Association of Vitamin D Supplementation and Normal Dietary Intake of Vitamin D With Changes in the Gut Microbiome and Markers of Colorectal Cancer Risk
Phase: NA
Conditions: Colorectal Cancer
Sponsor: Baylor University

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Study Protocol 
 
Project Title:  Randomized placebo controlled trial of vitamin D supplementation and normal 
dietary intake of vitamin D and changes in the gut microbiome and markers of colorectal cancer 
risk 
 
Short Title:  Vitamin D Microbiome Trial (VDMT) 
 
Principal Investigator:  
K. Leigh Greathouse 
Nutrition Sciences, Robbins College of Health and Human Sciences 
Baylor University 
 
Version Number: 3 
Version Date: March 3, 2022 
 
Confidential 
 
 
 
 

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Revision History 
Protocol 
Version 
Protocol 
Date 
Summary of Changes 
Consent 
Change? 
Consent 
Date? 
Version 1 
Nov 23, 2021 
 
New 
 
Version 2 
Jan 21, 2022 
Removed urine collection and extended study 
timeline and supplement consumption 
Yes 
 
Version 3 
Mar 3, 2022 
Added study visit and GPAQ 
Yes 
 
 
 
 
 
 
 
 
Contact Information 
 
Principal Investigator 
 
K. Leigh Greathouse, PhD, MPH, MS, RD 
Associate Professor, Nutrition Sciences 
Affiliate Professor of Biology 
Institute of Biomedical Studies 
Director, Didactic Program in Dietetics 
One Bear Place 97346 
Waco, TX  76798-7346 
Phone: 254-710-7619 
Email: Leigh_Greathouse@baylor.edu 
 
 
 

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Table of Contents 
Revision History ................................................................................................................................... 2 
1 
Introduction ................................................................................................................................. 5 
2 
Background, Rationale, and Aims ................................................................................................. 5 
2.1 
Relationship between Dietary Intake and the Microbiome .......................................................... 5 
2.2 
Vitamin D Intervention ................................................................................................................. 5 
3 
Experimental Design ..................................................................................................................... 7 
3.1 
Study Outline ................................................................................................................................ 7 
3.2 
Visit/Intervention Schedule .......................................................................................................... 8 
4 
Statistical Design and Microbiome Analysis .................................................................................. 8 
4.1 
Sample Size .................................................................................................................................. 9 
5 
Participant Selection .................................................................................................................... 9 
5.1 
Inclusion Criteria .......................................................................................................................... 9 
5.2 
Exclusion Criteria .......................................................................................................................... 9 
5.3 
Recruitment ............................................................................................................................... 10 
5.4 
Protections for the Use of Students as Research Subjects ......................................................... 10 
6 
Study Procedures ....................................................................................................................... 11 
6.1 
Demographics and Anthropometrics ......................................................................................... 11 
6.2 
Fecal Sample Collection .............................................................................................................. 12 
6.3 
Dietary Record Keeping .............................................................................................................. 12 
6.4 
Blood Draw ................................................................................................................................ 14 
6.5 
Follow-up ................................................................................................................................... 15 
6.6 
Microbiome Analysis .................................................................................................................. 15 
7 
Participant Withdrawal .............................................................................................................. 15 
8 
Assessment of Safety .................................................................................................................. 15 
8.1 
Safety of biological sample collection ........................................................................................ 15 
8.2 
Safety of dietary supplement  intervention ................................................................................ 15 
9 
Potential Benefits ....................................................................................................................... 16 
10 
Study Timeline/Duration ........................................................................................................ 16 
11 
Data Management .................................................................................................................. 16 
11.1 
Data Collection ........................................................................................................................... 16 
11.2 
Monitoring Plan ......................................................................................................................... 17 
12 
Administrative Considerations ................................................................................................ 17 
12.1 
Research Ethics Approval ........................................................................................................... 17 
12.2 
Confidentiality ............................................................................................................................ 17 
12.3 
Declaration of Interests .............................................................................................................. 18 

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13 
References .............................................................................................................................. 19 
 
 
 

