group is to develop evidence-based nutritional strategies for UC that 
compliment medical therapies. Our strategy is to link specific diets to their effects on microbiota 
functionality. Nevertheless, we understand that even when armed with the new powerful tools, 
identifying an effective diet that is also acceptable to patients will be a challenging and resource 
intensive journey. Therefore, in this proposal, which is our first step toward that ultimate goal, 
we will test dietary extremes in highly motivated, healthy volunteers. This will allow us to 
streamline our experimental toolkit and measure the magnitude of the potential effects on gut 
microbiota composition and function that can be achieved by controlling the diet, which will 
help to estimate the size of a formal clinical study in patients with UC. 
 
2 
STUDY HYPOTHESES AND OBJECTIVES 
2.1 
Hypotheses 
Our hypotheses are that excessive production of toxic sulfur-containing end products of 
microbial fermentation in the colon are major drivers of UC, and that it is possible to decrease 
their production by dietary means. 
 
2.2 
Primary Objectives 
The primary objectives of this study are: 
1. Compare low sulfur (Low-S) and high sulfur (High-S) diets with respect to fecal 
production of H2S and other end products of microbial fermentation, such as thiols, 
hydrogen gas (H2), and methane (CH4) (prediction: higher H2S with High-S diet). 
2. Determine whether the diet alters the fecal microbiota and the relative proportion of fecal 
SRB using 16S rRNA profiling and whole metagenome shotgun sequencing for species-
level resolution (prediction: higher SRB with High-S diet). 
 
2.3 
Secondary Objectives: 
1. Evaluate the tolerability and feasibility of high- and low-sulfur diet regimens. 
2. Measure changes in bile acid profiles associated with changes in dietary SAA intake. 
 
2.4 
Exploratory Objectives 
1. Assess changes in serum TMAO, carnitine, and phosphatidylcholine concentrations 
associated with changes in dietary SAA intake. 
 
2.5 
Study Outcome Measures 
2.5.1 Primary Outcome Measures 
Primary outcome measures of this study include:  
1. Concentration of H2S, CH4, H2, and methanethiol (CH3SH) in fecal headspace samples 
measured by gas chromatography spectrometry (GC).  
2. Changes in fecal bacterial composition (pre- vs. post-diet) associated with High-S vs. 
Low-S diets. 

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2.5.2 Secondary Outcome Measures 
1. Compliance with diet interventions and adverse event rates overall and between High-S 
vs. Low-S diets. 
2. Changes in gastrointestinal symptoms overall and within participants in High-S vs. Low-
S diets, by clinical assessment and adverse event monitoring. 
3. Changes in fecal bile acid profiles overall and within participants in High-S vs. Low-S 
diets, by liquid chromatography-mass spectrometry (LC-MS).  
2.5.3 Exploratory Outcome Measures 
1. Changes in serum concentrations of TMAO, carnitine, and phosphatidylcholine within 
participants and between High-S vs. Low-S diets. 
 
 
3 
RESEARCH DESIGN AND METHODS 
This is a randomized, 4- to 5-week crossover pilot study of 15 highly motivated healthy 
volunteers who will consume two 7-day intervention diets: 1) a diet low in sulfur-containing 
amino acids (Low-S diet), emphasizing plant-based foods and fat sources; and 2) a diet high in 
sulfur-containing amino acids (High-S diet), emphasizing animal protein and fat sources. A 14-
day washout period will follow each intervention, in which participants will eat their typical 
diets. Intervention order will be randomly assigned through a computer-generated number 
sequence. At the conclusion of the second intervention, participants will be instructed to resume 
normal dietary habits and will be tracked for an additional 7 days to monitor study endpoints and 
related variables. Frequent communication between participants, the registered dietitian, and 
other study investigators will be sustained throughout the trial to ensure compliance with dietary 
and data collection and to reduce attrition.  
 
Participants will be assessed prior to any dietary intervention to determine baseline 
demographics and characteristics, dietary habits, and measurements of endpoints of interest. 
High-S and Low-S meal plans will be designed by a registered dietitian trained in nutrient 
analysis, and detailed descriptions of each intervention will be discussed with participants. 
Participants meeting inclusion criteria will be consented at the screening clinic visit. Routine 
clinical examinations, assessments, laboratory evaluations, and sample collections will be 
completed at 5 clinic visits over a time period spanning approximately 35 days. 
 
3.1 
Study Participants and Recruitment 
Highly motivated participants will be recruited from the Minneapolis-St. Paul metropolitan area. 
All participants will comply with the following predetermined inclusion and exclusion criteria:   
3.1.1 Inclusion Criteria 
Participants will be eligible to participate in the study if all of the following conditions exist: 
1. Provide informed consent 
2. Ambulatory and community dwelling 
3. Able and willing to comply with the study schedule and procedures 
4. 18 - 80 years of age 

