Clinical trial NCT04415333
Conditions: Hypertension
Keywords: African American Blood pressure Gut Microbiome Short-chain fatty acid Butyrate
Sponsor: University of North Carolina, Chapel Hill
Start date: 2021-07-08; Completion date: 2021-11-02
Enrollment: 20 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
African Americans have the greatest burden of hypertension. Recently, the short-chain fatty acid, butyrate, has been reported to have some effect on blood pressure. Butyrate is not normally ingested since it is made by bacteria in the gut as a byproduct of fiber fermentation. In this proof of concept study, the investigators will investigate the effect of butyrate absorbed in the gut (via the participant self-administering an enema with butyrate) has on blood pressure.

Detailed description:
Description: African Americans (AA) have the greatest burden of hypertension. Recently, gut microbial dysbiosis (a term that describes a poorly diverse gut microbial profile and lower short-chain fatty acid (SCFA) production) has been linked to hypertension and may be involved in the pathogenesis of hypertension in African Americans. African Americans have been reported to have lower gut SCFA and SCFA can reduce blood pressure. This is a proof of concept pilot study to determine the relationship between gut SCFA and blood pressure (BP). Delivery of butyrate to the gut will be via enema.

Objectives: The objectives of this research are to 1. Identify gut microbial taxa (SCFA butyrate-producing microbes) and circulating butyrate levels associated with hypertension via a cross-sectional design in AA without and with hypertension. 2. Quantify the relationship between SCFA (butyrate) absorption into the bloodstream and subsequent changes in blood pressure in a 24-hour period after delivering butyrate into the gut via enema.

Participants: African Americans males and females ages of 30-50 without hypertension (normal BP/healthy control; systolic BP: 90-129/ diastolic BP: 60-89mmHg) and with hypertension (systolic BP: 130-159 mmHg/ diastolic BP: 80-99 mmHg) that do not take anti-hypertension medication will be recruited.

Description of Study: There are 2 groups; control (without hypertension; BP: 90-129/60-89mmHg; 5 male/5 female, n=10) and experimental (with hypertension; BP 130-159/80-99 mmHg; 5 male/5 female, n=10). Normotensive participants (control group) will be age (±1 year) and sex-matched to hypertensive group participants. All participants (control and experimental groups; 5 male/5 female) will provide stool and blood samples, and complete 24-hour (hr) ambulatory blood pressure (ABP) monitoring to compare the abundance of fecal butyrate-producing microbes, circulating (blood) butyrate concentrations, and average blood pressure responses during the day and overnight. Individuals in the control group will only participate in donating stool (1-time) and a blood sample (1-time) and wearing a 24-hr ABP monitor (1-day). In a crossover blinded randomized controlled pilot study, the 10 hypertensive AA subjects (5 male/5 female) will be randomized to self-administer a sodium butyrate (80mmol butyrate in 0.9% saline, 60 ml total) or control (low dose) butyrate (5mmol butyrate 0.9%, 60 ml total) enema 1 week apart (7 days). Subjects will provide a stool sample before each study day (2 total), have their BP measured via ABP monitor, submit to a blood draws (pre-enema-baseline), self-administer the randomized enema, submit to a 30 min \& 60 min post-enema blood draw, and wear the 24-hour ABP monitor for the remainder of that day. Subjects will be provided with written and verbal instructions on how to self-administer the enema. The dietary supplement (sodium butyrate) will be compounded by a local pharmacy (Custom Care Pharmacy - 109 Pisgah Church Rd. Greensboro, NC) into an enema at the concentrations listed above.

Arms: arm 1: After a randomized assignment in the crossover design, participants with hypertension will come to the testing office to self-administer the first enema with a concentration of 5 mmol sodium butyrate in a 0.9% saline solution (60 mL total). After a 7-days washout period, they will return to testing office to self-administer the other enema with a concentration of 80 mmol sodium butyrate in a 0.9% saline solution (60 mL total). arm 2: After a randomized assignment in the crossover design, participants with hypertension will come to the testing office to self-administer the first enema with a concentration of 80 mmol sodium butyrate in a 0.9% saline solution (60 mL total). After a 7-day washout period, they will return to the testing office to self-administer the other enema with a concentration of 5 mmol sodium butyrate in a 0.9% saline solution (60 mL total). arm 3: African Americans with normal blood pressure (control group) will not perform self-administration of the enema. They will submit to 1 blood draw and wear the 24-hour ambulatory blood pressure monitor for 1-day.
Interventions: intervention 1: Sodium Butyrate 5 mmol intervention 2: Sodium Butyrate 80 mmol
Intervention descriptions: intervention 1: Enema-based delivery of 5 mmol butyrate in 0.9% saline, (60 mL total) intervention 2: Enema-based delivery of 80 mmol butyrate in 0.9% saline, (60 mL total)

Protocol documents:
Prot_SAP_001.pdf: 
Effect of Gut Butyrate Delivery on Blood Pressure in 
African Americans with Hypertension 
NC TRaCS Grant #: 550KR181802 
National Clinical Trial (NCT) Identifier Number:  NCT04415333 
Principal Investigator: Marc D. Cook, PhD.; Ian Carroll, PhD. 
<IND/IDE> Sponsor: N/A 
Funded by: NC TRaCS Grant #: 550KR181802 
31 May 2018 


  
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Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. 1 
1 
PROTOCOL SUMMARY ............................................................................................................................... 1 
1.1 
Synopsis ................................................................................................................................................ 1 
1.2 
Schema ................................................................................................................................................. 4 
1.3 
Schedule of Activities (SoA) ................................................................ Error! Bookmark not defined. 
2 
INTRODUCTION ............................................................................................................................................ 5 
2.1 
Study Rationale .................................................................................................................................... 5 
2.2 
Background........................................................................................................................................... 5 
2.3 
Risk/Benefit Assessment .................................................................................................................... 6 
2.3.1 
Known Potential Risks ..................................................................................................... 6 
2.3.2 
Known Potential Benefits ................................................................................................ 6 
2.3.3 
Assessment of Potential Risks and Benefits ................................................................ 6 
3 
OBJECTIVES AND ENDPOINTS ................................................................................................................ 8 
4 
STUDY DESIGN ............................................................................................................................................. 8 
4.1 
Overall Design ...................................................................................................................................... 8 
4.2 
Scientific Rationale for Study Design.............................................................................................. 10 
4.3 
Justification for Dose ......................................................................................................................... 10 
4.4 
End of Study Definition ..................................................................................................................... 10 
5 
STUDY POPULATION ................................................................................................................................ 11 
5.1 
Inclusion Criteria ................................................................................................................................ 11 
5.2 
Exclusion Criteria ............................................................................................................................... 12 
5.3 
Lifestyle Considerations .................................................................................................................... 12 
5.4 
Screen Failures .................................................................................................................................. 12 
5.5 
Strategies for Recruitment and Retention ...................................................................................... 13 
6 
STUDY INTERVENTION ............................................................................................................................ 13 
6.1 
Study Intervention(s) Administration ............................................................................................... 13 
6.1.1 
Study Intervention Description ..................................................................................... 13 
6.1.2 
Dosing and Administration ............................................................................................ 14 
6.2 
Preparation/Handling/Storage/Accountability ................................................................................ 14 
6.2.1 
Acquisition and accountability ...................................................................................... 14 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 14 
6.2.3 
Product Storage and Stability ....................................................................................... 15 
6.2.4 
Preparation ...................................................................................................................... 15 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................................... 15 
6.4 
Study Intervention Compliance ........................................................................................................ 15 
6.5 
Concomitant Therapy ........................................................................................................................ 16 
6.5.1 
Rescue Medicine ............................................................................................................ 16 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 16 
7.1 
Discontinuation of Study Intervention ............................................................................................. 16 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 16 
7.3 
Lost to Follow-Up ............................................................................................................................... 17 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 17 
8.1 
Efficacy Assessments ....................................................................................................................... 17 
8.2 
Safety and Other Assessments ....................................................................................................... 19 
8.3 
Adverse Events and Serious Adverse Events ............................................................................... 21 


  
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8.3.1 
Definition of Adverse Events (AE) ............................................................................... 21 
8.3.2 
Definition of Serious Adverse Events (SAE) .............................................................. 22 
8.3.3 
Classification of an Adverse Event .............................................................................. 22 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 23 
8.3.5 
Adverse Event Reporting .............................................................................................. 23 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 24 
8.3.7 
Reporting Events to Participants ................................................................................. 24 
8.3.8 
Events of Special Interest ............................................................................................. 24 
8.3.9 
Reporting of Pregnancy ................................................................................................ 24 
8.4 
Unanticipated Problems .................................................................................................................... 24 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................. 25 
8.4.2 
Unanticipated Problem Reporting ................................................................................ 25 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................... 26 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 26 
9.1 
Statistical Hypotheses ....................................................................................................................... 26 
9.2 
Sample Size Determination .............................................................................................................. 26 
9.3 
Populations for Analyses .................................................................................................................. 26 
9.4 
Statistical Analyses............................................................................................................................ 27 
9.4.1 
General Approach .......................................................................................................... 27 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................................. 28 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 28 
9.4.4 
Safety Analyses .............................................................................................................. 28 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 28 
9.4.6 
Planned Interim Analyses ............................................................................................. 28 
9.4.7 
Sub-Group Analyses ..................................................................................................... 28 
9.4.8 
Tabulation of Individual participant Data .................................................................... 28 
9.4.9 
Exploratory Analyses ..................................................................................................... 28 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 28 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 28 
10.1.1 
Informed Consent Process ........................................................................................... 28 
10.1.2 
Study Discontinuation and Closure ............................................................................. 29 
10.1.3 
Confidentiality and Privacy ........................................................................................... 30 
10.1.4 
Future Use of Stored Specimens and Data ............................................................... 30 
10.1.5 
Key Roles and Study Governance .............................................................................. 31 
10.1.6 
Safety Oversight ............................................................................................................. 31 
10.1.7 
Clinical Monitoring .......................................................................................................... 31 
10.1.8 
Quality Assurance and Quality Control ....................................................................... 32 
10.1.9 
Data Handling and Record Keeping ............................................................................ 32 
10.1.10 
Protocol Deviations ........................................................................................................ 33 
10.1.11 
Publication and Data Sharing Policy ........................................................................... 33 
10.1.12 
Conflict of Interest Policy .............................................................................................. 33 
10.2 
Additional Considerations ................................................................................................................. 34 
10.3 
Abbreviations ...................................................................................................................................... 35 
10.4 
Protocol Amendment History ........................................................................................................... 37 
11 
REFERENCES ............................................................................................................................................. 38 
 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
Protocol #18-1680 
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STATEMENT OF COMPLIANCE 
 
This Phase I trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following:  
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
1 
 
PROTOCOL SUMMARY 
No text is to be entered in this section; rather it should be included under the relevant subheadings below. 
1.1 
SYNOPSIS  
Title: 
Effect of Gut Butyrate Delivery on Blood Pressure in African Americans 
with Hypertension 
Study Description: 
African Americans (AA) have the greatest burden of hypertension. Recently, 
gut microbial dysbiosis (a term that describes a poorly diverse gut microbial 
profile and lower short chain fatty acid (SCFA) production) has been linked 
to hypertension and may be involved in the pathogenesis of hypertension 
in African Americans. African Americans have been reported to have lower 
gut SCFA and SCFA can reduce blood pressure. This is a proof of concept 
pilot study to determine the relationship between gut SCFA and blood 
pressure (BP). Delivery of butyrate to the gut will be via enema.  
 
Objectives: 
 
The objectives of this research are to 1. Identify gut microbial taxa (SCFA 
butyrate-producing microbes) and circulating butyrate levels associated 
with hypertension via a cross-sectional design in AA without and with 
hypertension. 2. Quantify the relationship between SCFA (butyrate) 
absorption into the blood stream and subsequent changes in blood pressure 
in a 24-hour period after delivering butyrate into the gut via enema. 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
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Endpoints: 
 
 
 
 
Rationale: 
Primary: If gut butyrate is related to BP, we expect to observe a reduction 
in BP (measured by 24-hr Ambulatory Blood Pressure monitoring) after the 
butyrate enema but no change in BP after the control saline enema. 
 
In humans, butyrate producing microbial species are associated with blood 
pressure. Butyrate is absorbed in the colon, and once in the blood may exert 
beneficial effects on blood pressure. However, it is unclear which specific 
microbes are associated with blood pressure in African Americans or how 
much butyrate is absorbed into the blood which may affect BP.  
Study Population: 
 
 
  
Self-reported African Americans males and females ages of 30-50 without 
hypertension (normal BP/healthy control; systolic BP: 90-120/ diastolic BP: 
60-89mmHg) and with hypertension (systolic BP: 130-159 mmHg/ diastolic 
BP: 80-99 mmHg) that do not take anti-hypertension medication will be 
recruited. 
Phase: 
Phase I 
Description of 
Sites/Facilities Enrolling 
Participants: 
All data collection from human subjects will be conducted at North Carolina 
A&T State University (1 site). The studies will occur in the School of Nursing 
(Noble Hall) laboratory. The space is equipped for blood draws and private 
bathrooms for subjects to self-administer enemas. Nursing research staff 
will support the study by being present during each experiment, draw 
blood, and monitor participants. We will recruit subjects via posted fliers on 
campus, in the community, at our collaborating physicians primary care 
clinical hypertension practice (Veita Bland, MD.) and other local family 
practice clinics, and on a weekly radio health broadcast (“Its a Matter of 
Health”. Veita Bland, MD. Wednesdays 530pm). Dr. Veita Bland provide 
clearance and see participants if they do not have a primary care physician. 
She has a clinical research staff that coordinates the practice participation 
in other Phase II & III Clinical Research Studies. She will also review clinical 
data (blood pressures) and monitor any adverse reactions reported by the 
study staff and research participants.   
Description of Study 
Intervention: 
There are 2 groups; control (without hypertension; BP: 90-129/60-
89mmHg; 5 male/5 female, n=10) and experimental (with hypertension; BP 
130-159/80-99 mmHg; 5 male/5 female, n=10). Normotensive participants 
will be age and sex-matched to hypertensive group participants. In Aim 1: 
AA individuals with and without hypertension (5 male/5 female) will provide 
stool and blood samples, and 24-hour (hr) ambulatory blood pressure (ABP) 
monitoring, for comparison of fecal butyrate-producing microbes and 
circulating butyrate between AA with and without hypertension. Baseline 
comparisons of circulating butyrate, fecal butyrate produces, and 24-hr ABP 
measures will be made between the normal and hypertension groups. 
Individuals with normal BP will only participate in donating stool and blood 
samples and wearing a 24-hr ABP monitor. In Aim 2: In the crossover 
blinded randomized controlled pilot study, the 10 hypertensive AA subjects 
(5 male/5 female) will be randomized to self-administer a sodium butyrate 
(80mM butyrate in 0.9% saline, 60 ml total) or control saline (0.9%, 60 ml 
total) enema 1 week apart (7 days) in this crossover research design. 
Subjects will provide a stool sample before each study day (2 total), have 
their BP manually measured, be fitted with their ABP monitor, submit to a 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
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blood draws (pre-enema), self-administer the randomized enema, submit 
to a 30 min & 60 min post-enema blood draw, and wear a 24-hour ABP 
monitor for the remainder of the day. Subjects will be provided with written 
and verbal instructions on how to self-administer the enema. The dietary 
supplement (sodium butyrate) will be compounded by a local pharmacy 
(Custom Care Pharmacy - 109 Pisgah Church Rd. Greensboro, NC) into an 
enema at the concentrations listed above.  
Study Duration: 
 
 
 
 
Study Team: 
The timeline for study completion includes a recruitment plan to enroll the 
20 subjects (10 control normotensive and 10 stage-1 hypertensive subjects) 
in the 1st 3-4 months (~5-7 subjects per month) with the expectation of 
sample testing in months 5-8, data analyses and publication submission in 
months 9-12. 
Ian Carroll PhD, (Co-PI) Molecular Microbiologist. Dr. Carroll is an Assistant 
Professor at the UNC Chapel Hill Department of Nutrition. He will process, 
analyze and interpret microbiome data. Marc Cook PhD, (PI) Molecular 
Exercise Immunologist. Dr. Cook will facilitate every aspect of the 
intervention at NC A&T, collect data and samples, perform blood biomarker 
assays and analyze BP, blood, and microbiome data with Dr. Carroll. Veita 
Bland MD, (Collaborator) Primary Care Physician & Clinical Hypertension 
Specialist. Dr. Bland has her own private practice, affiliated with Cone 
Health Hospital system, and is experience in running Phase II and Phase III 
clinical trials concerning hypertension. She is the lead clinical contact, will 
lend her expertise in subject recruitment, analyses and interpretation of  
relevant clinical data collected (BP). 
 