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1 
Introduction 
Increase in early onset colorectal cancer (EOCRC) has been dramatic since 2012, with a 46% 
increase among those under 50. Recent data indicate a strong risk factor for EOCRC is 
insufficient dietary intake of vitamin D, and vitamin D insufficiency among adults in the US is 
42%, with those ages 20-39 with the highest prevalence. Evidence indicates that dietary and 
circulating vitamin D levels are regulators of immune homeostasis, in part by altering the gut 
microbiome. The human gut microbiome is the community of bacteria that reside within the 
human intestine, and their composition and function are strongly linked to CRC. Very little is 
known about the complex interaction of vitamin D with the microbiome over time in one 
individual, and its impact on markers of CRC risk. In order to produce robust analysis of these 
interactions, longitudinal samples with detailed dietary intake information from healthy human 
subjects consuming vitamin D or placebo are needed.  
2 
Background, Rationale, and Aims  
2.1 
Relationship between Dietary Intake and the Microbiome 
Aim 1:  To elucidate the complex relationship between the diet and microbiome using 
longitudinal data and mathematical modeling. 
 
The complex relationship between dietary intake and the microbiome, and the potential health 
implications of human exposure to microbial metabolites, are only beginning to be understood.1 
It is well known that altered dietary intake can trigger rapid, although transient, changes in the 
composition of the microbiome in as little as 1 to 2 days.2–4 The biggest factors in determining 
microbial response to diet are thought to include an individual’s starting microbiome, long-term 
dietary habits, and environmental exposures.5,6 However, there is some controversy over the 
extent to which long-term dietary patterns (i.e. vegan v. omnivore) influence the composition of 
the microbiome.6 
 
We still do not have a good understanding of how small dietary differences from day-to-day 
impact the microbiome. Recently the application of machine learning to a large dataset of 
microbiome, diet, anthropometrics, and blood test data was successfully used to predict blood 
glucose response to variable meal compositions.7 This finding suggests that personalized 
nutrition and optimal diets for health may be feasible in the future. A longitudinal dataset with 
accurately recorded dietary data and multiple samples over 12 weeks will provide valuable 
insight into the changes that occur at the individual level over time, while controlling for dietary 
trends and initial microbiome composition. 
2.2 
Vitamin D Intervention 
Aim 2:  To identify shifts in the composition of the human microbiome and CRC risk factors 
specifically caused by the consumption of Vitamin D supplements.  
 

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Vitamin D is one of 4 fat-soluble vitamins that are only obtained from dietary or supplementary 
intake, and which is necessary for healthy immune function, bone metabolism and electrolyte 
absorption.8–11 Since the 1980’s intake vitamin D containing foods, milk, eggs, fish and 
mushrooms has been declining in the United States.12 Most concerning has been the sharp 
decline in consumption of cow’s milk, which is a primary source of vitamin D, particularly among 
young females.13  Unfortunately, there has also been a sharp rise in EOCRC since 2000, from a 
rate of 5.9 cases/100,000 to 8.5 in 2018 (SEER). Projections for 2030 suggest 23% of rectal 
and 11% colon cancer will be diagnosed in US adults under the age of 50.14 Recently, however, 
a study using incident cases of EOCRC from the Nurses’ Health Study II, demonstrated a 50% 
reduced risk of EOCRC from total dietary intake of vitamin D >/= 450 IU/day vs <300 IU/day (HR 
= 0.40, 95% CI, 0.26-0.93).15  
 
Recent studies have determined the impact of habitual vitamin D intake and vitamin D 
supplementation on the human microbiome, and demonstrated beneficial modifications in 
composition.16 Specifically, 12 weeks of weekly oral dose of 50,000 IU vitamin D3 in 80 women 
increased the Bacteroides/Firmicutes ratio and increased probiotic bacteria Akkermansia and 
Bifidobacterium.16 It is yet unclear mechanistically how Vitamin D alters the composition and 
function of the microbiome, but several lines of evidence indicate a bi-directional interaction. 
Indirect effects of Vitamin D on the microbiota include systemic effects through the vitamin D 
receptor (VDR), which is expressed not only in the proximal colon but also functions as a 
transcription factor in a large number of genes that control immune response.17,18 Likewise, 
specific bacteria influence VDR expression in host cells.19,20  
 
In addition to vitamin D intake, the gut microbiome structure and function is also a significant 
risk factor for CRC development. Multiple studies demonstrate significant loss of diversity, and 
increases in pathogenic bacteria, Fusobacterium nucleatum and Bacteroides fragilis, are risk 
factors for CRC development and progression. Recent preliminary data using data from The 
Cancer Genome Atlas, demonstrated a distinct intra-tumor microbial composition in EOCRC vs 
late-onset, along with positive association between T regulator cells and inflammasome 
pathway.21 Thus, it is possible that the vitamin D insufficiency or deficiency may be also 
affecting gut microbiome structure and function negatively, increasing EOCRC risk.  
 