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5. BMI between 18.5 – 40.0 kg/m2 
6. Omnivorous or willing to consume animal products through duration of study 
3.1.2 Exclusion Criteria 
Subjects will be excluded from participation in the study if any of the following conditions exist: 
1. Antibiotic use within 3 months 
2. Planned antibiotic therapy within the period of the study, e.g., perioperative antibiotics. 
3. Use of sulfonamides or sulfasalazine, as these sulfur-containing medications may 
confound results 
4. Use of 5-aminosalicylates (5-ASA), as they are known to inhibit sulfidogenesis 
5. History of anaphylactic food allergies, e.g., peanuts, seafood. 
6. Food intolerances and allergies, including gluten sensitivity, lactose intolerance, and 
intolerance of high fiber dietary content. 
7. Strict vegan eating practices, with refusal to consume animal products. 
8. Planned use of oral probiotics while on study. 
9. Serious, concomitant illness that, in the opinion of the Investigator, would interfere with 
evaluation of safety or efficacy, or put the participant at risk of harm from study 
participation.  
10. Significant alcohol use, defined as > 20 g/day in females and > 30 g/day in males for a 
period of 3 months within one year prior to screening. 
11. Underlying chronic gastrointestinal disease that can cause diarrhea, including short bowel 
syndrome, irritable bowel syndrome, malabsorption, and celiac disease. 
12. History of partial or complete colectomy. 
13. History of malabsorptive bariatric surgery. 
14. Currently participating in another clinical study. 
15. Legally incompetent and unable to understand the study’s purpose, significance and 
consequences, and to make decisions accordingly. 
3.2 
Diet Intervention Design 
Two intervention diets will be designed by a registered dietitian trained in nutrient analysis. A 
registered dietitian will provide detailed instructions for each intervention diet, and educational 
materials will be developed to indicate foods with high and low contents of SAAs as well as 
example meal plans for participants to reference. Dietary instructions will be based on the 
following table:  
 
 
 
 
Table 1. Guidelines for intervention diets 

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High-S Diet (Emphasize animal-based foods) 
Low-S Diet (Emphasize plant-based foods) 
Meats: beef, pork, lamb, veal, venison, goat, 
processed meats (ham, bacon, bologna, salami, 
sausages, hot dogs, deli or lunch meats), poultry, 
seafood, shellfish 
Cooked lentils and beans, excluding soy beans or 
soy bean-derived products  
Dairy Products: cheese, milk, yogurt, ice cream, 
sour cream 
Dairy alternatives: almond milk, rice milk, hemp 
milk, coconut milk, cream substitutes 
Eggs  
Egg substitutes 
Animal-derived fats: butter, lard 
Plant-derived fats: oils (e.g., vegetable, olive, 
safflower, cottonseed, cod liver, coconut, peanut, 
sesame) 
Grains: white breads, pastas, rice 
Grains: whole wheat, brown rice, bulgur, buckwheat, 
barley, oats, fiber-containing breakfast cereals  
Limited intake of vegetables  
Fresh/frozen vegetables 
Limited intake of fruit  
All fruit  
Beverages: fruit juices, mineral water, sulfited 
wines, coffee, tea 
Beverages: fruit and vegetable juices, coffee, tea, 
distilled spirits, beer 
Soy Products: soy milk, tofu, tempeh, miso, soy 
crisps, dried soy beans 
Snack foods: granola bars, popcorn, pretzels, potato 
chips, rice cakes 
 
Nuts and seeds 
Condiments: mayonnaise, dairy-based salad 
dressings 
Condiments: oil-based salad dressings; ketchup, 
mustard, canned sauces and gravies 
 
During the Low-S diet intervention, participants will be advised to completely avoid foods high 
in SAAs, strictly eating foods included on the “Low-Sulfur Foods” list. During the High-S 
intervention, participants will be instructed to eat foods strictly from the “High-Sulfur Foods” 
list. Participants will record time and location of food intake and any deviations from the 
prescribed diet in the provided study log. Participants will have access to a registered dietitian 
throughout their participation in the study to assist with any issues regarding the trial or the diet. 
 
3.3 
Dietary Intake Assessment 
Dietary intake data will be collected before, during, and after the intervention period to estimate 
usual total energy intake and macro- and micronutrient composition. Two methods of dietary 
assessment will be used: food frequency questionnaires (FFQs) and 3-day diet records. The 
National Cancer Institute’s Diet History Questionnaire III (DHQ III) is a freely available web-
based FFQ. A web-based version of the DHQ III will be completed by participants at their 
baseline clinic visit (Day 0), in which they will be asked about their usual dietary intake over the 
past year.  
 
Participants will be instructed to record their daily intake of foods and beverages during each 7-
day intervention period. Throughout the intervention, participants will be instructed to maintain 
normal physical activity, sleep habits, and caffeine consumption (coffee and tea). A registered 
dietitian will be in contact with participants throughout the interventions to monitor compliance 
with the High-S and Low-S diets. Three-day diet records will be completed by participants prior 
to the intervention start date, once during each intervention, and twice during the washout period, 

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and one week after the conclusion of the second intervention diet to determine independent, ad 
libitum dietary habits. A registered dietitian trained and certified in the University of 
Minnesota’s Nutrient Data System for Research (NDSR) dietary interviewing and assessment 
protocols will instruct participants on methods for accurate quantification and recording of 
dietary intake. DHQ III data will be analyzed with Diet*Calc software developed by the National 
Cancer Institute. Analysis of 3-day diet records will be performed using NDSR data output.  
 