Description of Schema: 
Samples and Measures 
for each group. 
 
After consent, control subjects (without hypertension) will complete 
questionnaires, including 5-day dietary recall, which will be immediately 
followed by self-collection of a fecal sample (within a 24-hour period before 
their lab visit). experimental visit 1, control subjects will be fitted for their 
24-h ambulatory BP monitor and have one (1) blood draw (time 1) and will 
go about their day. They will return the ABP monitor the next day. This will 
complete their study. 
After consent, subjects with hypertension will complete questionnaires, 
including 3-day dietary recall, which will be immediately followed by self-
collection of a fecal sample (within a 24-hour period before their lab visit). 
At experimental visit 1, control subjects will have their BP manually 
measured and will be fitted for their 24-h ambulatory BP monitor, they will 
submit to a blood draw (pre) and then self-administer an enema (double 
blinded randomization to either a control or butyrate enema). After 
completing the enema, they will have subsequent blood draws at 30 
minutes (post-30) and 60 minutes (post-60) post enema. They will continue 
to wear the ABP monitor and will go about their day. They will return the 
ABP monitor the next day. Seven (7) days, but not longer than 21 days after 
the experimental visit 1, they will repeat the steps with the remaining 
enema experimental visit 2.  
In Aim 1 (cross-sectional design), between-group (control and 
hypertension) analyses will be performed from subject data collected at 
experimental visit 1. The analyses will include quantifying differences in 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
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mean hourly (day and night-time) BP measures, circulating butyrate levels 
and additional measures in section 8.2 (time 1), SCFA producing microbial 
biomass in stool samples, and dietary macro- and micro-nutrient intake. 
In Aim 2 (double-blinded crossover design), within-group comparisons will 
be analyzed between circulating butyrate and additional blood variables 
(section 8.2), mean hourly (day and night-time) BP measures, and fecal SCFA 
producing microbes.  
1.2 
SCHEMA 
 
 
Screening
Visit 1
•Total n=20 (10 normotensive and 10 hypertensive; 5 male and 5 female each group). A telephone screening for 
meeting inclusion criteria will be performed prior to Visit 1. Subjects with hypertension will need PCP clearance 
•Review inclusion and exclusion criteria, receive medical clearance documentation for hypertension subjects, and 
obtain informed consent. Urine pregnancy test for women of childbearing age.
•Obtain history and complete questionnaires: Demographic, physical activity. 
•Obtain Body Composition measures (weight, height, fat & fat-free mass) via bioelectrical impedence scale. 
Sample Collection and Randomization
Visit 2
•Normotensive (Control, n=10): Submit Stool sample, submit to 1 blood draw, wear 24-hr ABP monitor
•Hypertensive Group (n=10): Randomized to either enema (randomization.com) will be performed by only one 
member of our research team. Self-administer enema, blood draws, wear 24-hr ABP monitor.
Visit 3
•Normotensive: Return ABP monitor and End of Study.
•Hypertensive: Return ABP monitor. Will return for Visit 4.
•Review of adverse reactions or events.
Follow-up assessments of study endpoints and safety
Visit 4
•Hypertension Group (n=10): Repeat Visit 2 with remaining enema
Follow-up assessments of study endpoints and safety
Visit 5
•Hypertensive (n=10) Return ABP monitor and End of Study.
•Review of adverse reactions or events.
Follow-up Telephone Call 7 days and 30 days after participation and Review of Data: 12 month 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
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2 
 
INTRODUCTION 
2.1 
STUDY RATIONALE  
AA are grossly underrepresented in current and prospective studies of hypertension, although this group 
exhibits a significantly greater prevalence of endothelial dysfunction and hypertension. Hypertension is 
associated with genetic and environmental factors, such as cellular programming, diet, stress, and 
exercise. However, the gut microbiome has been linked to hypertension, and specific microbial profiles, 
characterized by low SCFA production, are associated with vascular disease. SCFAs may reduce BP by 
mitigating endothelial dysfunction. We are focusing on butyrate because research reports that gut 
butyrate is related to BP in humans and animals. 
2.2 
BACKGROUND  
Large studies have demonstrated that genetic variation accounts for <2% of population variation in BP, 
and that daily activities and environmental factors play a significant role in BP regulation. BMI has a 
significant impact on cardiovascular disease risk and BP, but these relationships are also variable. 
Regardless of these associations, recent attention has been focused on gut microbial composition, SCFA 
production (such as butyrate), and BP. Butyrate is only produced in the gut, via fermentation in the colon, 
and absorbed into the circulation where it activates receptors in blood vessels can reduces BP. In the first 
study performed in humans, Yang et al observed that hypertensive subjects exhibited reduced gut SCFA 
production that was related to hypertension. However, this lone human study did not address race/ethnic 
demographics, or stage of hypertension. 
As SCFAs are products of gut microbial metabolism, the results above suggest that the gut plays a 
role in BP control in humans. Oral administration of SCFAs reduced BP in mice, but this effect was 
attenuated in mice deficient in SCFA receptors Olfr78 and GPR41, which are present in EC and blood 
vessels. Further, prolonged (14d) butyrate infusion suppressed Angiotensin-II-induced hypertension. 
Recent studies show that butyrate-producing microbes, and gut butyrate concentrations, were lower in 
AA compared to native Africans and related to lower dietary fiber intake in AA. The result of low fiber 
intake is likely related to the prevalence of diseases, such as colon cancer incidence, glucose intolerance, 
and vitamin deficiency in the AA population. Decreased fecal butyrate content could indicate that more 
is absorbed by the gut. However, the above studies report that SCFA-producing microbes are less 
prevalent, suggesting a corresponding decrease in gut SCFA availability. 
The use of enemas for delivering drugs has been considered in clinical care. High doses of butyrate 
can disrupt intestinal epithelial cell barrier function in animals. Here, we propose to use a low dose (80 
mM) butyrate enema that has been reported safe in healthy volunteers and clinical populations. In respect 
to hypertension, gut SCFA concentration has been associated with BP control, but no clear mechanisms 
have been identified. In humans, we do not know whether SCFAs act outside of the colon (i.e., in the 
blood) to affect vascular function or work inside the colon, as butyrate has been shown to stimulate colon 
serotonin release and serotonin has some effect on BP. The latter mechanism is also poorly understood 
in humans. Thus, interventions to increase colonic butyrate are needed to determine its impact on 
vascular health. This proof of concept study will quantify the association between gut butyrate, circulating 
levels, and BP. Butyrate enemas have been used in healthy (Vanhoutvin SA et. al. 2009) and clinical 
populations of intestinal dysfunction (Steinhart, A.H. et al. 1996, Hamer et al. 2010) and have shown that 
delivery of butyrate, via enema, is safe and is not associated with any side effects. Further, there have 
been no reported side effects of oral ingestion of butyrate (Krokowicz et al. 2014).   
Innovation: Direct delivery of butyrate into the gut has been shown to be safe and novel approach 
to quantify gut butyrate absorption and its effect on BP. Ultimately, data generated from this study will 


Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with Hypertension 
 
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be used to identify specific microbial butyrate producing taxa related to hypertension for the development 
of clinical prebiotic and probiotic interventions to increase SCFA production in the gut. 
2.3 
RISK/BENEFIT ASSESSMENT   
2.3.1 KNOWN POTENTIAL RISKS  
Potential Risks: Potential Risks: We assess the potential risks in our studies to be the following: 1) Enema: 
risk of psychological discomfort with performing the enema is possible and frequent bowel movement 
after the enema is expected. 2) Blood drawing: risk of bruising and infection is minimal with blood draw. 
Minor discomfort with venipuncture is also common; 3) Risk associated with 24 hour ABP monitoring: 
Infrequent sleep disturbances have been reported when the cuff inflates; 4) Risk associated with collecting 
fecal samples: Risk includes psychological discomfort, potential embarrassment in collecting this sample, 
and awkwardness of collecting the sample; 5) Failure to maintain participant confidentiality risk: collection 
of private health information always poses a risk to patient confidentiality as we will be routinely collecting 
data concerning BP, fecal bacteria, and clinical blood measures. 
 
Protection against Risk: Concerning: 1). Enema: There are no known procedures for minimizing the risk of 
psychological discomfort in self-administering the enema. Subjects will have free access to clean and 
private restrooms to self-administer the enema and have subsequent bowel movements 2). Blood 
drawing: To minimize risks of the blood draw, a certified phlebotomist will use aseptic techniques to 
perform all blood draws. 3) 24-hour ABP monitoring: There are no procedures for minimizing the risk of 
infrequent sleep disturbances related to nighttime BP monitoring. BP will be taken every 30 minutes 
during wake hours and every hour during sleep hours 4). Risk associated with collecting fecal samples: To 
minimize risks, all personal protection equipment will be provided for the subject (gloves, collection 
containers). There are no known procedures for minimizing the risk of psychological discomfort in 
collecting the sample.  5). Failure to maintain subject confidentiality: To minimize risks, all information 
will be de-identified and handled in accordance with HIPPA guidelines. 
 
No Social, economic, or legal consequences have been identified as potential risks.  
2.3.2 KNOWN POTENTIAL BENEFITS  
 
Potential Benefits: Research participants may not receive a direct clinical benefit from this research study, 
particularly healthy control subjects. Subjects will have 24h ABP results, which that they would not 
normally have. Each participant will have a dietary assessment and be informed about which food choices 
would benefit their BP. Further, the subject’s primary physicians may have access to additional health-
related information (24hr ABP results), at the subject’s consent. The benefits to society will be the 
identification of strategies and novel therapeutic targets to reduce the risk of and attenuate hypertension 
in African American men and women in the North Carolina Triad area. Further, we will be able to inform 
the public about the benefits of gut health on clinical health outcomes. The risks to subjects are reasonable 
and minimal in relation to the anticipated benefits to participants and others because the potential 
benefits are primed to have a powerful impact on current and future health status of African Americans 
with hypertension.   
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
 
Psychological: 
Risk associated with the questionnaires: It is likely (10-25%) that subjects may feel a little uncomfortable, 
embarrassed, or incur emotional distress while sharing your personal health information or completing 
the questionnaires to assess stress or depression. To minimize these risks, subjects are free to skip any 
questions they do not wish to answer.  


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Emotional distress or Embarrassment: It is common (>25%) that subjects may experience embarrassment 
while collecting stool samples and/or self-administering an enema because of the personal nature of the 
sampling and administration of the enema rectally. In this study, subjects will be instructed (verbally and 
written) on how to perform the stool collection at home by themselves (provided all personal protection 
equipment) and return the sample to the laboratory. Also, subjects will be instructed (verbally and 
written) on how to properly self-administer the enema. Subject will also be given access to a private 
bathroom to perform the enema. There will also be an option for the subject to request the enema be 
administered by a nurse (collaborator). This will reduce the likelihood of embarrassment when 
participating in collecting the fecal sample (home) and enema self-administration (lab).  
Consequences of breach of confidentiality: This study involves the collection of private health 
information (health history, measurement of biological markers in blood, blood pressure, and fecal 
microbial samples) and poses a risk to subjects’ confidentiality. As a control for this risk, subject data will 
be de-identified by assigning each subject a study identification number. Files with subject information 
will be kept in locked office space within a locked storage cabinet. Electronic records will be kept on a 
laboratory computer (password protected) in an electronic file (e.g., RedCap, and/or Microsoft excel & 
Microsoft Word) that is also password protected. 
Physical: 
Medication (enema) side effects: An enema is primarily used to provide relief for occasional constipation. 
We expect that it will be very common (>50%) that subjects may have a bowel movement with 10 min of 
self-administering each enema (control (saline) or experimental (butyrate)). Side effects of a saline enema 
are reported to be abdominal pain, diarrhea, nausea, and vomiting. These side effects are often related 
to the severity of constipation individuals are hoping to alleviate. Since we are requiring a stool sample be 
collected within 24 hours of their data collection, we do not expect any subjects to be constipated. If they 
are, we will postpone their testing until the constipation issue is resolved. Enemas have also been used, 
clinically and experimentally, for drug delivery. Each active butyrate enema will consist of 60 mL (2.02 oz 
or 4 tablespoons) of an 80 mmol/L sodium butyrate solution titrated to a pH of 7.0 with sodium chloride 
added to maintain iso-osmolality. This concentration was not shown to "prompt any adverse effects in 
the treatment group and there were no withdrawals from drug toxicity". (Reference: Steinhart, A.H. et al. 
Aliment Pharmacol Ther 1996; 10: 729-736 and Hamer et al. Clinical Nutrition 2010 29: 738-744).  
Pain: Blood drawing: risk of bruising is common (>25%) and infection is rare (<1%) with blood draws. Minor 
discomfort with venipuncture is also common (>25%). Blood will be drawn by a nurse or phlebotomist 
who is skilled to minimize pain and discomfort during the procedure.  
Discomfort: The risk associated with 24-hour ambulatory BP monitoring has been reported to cause 
infrequent (1-10%) sleep disturbances when the cuff inflates. Because participants become familiar with 
the ABP monitor recordings during the day time, it is thought that participants are more familiar with it 
which reduces the occurrence of sleep disturbances.  
Stool collection: There is no reported data on the risk of infection with self-fecal collection. Mishandling 
the sample can lead to infections (1-10%). This would be extremely rare in fairly healthy people who do 
not have existing opportunistic gut bacterial infection (e.g., Clostridium difficile (C. diff)). To reduce the 
risk of infection, subjects will be provided with personal protective equipment (gloves, collection and 
storage materials) and written step-by-step instructions on how to collect the samples. They will also be 
educated on safe hand washing techniques to reduce this risk to only rare cases (<1%). Importantly, 
samples will be collected via the clean catch method and the swab method. Subjects will be given written 
instructions (and equipment-gloves and toilet hat) to safely catch the bowel movement and store in a 
container with a screw top lid. Also, subjects will use a sterile swab and apply it to used toilet paper after 
having a bowel movement and store. They will handle their stool with the appropriate provided protective 
equipment to obtain the sample, which will also reduce the risk infection. They will store the samples in 


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their respective containers, place them in a bag, and place it in their freezer until they transport the 
sample (in a container with freezer packs) to the lab the next morning for their study.  
 