Although dietary vitamin D supplementation has been used in the clinical setting for decades, 
the effect of supplementary vitamin D consumption on the structure of the microbiome has not 
been studied in humans in fine scale or with concomitant adjustment for dietary intake. 
Understanding the interaction of vitamin D with the microbiome in humans could lead to 
important advancements in the understanding of how vitamin D together with diet impacts the 
microbiome composition, and ultimately, risk of EOCRC. This study has the potential to lay the 
ground work for an adjunctive therapy to manipulate the microbiome to reduce risk of EOCRC. 
This proposed study is designed to evaluate the effect of vitamin D supplementation on the 
normal structure of the microbiome and data will not be used to diagnose, prevent, cure 
or treat disease.  

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3 
Experimental Design 
Study participants will complete 16 days of active study involvement after providing informed 
consent on day 0, which will be conducted over a period of 12 weeks. There will be 4 in-person 
visits, including the consent visit where the participants will have contact with the researchers. 
Visit 1 will be the consent visit and participants who consent will receive training on how to 
collect stool samples, take supplements and dietary record keeping with an online diet record 
tool called ASA24. Visit 1 will include a fasting blood draw and body composition measurement. 
At visit 1 participants will receive stool collection kits for week 1 and their supply of dietary 
intervention supplements (gummies) as well as written instruction on how many gummies to 
take each day during the intervention period. They will also complete a food frequency 
questionnaire, the DHQIII online. At visit 2 participants will receive collection kits for week 2 and 
their supply of dietary intervention gummies as well as written instruction on how many 
gummies to take each day during the intervention period. At study visit 3, participants will 
complete a fasting blood draw, stool kit for Study day 16, and intervention gummies. At visit 4, 
this will conclude the study. Participants will complete a fasting blood draw, body composition 
measurement, dietary recall and mental health survey. The process for returning fecal samples 
is described below in Section 6.2. 
3.1 
Study Outline 
Study Day 0 (Study Visit 1) 
• 
Fasted blood draw 
• 
Body composition 
• 
DHQIII online 
• 
Stool history 
• 
Mental health questionnaires  
  
Study Days 1-14  
• 
Consume vitamin D or placebo as gummies for 14 days 
• 
Consume normal diet and keep detailed dietary records using ASA24 (daily 24-hour 
recalls) 
• 
Collect 1 stool sample per day for 14 days 
• 
Study Visit 2/Study day 7 – delivery of 2nd week of stool collection kits 
 
Study Day 15 (Study Visit 3) 
• 
Fasted blood draw  
• 
Provide vitamin D or placebo gummies to last through week 9 
• 
Stool history 
• 
Post self-perceived stress scale 
 
Study Day 16 (Study Visit 4) 
• 
Provide remainder vitamin D or placebo gummies to last through week 12 
• 
Provide stool collection kit for Study day 16 

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Study Day 16 (Study Visit 5) 
• 
Fasted blood draw  
• 
Stool history 
• 
ASA24 dietary record 
• 
Body composition 
• 
Collect final stool sample 
• 
Post self-perceived stress scale 
• 
Global Physical Activity Questionnaire (GPAQ) 
3.2 
Visit/Intervention Schedule 
Study Visit Number 
1 
 
 
 
 
 
 
2 
 
 
 
 
 
 
 
3 
4 
5 
Study day (0-15) 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Week of study 
1 
2 
 
 
 
 
 
2 
 
 
 
 
 
 
3 
 
 
12 
Decide if you want 
to participate 
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Study training 
!"
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study checkup 
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Complete 
questionnaires 
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Measure body 
composition 
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Collect stool 
 
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Record diet 
 
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Take dietary 
supplement 
 
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Fasting blood draw 
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4 
Statistical Design and Microbiome Analysis 
This longitudinal study will allow for the use of each subject as their own control. We will perform 
testing for associations between diet, taxa, groups of bacterial taxa (and their functional 
capacities) with metadata parameters in different combinations. We will test the null hypotheses 
that there are no associations between dietary intake variability and microbiome composition 
within and between subjects, and between dietary intervention groups. Finally, we will test the 
null hypothesis that vitamin D has no effect on the microbiome. 
 