3.3.1 Tolerability and Feasibility 
There are many difficulties associated with examining the effects of dietary components on 
health, and subject compliance is of major importance to the outcome of any dietary trial. It is 
extremely difficult to control for subject compliance completely; however, daily diet records 
during each intervention and 3-day diet records in each standard diet phase, coupled with 
frequent contact by study staff, will improve participant adherence.  
 
Participants will be provided with a study log at the baseline clinic visit (Day 0). Data recorded 
in the study log will be reviewed by study investigators at each contact with participants and will 
be recorded for data analysis. Participants will be instructed to record the following during each 
day of the study period: 
 Adverse events, with specific attention paid to gastrointestinal symptoms including 
bloating, gas, abdominal pain, constipation, and loose stools 
 New medications and/or any medication use – dose and frequency of administration 
 Time of bowel movements and stool consistency 
 Physical activity 
 
Since the intention of this project is to generate pilot data for a longer randomized trial, 
approximating compliance for a prolonged period of time (≥ 3 months) is necessary for the 
design of future interventions. A questionnaire will be developed to assess participants’ input on 
tolerability and feasibility for each dietary intervention. 
 
3.4 
Sample Collection 
3.4.1 Fecal Samples 
Fecal samples will be collected to assess gut microbial composition (via 16S sequencing) and to 
evaluate concentrations of fecal gases related to sulfur metabolism (via GC). Participants will 
collect samples in disposable commode specimen containers placed under the toilet seat prior to 
a bowel movement. Samples collected from the center of the specimen will be used for DNA 
sequence analysis. Larger collection tubes will be provided for intact stool samples for fecal 
headspace analysis and dry weight determination. The remainder of fecal specimens collected 
on-site will be transferred into sterile containers. Specimens will be put in the freezer until 
transfer to the investigator’s lab. Full samples will be collected at designated study visits. 
Participants will also be asked to collect intermittent samples in collection tubes throughout the 
study. These will be collected on days (±2 days) -4, -2, 3, 5, 9, 11, 16, 18, 23, 25, 30, and 32. 
These samples will be stored in the participant’s freezer and collected at each study visit. 
Participants will also record all bowel movements in their study log. 
 

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3.4.2 Urine Samples 
24-hour urine collections will take place per PI discretion at baseline (Day 0; Day 0±2 days), at 
the end of the first diet intervention (Day 7; Day 5±2 days), at the end of the washout period 
(Day 21; Day 19±2 days), and at the end of the second diet intervention (Day 28; Day 26±2 
days). Participants will collect all urine for a 24-hour period in a collection bottle(s), which will 
be stored in the refrigerator. Participants will be asked to note the exact time they begin and 
complete their urine collection. The entire 24 hour sample (all urine containers) will be returned 
after completion of the 24-hour period. Samples collected will be used for urine sulfate analysis.  
 
3.4.3 Blood Samples 
Blood (10 mL) will be collected per PI discretion in the clinic at visits 1, 2, 3, 4, 5, and 6 in 
Vacutainer Blood Collection Tubes. Blood for clinical laboratory assessment will be collected 
into tubes containing lithium heparin as anticoagulant. The tubes will be inverted carefully 3-5 
times to mix blood and anticoagulant, and kept at room temperature until centrifugation, which 
will be done within 15 minutes of collection. Blood for measurement of TMAO and metabolites 
will be collected into a gel-barrier tube and allowed to clot for 30 minutes. Centrifugation of all 
samples will be carried out for 15 minutes at 1400 RCF (relative centrifugal force) at refrigerated 
temperature. The top layer (serum or plasma) will be collected and frozen at -80oC. 
 
3.5 
Gas Concentration Measurement 
Gases produced by fecal samples will be analyzed by GC. Approximately 10g of fecal material 
will be transferred to a gas-tight septum jar and incubated at 37oC for 2 hours. After incubation, 
5mL gas samples will be removed with a syringe and injected into 10ml headspace vials, pre-
purged with nitrogen gas. H2S and CH3SH concentration will be measured using a gas 
chromatograph outfitted with a sulfur chemiluminescence detector (GC-SCD). CH4 
concentration will be measured using a gas chromatograph with a flame ionization detector (GC-
FID) and CO2/H2 is quantified using a gas chromatograph equipped with a thermo-conductivity 
detector (GC-TCD). Calibration gases will be used as standards for all quantifications. All 
measurements will be normalized to mL per gram (dry weight) fecal material. Dry weight is 
calculated by taking a known mass of wet fecal material, baking it at 95oC under vacuum for ≥ 
16 hours, weighing the dried fecal material and calculating the ratio of dry weight / wet weight. 
 
3.6 
16S Sequencing 
Fecal bacteria composition will be characterized using high throughput 16S rRNA gene sequence 
analysis. 
3.6.1 DNA Extraction from Fecal Samples 
DNA will be extracted from ~0.25g aliquots of fecal samples using a PowerSoil DNA Extraction 
Kit (Mo Bio Laboratories, Carlsbad, CA) performed according the manufacturer’s instructions. 
Approximately 100 ng of DNA will be used for PCR amplification of the 16S rRNA gene in 
samples. 
 