No Social, economic, or legal consequences have been identified as potential risks. 
 
3 
OBJECTIVES AND ENDPOINTS 
 
 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
The primary objective is to 
demonstrate that the gut butyrate 
concentration is indirectly 
proportional to BP (greater gut 
butyrate leads to reduction in BP). 
 
 
In this proof of concept pilot study, 
the primary clinical endpoint is to: 
 
1. Quantify the effect of 
butyrate into the gut (via 
enema) on BP measured over 
a 24-hour period.  
We hypothesize there will be a 
significant reduction in mean hourly 
BP, some time during the daytime 
and/or nighttime (dipping) 24-hour 
ABP monitoring period, ONLY after 
the butyrate enema. Also, we will 
quantify the effect of butyrate on 
blood biomarkers (inflammation, 
oxidative stress, and nitric oxide 
levels) that may correspond with 
lower BP in Hypertensive subjects. 
 
Data does not exist for estimating the 
effect size of the proposed treatment 
(cross-sectional or randomized double 
blinded cross-over study), which itself 
will be used to determine appropriate 
ranges of effect sizes for subsequent 
studies. We consulted Dr. Michael 
Love (UNC-CH NC TraCS 
Biostatistician). 
 
Delivering the SCFA 
butyrate into the gut 
(via enema) and 
quantifying its 
absorption into the 
blood stream (where it 
is normally produced 
and absorbed), will aid 
in determining the role 
of gut SCFA production 
in the regulation of BP, 
and circulating 
biomarkers related to 
BP. We expect a 
reduction in BP within 
24 hours of delivery of 
gut butyrate only. We 
also expect a reduction 
in blood biomarkers 
related to BP after the 
butyrate experiment 
only.   
 
 
4 
STUDY DESIGN  
 
4.1 
OVERALL DESIGN 
 


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• 
In this proof of concept pilot study, our central hypothesis is that low gut butyrate availability is 
related to hypertension in African Americans (AA), and that acutely increasing gut butyrate (via a 
one-time butyrate enema) will translate into greater circulating butyrate and an acute and 
measurable reduction in BP. There are 2 groups; normotensive – control group (n=10; 5 men & 5 
women) and hypertensive – experimental group (n=10; 5 men & 5 women). 
• 
 This is a Phase I clinical trial that will utilize a one-time dose dietary supplement (butyrate) 
compounded in an enema to determine the effect of gut butyrate delivery on absorption and BP 
in AA men and women with mild to moderate hypertension.  
• 
In Aim 1, we will complete a cross-sectional study to quantify the differences in fecal SCFA 
microbes, circulating butyrate, and define associations between butyrate producing microbes, 
circulating butyrate, and BP between normotensive and hypertensive participants. Normotensive 
participants (control group) will submit a fecal sample with dietary records, have their BP 
measured, donate a blood sample, and wear a 24-hour ambulatory BP monitor for the remainder 
of the day. In Aim 2, we will perform a randomized double blinded placebo controlled cross-over 
study to determine the effect of a butyrate enema on BP in participants with mild-to-moderate 
hypertension. Participants with hypertension will perform the experiment by submitting a stool 
sample and dietary records, then have their resting BP measured manually before  on their non-
dominate arm. After the manual BP measurement, subjects will have the ambulatory BP (ABP) 
monitor placed on their non-dominant arm and undergo a blood draw. Subjects will then self-
administer an enema (random control or butyrate), have their blood drawn 2 additional times 
within 1 hours (at 30 min and 60 min post enema) and wear a 24-hour ABP monitor for the 
remainder of the day. The monitor will measure BP every 30 minutes during waking hours (until 
10pm) and every 1 hour during sleep hours (10pm-7am). The BP monitor will be removed when 
the subject returns the monitor the next morning. Seven (7) days, but no longer than 21 days, 
after performing the 1st experiment, participants will repeat the study with the remaining enema.  
• 
Measurements in blood and stool: In each blood sample collected, we will measure plasma 
butyrate levels (mmol/L; performed in the Biomarker Mass Spectrometry Core in the Gillings 
School of Global Public Health at UNC-CH). In Dr. Cook laboratory, we will measure plasma 
markers related to BP ((IL-1β, CRP), oxidative stress (8-Isoprostane), nitric oxide (quantifying total 
nitrate/nitrite), and serotonin) via Elisa. Rationale for the additional blood biomarkers are that 1. 
Hypertension is joined with elevated inflammation, greater oxidative stress, and lower nitric oxide 
availability (endothelial dysfunction) and 2. butyrate is anti-inflammatory and may increase nitric 
oxide availability (improve endothelial function) and subsequently be followed by lower BP. We 
expect hypertensive subjects to have greater inflammation, oxidative stress, and lower nitric 
oxide levels compared to control subjects. Serotonin has been shown to be a byproduct of 
microbial metabolism and may be related to hypertension. We expect lower levels of IL-1 β and 
CRP, oxidative stress, and increased nitric oxide after the butyrate enema and no change after the 
control enema. The stool samples collected will be delivered to and processed by Dr. Carroll and 
analyzed in the UNC-CH Microbiome Core. Dr. Carroll will perform bioinformatic microbiome 
analyses of the raw sequence data. 
• 
Dietary data analyses: Dietary intake will be assessed by three 24-h dietary recalls, collected with 
dietary recall sheets and either in person or by telephone using Nutrition Data System for 
Research (NDSR, University of Minnesota) software, using a multiple pass system. This method 
has been previously validated against doubly labeled water and has been proven to be accurate 
for assessing dietary intake in groups (Blanton et al 2006). 


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• 
To minimize bias in the methodology: 1. The PI’s of this project will not consent any participant, 
but other research study personnel will obtain consent. 2. Randomization of experimental enemas 
(control and butyrate) will be performed by the compounding pharmacy and the key will be 
provided to the PI’s at the completion of the study; 3: Sample testing (fecal microbiome and SCFA 
in blood) will be completed by core facilities at UNC-CH. RANDOMIZATION PROCESS 
• 
The experimentation will occur at a single site (NC A&T) but sample testing and data analyses will 
occur at 2 sites (NC A&T and UNC-Chapel Hill). 
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
Gut microbial imbalance and low SCFA production is implicated in hypertension and other diseases in AA, 
which are characterized by decreased gut SCFA production. This is a randomized crossover design where 
participants with hypertension will be their own control to quantify the effect of gut butyrate on BP. 
Subjects with hypertension will perform both enema treatments (control saline enema or experimental 
butyrate enema) no sooner than 7 days, but no later than 21 days, apart. We expect to quantify 
measurable increases in circulating butyrate in the blood that will correlate with measurable decreases in 
BP after the butyrate enema and not the control saline enema. In Aim 1, comparisons between 
normotensive and hypertensive participants will be made concerning fecal microbial characteristics and 
SCFA producing bacterial biomass, circulating (blood) butyrate levels, and 24-hour BP (averaged every 
hour during waking and nighttime hours). The control group will be age (±1 year) and sex matched to the 
hypertension group participants. Rationale for Length of Washout Period: We propose no less than a 7 
days between self-administration of the enemas to be a sufficient washout period because the laxative 
effects of the enema are normally immediate (within 30 minutes if no bowel obstruction is present which 
will not be likely as subjects will need to have a bowel movement the day before or morning of the 
experiment), transient (not lasting longer than a 24 hour period if no significant constipation), and 
absorption and metabolism of gut butyrate is expected to be fairly quick (as colonocytes will absorb). Also, 
we expect circulating butyrate levels to exert their effect (if any) on BP within a 24-hour period. 
Participation in the study will be halted if subjects have any issues with constipation and cannot provide 
a stool sample before the experiment.   
4.3 
JUSTIFICATION FOR DOSE 
Enemas have also been used, clinically and experimentally, for drug delivery. Each active butyrate enema 
will consist of 60 mL (2.02 oz. or 4 tablespoons) of an 80 mmol/L sodium butyrate solution titrated to a 
pH of 7.0 with sodium chloride added to maintain iso-osmolality. This concentration was not shown to 
"prompt any adverse effects in the treatment group and there were no withdrawals from drug toxicity". 
(Reference: Steinhart, A.H. et al. Aliment Pharmacol Ther 1996; 10: 729-736 and Hamer et al. Clinical 
Nutrition 2010 29: 738-744). Participants with hypertension will only perform 1 control enema and 1 
butyrate enema seven days apart.  
 
 
4.4 
END OF STUDY DEFINITION 
Normotensive participants (control) will be considered to have completed the study after he or she has 
completed Visit 3 (shown in Schedule of Activities section 1.2). 
 
Hypertension participants will be considered to have completed the study after he or she has completed 
all phases of the study at Visit 5 (shown in Schedule of Activities section 1.2) 
 


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5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
 
Normotensive (control subjects without hypertension): In order to be eligible to participate in this 
study, an individual must meet all of the following criteria (which will be assessed after an initial 
telephone interview and at Visit 1 (screening and consent visit): 
1. Provision of signed and dated informed consent form 
2. Be an African American adult (man or woman) between 30 - 50 years of age with normal blood 
pressure (never diagnosed with hypertension) (systolic: 90-120 and diastolic: 60-89 mmHg). 
3. Body Mass Index of 18.5-30 kg/m2. 
4. Not have any other diagnosed cardiovascular disease 
5. Not exercise regularly (Participate in less than 60 minutes of exercise/week)  
6. Not be pregnant or be lactating 
7. Be free of active diseases that affect your intestines (i.e., chronic constipation, diarrhea, Crohn’s 
disease, ulcerative colitis, irritable bowel syndrome, diverticulosis, stomach or duodenal ulcers, 
diabetes, hepatitis, HIV, and cancer) 
8. Have not taken antibiotics in the past 3 months 
9. Have not been regularly taking medications that impact intestinal function (i.e., laxatives, 
enemas, anti-diarrheal agents, narcotics, antacids, antispasmodics, antidepressants, 
anticonvulsants, antibiotics, herbals, homeopathy, and home remedies) or fiber supplements. 
10. Have no plans of travel out of town during the study periods. 
11. Agreement to adhere to Lifestyle Considerations (see section 5.3) throughout study duration 
 
Hypertension subjects not on anti-hypertension medication (intervention group). The pool of subjects 
that do not take hypertension medication may be limited. If we encounter a heavily medicated subject 
population, we may include subjects taking diuretics only: In order to be eligible to participate in this 
study, an individual must meet all of the following criteria (which will be assessed after an initial 
telephone interview and at Visit 1 (screening and consent visit): 
1. Provision of signed and dated informed consent form. Letter of clearance or signature of PCP on 
informed consent. 
2. Be an African American adult (man or woman) between 30 - 50 years of age with stage-1 to 
stage-2 hypertension (systolic: 130-159 and diastolic: 80-99 mmHg). 
3. Not taking any anti-hypertension medications (although we may enroll individuals only taking a 
diuretic where resting BP levels are within the range of stage-1 hypertension: systolic BP 130-
140 mmHg. Subjects can resume taking the diuretic after they remove the monitor).  
4.  Body Mass Index of 18.5-30 kg/m2 
5. Not have any other diagnosed cardiovascular disease 
6. Not exercise regularly (Participate in less than 60 minutes of exercise/week)  
7. Not be pregnant or be lactating 
8. Be free of active diseases that affect your intestines (i.e., chronic constipation, diarrhea, Crohn’s 
disease, ulcerative colitis, irritable bowel syndrome, diverticulosis, stomach or duodenal ulcers, 
diabetes, hepatitis, HIV, and cancer) 
9. Have not taken antibiotics in the past 3 months 
10. Have not been regularly taking medications that impact intestinal function (i.e., laxatives, 
enemas, anti-diarrheal agents, narcotics, antacids, antispasmodics, antidepressants, diuretics, 
anticonvulsants, antibiotics, herbals, homeopathy, and home remedies) or fiber supplements. 


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11. Have no plans of travel out of town during the study periods. 
12. Agreement to adhere to Lifestyle Considerations (see section 5.3) throughout study duration 
 
5.2 
EXCLUSION CRITERIA 
 
An individual who meets any of the following criteria will be excluded from participation in this study: 
 
1. Exercise more than 60 minutes per week for more than 4 consecutive weeks.  
2. Diagnosed with  stroke, history of myocardial infarction (heart attack); liver, lung, or kidney 
diseases; peripheral vascular disease or cancer within the last 6 months.  
3. Presence of metabolic disease (diabetes mellitus), inflammatory diseases (e.g., inflammatory 
bowel diseases, rheumatoid arthritis, and systemic lupus erythematosus); kidney stones or 
gallbladder problems; diagnosed liver, lung or kidney diseases; 
4. Pregnancy, lactation, or actively trying to conceive. 
5. Taking anti-hypertension medications (i.e., calcium channel blockers, ACE inhibitors, 
angiotensin- receptor blockers, β-blockers, vasodilators, etc.) other than diuretics (e.g.,  
hydroclorothiazide, chlorothiazide, furosemide, etc) or medications known to affect 
inflammation or metabolic function (anti-inflammatories, statins, thyroid medication) in the past 
1 month. If diuretics are used, subjects may able to participate if they agree to refrain from 
taking their diuretic the day of the experiment. In this instance, resting systolic BP while on their 
medication will still need to be greater than 130 mmHg. 
6. Current smoker or tobacco use within the last 10 years 
 
5.3 
LIFESTYLE CONSIDERATIONS 
 
During this study, participants are asked to: 
• 
Refrain from consuming alcohol once their testing period starts (3 days before you collect your 
fecal sample and your Visit 2.) 
• 
Not change their eating habits. 
• 
Participants must not change their exercise habits (start to exercise or exercise more than 60 
min per week during the study). 
• 
Participants taking diuretics may be asked to refrain from taking their medication on the day of 
the experiment.  
 