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Primary Outcome:  Microbiome composition and the relationship with dietary and supplemental 
intake of vitamin D and serum vitamin D levels 
 
Secondary Outcomes: Circulation inflammatory markers (e.g. TNFa, IL-8, CRP); markers of 
gut permeability (e.g. zonulin, intestinal fatty acid binding protein, soluble CD14, 
lipopolysaccharide and lipopolysaccharide-binding protein); glucose and insulin; and other 
nutritional and microbial metabolites. 
4.1 
Sample Size 
We will enroll 40 adults in this study. The adults will be randomized, with 20 individuals per 
study arm, to receive either vitamin D (4000 IU daily) or placebo during the intervention period. 
To control for initial vitamin D (25(OH) D3) levels at baseline (including those with 
hypovitaminosis), subjects will be randomized using 1:1 matching based on vitamin provided 
from blood samples at study visit 1. Study PI will be blinded to subject randomization. We will 
obtain 15 stool samples and 15 24-hour dietary recalls from each individual, for a total of 561 
stool samples and 561 dietary records. We will obtain pre- and post- intervention blood samples 
from each individual for a total of 81 blood samples. We performed a power analysis using 
vitamin D as the primary exposure, and the top 10 dominant microbial taxa as the primary 
outcome. Using monte carlo simulation of microbial taxa distributed according to a lognormal 
distribution, and a non-parametric Mann-Whitney U test for significant differentiation, we found 
that this sample size is sufficient to detect a change of ≥ 1.24 standard deviations in any one of 
the dominant taxa. This analysis accounted for multiple hypothesis testing using Bonferroni 
correction. 
5 
Participant Selection 
5.1 
Inclusion Criteria 
• 
Adult 18 years or older 
 
Exclusion Criteria 
• 
Women who are currently pregnant or breastfeeding 
• 
Use of antibiotics within the last 2 weeks 
• 
Use of supplementary vitamin D within the last month 
• 
Self-reported, pre-existing history of inflammatory bowel disease, heart disease or 
diabetes 
• 
Students under 18 
• 
Tanning/sun exposure > 60 min at a time in last 4 weeks  
• 
No phenobarbital, carbamazepine, spironolactone chronic therapy; or steroid use within 
the last 2 weeks 
• 
Severe allergy to ingredients found in supplements 
 

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It might be possible that some students enrolled in BIO 1125 may be under the age of 18, 
however, these students will be excluded from study enrollment as we are only studying the 
effects on adults as part of this research.  
5.2 
Recruitment 
Participants will be recruited from the Baylor University and the surrounding communities. This 
proposed study is planned to provide data for use in a undergraduate research engagement 
course (BIO 1125) at Baylor University. Citizen science is a growing movement that increases 
the involvement of everyday people and students in the collection and analysis of data in 
collaboration with professional scientists. Healthy adult participants will be recruited for this 
study from the University and surrounding community, as well as from students who express 
interest in taking this citizen science undergraduate research engagement course. Subjects will 
not be required to take the course to participate in the study, and students will not be required to 
participate in the study to take the course. 
5.3 
Protections for the Use of Students as Research Subjects 
Analysis of the proposed research study data will take place as part of a newly redesigned 
course at Baylor University offered through the Biology Department, and some of the 
participants will be recruited from the pool of students who express interest in taking this class. 
De-identified data generated from the research study will be used by the students in the course 
to learn about computational biology and the microbiome. Identifying metadata such as gender, 
race, and age will not be shared with the class. Leigh Greathouse, this study’s principal 
investigator (PI) and adjunct course instructor, has extensive experience and involvement with 
other human microbiome projects.  
 