3.6.2 Amplification of 16S rRNA Gene and Sequencing 
The V5+V6 hypervariable regions of the 16s rRNA gene will be amplified using primers specific 
for that region.  Amplicons obtained from individual samples will be pooled in equimolar ratios 

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prior to sequencing.  Paired end sequencing will be performed using the Illumina MiSeq 
(Illumina Inc, San Diego, CA) platform at the University of Minnesota Genomics Center 
(UMGC).  Sequence data will be analyzed using the MOTHUR program30 for taxonomic 
assignment and alpha/beta diversity.  Sequence datasets will be compared by principle 
component analysis. 
 
3.7 
Fecal Bile Acid Analysis  
Bile acids will be detected and quantified using liquid chromatography – mass spectrometry 
(LC-MS). Bile acids will be extracted from fecal material by sonication in acetonitrile, with the 
resulting extract injected directly into the LC-MS.  All quantifications will be based on a C13 
labeled internal standard and standard curves generated from known concentrations of each bile 
acid. 
 
The investigator’s lab will store all of the stool samples as well as the urine and blood samples 
collected for metabolomics.  This laboratory is located at: 
 
Wallin Medical Biosciences Building 
      University of Minnesota 
      2101 6th Street SE 
      Minneapolis, MN 55414 
 
3.8 
Treatment Assignment 
This is a randomized, open-label crossover study. Each participant will receive both dietary 
interventions. The sequence in which participants receive the interventions will be determined 
through computer-generated random number assignment prepared by the study statistician.  
 
3.9 
Withdrawal 
3.9.1 Reasons for Withdrawal 
A study participant will be discontinued from the study for: 
 Completion of the study; 
 Request by participant to terminate participation; 
 Requirement for prohibited concomitant medication or treatment; 
 Unable to comply with requirements of the protocol; 
 Lost to follow-up; 
 At the request of the institutional review board (IRB); 
 The participant’s well-being, based on the opinion of the investigator. 
 
Participants who are discontinued from further study agent/interventions will be followed for 
safety until completion of the normal visit schedule. Participants will be contacted by phone 
within one week after withdrawal, and again after one month. 
3.9.2 Handling of Withdrawal 
Participants will be encouraged to complete the study; however, they may voluntarily withdraw 
at any time. The sponsor-investigator will provide a written explanation of the reason for 

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withdrawal in a source document and the reason will be recorded on a case report form. 
Participants will be asked for permission to continue scheduled evaluations, and complete an 
end-of-study evaluation. Medical care that may be required for management of adverse events in 
the course of the study will be charged to the medical insurance of the participant.  
3.10 Study Termination 
If the sponsor-investigator or appropriate regulatory officials discover conditions arising during 
the study that indicate that the study should be halted, this action may be taken after appropriate 
consultation between the  sponsor-investigator, study statistician, and independent medical 
monitor. Conditions that may warrant termination of the study include, but are not limited to, the 
following: 
 The discovery of an unexpected, serious, or unacceptable risk to the participants enrolled 
in the study, 
 A decision on the part of the sponsor-investigator to suspend or discontinue testing or 
evaluation of the intervention. 
 
4 
STUDY PROCEDURES AND EVALUATION SCHEDULE 
4.1 
Clinical/Laboratory Evaluations and Study Schedule 
Consent will be obtained before any clinical evaluations are performed. There is one screening 
visit and 6 visits planned over the approximately 1 month study duration for each subject. The 
study will last until every evaluable subject has completed the Safety Follow-Up Visit. (See 
Schedule of Events in the Appendices). Of note, Vital signs and collection of blood samples will 
be completed based on PI discretion, except for the Screening and Safety Follow-up Visit. 
4.2 
Initial Screening Visit  
The study will be explained to potential participants qualified from initial recruitment. Consent 
will be obtained at this time that will go over all the procedures and studies involved. During this 
visit the medical history will be collected and inclusion/exclusion criteria will be thoroughly 
reviewed. The following procedures will occur after the consent form is signed: 
 Thorough review of the study, including involved procedures 
 Medical history, including medications over the previous 12 months, and demographics 
 Vital signs (heart rate, blood pressure, respiration rate) 
 Height and weight measurement 
 Review of methods used to record and measure food consumption during the study, to 
be conducted by dietitian. 
 Three-day diet record is initiated following this visit, to be completed prior to visit 1 
  
(baseline). 
 Urine collection kit provided, to be completed prior to visit 1 
 Link provided for web-based NCI Diet History Questionnaire III (DHQ III) to assess the 
baseline diet of subjects prior to entry into the study 
 
4.3 
Visit 1 - Baseline (Day 0) 
The following procedures are conducted during this visit: 
 Measurement of vital signs  
 Weight measurement 

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 Collection of blood samples for clinical labs and research measurements 
 Fecal sample collection 
 24-hour urine sample content collection 
 Dietitian consult and review of 3-day diet record 
 Assignment of first intervention diet, to begin immediately after conclusion of visit 
 Receive instructions on completing adverse events diary card 
 Review of adverse events and concomitant medications 
 