If the participant cannot adhere to these lifestyle considerations, they may be withdrawn from the 
study. 
 
5.4 
SCREEN FAILURES 
 
Screening Failures: We will account for the total number of potential subjects contacted, and those 
actually enrolled in the study. Individuals who do not meet the inclusion/exclusion criteria for 
participation in this trial (screen failure) will be excluded. Individuals who do not agree to perform every 
aspect of the study, refuse to adhere to the lifestyle considerations during the study, or take longer than 
7 days to sign and return the informed consent (after participating in the initial telephone screening 
and/or Visit 1 informed consent, respectively), may be rescreened if they decided to participate later. 
Rescreened participants will be assigned the same participant number as for the initial screening. 


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5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
We will recruit 10 normal-to-overweight (BMI 18.5-30 kg/m2) African American (AA) adults (5 male/5 
female, 30-50 y.o.) with normal BP (without hypertension) (90-120/60-89mmHg) and 10 age- and gender-
matched normal-to-overweight AAs with stage-1 and stage 2 hypertension (130-159/80-99 mmHg). AA 
with hypertension will perform the intervention, while acting as their own control, to determine the effect 
of gut butyrate on BP. This is a single site study where participants will be recruited from the NC A&T 
community, surrounding area, and from the private practice of our collaborating physician Dr. Bland. Fliers 
will be posted on campus, in community centers, and at Dr. Bland’s office. Dr. Bland will also recruit during 
her weekly radio show “It’s a Matter of Health” on NC A&T radio station 90.1 FM. Subjects will respond 
to fliers and contact the research staff via email or phone. Our group is experienced in culturally sensitive 
methods of subject recruitment in AA, via strong community relationships and health education, and we 
expect no issues with recruitment. Also, the sample size is small (n=10) in the intervention portion and we 
do not expect that we will exhaust the subject pool that may complete the study. We will work to minimize 
non-compliance and provide monetary incentive in this short study. Incentives will occur in the form of 
$100 gift cards for completing each portion of the study. For control subjects, they can receive $100 for 
completing the study (at Visit 3). Hypertension subjects can receive up to $200 for completing the study 
($100 after completing the enema, blood draws, submitting fecal sample, and returning the ABP monitor 
at Visit 3 and Visit 5).  
 
6 
STUDY INTERVENTION 
 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
Participants with hypertension will  self-administer 2 enemas, 7 days apart. 1 enema will be a 0.9% control 
saline enema (60 ml) and the other enema (experimental) will contain sodium butyrate at a concentration 
of 80 mM in 60ml total volume. Both the subjects and the PI’s will be blinded to the order the enemas are 
performed (double blinded crossover design). One member of the research staff will perform the blinding 
and will release the key at the conclusion of the study (all participants completed). Both enemas will be 
made/compounded at Custom Care Pharmacy (Greensboro, NC) for quality control and matched for 
sodium concentration. 
 
Justification for IND exemption: According to the Guidance for Clinical Investigators, Sponsors, and IRBs 
IND Guidelines, the use of the sodium butyrate enema would be classified as exempt from the need for 
FDA review and regulation for this study. Sodium Butyrate is a short chain fatty acid that is commercially 
available as a dietary supplement. Delivery of butyrate, via enema, has been used and reported in 
published clinical studies. Two studies have used the butyrate enema to quantify its effect on gut 
inflammation (Hamer et al. Clin Nutr 2010, 29:738-44; Steinhart et al. Aliment Pharmacol Ther 1996, 
10:729-36. In these studies, there were no reported adverse reactions or side effects in its use in the 
clinical populations. 
Rationale: Although butyrate is commercially available in ingestible forms (powder and pills), we do not 
normally ingest butyrate. Certain bacteria in our gut (colon) produce it from fermenting food products 
(fiber). The level of butyrate producing bacteria has been associated with blood pressure (BP) and it is 
believed that greater butyrate production in the gut is associated with better cardiovascular health and 


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lower BP. The butyrate produced in the gut is absorbed by it into our blood stream, just as other nutrients. 
Our purpose in utilizing the butyrate enema is to deliver a measurable, and safe, amount of butyrate into 
the colon (where it is naturally produced). We are not using it to diagnose, treat, or cure high BP 
(hypertension). We are only using this delivery method to quantify how much is absorbed into the blood 
stream and measure if it has any effect on BP in individuals with moderately elevated BP.  
 
The enemas will be compounded by a local pharmacy based on concentrations previously used in 
published studies. Our collaborating study physician will write a prescription for the placebo (0.9% saline, 
as found over-the-counter) and butyrate enema  to be made. Each active butyrate enema will consist of 
60 mL (2.02 oz or 4 tablespoons) of an 80 mmol/L sodium butyrate solution titrated to a pH of 7.0 with 
sodium chloride added to maintain iso-osmolality. The compounding pharmacy will label each enema with 
the following wording: “Caution: New Drug—Limited by Federal (or United States) law to investigational 
use,” in conjunction with the regulations found in 21 CFR 312.6. The study is a double-blinded randomized 
controlled trial.  
 
Randomization will be performed by 1 member of the research team at the beginning of the study. 
Enemas will be assigned a letter (e.g., A or B) and each letter will be placed in an envelope. Each time a 
subject completes consent, the research staff responsible for randomization will randomly choose an 
envelope which will coincide with which enema they will receive first at Visit 2 (A or B). They will receive 
the other enema at their Visit 4. That staff member will reveal the key upon the conclusion of the study.  
 
6.1.2 DOSING AND ADMINISTRATION 
Participants with hypertension will complete 2 studies, 1 with each enema (control saline or butyrate) in 
a random order. Dosing was based on previous studies that report no adverse events associated with the 
sodium butyrate enema below 80 mmol/L concentration. Enemas will be performed in the morning 
(before 9am). We will educate participants on how to efficiently perform the self-administered enema, to 
reduce variability, and measure remaining solution to calculate dose delivered. Nurses will be present to 
aid in administering enema’s if participants are uncomfortable with doing this themselves. The PI and 
nurses will be present during the entire experiment time.  BP will be monitored, via the 24-hour ABP 
monitor, for 1 ½ hours after the enema in the laboratory and then the subject will go home with an ABP 
monitor and will wear it until they return it the following day (see procedures in section 4.1, bullet 3).  
 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
 
Subjects with hypertension will need medical clearance from their primary care physician (PCP) before 
they can be included in the study (see Schema 1.2 – Visit 1 and Inclusion - Section 5.1). If they do not have 
a PCP, they will be referred to Dr. Bland, our collaborating and study physician to provide medical 
clearance. Dr. Bland will write the prescription for the enemas to be compounded by the pharmacy for all 
participants. The pharmacy will generate both enemas and research staff (not the PI’s) will pick up the 
prescriptions. Randomization process is described in section 6.1.1.   
 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 


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Both enemas (control and butyrate) will be in an enema applicator bottle (60ml total volume). Sodium 
butyrate is available for purchase as a dietary supplement. We will supply the pharmacy with order 
information for its purchase for them to use. The compounding pharmacy will label the package with 
Enema A or B. One will be the experimental enema with butyrate and one will be the control. Only the 
pharmacy and the research staff member responsible for the randomization will know which enema A 
and B are. The pharmacy will also label the enema with this statement:  Caution: New Drug—Limited by 
Federal (or United States) law to investigational use” in accordance with the regulations found in 21 CFR 
312.6. 
 
6.2.3 PRODUCT STORAGE AND STABILITY 
Both enemas will be stored at room temperature by the pharmacy and by the researchers until giving 
them to the participants on the testing day.  Again, their randomization schedule will be completed once 
they complete informed consent (at Visit 1) and have a random envelope chosen for their treatment 
enema. They will receive that enema, either A or B, at Visit 2 and the other enema at Visit 4.  
 
 
6.2.4 PREPARATION 
Preparation of both enemas will be done by the compounding pharmacy. The study staff will not need to 
do any preparation to the products other than mixing (shaking well) before giving them to the subjects 
before self-administration.  
 
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
Aim 2 is a double blinded randomized placebo-controlled crossover trial. Only the participants with 
hypertension will perform the enema in a randomized order. The subjects, nor the PI’s, will know which 
enema the subjects are receiving. Randomization will be performed by 1 member of the research team at 
the beginning of the study whom is also not involved in any informed consent process. Enemas will be 
assigned a letter (e.g., A or B) and each letter will be placed in an envelope (blinded allocation 
concealment). Each time a subject completes consent, the subject will be presented with a stack of 
envelopes will choose an envelope which will contain a card coinciding with which enema they will receive 
first at Visit 2 (A or B). They will receive the first enema at Visit 2 and the remaining enema at their Visit 
4. The research team member will reveal the key upon the conclusion of the study.  
 
Since there has been no reported side effects of the butyrate enema, we do not anticipate being able to 
identify which enema was administered because of side effects. The only side effect for both would be a 
bowel movement. We expect all participants to have a movement within 10 minutes of the 
administration.  
 
 
6.4 
STUDY INTERVENTION COMPLIANCE 
 
Adherence to the protocol will consist of participants completing the following: 
Control subjects (without hypertension): 
1. Completing the 3-day dietary recall and submitting a fecal sample immediately before (day prior) 
or on the testing day. 
2. Submitting to a manual resting BP measurement and a (1) blood draw. 


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3. Wearing the 24-hour ambulatory blood pressure monitor after the blood draw and returning the 
monitor with activity diaries on the following day.  
 
Hypertension subjects: 
1. Completing the 3-day dietary recall and submitting a fecal sample immediately before (day prior) 
or on each experimental testing day (two in total). 
2. Submit to a manual BP measurement and blood draw (time 0) then self-administer the enema 
and submitting to subsequent manual BP measurements and blood draws (time 30min & 60 min) 
after the enema. 
3. Wearing the 24-hour ambulatory blood pressure monitor after the blood draws and returning the 
monitor with activity diaries on the following day. 
  
Adherence to the protocol will be encouraged with a monetary payment after completion of the steps 
above and returning the monitor. 
 
6.5 
CONCOMITANT THERAPY 
Not Applicable 
 
6.5.1 RESCUE MEDICINE 
Not Applicable 
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
The proposed project is a phase I clinical trial that does not have multiple sites. There is no inclusion of 
vulnerable populations and does not involve “high risk” procedures. This is an uncomplicated study that 
includes enemas as the sole experimental intervention.  With this, the PI, study physician, and nursing 
collaborators will primarily be responsible for monitoring the safety of each participant. The PI will be 
responsible for reviewing overall study progress and minimizing research associated risk by monitoring 
activities of the research and updating records on a weekly basis with study team members. Dr. Cook, in 
consultation with the study physician (Dr. Bland), will report any adverse events to the IRB. Any sign, 
symptom, or abnormal assessment during the study will be immediately reported to Dr. Bland via phone, 
when she is not present, whom will assess the event at that time. Every effort will be made to determine 
the cause of any events that occur during the subjects’ participation in the study to determine if the 
adverse event was related to study activities. Under the direction of the study physician and IRB, we will 
suspend any activity that related to any consistent signs of initiating adverse events in our subject 
population. Whenever appropriate, we will amend informed consent and/or study protocol documents in 
response an adverse event. 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following reasons: 
 
• 
Pregnancy 
• 
Significant study intervention non-compliance  


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• 
If any clinical adverse event (AE), other medical condition, or situation occurs such that continued 
participation in the study would not be in the best interest of the participant 
• 
Participant develops any infection that requires antibiotic prescription and usage 
• 
Disease progression which requires discontinuation of the study intervention 
• 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• 
Participant with Hypertension unable to complete the enemas within 21 days of each other. 
The reason for participant discontinuation or withdrawal from the study will be recorded on their Case 
Report Form (CRF). Subjects who sign the informed consent form and are randomized but do not receive 
the study intervention may be replaced.  Subjects who sign the informed consent form, and are 
randomized and receive the study intervention, and subsequently withdraw, or are withdrawn or 
discontinued from the study, will be replaced. 
7.3 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if he or she fails to return for the data collection or 
experimental scheduled visits for 2 weeks and is unable to be contacted by the study site staff.  
 
The following actions must be taken if a participant fails to return for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit within 1 week of 
contact and counsel the participant on the importance of maintaining the assigned visit schedule 
and ascertain if the participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, 
a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts will be documented in the participant’s study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
EFFICACY ASSESSMENTS  
Telephone screening  
1. Once subjects respond, via email or phone, to recruitment materials, we will assess if they meet 
the inclusion criteria for either group via phone (see telephone screening questionnaire). After 
completion of the questionnaire, the screener will be able to make the potential subject aware of 
their eligibility. The telephone screener will be able to let the. We will mail their informed consent 
or schedule a time for the participant to pick one up and schedule their Visit 1 at that time. 
Visit 1: Screening visit – Informed consent and questionnaires 
1. Medical history, physical activity, and dietary recall questionnaires: The PI (Dr. Marc Cook) will 
review the medical history questionnaire to ensure all the questions and their answers regarding 


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the participant’s health, current medication use, medical history and exercise pattern are clear. 
The PI will also confirm that their PCP has agreed with their participation in the study. Participants 
will also fill out a physical activity readiness questionnaire (PAR-Q) to determine the amount of 
time they exercise currently. The PI will review these documents to ensure eligibility to participate 
in this study.  Participants will be coached on how to complete their food and beverage intake for 
the 3 days following this initial screening. We will provide the paper forms to complete the records 
before they submit the stool samples. Pre-menopausal women will be asked to confirm they are 
not pregnant by taking an over-the-counter pregnancy test we will provide. This is the only time 
a participant will be asked to take a pregnancy test. A member of the research team will assess 
that the result is negative for them to proceed through the study. 
2. Manual blood pressure will be recorded 3 times within 3 minutes, on the participants non-
dominant arm, after sitting for 5 min. An average of the last 2 BP readings will be recorded as their 
resting BP. This BP measurement will also confirm that they  are Normotensive (BP ≤129 mmHg 
systolic) or have Hypertensive (BP ≥ 130 mmHg systolic). 
3. Body Composition: Height and body weight measured on a scale that will determine body weight, 
body fat mass, body muscle mass, and total body water. The scale requires standing on the scale 
barefoot and hold on to the machine. The test takes approximately 2 minutes to complete the 
measurement. 
Visit 2: Measurements and Sample collection 
1. Body Composition (both groups): Body weight will be measured on a scale that will determine 
body weight on the same scale as before. The test takes approximately 2 minutes to complete the 
measurement. This is to ensure no significant shifts in body composition (weight gain or loss ±2.2 
lbs. or 1 kg) since their visit 1.  
2. Stool sample collection (both groups): Participants will submit a stool sample collected at home 
within 24-hours of this visit (collected the day before or the morning of the experiment) and will 
submit the 3-day dietary recall. Stool samples will be collected by the clean catch method and the 
swab method. Participants will use the protective equipment given (gloves, toilet hat, spoon, and 
sterile container) to collect a spoonful stool sample for fecal butyrate analysis. Participants will 
also apply a sterile swab to used toilet paper to collect the sample for fecal microbiome analyses 
and place in the sterile container. If there is minimal residue on the toilet tissue after wiping, the 
subject will need to gently rub the tip of the sterile swab across their stool sample collected, via 
the clean catch method, and place in the sterile container. 
3. Placement of 24-hour ABP monitor (both groups): Participants will be fitted for their ABP monitor, 
rest for 5 minutes, and have their resting BP measured 3 consecutive times. The cuff will remain 
on their non-dominant arm throughout the experiment and until they return the monitor the next 
day. 
4. Blood sampling (both groups): Blood will be taken from the antecubital (forearm) vein by a 
phlebotomist or nurse. A small amount (about 3 tablespoons) of blood will be taken. Plasma 
butyrate will be measured by the UNC-CH  Biomarker Mass Spectrometry Core in the UNC Gillings 
School of Global Public Health.  Dr. Cook will measure biomarkers in plasma (IL-1β, CRP), oxidative 
stress (8-Isoprostane), nitric oxide (quantifying total nitrate/nitrite), and serotonin via Elisa (R&D-
Abcam) in his laboratory.  
a. In hypertensive subjects only: Blood draw will occur right before they complete the 
enema. After the enema, participants will have less blood taken (about 2 tablespoons) at 
30 minutes and 60 minutes (1 hour) after administering the enema.  