Importantly, participation in this research study and course will present a unique learning 
opportunity for students to be involved in citizen science. We believe that the students will be 
highly invested in learning about data analysis techniques and methodology in part because 
they will be analyzing their own data. We also believe that the act of participating in the research 
study is an excellent opportunity for the students in the class who are considering careers in 
biomedical research and medicine.  
 
Our study will include a dietary intervention, students will analyze the complete de-identified 
dataset, and we plan use the data generated for publication.  
 
Study participates with be provided a mental health resource sheet should they need to seek 
professional help at any time during the study.  
 
 
To prevent coercion of the study research participants, the following protections are in 
place: 
 

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1. Students who express interest in the course during the recruitment period will be able to 
register regardless of their intent to participate in the study.  Prior to the start of class, 
students will be informed that the class will use data generated from this study. They will 
be offered the opportunity to participate in study as well as a copy of the consent form.  
They will be asked to provide informed consent for the research study before the 
beginning of the course.  
a. Students who do not consent to take part in the research study or who are 
excluded from participating in the research study will still be given permission to 
take the class. 
b. Study participation will be open to all members of the University community and 
public who are not interested or eligible to take the course.  
2. Gathering of informed consent will be completed by a study staff member and not the 
study PI/course instructor.  
3. The study PI/course instructor will be blinded to the identity of the participants in the 
study until completion of the course to prevent any bias in grading.  
a. Students will not be graded for their participation in the research study and their 
completion of the study will not affect their success in the course.  
4. Students will not be compensated for their participation in the research study and will 
receive no direct benefits from participation.  
5. The course will be elective. 
 
All data generated from the research study will be de-identified before use in the class. 
Individuals who choose to participate in the research study can elect to receive a personalized 
report about their microbiome composition and dietary intake. This report will be generated at 
the end of the course after final grades have been distributed to keep all potentially identifying 
information private throughout the duration of the course. If a student completes the research 
study and then drops the course they may still elect to receive their personalized report. 
Standard Baylor University policies for dropping courses will still apply to the academic portion 
of the course.  
6 
Study Procedures 
6.1 
Demographics and Anthropometrics 
At visit 1, basic demographic data will be collected including age, gender, race/ethnicity, and 
medication use. Participant weight will be measured to the nearest kg and height will be 
measured to the nearest cm at using an electronic scale and wall mounted tape-measure. Body 
composition will be measured using the InBody device, which is a bioelectrical impedance 
device that measures percent body fat and lean muscle, and has been used by Dr. Greathouse 
in other IRB approved studies at Baylor University. For students and study participants who 
have mental or physical issues where this would not be appropriate, such as a history of eating 
disorders, the option to just provide self-reported height and weight will be offered.  

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6.2 
Fecal Sample Collection 
Fecal collection will be completed by self-sampling. Self-sampling has proven to be highly 
successful based on Dr. Dan Knights’ American Gut Project collaboration utilizing self-sampling 
of fecal material from 7,000 individuals.22 The participant will be trained on the self-sampling 
procedure after providing consent at visit 1. Briefly, the participant will collect a small quantity of 
fecal material using a provided collection kit once per day during each day of the study that they 
produce a stool. Participants will be instructed on proper sample storage until the sample is 
returned at a study visit.  
 
To avoid room-temperature storage bias, students and members of the University community 
who participate in the study will be asked to drop off samples daily at one of two on-campus 
locations. Stools samples will be stored at -80 within 24 hours of drop off. Participants who do 
not visit the University campus each day will be given FedEx stamped addressed envelopes 
and asked to drop samples in the mail daily. The FedEx labels will be for 24 hour delivery 
using special type B labels intended for use with biospecimens. Further, these stool 
collection kits and methods are the same methods used with the American Gut Project 
kit and study (N=15,096), which conducted extensive temperature bias controls 
(McDonald et al. mSystems, 2018).  
 