4.4 
Visit 2 (Day 7 ± 2 days; Final day of first diet intervention) 
 Vital signs  
 Weight  
 Collection of blood samples for clinical labs  
 Collection of stool sample for research measurements 
 Review of 3-day diet record with dietitian 
 Review of adverse events and concomitant medications 
 24-hour urine sample content collection 
 Urine collection kit provided, to be completed prior to visit 3 
 Review of adverse events and concomitant medications 
 
4.5 
Visit 3 (Day 14 ± 2 days) 
 Vital signs  
 Weight  
 Collection of blood samples for clinical labs and research measurements 
 Collection of stool sample for research measurements 
 24-hour urine sample content collection 
 Review of 3-day diet record with dietitian 
 Review of adverse events and concomitant medications 
 
4.6 
Visit 4 (Day 21 ± 2 days; First day of second diet intervention) 
 Vital signs  
 Weight  
 Collection of blood samples for clinical labs  
 Collection of stool sample for research measurements 
 Assignment of second intervention diet, to begin immediately after conclusion of visit 
 Review of 3-day diet record with dietitian 
 Review of adverse events and concomitant medications 
 Urine collection kit provided, to be completed prior to visit 5 
 
4.7 
Visit 5 (Day 28 ± 2 days; Final day of second diet intervention) 
 Vital signs  
 Weight  
 Collection of blood samples for clinical labs and research measurements 
 Collection of stool sample for research measurements 
 24-hour urine sample content collection 

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 Review of 3-day diet record with dietitian 
 Review of adverse events and concomitant medications 
4.8 
Visit 6 (Day 35 ± 2 days; Follow-up visit) 
 Vital signs  
 Weight  
 Collection of blood samples for clinical labs and research measurements 
 Collection of stool sample for research measurements 
 24-hour urine sample content collection 
 Review of 3-day diet record with dietitian 
 Review of adverse events and concomitant medications 
 
 
4.9 
Unscheduled Visits 
If at any time an unscheduled visit, including phone calls, should occur throughout the duration 
of the study, the following procedures will be completed: 
 Adverse Events 
 Concomitant medications 
 Ask for any concerns about study procedures 
 Physical examination and review of medical history as needed 
 Order any lab tests as clinically indicated 
 
4.10 Participant Compensation 
Participants can receive up to a total of $150 for participation in the study; $25 paid for the 
completion of each study visit (a total of 6 study visits).  Payment will be received in the form of 
gift cards. 
4.11 Potential Risks  
4.11.1 Risk of gastrointestinal symptoms 
Changes in dietary intake can trigger gastrointestinal symptoms. However, these effects are 
typically minor in nature and occur in a small percentage of healthy volunteers. 
4.12 Known Potential Benefits 
IBD is a very complex condition and dietary intervention is only one component of a treatment 
and prevention plan. This study will generate information necessary to carry out a follow-up 
study specifically designed for patients with UC. We hope it will constitute an important step 
toward understanding the mechanisms of this disorder and contributions of diet and the gut 
microbiota to its pathophysiology. The potential benefits, therefore, are primarily societal in 
terms of enhancing understanding and ultimately finding better ways to treat this condition. 
4.13 Risk/Benefit Ratio 
Overall the risk of participating in this study is small. The benefit is primarily societal. 
 
5 
DATA SAFETY AND MONITORING PLAN 
5.1 
Definitions 
Adverse Event (AE) 

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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 will be regarded as 
adverse events.  Abnormal results of study procedures are considered to be AEs if the 
abnormality: 
 
 Results in study withdrawal 
 Is associated with a serious adverse event (SAE) 
 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. 
 
Adverse Reaction 
An adverse reaction is any adverse event caused by the investigational agent.  Adverse reactions 
are a subset of suspected adverse reactions. 
 
Suspected Adverse Reaction 
A suspected adverse reaction is an adverse event for which there is a reasonable possibility that 
the investigational agent caused the adverse event. 
 
Serious Adverse Event (SAE) 
A serious adverse event (SAE) is any adverse event that is: 
 Fatal 
 Life-threatening 
 Requires or prolongs a hospital stay 
 Results in persistent or significant disability or incapacity 
 A congenital anomaly or birth defect 
 
Important medical events are events that may not be immediately life-threatening, but are clearly 
of major clinical significance and may be SAEs.  They may jeopardize the subject, and may 
require intervention to prevent one or the other serious outcomes noted above.   
 
Hospitalization  
Hospitalization shall include any initial admission (even if less than 24 hours) to a healthcare 
facility as a result of a precipitating clinical adverse event; to include transfer within the hospital 
to an intensive care unit.  Hospitalization or prolongation of hospitalization in the absence of a 
precipitating, clinical adverse event (e.g., for a preexisting condition not associated with a new 
adverse event or with a worsening of the preexisting condition; admission for a protocol-
specified procedure) is not, in itself, a serious adverse effect. 
 
Expected Adverse Event 
Expected adverse events are those that are known to be associated with or have the potential to 
arise as a consequence of participation in the study. 
 
Unexpected Adverse Event 

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An adverse event or suspected adverse reaction is considered unexpected if it is not listed in the 
Protocol at the specificity or severity that has been observed. 
 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
An adverse event that in the opinion of the Principal Investigator is unexpected and related to the 
investigational agent. 
 