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5. In Hypertensive subjects only: Blood pressure will be measured manually and then after the 
placement of the ABP monitor on their non-dominant arm before the enema, manually before 
the 30 min blood draw and manually before the 60 min blood draw. The PI and nursing research 
staff will ensure that participants are not experiencing any symptoms related to the treatment, 
as well as make sure their BP is within the normal range for them. Participants will be monitored 
for longer if they exhibit a ≥ 20 mmHg change in their BP along with symptoms (presyncope). If 
Dr. Bland is not present, she will be called and alerted to the event immediately. 
6. 24-hour blood pressure monitoring (both groups): will be obtained via Mobil-O-Graph® 24h PWA 
(IEM) ambulatory blood pressure (ABP) monitoring device. Subjects will be fitted with the non-
invasive monitor (Mobil-O-Graph BP PWA) to measure BP and arterial stiffness for 24-hrs on the 
day they perform each enema. The 24-hr period will begin after completion of the enema and 
blood draws. The BP cuff will be fitted to participant’s non-dominant arm with cuff size 
determined by upper arm circumference. BP measurements will be obtained at 30-min intervals 
during the day (700–2200 hours) and 60-min intervals at night (2200–700 hours). Participants will 
be instructed not to exercise before or during the monitoring period and to pause momentarily 
and maintain their body position during each BP measurement. They will also be given 
instructions (verbal and written) on how to keep a blood pressure diary, where they will record 
their activities at the time the monitor was recording the blood pressure. 
Visit 3: Return the 24-hour BP monitor (both groups). Participants will receive compensation at this time. 
This will complete the control (Normotensive subjects study) 
Visit 4: Hypertension participants only: Repeat Visit 2 by completing the other enema. 
Visit 5: Return the 24-hour BP monitor. You will receive compensation at this time. 
 
8.2 
SAFETY AND OTHER ASSESSMENTS 
• 
Assessment of Eligibility. Preliminary review of eligibility will occur during their initial telephone 
screening performed by research staff. They can be excluded from the study then if then if they do 
not meet all the inclusion criteria. If it is appropriate to continue, they will be sent (or can pick up) 
an informed consent form prior to their official Visit 1 to review and have their primary care 
physician (PCP) review. Participants with hypertension will need to obtain approval from their PCP 
to ensure they are healthy enough to participate in this study.  We expect most, if not all, of our 
participant pool to be recruited from Dr. Bland’s practice. They will not be required to have a full 
physical.  
o If the participant has a PCP, the participant will be given their informed consent for their 
PCP to review the study protocols. The PCP will acknowledge that the patient has mild-to-
moderate hypertension (within the ranges specified in the informed consent) and there 
are no contraindications to their participation. The PCP will provide the research team 
with a letter stating that they have reviewed the study information and they approve of 
their patient participating in the study.  
o If the participant does not have a PCP, they will be referred to Dr. Bland. She will review 
their history and provide an assessment their ability to participate by confirming they do 
have mild-to-moderate hypertension and there are no contraindications to their 
participation. This will be done free of charge to the participant. However, because they 
do not have a regular PCP, they may become a patient of Dr. Bland’s (or referred to 


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another physician) which they would pay for services. This is not prerequisite on them 
participating in the study.  
o After this, the PI will assess if the participant continues to meet all the inclusion criteria to 
be admitted into the study.  
o Dr. Bland will review the provided medical history form of each participant and confirm 
their PCP has given consent for them to participate before writing the prescription for the 
enema’s to be compounded by the Pharmacy.  
• 
Vital signs. Blood pressure will be monitored every 30 minutes while they are in the lab during the 
experiment and for 24-hours after they self-administer the enema (every 30 minutes during 
waking hours and every hour while they are asleep). If the participant experiences any AE’s during 
the experiment (in the lab), the research team will contact Dr. Bland immediately, via phone, if she 
is not present. If they experience any AE’s after leaving, participants will be directed to 
immediately contact the research team (PI or nurses) to assess and we will contact Dr. Bland if 
needed. If they experience any SAE’s they will be directed to seek emergency services and Dr. Bland 
will be notified immediately.    
• 
Biological specimen collection and laboratory evaluations. We will collect stool samples and 
blood. Bioinformatics on gut microbial composition (diversity, richness, biomass) and identification 
of specific SCFA producing microbes will be performed and compared between AA with and 
without hypertension. Blood will be collected to measure circulating butyrate levels at rest and 
after each enema (in hypertension group only). Gut microbial bioinformatics and blood butyrate 
concentrations will be measured by Microbiome Core and Biomarker Mass Spectrometry Core 
laboratory facilities, respectively, at UNC-Chapel Hill. In Dr. Cook’s laboratory, we will also 
measure biomarkers related to vascular health and blood pressure via enzyme linked 
immunosorbent assays (Elisa) (e.g., inflammatory cytokines (C-reactive protein & interleukin-1β), 
nitric oxide, endothelin-1, oxidative stress (8-isoprostane), and serotonin). Each of these measures 
has been identified to be related to elevated BP in humans. Butyrate, being anti-inflammatory, 
and the abundance of microbes that produce butyrate are likely related to BP. Each of these 
measures, along with body composition measures and macronutrient (carbohydrate – with soluble 
and insoluble fiber, fat, & protein) of 3-day dietary recall will be utilized to help explain gut 
microbial composition, circulating biomarkers, and the effect of butyrate enema on BP in the 24 
hour period. Dr. Carroll will perform bioinformatic statistical analyses in his laboratory. 
Comparative statistical analyses will be performed to define the relationship between BP, gut 
microbial composition, circulating butyrate, and biomarkers of vascular health. 
• 
Counseling procedures, including any dietary or activity considerations. At the screening visit (1), 
participants will be counseled on how to complete their dietary recall.  
• 
Administration of the enema. Participants, at Visit 1 and 2 (right before administration), will be 
educated on how to self-administer the enema. They will be provided with a private bathroom 
space to administer it. Research staff will be close by to support the participant if needed. If the 
participant is uncomfortable with self-administering the enema, a research staff who is a nurse 
may administer it for them if the participant chooses. After the administration, the participant will 
recap the enema applicator bottle and return it to the research staff to measure how much they 
were able to deliver.  
• 
Assessment of study intervention adherence.  
Adherence to the protocol will consist of participants completing the following: 
Control subjects (without hypertension): 
a. Completing the 3-day dietary recall and submitting a fecal sample before the testing day. 
b. Submitting to a blood draw. 


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c. Wearing the 24-hour ambulatory blood pressure monitor after the blood draws and 
returning the monitor with activity diaries on the following day.  
Hypertension subjects: 
a. Completing the 3-day dietary recall and submitting a fecal sample before each 
experimental testing day. 
b. Self-administering the enema and submitting to a blood draws. 
c. Wearing the 24-hour ambulatory blood pressure monitor after the blood draws and 
returning the monitor with activity diaries on the following day. 
  
Adherence to the protocol will be encouraged with a monetary payment after completion of the steps 
above and returning the monitor. 
o Same as Study Intervention Compliance, section 6.4 
• 
Administration of questionnaires or other instruments. Participants will keep a dietary record for 
3 days before they submit any stool sample. They will also be provided with a paper diary to record 
their activities when their blood pressure was being recorded by the ambulatory BP monitor. 
Subjects will also complete a “perceived stress” questionnaire in effort to assess the current levels 
of perceived psychological stress. Heightened stress has been associated with higher blood 
pressure (Hamer M. 2012 Psychosom. Med. Nov-Dec;74(9):896-903) and reduced gut microbial 
diversity (Moloney RD. et al. 2014 Feb;25(1-2):49-74). 
• 
Assessment of adverse events (AE’s). Research staff (the PI and Nursing Faculty at NC A&T) will 
be present during the experiment, will draw blood, and will assess any adverse events witnessed 
or reported by the participant (specifically unfavorable changes in BP, although this phenomenon 
and others have not been previously reported). Dr. Bland will be notified, via phone of she is not 
present, immediately. We will also see the participants the following day when they return their 
ABP monitor. At that time, we will collect their BP diary and follow up with any issues reported by 
the participant. Participants will be instructed to discontinue the use of the ABP monitor if any 
significant skin irritation occurs that is not bearable.  
o Follow-up. Study staff (Dr. Cook and Nurses) will record all reportable events with start 
dates occurring any time after informed consent is obtained until 7 (for non-serious AEs) 
or 30 days (for SAEs) after the last day of study participation. We will instruct participants 
to inform us (phone call or email) if they feel they are experiencing symptoms that may 
be related to their participation in this study. We will also contact them, via their 
preferred contact method (phone or email) at days 7 and 30 post study to inquire about 
them experiencing AE or SAEs. At each study visit, we will inquire about the occurrence 
of AE/SAEs since the last visit.  Events will be followed for outcome information until 
resolution or stabilization. 
 
   
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
In this study, an Adverse Event means any untoward medical occurrence associated with the use of the 
study specific intervention tools (enema, sample collections, BP monitor) in the participants, or whether 
or not an event occurs while the participant is actively enrolled in the study and requires assessment 
that the AE could be intervention-related. 
 


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8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
An Adverse Event or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or funding agency, it results in any of the following outcomes: death, a life-threatening 
adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or 
significant incapacity or substantial disruption of the ability to conduct normal life functions, or a 
congenital anomaly/birth defect. Important medical events that may not result in death, be life-
threatening, or require hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the participant and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the development of drug dependency or 
drug abuse. 
 
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
 
8.3.3.1 SEVERITY OF EVENT 
 
For adverse events (AEs) not included in the protocol defined grading system, the following guidelines will 
be used to describe severity.  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
 
All adverse events (AEs) will have their relationship to study intervention assessed by the clinician (Dr. 
Bland) who may examine and evaluate the participant based on temporal relationship and his/her clinical 
judgment. The degree of certainty about causality will be graded using the categories below. In a clinical 
trial, the study product will always be suspect.  
 
• 
Related – The AE is known to occur with the study intervention, there is a reasonable possibility 
that the study intervention caused the AE, or there is a temporal relationship between the study 
intervention and event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study intervention and the AE. 
• 
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study intervention 
and event onset, or an alternate etiology has been established. 
 


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8.3.3.3 EXPECTEDNESS  
 
Dr. Veita Bland, MD and the PI Dr. Marc Cook, will be responsible for determining whether an adverse 
event (AE) is expected or unexpected.  An AE will be considered unexpected if the nature, severity, or 
frequency of the event is not consistent with the risk information previously described for the study 
intervention. Dr. Cook and clinical research nursing staff will monitor participants during and immediately 
after the experiments. 
 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention of 
study personnel during study visits and interviews of a study participant or upon review by the research 
staff. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate case report form (CRF). Information to be collected includes event description, time of onset, 
clinician’s assessment of severity, relationship to study product (assessed only by those with the training 
and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring 
while on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
 
Hypertension (>130/80mmHg), which will be present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study (increase in BP to greater than or equal to 180/100 mmHg), it 
will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event 
at each level of severity to be performed. AEs characterized as intermittent will be documented at onset 
and duration of each episode. 
 
Study staff (Dr. Cook and Nurses) will record all reportable events with start dates occurring any time after 
informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation. We will instruct participants to inform us (phone call or email) if they feel they are 
experiencing symptoms that may be related to their participation in this study. We will also contact them, 
via their preferred contact method (phone or email) at days 7 and 30 post study to inquire about them 
experiencing AE or SAEs. At each study visit, we will inquire about the occurrence of AE/SAEs since the 
last visit.  Events will be followed for outcome information until resolution or stabilization. 
 
8.3.5 ADVERSE EVENT REPORTING  
Dr. Cook, Dr. Bland (study physician), and nursing collaborators present will primarily be responsible for 
monitoring the safety of each participant. The PI will be responsible for reviewing overall study progress 
and minimizing research associated risk by monitoring activities of the research and updating records on 
a weekly basis with study team members. Dr. Cook, in consultation with the study physician, will report 
any adverse events to the IRB and Funding agency. Any sign, symptom, or abnormal assessment during 
the study will be immediately reported to subject and their primary physician for follow-up assessment. 
Every effort will be made to determine the cause of any events that occur during the subjects’ 
participation in the study to determine if the adverse event was related to study activities. Under the 


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direction of the study physician and IRB, we will suspend any activity that related to any consistent signs 
of initiating adverse events in our subject population. Whenever appropriate, under the direction of the 
IRB, we will amend informed consent and/or study protocol documents in response an adverse event. 
Participants will have hypertension, but BP’s will be measured every 30 (wake hours) to 1 hour (sleeping 
hours). We will review the BP immediately upon the return of the ABP monitor.  
 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
 
The PI and/or study physician (Dr. Bland) will immediately report any serious adverse event to the IRB and 
funding agency, whether or not considered study intervention related, including those listed in the 
protocol or investigator brochure and must include an assessment of whether there is a reasonable 
possibility that the study intervention caused the event. Study endpoints that are serious adverse events 
(e.g., all-cause mortality) will be reported in accordance with the protocol unless there is evidence 
suggesting a causal relationship between the study intervention and the event (e.g., death from 
anaphylaxis). In that case, the investigator will immediately report the event to the IRB and funding 
agency. 
 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site investigator 
deems the event to be chronic or the participant is stable. Other supporting documentation of the event 
may be requested by the Data Coordinating Center (DCC)/study sponsor and should be provided as soon 
as possible. 
 