We anticipate that not all participants will pass a stool each day. Participants who do not pass a 
stool each day, will be asked to record their dietary intake normally for that day, but to skip the 
stool collection and to return an empty collection kit for the day(s) they do not pass a stool.  
6.3 
Dietary Record Keeping 
The DHQIII utilized for the study will be the version that takes account of the subject’s dietary 
history one year prior (with portion size) to study entry; the online tool DHQIII was created by 
the National Cancer Institute. The daily dietary questionnaire will be based on a 24-hour dietary 
recall. Participants will record everything they eat and drink each day during the study. Each 
participant will undergo training to complete dietary records at visit 1 after providing consent. 
Participant training will include encouragement to actually measure foods eaten using 
measuring spoons and cups whenever possible. Participants will record their intake using 24-
hour dietary recalls with an online tool (Automated Self-Administered 24-Hour Recall [ASA24]-
2016. Bethesda, MD: National Cancer Institute). The ASA24 is based on the United States 
Department of Agriculture’s (USDA) Automated Multiple-Pass Method (AMPM), which has been 
validated. The ASA24 system provides secure, password-protected diet entry for participants 
and de-identified, password-protected data access for researchers. The system does not ask for 
or store any personal identifying information. Dietary records will be analyzed for nutrient 
content using the ASA24-generated output which is based on the USDA Food and Nutrient 
Database for Dietary Studies (FNDDS). From the DHQIII, the Healthy Eating Index (HEI-2016) 
will be calculated, which is used to determine of the overall diet quality. The HEI is calculated 
based on 12 components which each have an individually assigned score to give a maximum 
score of 100. Those individuals having a score above 80 are considered to have a healthy diet, 

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whereas those below 50 are considered to have a poor dietary intake, and do not meet 75% of 
the recommended dietary allowances. 
 
6.4 
Dietary Supplement Intervention 
The recommended dose of vitamin D for adults is 600 IU/day and has an upper limit of 4000 
IU/day, with clinical intervention to replete deficiency being 50,000I IU/day. We have set our 
dose at the upper daily limit, established by the Institute of Medicine, since this study is not 
mean to replete a clinical deficiency. The supplement will be provided as Nordic Naturals 
vitamin D3 gummies, which have been certified by a third party with Good Manufacturing 
Procedures. For these gummies, 4 gummies will provide 4000 IU of vitamin D3. Subjects will 
consume 4 gummies at the largest meal of the day for 12 weeks. Subjects who are assigned to 
the placebo group will be provided with placebo gummies, Yum Earth Organic Vegan Fruit 
Snacks, which have the same composition as the supplement but no vitamin D3. Both will be 
put in bottles without manufacture labeling so as to disguise the vitamin D from placebo. If for 
any reason we are not able to get these placebo products, we have the ability to print 3D molds 
and make our own placebo gummies, which we have done before in our food lab. See Table 1 
for list of ingredients. 
 
 
6.4 
Mental Health Assessment 
Mental and physical health:  Mental health will be measured with several instruments: First, is 
the College Undergraduate Stress Scale, which assesses life stressors similar to the Holmes 
Rahe scale, but adapted for college students (Renner & Mackin, 1998). In addition, the Medical 
Outcomes Study Questionnaire Short Form 36 Health Survey (SF-36; Ware & Gandek, 1998) 
Table 1. Ingredients list of vitamin D3 and placebo supplements
Vitamin D
Placebo
Ingredients
vitamin D (cholecalciferol)
Organic rice syrup
Organic tapioca syrup
Organic cane sugar
Organic cane sugar
Pectin
purified water
Citric Acid
pectin
Ascorbic Acid
citric acid
Natural flavors colored with 
organic concentrate (Apple, 
carrot, 
pumpkin,blackcurrent)
furamic acid
Organic sunflower oil
natural flavor
Organic carnauba wax
sodium citrate dihydrate
fruit and vegetable juice (color)
Allergens
Gluten Free
Vegan
Contains milk derivatives
Gluten Free
Non-GMO
Non-GMO

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will be used to assess general health. This scale is highly reliable with estimates of both 
physical and mental health typically above 0.90.  
 
Depression and anxiety: will be assessed with the Hospital Anxiety and Depression Scale 
(Stern, 2014). This scale has been validated in patient populations and general community 
settings. It is one of the National Institute for Health and Care Excellence (NICE) recommended 
tools for diagnosis of depression and anxiety.  
 