Assessment of Severity 
The sponsor-investigator will make an assessment of severity for each AE and SAE reported 
during the study. The assessment will be based on the sponsor-investigator’s clinical judgment. 
The severity of each AE recorded will be assigned a severity based on Common Terminology 
Criteria for Adverse Events (CTCAE) grading. 
 
Assessment of Causality 
The sponsor-investigator will estimate the relationship between the investigational agent and the 
occurrence of each AE or SAE by using his best clinical judgment. Other elements, such as the 
history of the underlying disease, concomitant therapy, other risk factors, and the temporal 
relationship of the event to administration of the investigational agent, will be considered and 
investigated.  
 
An SAE may be recorded when the sponsor-investigator has minimal information to include in 
the initial report.  The sponsor-investigator may change his opinion of the causality in light of 
follow-up information, with subsequent amendment of the SAE report.  
 
 
 
 

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Categories 
Definition 
Definitely related 
This relationship suggests that a definite 
causal relationship exists between the 
administration of the investigational agent 
and the AE, and other conditions 
(concurrent illness, progression/expression 
of disease state, or concurrent medication 
reaction) do not appear to explain the 
event. 
Probably related 
This relationship suggests that a reasonable 
temporal sequence of the event with 
investigational agent administration exists 
and, based upon the known or previously 
reported adverse reactions, or judgment 
based on the investigator’s clinical 
experience, the association of the event 
with the investigational agent seems likely. 
Possibly related 
This relationship suggests that treatment 
with the investigational agent may have 
caused or contributed to the AE (i.e., the 
event follows a reasonable temporal 
sequence from the time of investigational 
agent administration and/or follows a 
known response pattern to the 
investigational agent but could also have 
been produced by other factors.) 
Not related 
This relationship suggests that there is no 
association between the investigational 
agent and the reported event. 
 
5.2 
Recording of Adverse Events 
At each contact with the subject, the investigator will seek information on adverse events by 
specific questioning, reviewing the adverse events diary card, and, as appropriate, by 
examination. Information on all AEs will be recorded immediately in the source document and 
the appropriate AE module of the case report form (CRF). All clearly related signs, symptoms, 
and abnormal diagnostic procedures results will be recorded in the source document. 
 
All AEs occurring during the study period will be recorded. The clinical course of each event 
will be followed until resolution, stabilization, or until it has been determined that the study 
treatment or participation is not the cause. AEs still unresolved at the conclusion of the study will 
be followed up on for one month. SAEs still ongoing at the end of the study period will be 
followed up to determine the final outcome. Any SAE that occurs after the study period and is 

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considered to be possibly related to the study treatment or study participation will be recorded 
and reported immediately. 
 
Expected adverse events include: 
 Events associated with venipunctures. 
o Discomfort and slight bruising. 
 Events associated with dietary modification. 
o Changes in bowel habits, including diarrhea and/or constipation 
 
Possible, although unlikely, adverse events that may be encountered include: 
 Events associated with venipunctures. 
o Bruising 
o Bleeding 
o Lightheadedness, fainting 
o Infection at the venipuncture site 
o Nausea 
o Anxiety 
o Swelling at the venipuncture site 
 
5.3 
Adverse Event Management 
If an adverse event occurs that requires clinical management, the subject will be evaluated at an 
unscheduled study visit with a physical exam. Tests and treatments that may be clinically 
indicated will be ordered.  
5.4 
Reporting of Serious Adverse Events 
5.4.1 IRB Notification by Sponsor Investigator 
Reports of all serious adverse events (including follow-up information) must be submitted to the 
IRB within the reporting timeline requirements, if the SAE falls under the UPIRTSO guidelines.  
Copies of each report and documentation of IRB notification and receipt will be kept in the 
Clinical Investigator’s binder. 
5.4.2 UPIRTSO Events 
Upon first learning of a UPIRTSO event, investigators are required to submit a report of the 
applicable event(s) to the IRB within the required reporting timeline. 
 
5.5 
Temporary Interruption of Intervention in an Individual Subject 
If a participant does not or cannot comply with the assigned diet intervention, further eligibility 
will be assessed by the Principal Investigator. A participant may be able to continue in the study 
if the interruption is temporary (1-2 days).  
 
6 
STATISTICAL CONSIDERATIONS 
6.1 
Sample Size Considerations 
This feasibility study plans to enroll 17 participants who will be assigned to receive two dietary 
interventions in a random order. We anticipate that 2 subjects may drop out from the study for 
various reasons, including inability to comply with the study visit schedule or new medical 

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problems unrelated to the study, e.g., use of antibiotics for an infection. The intention of this 
study is to generate data which will inform the sample size necessary for a larger trial, so power 
calculations have not been performed. 
 
We will compare primary, secondary, and exploratory outcome measures within each 
intervention (Day 0 vs. Day 7) and between the two interventions (High-S vs. Low-S) using 
paired t-tests. 
6.2 
Randomization 
This is a randomized, open-label crossover study. Each participant will receive both dietary 
interventions. The sequence in which participants receive the interventions will be determined 
through computer-generated random number assignment prepared by the study statistician.  
 