The PI will be responsible for notifying the IRB (via phone call and email to the scientific review officer) of 
any unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no later 
than 7 calendar days after the IRB’s initial receipt of the information.   
 
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
 
Current and future participants will be notified of all unexpected events that occur by the research staff. 
For example, these may include frequent bowel movements or a measurable increase in BP or 
hypotensive event (BP measured below systolic BP 90 mmHg and/or diastolic BP 60 mmHg) after self-
administration of the enema. These have not been previously reported but are possible. After 
consideration of any unexpected event by the IRB, we will follow their instruction on whether they 
determine it is in the best interest to notify current and future participants by updating the informed 
consent risks. This may require the current subjects (signed consent forms) to reconsent to the study to 
allow them the opportunity to decide if they still want to continue their participation. 
 
8.3.8 EVENTS OF SPECIAL INTEREST  
Not Applicable 
 
8.3.9 REPORTING OF PREGNANCY  
Not Applicable 
 
 
8.4 
UNANTICIPATED PROBLEMS 


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8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
If any UP arises, we will report problems to the IRB and grant agency and follow their instructions to 
addressing these UPs, which may include but are not limited to: Modifying the inclusion/exclusion criteria 
and informed consent to mitigate the newly identified risks, provide additional verbal and written 
information concerning the new identified risks, and suspend the enrollment of new participants or 
halting the study procedures for enrolled participants, if necessary.   
8.4.2  UNANTICIPATED PROBLEM REPORTING  
The principal investigators and/or study physician will report unanticipated problems (UPs) to the 
reviewing Institutional Review Board (IRB) and grant funding agency. The UP report will include the 
following information: 
 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:   
 
• 
UPs that are serious adverse events (SAEs) will be reported to the IRB and funding agency within 
3 days of the investigator becoming aware of the event.  
• 
Any other UP will be reported to the IRB and study sponsor within 7 days of the investigator 
becoming aware of the problem.  
• 
All UPs will be reported to appropriate institutional officials (as required by an institution’s written 
reporting procedures), the supporting agency head (or designee), and the Office for Human 
Research Protections (OHRP) within 7 days of the IRB’s receipt of the report of the problem from 
the investigator. 
 
 


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8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
Participants will be informed about UPs within 24 hours. We expect participants experiencing UPs to 
inform us as soon as possible. At that time, we determine if that is an unanticipated problem within 24 
hours. If it significantly affects their activities of daily living during or within 14 days of their participation, 
we will notify current and future participants in writing, and by updating the informed consent, to make 
them aware of this new UP. We will also immediately follow-up with subjects who completed their 
participation to inquire whether they have experienced the UP at any time during and up to 14 days after 
their participation. 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STATISTICAL HYPOTHESES 
Our central hypothesis is that low gut butyrate availability is related to hypertension in AA, and that 
acutely increasing gut butyrate will translate into greater circulating butyrate and an acute and 
measurable reduction in BP. 
 
1. We expect AA with hypertension will have significantly (p< 0.05) lower SCFA (butyrate) producing 
microbes in their stool samples, compared to their normotensive age and sex matched peers. We expect 
that AA with hypertension will have greater circulation of inflammatory biomarkers, oxidative stress, and 
lower nitric oxide, compared to the control group. 
 
2. We expect circulating butyrate to be significantly lower in AA with hypertension which will be inversely 
related to BP levels (low circulating butyrate = higher BP).  
 
3. In the hypertension group, we expect the butyrate enema to increase circulating butyrate levels and 
elicit an acute reduction in BP within 24 hours of self-administration of the butyrate enema. We expect 
no change in circulating butyrate levels or BP after the control (saline) enema.    
 
9.2 
SAMPLE SIZE DETERMINATION 
Power Analysis and Sample size estimate: This is a proof of concept pilot study. Data does not exist for 
estimating the effect size of the proposed treatment (cross-sectional or cross-over study), which itself will 
be used to determine appropriate ranges of effect sizes for subsequent studies. We consulted Dr. Michael 
Love (UNC-CH NC TraCS Biostatistician). Sample size for this study was influenced by cost of study (subject 
renumeration and sample analyses) and the ability to feasibly recruit and retain subjects within the grant 
timeline and complete data analyses the study in the 1-year grant period. Further, VANHOUTVIN SA et al. 
(reference in reference section) recruited 11 subjects (10 completed) in their study on the effects of 
butyrate enema on visceral perception and safety.  
 
9.3 
POPULATIONS FOR ANALYSES 
Aim 1: 10 normal-to-overweight (BMI 18.5-30 kg/m2) AA adults (5 male/5 female, 30-50 y.o.) with normal 
BP (90-129/60-89mmHg) and 10 age- and gender-matched normal-to-overweight AAs with stage-1 
hypertension (130-159/80-99 mmHg) will provide fecal and blood samples to quantify differences in the 
abundance of fecal butyrate-producing microbes and circulating butyrate between AA with- and without 
hypertension. We hypothesize that participants with normal BP, when compared to individuals with 


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hypertension will have a greater abundance of butyrate producing microbes in their stool and greater 
plasma butyrate concentrations.  
 
Aim 2: In this crossover blinded randomized controlled trial, over two visits that are 1 week apart, the 
same 10 hypertensive AAs will be randomized to self-administer a sodium butyrate (80mM butyrate in 
saline) or control saline enema in this paired research design. Butyrate enemas are well tolerated in 
human studies. BP will be manually monitored immediately after the enema, and a 30 min and 60 min 
post enema. At these times, post-enema blood will be collected to determine plasma butyrate 
concentrations. Also, participants will wear a 24h ABP monitor to measure oscillations in BP after the 
enema. We hypothesize that only the butyrate enema will elicit a reduction in BP within the 24-hour 
period after the enema. We expect to see no significant change in BP after the control enema. 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
Statistical Analyses: Aim 1: In this age and sex-matched cross-sectional design study (differences between 
individuals with normal BP and hypertension), paired t-test will be used to determine differences 
circulating blood butyrate, blood biomarkers (inflammatory markers, oxidative stress, nitric oxide levels – 
section 8.2), the relative abundance of butyrate producing fecal microbes. ANOVA with covariate analyses 
(i.e., BMI, intake of dietary fiber (grams), mean difference in resting BP between groups) will be used to 
determine these variables relationship to blood butyrate, blood biomarkers, butyrate producing 
microbes, and BP between the control and hypertension groups. Stratification method will be utilized to 
analyze results stratified by age (30-40 yr old and 40-50 yr old) and BMI (normal to overweight). 
 
Aim 2: Repeated measures ANOVA will be employed to determine the relationship between enema 
(butyrate and saline) treatment on circulating butyrate (0, 30, 60 min), biomarkers, and hourly mean (day-
time and night-time) systolic and diastolic  BP measured via 24-hr ABP monitoring. Utilizing a generalized 
linear regression model analyses, we will utilize variables (i.e., dietary carbohydrate, fat, protein, and fiber 
intake; blood biomarker values) to estimate their relationship with BP. Magnitude of change in blood 
butyrate after each enema will be calculated to perform analyses to quantify the relationship between 
blood butyrate changes and BP changes. 24-hr ABP data will be averaged for each hour. Analyses of these 
measures will be performed following Edwards & Simpson Blood Press Monit. 2014 June ; 19(3): 153–163. 
Stratification method will be used to quantify any effect of “stage of hypertension (systolic: 130-139   and 
140-149, 150-159)” on magnitude of change in BP after the treatments. Sensitivity analyses (including 
correlation, regression, and subjective) will be performed to explore extreme or questionable results in 
changes in BP utilizing appropriate variables and covariates (e.g., change in circulating butyrate, dietary 
fiber intake).   
 
Overall: Data will be considered statistically significant at a p≤0.05 and all statistical methods will be 
tabulated with their corresponding confidence intervals. Hypothesis testing with a p≥0.05 will be 
considered not statistically significant and will be reported as inconclusive. Pearson r and non-parametric 
Spearman Rho correlation analysis will be used to determine relationships between enema, circulating 
butyrate, and difference/change values of BP.  
 
Microbiome Analyses. We will perform bioinformatic microbiome analyses of the raw sequence data. 
PICRUSt will be employed to identify differentially abundant genes in samples. Predictive functional 


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profiles will be used to identify and quantify SCFA and butyrate producers. Comparisons will be made 
between normotensive and hypertensive subjects using Fishers exact tests. With this, we will identify the 
relative abundances of specific butyrate-producing microbial taxa to quantify their association with BP. 
Relative abundance values of these taxa will be input in the statistical models explained in Aims 1 & 2.    
 
Missing Data: Depending on the missing variable we will consider utilizing regression analyses, pairwise 
deletion analyses, or multiple data imputation methods.  
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
Please see section 9.4.1. 
 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
N/A 
 
9.4.4 SAFETY ANALYSES 
N/A 
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
Descriptive statistics (mean ± SEM) will be reported for all subject characteristics (besides gender) and 
outcome variables (BP, blood butyrate and biomarkers). Correlative statistics (Pearson r, Spearman Rho, 
Fisher Exact Tests) will be used to determine the relationship between circulating butyrate, microbial SCFA 
producing biomass, and resting BP, as well as average hourly BP measured with the ABP monitor. 
 
9.4.6 PLANNED INTERIM ANALYSES  
This intervention is not being used to treat or cure hypertension. Therefore, we will not be performing 
any interim analyses of efficacy or appropriateness of treatment using this experimental design.  
 
9.4.7 SUB-GROUP ANALYSES 
African Americans are the sole population being studied in this proposal. We do not intend to perform 
sub-group analyses (e.g., sex) in this small pilot study. The participants will be sex, age, and BMI matched. 
These data will be used to perform power analyses for subsequent studies.  
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
Group means, not individual participant data, of all variables will be reported.  
 
9.4.9 EXPLORATORY ANALYSES 
Not Applicable 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 


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10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
 
Consent forms describing in detail the study intervention, study procedures and assessments, risks and 
benefits, compensation and cost to participants, and confidentiality will be provided to the participant 
before starting any procedures or collecting any data. If the participant has hypertension, they will be 
provided with a consent form to discuss with their physician. Informed consent will be completed at Visit 
1 (consent visit), which is required prior to starting intervention/administering study intervention.  
 
The following consent materials are submitted with this protocol: 1. Control subject (normotensive) and 
Hypertension subject informed consents. Also included are questionnaires (health history, physical 
activity, demographic, telephone), diaries (3-day dietary recall, 24-hour ambulatory blood pressure diary), 
recruitment materials (email blast, radio advertisement, and flyer), and informational forms (swab 
method, ambulatory blood pressure monitor booklet).  
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study 
and continues throughout the individual’s study participation. Consent forms will be Institutional Review 
Board (IRB)-approved and the participant will be asked to read and review the document. The 
investigator/research staff will explain the research study to the participant and answer any questions 
that may arise. A verbal explanation will be provided in terms suited to the participant’s comprehension 
of the purposes, procedures, and potential risks of the study and of their rights as research participants.  
Participants will have the opportunity to carefully review the written consent form and ask questions prior 
to signing. The participants will have the opportunity to discuss the study with their family or surrogates 
or think about it prior to agreeing to participate. The participant will sign the informed consent document 
prior to any procedures being done specifically for the study. Participants will be informed that 
participation is voluntary and that they may withdraw from the study at any time, without prejudice. A 
voluntary participation/withdrawal statement is included in the informed consent. A copy of the informed 
consent document will be given to the participants for their records. The informed consent process will 
be conducted and documented in the source document (including the date), and the form signed, before 
the participant undergoes any study-specific procedures. The rights and welfare of the participants will 
be protected by emphasizing to them that the quality of their medical care will not be adversely affected 
if they decline to participate in this study. 
 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Study oversight: The research study staff will have weekly meetings to assess the study progress 
and review, as a group, any issues that arise (including adverse events). Under the counsel of Dr. Bland 
(after she comprehensively reviews the AE’s or SAE’s) and the IRB, we will act accordingly to halt any 
procedures scheduled to be performed. If the study is prematurely terminated or suspended, the PI (Dr. 
Cook) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will 
provide the reason(s) for the termination or suspension. Written notification, documenting the reason for 
study suspension or termination, will be provided to all study participants, funding agency and sponsor, 
and any regulatory authorities.  Study participants will be contacted, as applicable, and be informed of 
changes to study visit schedule. 
  
Circumstances that may warrant termination or suspension will be assessed by Dr. Bland and include, but 
are not limited to: 


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• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and 
satisfy the funding agency, IRB, and/or any other regulatory authorities. 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
 
This study is funded through NC TraCS, which is funded through the NIH CTSA award. Therefore, this 
pilot project will automatically have a Certificate of Confidentiality. 
 
Participant confidentiality and privacy is extremely important and will be upheld by the research team. All 
information and data collected generated will be held in strict confidence. Confidentiality will be extended 
to cover testing of biological samples in addition to the clinical measures (blood pressures) by assigning 
subject ID numbers. No information concerning the study or the data will be released to any unauthorized 
third party (e.g., personal PCP) without prior written approval to the research staff.  
 
All research activities will be conducted in as private a setting as possible. 
 
Study staff, authorized representatives of the funding agency, and representatives of the Institutional 
Review Board (IRB) and/or regulatory agencies may inspect all documents and records required to be 
maintained by the investigator, for the participants in this study. We will comply with requests for access 
to such records when necessary. 
 
The study participant’s contact information will be securely stored for internal use during the study. At 
the end of the study, all records will continue to be kept in a secure location for as long a period as dictated 
by the reviewing IRB, Institutional policies, or funding agency requirements (no less than 4 years after the 
study ends). 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored in a password protected software (RedCap) and statistical analyses files 
 (Graphpad, SPSS) on a password protected computer. This will not include the participant’s contact or 
identifying information. Rather, individual participants and their research data will be identified by a 
unique study identification number. At the end of the study, all study databases will remain de-identified 
and stored indefinitely.  
 
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
 
De-identified data (labeled with the subjects unique ID) collected for this study will be analyzed and stored 
at North Carolina A&T State University and some biological samples (blood and fecal) at University of 
North Carolina – Chapel Hill Core Facilities. After the study is completed, de-identified results from the 
study will be transmitted to and stored in password protected files (RedCap) on a password protected 


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computer, for use by other researchers including those outside of the study. Participants will be notified 
of this in the informed consent.  
 
With the participant’s approval (via informed consent) and as approved by the IRBs, de-identified 
biological samples will be stored as defined above. These samples will be used to research the causes of 
hypertension, its association with cardiovascular disease risk, and related complications to improve 
treatment strategies.  
 