Perceived Stress Scale: The Perceived Stress Scale(PSS) is a validated scale that measures 
the degree to which respondents find their lives “unpredictable, uncontrollable, and 
overloading.” Reliability of this scale has been established to be greater than 0.86. As well 
content, predictive, and concurrent validity have been established for this measurement tool in 
adolescents and adults (Cohen S, Kamarck T,  & Mermelstein R. 1983; Yarcheski A, & Mahon 
N. 1999) 
 
Alcohol and drug use: We will use the World Health Organization’s tool for screening alcohol, 
tobacco & drug use (Newcombe, Humeniuk & Ali, 2005). This screening instrument is used to 
assess use of a range of substances including tobacco, stimulants, depressants, 
hallucinogenics, and opioids. 
 
All of these scales have been used by Dr. Greathouse in previous studies approved by the 
Baylor University IRB for use in studies with University students.  
 
6.5       Physical Activity 
Physical activity will be assessed using the Global Physical Activity Questionnaire (GPAQ), 
which was designed by the World Health Organization. The GPAQ is a valid measure of 
moderate to vigorous activity among healthy adults (Cleland et al. 2014). The GPAQ consists of 
16 questions that estimate an individual’s level of physical activity in 3 domains (work, transport 
and leisure time). We will used this tool to measure average regular levels of physical activity in 
our participants.  
6.6    Blood Draw 
Blood will be drawn by a licensed phlebotomist from a peripheral arm vein at the designated 
visits through standard sterile methods. At each blood draw, ~15mL of blood of blood will be 
collected. Participants will be asked to fast for at least 8 hours prior to the blood draw. 
Participants will remain in a seated position throughout the procedure and be observed for light-
headedness, bleeding, and bruising before, during, and after the procedure. If a participant 
experiences light-headedness they will be given the option to recline or lay down and continue 
to be monitored until the light-headedness resolves. If the phlebotomist is unable to successfully 
locate a vein after three attempts, the blood draw will not be completed. 

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6.7      Follow-up 
Subjects enrolled study may elect to be contacted by telephone, text, or email daily during the 
data collection period as a reminder to collect a sample, complete a dietary record, and/or to 
check on their progress.  
6.8     Microbiome Analysis 
We will use RNA gene sequencing or 16S gene amplicon sequencing or shotgun DNA 
sequencing as well as novel methods in development relying on sequencing a subset of the 
fecal microbial RNA or DNA to accurately characterize taxonomic composition of the 
microbiome.  
7 
Participant Withdrawal 
Participants may withdraw voluntarily at any time.  
 
Participants may be withdrawn from the study by the PI for the following reasons: 
• 
The participant is diagnosed with a disease that may affect their ability to pass feces. 
• 
The study is stopped.  
 
If a subject withdraws from the study, no further data will be collected on the subject. The data 
that have been collected to that point will continue to be used unless the subjects notifies the PI.  
8 
Assessment of Safety 
8.1 
Safety of biological sample collection 
Collecting fecal samples is a low risk procedure. Fecal collection kits will contain gloves for 
participants to wear while collecting their own samples when the chance of contact with stool is 
highest. The sampling kit contains ammonium sulfate, and participants will be instructed not to 
ingest this substance. Participants will be instructed to wash their hands thoroughly after 
completing each stool collection.  
 
Fasting blood draws a generally considered minimal risk procedures. There is a small risk of 
pain and discomfort during the blood draw and it is possible that bruising at the site of the draw 
will occur.  
 
8.2 
Safety of dietary supplement intervention 
Vitamin D occurs naturally in low amounts in foods, including cow’s milk, fish, eggs and 
mushrooms. Most commercially available vitamin D supplements are made from processing 
sheep wool to extract lanolin. This product is then saponified and refined using solvent washes. 
This crude cholesterol is then subjected to a series of chemical reaction to produce 7-
dehydrocholestrol, which is then exposed to UV light to form vitamin D3. The upper limit of 

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vitamin D intake as set forth by the Institute of Medicine and USDA is 4000 IU/day for adults 
over 19. Safety testing for various doses of vitamin D has been previously conducted in healthy 
adults at 3 doses, 400, 4000, and 10,000 IU/day, with 4000 IU dose produce milder 
hypercalcemia in 3% of subjects (N=373) ages 50-70.23 Thus, this does has been deemed safe 
in adults for long-term intake.  
 