6.3 
Blinding 
The clinical co-investigators will remain blinded to the intervention. Due to the nature of the 
intervention, the PI and other personnel giving dietary instructions, and participants will not be 
blinded. All investigators will be blinded to the randomization schedule and measured endpoints.    
 
6.4 
Planned Interim Analyses 
Independent monitoring of the clinical study for compliance will be conducted periodically (at a 
minimum of every 6 months) by qualified staff of the University of Minnesota in accordance 
with the established monitoring plan. No interim analyses are planned for this study. 
 
6.5 
Safety Review 
To minimize risk, cumulative safety data will be reviewed by the sponsor-investigator and an 
independent medical monitor. Medical monitoring will be initiated by the occurrence of adverse 
events as they arise. This safety monitoring will include careful assessment and appropriate 
reporting of AEs as noted above. Medical monitoring will include a regular assessment of the 
number and type of SAEs. Study enrollment and dosing will be stopped and an ad hoc review 
will be performed if any of the following occurs: 
1. Occurrence of a life-threatening allergic/hypersensitivity reaction (anaphylaxis), 
manifested by bronchospasm with or without urticaria or angioedema;  
2. An overall pattern of symptomatic, clinical, or laboratory events that the sponsor-
investigator, scientific liaisons, regulatory affairs manager and statistician consider 
associated with the intervention and that may appear minor in terms of individual events 
but that, collectively, may represent a serious potential concern for safety; 
3. Any SAE that is possibly, probably, or definitely related to the intervention; 
4. Two or more subjects experience the same CTCAE Grade 3 or higher adverse event that 
is possibly, probably, or definitely related to study product administration. 
 
7 
DATA AND RECORD KEEPING 
7.1 
Data Capture Methods 
Data for this study will include safety, laboratory and outcome measures. Clinical data (including 
AEs and concomitant medications) and clinical laboratory data will be managed by the principal 

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investigator, co-investigators, study coordinator, and biostatistician. Data will be entered into 
REDCap, an internet data entry system provided by the Clinical and Translational Science 
Institute. The data system includes password protection and internal quality checks to identify 
data that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly 
from the source documents.  
 
7.2 
Source Documents 
A source document is defined as the location where study-related data are initially recorded.  
Source documents for this study will include hard copy paper and/or electronic forms, laboratory 
printouts, and medical records onto or into which data will first be recorded. 
 
7.3 
Study Records Retention 
Per University of Minnesota policy all documents concerning the use of human subjects in 
research will be maintained for at least 3 years from completion of IRB-related work and at least 
6 years for HIPAA. No record will be destroyed without the written consent of the sponsor-
investigator. 
 
The sponsor-investigator will permit authorized representatives of the University of Minnesota 
and regulatory agencies to examine (and when required by applicable law, copy) clinical records 
for the purposes of clinical site monitoring, quality assurance reviews, audits, and evaluation of 
the study safety and progress. 
 
7.4 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol. The noncompliance 
may be either on the part of the subject, the investigator, or the study site staff. As a result of 
deviations, corrective actions are to be developed by the site and implemented promptly.  
 
When a deviation from the protocol is necessary for an individual subject, the Investigator must 
complete a description of the deviation from the protocol and justification on the Protocol 
Deviation Form. It will not be considered a protocol deviation if a subject is unable to provide a 
stool sample at any visit that requires a stool sample.  
 
8 
QUALITY CONTROL AND QUALITY ASSURANCER 
8.1 
Dietary Intake 
Agreement of participants’ short- and long-term dietary intake will be assessed through 
comparison between NDSR and DHQ databases at baseline and Visit 6.   
 
9 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
9.1 
Ethical Standard 
The investigator(s) will ensure that this study is conducted in conformity with the principles of 
The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Participants 
of Research of The National Commission for the Protection of Human Participants of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 312, 

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and/or ICH E6; 62 Federal Regulations 25691 (1997). The University of Minnesota holds a 
current federal-wide assurance (FWA) issued by OHRP for federally funded research. 
 
9.2 
Institutional Review Board 
The Human Participants Protection Program at the University of Minnesota will be asked to 
review and approve this protocol, associated consent documents, and recruitment materials. 
Approval of any amendments to the protocol or consent materials will also be requested before 
they are implemented.  
 
10 INFORMED CONSENT PROCESS 
The informed consent process will be initiated before a volunteer agrees to participate in the 
study and will continue throughout the individual’s study participation. The participant will sign 
the informed consent document before any procedures are undertaken for the study. A copy of 
the signed informed consent document will be given to the participant for his/her records. The 
consent will explain that participants may withdraw consent at any time throughout the course of 
the trial.  Extensive explanation and discussion of risks and possible benefits of this investigation 
will be provided to the participants in understandable language. Adequate time will be provided 
to ensure that the participant has time to consider and discuss participation in the protocol.  
 
The consent form will describe, in detail, the study interventions / procedures and risks / benefits 
associated with participation in the study. The rights and welfare of the participants will be 
protected by emphasizing that their access to and the quality of medical care will not be 
adversely affected if they decline to participate in this study. 
 