When the study is completed, access to study gut microbial data and/or samples will be stored in 
Dataverse.org. All participants will receive their individual BP data at the conclusion of the study in a paper 
packet. BP data will consist of the average (every hour) awake and sleeping BPs recorded via ABP monitor. 
These data may help to inform themselves or their PCP’s about potential treatment strategies or lifestyle 
changes (diet and exercise) that may benefit them.  
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
Provide the name and contact information of the Principal Investigator and the Medical Monitor. 
Principal Investigator 
Principal Investigator 
Medical Monitor 
Marc Cook, PhD 
Ian Carroll, PhD 
Veita Bland, M.D. 
North Carolina A&T SU  
UNC- Chapel Hill 
Bland Clinic P.A.  
405 Benbow Rd. Corbett Sport 
Ctr 
Greensboro, NC 27411 
111 Mason Farm Road 7312C 
MBRB. Chapel Hill, NC 27599 
1317 N Elm St # 7 Greensboro, 
NC. 27401 
336-285-3547 
(919) 843-4893 
(336) 373-1557 
mdcook@ncat.edu 
ian_carroll@med.unc.edu 
spice1nc@aol.com 
 
 
10.1.6 SAFETY OVERSIGHT 
Safety oversight will be under the direction of the research study staff composed of individuals with the 
appropriate expertise, including Dr. Bland (M.D. Clinical Hypertension Specialist), PI’s, and the research 
staff collaborators (nurses). Research staff will meet on a weekly basis for data collection review, 
assessment of protocol and procedures, and assessment of safety of the study.  
 
10.1.7 CLINICAL MONITORING 
 
Site monitoring will be conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of the 
trial is in compliance with the currently approved protocol/amendment(s), with International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory requirement(s).  
 
• 
Monitoring for this study will be performed by Dr. Marc Cook, PhD, nursing collaborators (Dr. 
Tiffany Morris, PhD. and Dr. Ruthie Rogers, DNP), and overseen by Dr. Veita Bland, M.D. Dr. Bland 
will review all clinical data collected, assess any reported AE’s and determine their severity and 
potential relatedness to the study experiment (if there is more than 7 days after an experiment 
was completed). She will follow-up with participants to assess their health (via physical), if 
necessary. 


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• 
At this one site, the research team will meet every week to review data collected and transfer 
data into all password protected electronic storage forms (e.g., RedCap and statistical analyses 
data). 
• 
Safety monitoring will be in real time (monitoring BP and symptoms for no less than 1.5 hours 
after enema administration) and follow-ups at least 2 times (no later than 7 and again at 30 days) 
after the completion of their study.  
• 
Independent audits will not be conducted in this study as all research staff will meet on a weekly 
basis (and more frequently if needed) to review data, study progress, and any unexpected events. 
 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
 
We will perform internal quality management of study conduct, data and biological specimen collection, 
documentation and completion. Research staff will meet weekly to review all data collected and oversee 
the data entry. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated between the data entry research staff and the PI/clinical research staff for 
clarification/resolution. 
 
The investigational site will provide direct access to all source data/documents and reports for the 
purpose of monitoring and auditing by the funding agency, and inspection by local and regulatory 
authorities. 
 
10.1.9 DATA HANDLING AND RECORD KEEPING  
 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
 
Data collection is the responsibility of the research staff under the supervision of Dr. Marc Cook. The 
investigator will be responsible for ensuring the accuracy, completeness, legibility, and timeliness of the 
data reported. 
 
All source documents will be completed in a neat, legible manner to ensure accurate interpretation of 
data. Data will be transferred into a password protected data managing software (RedCap)  no more than 
7 days after data is collected. All data that is collected will be entered into RedCap on a weekly basis. 
 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case report form 
(eCRF) derived from source documents will be consistent with the data recorded on the source 
documents.  
 
Clinical data (including adverse events (AEs) and unanticipated problems) and clinical laboratory data will 
be entered into a password protected file (on a password protected computer). Clinical data will be 
entered directly from the source documents. 
 
 
10.1.9.2 STUDY RECORDS RETENTION  


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Study paper documents with identifiers will be retained for a minimum of 4 years in a locked file cabinet 
in a locked office space. After 4 years, all paper documents will be shredded. De-identified data will be 
stored in a password protected file on a password protected computer indefinitely.  
 
10.1.10 
PROTOCOL DEVIATIONS  
 
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonisation Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, the investigator, or the study site staff. As a 
result of deviations, corrective actions will be developed and implemented promptly.  
 
It is the responsibility of the PI (Dr. Marc Cook) to use continuous vigilance to identify and report 
deviations within 3 working days of identification of the protocol deviation, or within 3 working days of 
the scheduled protocol-required activity.  All deviations will be addressed in study source documents, 
reported to UNC-CH and NC A&T Institutional Review Board (IRB)’s.  Protocol deviations will be sent to 
the reviewing IRB per their policies. The principal investigator will be responsible for knowing and adhering 
to the reviewing IRB requirements.  
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
 
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations: 
 
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. We will submit final peer-reviewed journal manuscripts that 
arise from NIH funds to the digital archive PubMed Central upon acceptance for publication. 
 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-reviewed 
journals.  Data from this study may be requested from other researchers 5 years after the completion of 
the primary endpoint by contacting Dr. Marc Cook (mdcook@ncat.edu) at NC A&T State University.  
 
In addition, this study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human or non-human genomic data, as well as the use of these 
data for subsequent research. These data will include fecal microbial genome sequence data. 
 
 
 
10.1.12 
CONFLICT OF INTEREST POLICY 
 
[The independence of this study from any actual or perceived influence. The PI’s, or identified research 
staff, do not have any conflicts of interest to report.  Any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed by removing the research staff personnel with the conflict of interest. Furthermore, persons 


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who have a perceived conflict of interest will be required to have such conflicts managed in a way that is 
appropriate to their participation in the design and conduct of this trial (e.g., removing them from a 
portion of the study).  The study leadership in conjunction with NIH/NC TRaCS has established policies 
and procedures for all study group members to disclose all conflicts of interest and will establish a 
mechanism for the management of all reported dualities of interest. 
 
10.2 ADDITIONAL CONSIDERATIONS 
Listed Below 
 
1. Saline or sodium butyrate enema could raise BP in individuals with salt-sensitive hypertension. 
However, there has been no association between a one-time enema increasing BP. In fact, receiving an 
enema may elicit a vagal response to lower BP.  
 
2. Both enemas will be purchased from Custom Care Pharmacy (Greensboro) for quality control and 
matched for sodium concentration.  
 
3. Biobanking of blood and fecal microbial samples will allow for further analysis of factors that are not 
addressed in this study, such as measuring additional SCFA’s (acetate, propionate)  
 
4. We will educate participants on how to efficiently perform the self-administered enema, to reduce 
variability, and measure remaining solution to calculate dose delivered.  
 
5. We will work to minimize non-compliance and provide monetary incentive in this short study.  
 
6. Our group is experienced in culturally sensitive methods of subject recruitment in AA, via strong 
community relationships and health education, and we expect no issues with recruitment.  
 
7. The pool of subjects that do not take hypertension medication may be limited. If we encounter a heavily 
medicated subject population, we may include subjects taking diuretics only. 
 
 


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10.3 ABBREVIATIONS 
The list below includes abbreviations utilized in this template.  However, this list should be customized for 
each protocol (i.e., abbreviations not used should be removed and new abbreviations used should be 
added to this list). 
 
AA 
African American 
AE 
Adverse Event 
ABP 
Ambulatory Blood Pressure 
ANCOVA 
Analysis of Covariance 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS 
Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NC TRaCS 
North Carolina Translational and Clinical Sciences 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PCP 
Primary Care Physician 
PI 
Principal Investigator 
QA 
Quality Assurance 


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QC 
Quality Control 
SAE 
Serious Adverse Event 
SCFA 
Short Chain Fatty Acid 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
 
 
 


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10.4 PROTOCOL AMENDMENT HISTORY 
The table below is intended to capture changes of IRB-approved versions of the protocol, including a 
description of the change and rationale. A Summary of Changes table for the current amendment is 
located in the Protocol Title Page.  
 
Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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11 REFERENCES  
Journal Citations:  
Butyrate in Humans 
• 
Steinhart, A.H. et al. Aliment Pharmacol Ther 1996; 10: 729-736 Treatment of left-sided 
ulcerative colitis with butyrate enemas: a controlled trial. 
• 
Vanhoutvin SA et. al. Neurogastroenterol Motil 2009, 21:952-e76. The effects of butyrate 
enemas on visceral perception in healthy volunteers. 
 
Hamer et al. Clinical Nutrition 2010 29: 738-744. Effect of butyrate enemas on inflammation and 
antioxidant status in the colonic mucosa of patients with ulcerative colitis in remission 
• 
Krokowicz et al. Int J Colorectal Dis (2014) 29:387–393. Microencapsulated sodium butyrate 
administered to patients with diverticulosis decreases incidence  of diverticulitis—a prospective 
randomized study 
Effect of Exercise on SCFA microbes in the gut 
• 
Cook MD et al. Immunol Cell Biol. 2016 Feb;94(2):158-63. Exercise and gut immune function: 
evidence of alterations in colon immune cell homeostasis and microbiome characteristics with 
exercise training. 
• 
Allen, JM et al. Med Sci Sports Exerc. 2018 Apr;50(4):747-757. Exercise Alters Gut Microbiota 
Composition and Function in Lean and Obese Humans. 
Dietary Analyses 
• 
Blanton CA et. al. 2006 J Nutr. 136(10):2594-9. The USDA Automated Multiple-Pass Method 
accurately estimates group total energy and nutrient intake. 
Stress and Cardiovascular Disease and Gut Microbiome 
• 
Hamer M. Psychosom Med. 2012 Nov-Dec;74(9):896-903. Psychosocial stress and cardiovascular 
disease risk: the role of physical activity. 
• 
Moloney RD et. al. Mamm Genome. 2014 Feb;25(1-2):49-74. The microbiome: stress, health and 
disease. 
Ambulatory Blood Pressure Monitoring 
• 
Edwards LJ and Simpson SL. Edwards & Simpson Blood Press Monit. 2014 June ; 19(3): 153–163. 
An Analysis of 24-Hour Ambulatory Blood Pressure Monitoring Data using Orthonormal 
Polynomials in the Linear Mixed Model. 

ICF_000.pdf: 
Informed Consent 
Effect of Gut Butyrate Delivery on Blood Pressure in African 
Americans with Hypertension 
NCT number NCT04415333 
Document Date 07/01/2021 


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Study Title: Effect of Gut Butyrate Delivery on Blood Pressure in African Americans with 
Hypertension 
Principal Investigator: Marc D. Cook (NC A&T); Ian Carroll (UNC-CH) 
 
Concise Summary 
The purpose of this research study is to determine the effect of butyrate, a short chain 
fatty acid produced in the gut (intestines) on blood pressure (BP). As a participant with 
hypertension (elevated blood pressure; systolic: 130-159 and diastolic: 80-99 mmHg) you will 
undergo screening that includes medical health history, physical activity behavior, and dietary 
habits questionnaires, blood pressure measurement and a body composition measurement on 
a digital scale to assess your eligibility for this study. Once screening is complete, you will be 
required to undergo testing on 2 separate days (7 days apart). The testing days will include 
submitting a fecal sample obtained by you at home, self-administer an enema, submitting to 
blood draw after the enema, and wearing a blood pressure monitor for 24 hours after the blood 
draw. This sequence will be completed twice and the sequence of enema you self-administer 
will be randomized to a saline enema with a very small amount of butyrate and a saline enema 
that contains moderate amount of butyrate. You will return the monitor on the following day 
and this will complete your study. The screening visit and each testing visit (2 testing visits 
total) will take about 1.5 hours and total study duration of about 2 weeks (14 days). 
This study is for research purposes and may or may not provide benefits to you. You 
may receive a sense of fulfillment from personally contributing to medical knowledge about 
how the bacteria in your gut affect your blood pressure. The risks of this study are outlined 
below. However, the greatest risks include emotional distress associated with collecting your 
own fecal sample and self-administering an enema, pain associated with blood draw, irritation 
or discomfort with wearing a blood pressure monitor for 24 hrs, and loss of confidentiality. If 
you are interested in learning more about this study, please continue to read below. 
 
Purpose of the Study  
You have been asked to participate in a research study about the effects of the short 
chain fatty acid, butyrate, on blood pressure in African Americans with Hypertension. You have 
been asked because you are an African American between the ages of 30-50 years old and 
have hypertension (moderately elevated blood pressure; systolic: 130-159 and diastolic: 80-
99 mmHg). 
African Americans have hypertension (high blood pressure) more often than any other 
population in the United States.  This makes hypertension one of the most important health 
concerns in the African American community.  Most of the time, African Americans get 
hypertension at an earlier age and it causes more damage.   
Recently, the bacterial make-up of the intestines (gut) has been related to elevated 
blood pressure. Researchers believe that specific types of bacteria that breakdown fiber and 
make a short chain fatty acid called butyrate may be lower in African Americans with 
hypertension. The only way we get butyrate in our bodies is when the bacteria make it from 
certain carbohydrates (fiber) in our intestines and we absorb it into our blood.  
The purpose of study is to measure differences in the gut bacteria that make butyrate 
in the stool, and measure how much butyrate is in the blood, in African Americans with and 
without elevated blood pressure. To study this, we are asking you donate stool and blood 


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samples, self-administer one enema that contains a low amount of butyrate (5 mmol) and one 
enema that contains a higher and safe amount of butyrate (80 mmol) seven days apart, and 
wear a blood pressure monitor for up to 24 hours. *Note* You are being asked to act as your 
own control by completing the self-administration of the enemas to determine if butyrate has 
any effect on your blood pressure. This is the reason for completing the 2 enemas in a random 
order, 1 week apart. The principal investigators nor you will know the order in which you 
receive the enema. Your blood will be drawn to measure the amount of butyrate in your blood 
(before and after each enema) and you will wear a portable blood pressure monitor for up to 
24 hours after each enema, which will measure your BP throughout the day.  
The primary researcher involved in this study is Dr. Marc Cook from the Department of 
Human Performance and Leisure Studies at NC A&T and Dr. Ian Carroll in the Department of 
Nutrition at the University of North Carolina at Chapel Hill (UNC-CH). If you do not have a 
primary care physician, the physician you will be referred to Dr. Veita Bland M.D. (study 
physician) of the Cone Health Hospital affiliated Bland PA Clinical Hypertension Clinic at 1317 
N Elm St # 7, Greensboro, NC 27401, Phone: (336) 373-1557. Please ask the staff to explain 
any words or information that you do not clearly understand. You may take home an unsigned 
copy of this consent form to think about or discuss with family or friends before making your 
decision. If you do not qualify for the study, none of your information obtained in the screening 
will be stored. Approximately 20 subjects may be involved in this research at NC A&T. 
 