9 
Potential Benefits 
Subjects will not personally benefit from giving specimens. The results are not expected to be 
clinically meaningful to the study participants. The research will benefit the larger research 
community by enabling the development of better methods to relate dietary intake to the 
microbiome.  
 
Participants may request an electronic information packet with their individual results via email. 
Results will include the composition of their gut (most abundant taxa) as well as other relevant 
individual markers and summary information of dietary intake. To maintain confidentiality, 
personalized information packets will be sent (at the completion of the course associated with 
this study) by the study PI. Also included in this informational packet will be a comparison of that 
individual’s microbial community to those of other study participants, in de-identified aggregate. 
This may include a graphical representation of ecological distance to other patients, comparison 
diversity scores, and other summary statistics. This will be accompanied by a disclaimer that 
this information should be used for research purposes only and is not a reliable clinical test for 
any purpose. Participants will be notified via email upon completion of the study of this 
information’s availability upon request. 
10 
Study Timeline/Duration 
Recruitment for the study will begin during January 2022 with the data collection completed by 
December 2022.  
11 
Data Management 
11.1 Data Collection 
De-identified participant data will be entered into a secure database, stored on a password-
protected server with full disk encryption, behind both Baylor University secure firewall. DHQIII, 
and 24-hour dietary recall data collected using ASA24 are secured at the hosting site using 
industry standard security controls, including firewalls and encryption. All data entered into the 
DHQIII and ASA24 system at the Respondent's computer are encrypted by the internet browser 
(e.g., Internet Explorer, Firefox) before they are transmitted to DHQIII or ASA24’s servers using 
Secure Socket Layer (SSL) Technology.  

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11.2 Monitoring Plan 
This is a minimal risk trial. Data will be monitored by the PI. Sample acquisition carries minimal 
to no known medical risk to the subjects. The nature of risk for accessing private records will be 
minimal to none, since all data collected will be de-identified. Possible invasion of privacy may 
occur as other participants may observe the fecal specimen being dropped off. Interviews will be 
conducted in private rooms or sectioned-off areas in order to maintain privacy of our 
participants. Participant data will be de-identified. As a result of the de-identification process, 
individuals participating will be assigned a unique identifier number. This unique identifier will be 
used to link de-identified samples and data collected from the interview (data listed in the Data 
Collection section). Study coordinators will have access to the link during the data collection 
period. The study PI will only have access to the link to the unique identifier after the course 
completion to protect the identity of student study participants during the course. The identifiers 
will be maintained in a database on a secure computer protected with password access and full-
disk encryption. If a breach in confidentiality occurs, we will report the breach to the Institutional 
Review Board (IRB) and the HIPPA Privacy Officer as per institutional policy. 
12 
 Administrative Considerations 
12.1 Research Ethics Approval 
The study will be conducted in accordance with the International Conference on Harmonization 
(ICH) for Good Clinical Practice (GCP), the Declaration of Helsinki, and the appropriate 
regulatory requirement(s).  Baylor University IRB will review all appropriate study documentation 
in order to safeguard the rights, safety and well-being of the participants.  The study will only be 
conducted at sites where IRB approval has been obtained.  The protocol, informed consent, 
written information given to the patients, annual progress reports, and any revisions to these 
documents will be provided to the IRB by the investigator.  Essential clinical documents will be 
maintained to demonstrate the validity of the study and the integrity of the data collected.  
Master files will be established at the beginning of the study, maintained for the duration of the 
study and retained according to the appropriate regulations. 
12.2 Confidentiality 
Information about study subjects 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 subject authorization informing the subject of the following:  
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 

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should be made to obtain permission to collect at least vital status (i.e. that the subject is alive) 
at the end of their scheduled study period. 
12.3 Declaration of Interests 
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.) will 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. The following situations include conflicts of interest: when substantial 
changes in business or financial interests occur, when an activity that presents a potential 
conflict of interest is anticipated, or when submitting an application for research support or 
technology transfer, submitting research protocols to the IRB, or receiving financial 
contributions. All Baylor University investigators will follow the University conflict of interest 
policy. 
 
 

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13 
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