10.1 Participant Confidentiality 
Participant confidentiality is strictly held in trust by the participating investigator, his staff, and 
their agents. This confidentiality includes documentation, investigation data, participant’s 
clinical information, and all other information generated during participation in the study. 
 
No information concerning the study or the data generated from the study will be released to any 
unauthorized third party without prior written approval of the sponsor and the participant. 
 
The study monitor or other authorized representatives of governmental regulatory agencies may 
inspect all documents and records required to be maintained by the investigator, including but 
not limited to, medical records (office, clinic, or hospital) and pharmacy records for the 
participants in this study. The clinical study site will permit access to such records. 
 
10.2 Principal Investigator Responsibility when Participant Withdraws or is Discontinued 
If a participant terminates the study early, and is willing, the tests and procedures that would 
occur at all forthcoming study visits will be completed. 
 
10.3 Future Use of Stored Specimens 
Blood samples collected during the study will be sent to and processed by hospital laboratory 
personnel at University of Minnesota Medical Center, Fairview. Blood will be discarded. 

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11 PUBLICATION POLICY 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials-registration policy as a condition for publication. This policy requires that all clinical 
trials be registered in a public trials registry such as ClinicalTrials.gov, which is sponsored by the 
National Library of Medicine. This protocol will be registered on ClinicalTrials.gov.  
 
 

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inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 
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2. Kappelman MD, Rifas-Shiman SL, Porter CQ, et al. Direct health care costs of crohn's disease 
and ulcerative colitis in US children and adults. Gastroenterology. 2008;135(6):1907-1913.  
 
3. Benchimol EI, Manuel DG, Guttmann A, et al. Changing age demographics of inflammatory 
bowel disease in ontario, canada: A population-based cohort study of epidemiology trends. 
Inflamm Bowel Dis. 2014;20(10):1761-1769.  
 
4. Burisch J, Jess T, Martinato M, Lakatos PL, ECCO -EpiCom. The burden of inflammatory 
bowel disease in europe. J Crohns Colitis. 2013;7(4):322-337.  
 
5. Burisch J, Pedersen N, Cukovic-Cavka S, et al. East-west gradient in the incidence of 
inflammatory bowel disease in europe: The ECCO-EpiCom inception cohort. Gut. 
2014;63(4):588-597.  
 
6. Kappelman MD, Moore KR, Allen JK, Cook SF. Recent trends in the prevalence of crohn's 
disease and ulcerative colitis in a commercially insured US population. Dig Dis Sci. 
2013;58(2):519-525.  
 
7. Cosnes J, Gower-Rousseau C, Seksik P, Cortot A. Epidemiology and natural history of 
inflammatory bowel diseases. Gastroenterology. 2011;140(6):1785-1794.  
 
8. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human 
gut microbiome. Nature. 2014;505(7484):559-563. 
 
9. Roediger WE, Duncan A, Kapaniris O, Millard S. Reducing sulfur compounds of the colon 
impair colonocyte nutrition: Implications for ulcerative colitis. Gastroenterology. 
1993;104(3):802-809.  
 
10. Levine J, Ellis CJ, Furne JK, Springfield J, Levitt MD. Fecal hydrogen sulfide production in 
ulcerative colitis. Am J Gastroenterol. 1998;93(1):83-87. 
 
11. Magee EA, Richardson CJ, Hughes R, Cummings JH. Contribution of dietary protein to 
sulfide production in the large intestine: An in vitro and a controlled feeding study in humans. 
Am J Clin Nutr. 2000;72(6):1488-1494. 
 
12. Magee E, Richardson C, Cummings J. Dietary precursors of sulphide in the human large 
intestine. Proc Nutr Soc. 2001(60):16A. 
 

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13 Appendix 1. Schedule of Events 
Procedures 
Screening 
Baseline       
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
  
 
0 
Day 7 
Day 14 
Day 21 
Day 28 
Day 35 
Obtain consent 
X 
  
  
  
  
  
 
Medical history 
X 
  
  
  
  
  
 
Vitals* 
X 
X  
 X 
X 
 X 
X 
X 
Height 
X 
  
  
  
  
  
 
Weight 
X 
X  
X 
X 
X  
X 
X 
Clinical labs1*  
 
X 
 X 
X 
X 
X 
X 
Fasting blood for TMAO and metabolites* 
 
X 
 
X 
 
X 
X 
Provide stool sample** 
 
X 
 X 
X 
X 
X 
X 
24-hour urine collection* 
 
X 
X 
X 
 
X 
X 
Diet assignment 
 
X 
 
 
X 
 
 
Dietary History Questionnaire 
  
X 
  
  
  
  
 
Provide food diary 
X 
X 
X 
X 
X 
X 
X 
Review food diary 
  
X 
X 
X 
 X 
X 
X 
Adverse events/Concomitant medications 
 
X 
X 
X 
X 
X 
X 
        1Clinical labs include: fasting lipid panel and C-reactive protein (CRP)  
     *Optional based on PI discretion 
     **Intermittent stool samples (in frozen collection tubes) will be dropped off at each respective visit following collection. Samples will be 
collected on days (±2 days) -4, -2 3, 5, 9, 11, 16, 18, 23, 25, 30, and 32.