This research is sponsored by the North Carolina Translational & Clinical Sciences Institute 
(NC TraCS) Award # 550KR181802. 
 
Procedures 
If you qualify and choose to participate in this study, you will be asked to complete an informed 
consent and questionnaires providing information on your health history, physical activity, 
race/ethnic background demographics, and dietary habits (dietary recall), and have your blood 
pressure and body composition (body weight, height, fat mass, muscle mass) measured at 
Visit 1. At Visit 2, you will be asked to provide your home collected stool sample and 3-day 
dietary recall, self-administer a saline enema (control: contains 0.9% saline as you would find 
in the store) or a saline enema that contains sodium butyrate(experimental: contains 0.9% 
saline and the short-chain fatty acid butyrate at a concentration of 80 millimolar), provide blood 
samples, and wear a 24-hour ambulatory BP monitor for the rest of the day. At Visit 3, you will 
return the 24-hour BP monitor the day after Visit 2. At Visit 4, 7 days after Visit 2, you will be 
asked to repeat Visit 2 and complete the study by administering the other enema. At Visit 5, 
you will return the 24-hour BP monitor. At Visits 2 & 4, neither you or the research assistants 
will know which enema you will receive, and you will be your own control subject by performing 
both enemas.  
Your participation is expected to take about 2 weeks. Stool samples will be obtained by 
you at home and brought to the laboratory, along with your 3-day dietary recall, the morning 
you are scheduled to perform the enema and have your blood drawn. Afterward, you will leave 
while wearing a portable BP monitor, wear it for the remainder of the day (24 hours) and return 
it to the laboratory the next day. Dr. Vieta Bland, the physician and clinical hypertension 
specialist, will write a prescription for the enema’s and they will be made at the Custom Care 
Pharmacy (located at 109-A Pisgah Church Road, Greensboro, NC 27455). We will pick them 


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up for you and have them at your Visit 2 & 4. Enema’s are commonly used to relieve 
constipation, but they have also been used to deliver drugs into the intestines. Our purpose in 
using the butyrate enema is to determine how much butyrate gets absorbed into your blood 
and to measure butyrate’s effects on your BP. Again, the bacteria in our gut naturally produces 
butyrate for us, but if we have reduced bacteria in our gut that make it, this may be related to 
elevated BP. At Visits 3 & 5, you will receive compensation for returning the BP monitor and 
completing that portion of the study.  
Participants in this study should: 
 
a) Be an African American adult (man or woman) between 30 - 50 years of age with 
hypertension (systolic: 130-159 and diastolic: 80-99 mmHg). 
b) Not taking any anti-hypertension medications (although we may enroll individuals only 
taking a diuretic. BP will be assessed in the morning after 24-hour fast from their 
medication and no medication will be taken within 24 hours of their study visit or during 
the study. They can resume taking the diuretic after they remove the monitor). 
c)  Body Mass Index of 18.5-30 kg/m2 
d) Not have any other diagnosed cardiovascular disease 
e) Not exercise regularly (Participate in less than 60 minutes of exercise/week)  
f) Not be pregnant or be lactating 
g) Be free of active diseases that affect your intestines (i.e., chronic constipation, diarrhea, 
Crohn’s disease, ulcerative colitis, irritable bowel syndrome, diverticulosis, stomach or 
duodenal ulcers, diabetes, hepatitis, HIV, and cancer) 
h) Have not taken antibiotics in the past 3 months 
i) Have not been regularly taking medications that impact intestinal function (i.e., 
laxatives, enemas, anti-diarrheal agents, narcotics, antacids, antispasmodics, 
antidepressants, diuretics, anticonvulsants, antibiotics, herbals, homeopathy, and home 
remedies) 
j) Have no plans of travel out of town during the study periods. 
k) Agreement to adhere to Lifestyle Considerations listed within this informed consent 
throughout study duration 
 
If you agree to participate, you will be asked to avoid alcohol 48 hours before your data 
collection appointment and not to change your eating habits. This is because changing 
your diet can affect the types of bacteria found in your intestines. If you are taking 
medication, you will be asked to continue taking them throughout the study period.  
 
If you qualify for the study, you will be asked to do the following: 
 
1. Complete questionnaires about your health history, racial/ethnic background, physical 
activity levels, and dietary habits. If you are a woman, you will be asked to verify you are 
not pregnant by performing a urine pregnancy test. Risks associated with the 
questionnaires and pregnancy test: You may feel a little uncomfortable sharing your 
personal health information.  You are free to skip any questions you do not wish to 
answer. If you are female and test positive during the pregnancy test you may be 
surprised or experience distress if you were unaware. 


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2. Perform body composition measurements that will be done by stepping on a scale for 2 
minutes. Risks associated with this procedure are minimal. You may be embarrassed 
to learn what your body composition (body weight, fat mass, and lean mass) is if it is 
unfavorable to you. This measurement will be taken by 1 study staff member and will be 
confidential, just as all your data. 
3. Collect a stool sample at home and bring to the laboratory at Visit 2 and 4 (which will be 
1 week apart). You will be provided with everything you need to collect the sample 
safely. Risks associated with stool sample collection include experiencing emotional 
stress related to working with your own stool. Mishandling the sample can lead to 
infections, however, personal protection equipment will be provided, and safe hand 
washing technique will be taught to reduce this risk to only rare cases. 
4. Donating blood samples for the assessment of circulating butyrate and other biomarkers 
in your blood. Blood samples will be collected by a phlebotomist or a nurse. Risks 
associated with a blood draw include mild pain, bleeding, bruising at the site of the draw 
and rarely, infection. 
5. Self-administer an enema, in a random order (one at Visit 2 and one, 7 days later, at 
Visit 4). Risks associated with the enema include experiencing emotional stress related 
to performing the enema, especially if you have never done so before. We will provide 
you with instructions on how to perform the enema effectively. If you are uncomfortable 
with performing the enema, a nurse associated with the study can provide you with 
assistance and administer it. It is also expected that you may have a bowel movement 
within 10 minutes of completing the enema. You will be given access to a private 
bathroom after the enema. You may request to have a study nurse administer the 
enema. 
6. Have your blood pressure monitored with a monitor you will wear for 24 hours on the 
day you perform the enema. Risks associated with wearing a 24-hr BP monitor include 
discomfort while wearing and possible loss of sleep if you are awakened during the 
night when measurements are recorded each hour. It is expected that you will become 
used to the monitor throughout the day, which will likely reduce sleep disturbances 
overnight. In addition, you may feel embarrassed or self-conscious while wearing the 
BP monitor. 
 
Procedures and Assessments 
Visit 1: Screening visit – Informed consent, questionnaires, blood pressure, and body 
composition 
1. Medical history, physical activity, and dietary recall questionnaires: We will review the 
medical history questionnaire to ensure all the questions and their answers regarding 
your health, current medication use, medical history and exercise pattern are clear. This 
is done to ensure you are eligible to participate in this study. You will also fill out a 
physical activity readiness questionnaire (PAR-Q) to determine the amount of time you 
exercise currently. You will also be asked to complete a dietary food record. For this, 
you will record your food and beverage intake for the 5 days following the initial 
screening. You will be provided the paper forms to complete the records before you 
return to submit your samples. Pre-menopausal women will be asked to confirm they 


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are not pregnant by taking an over-the-counter pregnancy test we will provide. This is 
the only time you will be asked to take a pregnancy test.  
2. Blood Pressure (BP): Blood pressure will be measured after lying down for 5 minutes 
undisturbed. A blood pressure cuff will be placed on the right arm and connected to an 
automated BP monitor that will measure your blood pressure two times (approximately 
1 minute apart). 
3. Body Composition: Your height and body weight measured on a scale that will 
determine your body weight, body fat mass, body muscle mass, and total body water. 
The scale requires you to stand on the scale barefoot and hold on to the machine. The 
test takes approximately 2 minutes to complete the measurement. 
 
Visit 2: Measurements and Sample collection 
 
1. Body Composition: Your body weight will be measured on a scale that will determine 
your body weight on the same scale as before. The test takes approximately 2 minutes 
to complete the measurement. 
2. Stool sample collection: You will be asked to submit a stool sample you collected at 
home within 24-hours of this visit (collected the day before). You will also need to 
submit your 3-day dietary recall. 
3. Blood sampling: Blood will be taken from the antecubital (forearm) vein by a 
phlebotomist or nurse. A small amount (about 3 tablespoons) of blood will be taken right 
before you complete the enema (baseline). You will have less blood taken (about 2 
tablespoons) 30 minutes and 60 minutes (1 hour) after you complete the enema  
4. 24-hour blood pressure monitoring: We will introduce you to the 24-hour ambulatory 
blood pressure monitoring device that you will be sent home with to perform the 
measurements. You will be fitted with a blood pressure cuff and sent home with 
instructions on how to use it. You will also be given instructions (verbal and written) on 
how to keep your blood pressure diary, where you will record your activities at the time 
the monitor was taking your blood pressure. 
 
Visit 3: Return the 24-hour BP monitor. You will receive compensation at this time. 
 
Visit 4: Repeat Visit 2 by completing the other enema. 
 
Visit 5: Return the 24-hour BP monitor. You will receive compensation at this time. 
 
Follow-up procedures 
There are no follow-up experimental procedures after participants have completed the study. 
Follow-up telephone calls will be made 7 days and 30 days after participation to ask about your 
overall health. You will be contacted to return to the lab to receive a packet and explanation of 
your 24-hour ABP individual results at the end of the study. Further, you may be contacted to 
share the publication(s) of the results which will contain your unidentifiable data. 
 
Risks and Discomforts 


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The following risks are related to your participation in this research study have been explained 
in above and can be reviewed at this time. Additional risks may include social risks related to 
confidentiality. If you are faculty or staff of NC A&T, it is likely (10-25%) that your work peers 
may discover that they are participating in a research study. To reduce this risk may make 
adjustments in scheduling your experiments (weekends if requested) to reduce any potential 
interactions with your work peers on campus. 
 
To minimize the risk of breach of privacy or confidentiality, all health information and blood 
samples collected will be stored coded as to not be able to identify you. 
 
If you think you are having a bad reaction during or within 1 week from the end of the study, 
immediately contact Dr. Marc Cook (336-285-3547, mdcook@ncat.edu). 
 
Benefits 
This study is for research purposes and may or may not provide benefits to participants. You 
may receive a sense of fulfillment from personally contributing to medical knowledge about 
ways of improving gut health and blood pressure. We hope to learn more about how specific 
types of bacteria in the gut affect blood pressure in African Americans.  
 
Compensation or Costs to Study Participants  
For your participation in this study, you will receive compensation totaling $300 total ($150 
each time you complete an experiment and return the 24-h ABP monitor). You may decide to 
leave the study at any time, however, no monetary compensation will be provided unless you 
have donated all samples and completed BP monitoring for an experiment. This includes 
donating the stool sample with the 3-day dietary recall, performing an enema and donating the 
blood samples, and wearing and returning the portable BP monitor.  You will not pay for any 
testing or procedures associated with this study. You will not be compensated for your travel to 
and from the laboratory. 
 
Certificate of Confidentiality 
What is a Certificate of Confidentiality? 
This research is covered by a Certificate of Confidentiality. With this Certificate, the 
researchers may not disclose or use information, documents or biospecimens that may identify 
you in any federal, state, or local civil, criminal, administrative, legislative, or other proceedings 
in the United States, for example, if there is a court subpoena, unless you have consented for 
this use. The Certificate cannot be used to refuse a request for information from personnel of a 
federal or state agency that is sponsoring the study for auditing or evaluation purposes or for 
information that must be disclosed in order to meet the requirements of the federal Food and 
Drug Administration (FDA). 
  
The Certificate of Confidentiality will not be used to prevent disclosure as required by federal, 
state, or local law, such as mandatory reporting requirements for child abuse or neglect, 
disabled adult abuse or neglect, communicable diseases, injuries caused by suspected 
criminal violence, cancer diagnosis or benign brain or central nervous system tumors or other 
mandatory reporting requirement under applicable law. The Certificate of Confidentiality will 


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not be used if disclosure is for other scientific research, as allowed by federal regulations 
protecting research subjects or for any purpose you have consented to in this informed 
consent document. 
  
You should understand that a Certificate of Confidentiality does not prevent you from 
voluntarily releasing information about yourself or your involvement in this research. If an 
insurer, employer, or other person obtains your written consent to receive research 
information, then the researchers may not use the Certificate to withhold that information. 
 
Confidentiality 
Your samples will be coded with an identification number (rather than your name or initials) to 
maintain your confidentiality. Any questionnaires or records that identify you, and this consent 
form signed by you, may be inspected by the Department of Health and Human Services 
(DHHS) agencies, the North Carolina A&T State University and University of North Carolina 
Institutional Review Boards.  Because of the need to release information to these parties, 
absolute confidentiality cannot be guaranteed. However, to the extent permitted by the 
applicable laws and regulations, records identifying you will be kept confidential and will not be 
made publicly available. In general, we will not tell anyone any information about you. The 
results of this study may be presented at research meetings or in publications, but your identity 
will not be disclosed in those presentations.  
Your records will be kept in a locked cabinet in a locked office space with access limited 
to essential research staff. Research data collected will be transferred to a password protected 
excel file on a password protected laboratory computer. Access will only be given to essential 
research staff that will update these files. Records stored in this manner will be coded and will 
not be able to identify you in any way. These electronic files will be kept indefinitely. Biological 
specimens (blood and stool samples or bacterial DNA isolated from those stool samples) will be 
stored in a freezer, indefinitely. No one will be able to identify you from these samples, as they 
will be stored with the coded ID numbers. 
Information that identifies you personally will not be released without your written 
permission (for example, to your primary care physician), and if the results of this research are 
published or discussed in conferences, no information will be included that would reveal your 
identity. We are required to keep all data and records for at least four years after the close of 
the study. All paper records that may identify you as a participant in the study will be destroyed 
(via shredding) at that time. 
 
Questions about the Study 
If you have any questions about your involvement in this project, you may contact Dr. Cook at 
336-285-3547 or by email at mdcook@ncat.edu. If you have any study-related concerns or any 
questions about your rights as a research study participant, you may contact the Office of 
Research Compliance and Ethics at North Carolina A&T State University at 336-285-2961. 
 
Voluntary Participation/Withdrawal 
Your participation is voluntary, and you may end your participation at any time. Refusing to 
participate or leaving the study at a later time will not result in any penalty or loss of benefits to 
which you are entitled. 


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Statement of Consent 
I have read the above information and have received answers to any questions I had. I am at 
least 18 years of age or older and voluntarily consent to take part in this research study. 
 
 
Participant’s Name (Printed):  
 
 
 
 
 
 
 
 
 
 
 
Participant’s Signature:  
 
 
 
 
 
 
  Date:  
 
 
 
 
Researcher’s Signature:  
 
 
  Date: