Dietary Nitrate for Heart Failure
Phase: PHASE1
Conditions: Heart Failure, Hypertension, Pulmonary
Sponsor: Washington University School of Medicine

 
 
 
 
 
 
1 
 
 
 
 
 
Protocol 
NCT01682356 
December 7, 2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
2 
 
 
 
Dietary Nitrates for Heart Failure 
 
 
 
Principal Investigator:  Linda R. Peterson, MD 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
3 
TABLE OF CONTENTS 
 
A 
INTRODUCTION .......................................................................................................................... 4 
A1 
STUDY ABSTRACT .................................................................................................................... 5 
A2 
PRIMARY HYPOTHESIS .............................................................................................................. 5 
A3 
PURPOSE OF THE STUDY PROTOCOL .......................................................................................... 5 
B 
BACKGROUND ............................................................................................................................ 6 
B1 
PRIOR LITERATURE AND STUDIES .............................................................................................. 6 
B2 
RATIONALE FOR THIS STUDY..................................................................................................... 7 
C 
STUDY OBJECTIVES .................................................................................................................. 8 
C1 
PRIMARY AIM .......................................................................................................................... 8 
C2 
SECONDARY AIM ................................................................. ERROR! BOOKMARK NOT DEFINED. 
C3 
RATIONALE FOR THE SELECTION OF OUTCOME MEASURES ... ERROR! BOOKMARK NOT DEFINED. 
D 
INVESTIGATIONAL AGENT ..................................................................................................... 8 
D1 
PRECLINICAL DATA .................................................................................................................. 8 
D2 
CLINICAL DATA TO DATE ......................................................................................................... 9 
D3 
DOSE RATIONALE AND RISK/BENEFITS...................................................................................... 9 
E 
STUDY DESIGN ............................................................................................................................ 9 
E1 
OVERVIEW OR DESIGN SUMMARY ............................................................................................. 9 
E2 
SUBJECT SELECTION AND WITHDRAWAL ................................................................................... 9 
2.a 
Inclusion Criteria ................................................................................................................ 9 
2.a 
Exclusion Criteria................................................................................................................ 9 
2.b 
Ethical Considerations ....................................................................................................... 10 
2.c 
Subject Recruitment Plans and Consent Process................................................................. 10 
2.d 
Randomization Method and Blinding.................................................................................. 10 
2.e 
Risks and Benefits .............................................................................................................. 10 
2.f 
Early Withdrawal of Subjects ................................................................................................. 10 
2.g 
When and How to Withdraw Subjects ................................................................................. 10 
2.h 
Data Collection and Follow-up for Withdrawn Subjects ..................................................... 10 
E3 
STUDY DRUG ......................................................................................................................... 10 
3.a 
Description ........................................................................................................................ 10 
3.b 
Treatment Regimen ............................................................................................................ 10 
3.c 
Method for Assigning Subjects to Treatment Groups........................................................... 11 
3.d 
Preparation and Administration of Study Drug ................................................................... 11 
3.e 
Subject Compliance Monitoring ......................................................................................... 11 
3.f 
Prior and Concomitant Therapy ............................................................................................. 11 
3.g 
Packaging ......................................................................................................................... 11 
3.h 
Blinding of Study Drug ...................................................................................................... 11 
3.i 
Receiving, Storage, Dispensing and Return ............................................................................ 11 
F 
STUDY PROCEDURES .............................................................................................................. 11 
F1 
SCREENING FOR ELIGIBILITY ....................................................................................................... 11 
F2 
SCHEDULE OF MEASUREMENTS ................................................................................................... 11 
F3 
SAFETY AND ADVERSE EVENTS ................................................................................................... 13 
3.a 
Safety and Compliance Monitoring .................................................................................... 13 
3.b 
Medical Monitoring ........................................................................................................... 13 
i 
Investigator only ............................................................................................................................... 13 

 
 
 
 
 
 
4 
ii 
Independent expert to monitor ........................................................................................................... 13 
iii 
Institutional Data and Safety Monitoring Board ................................................................................. 13 
iv 
Independent Data and Safety Monitoring Board ................................................................................. 13 
3.c 
Definitions of Adverse Events ............................................................................................. 13 
3.d 
Classification of Events ...................................................................................................... 13 
i 
Relationship ..................................................................................................................................... 13 
ii 
Severity ............................................................................................................................................ 13 
iii 
Expectedness .................................................................................................................................... 13 
3.e 
Data Collection Procedures for Adverse Events ................................................................. 14 
3.f 
Reporting Procedures ............................................................................................................ 14 
3.g 
Adverse Event Reporting Period......................................................................................... 14 
3.h 
Post-study Adverse Event ................................................................................................... 14 
F4 
STUDY OUTCOME MEASUREMENTS AND ASCERTAINMENT ........................................................... 14 
G 
STATISTICAL PLAN ................................................................................................................. 14 
G1 
SAMPLE SIZE DETERMINATION AND POWER ............................................................................ 14 
G2 
INTERIM MONITORING AND EARLY STOPPING .......................................................................... 14 
G3 
ANALYSIS PLAN ..................................................................................................................... 14 
G4 
STATISTICAL METHODS .......................................................................................................... 14 
G5 
MISSING OUTCOME DATA ....................................................................................................... 14 
G6 
UNBLINDING PROCEDURES ..................................................................................................... 15 
H 
DATA HANDLING AND RECORD KEEPING ......................................................................... 15 
H1 
CONFIDENTIALITY AND SECURITY ........................................................................................... 15 
H2 
TRAINING ............................................................................................................................... 15 
H3 
CASE REPORT FORMS AND SOURCE DOCUMENTS ..................................................................... 15 
H4 
RECORDS RETENTION ............................................................................................................. 15 
H5 
PERFORMANCE MONITORING .................................................................................................. 15 
I 
STUDY MONITORING, AUDITING, AND INSPECTING ...................................................... 15 
I1 
STUDY MONITORING PLAN ......................................................................................................... 15 
I2 
AUDITING AND INSPECTING......................................................................................................... 15 
J 
STUDY ADMINISTRATION ...................................................................................................... 15 
J1 
ORGANIZATION AND PARTICIPATING CENTERS ............................................................................ 15 
J2 
FUNDING SOURCE AND CONFLICTS OF INTEREST .......................................................................... 15 
J3 
COMMITTEES .............................................................................................................................. 16 
J4 
SUBJECT STIPENDS OR PAYMENTS ............................................................................................... 16 
J5 
STUDY TIMETABLE ..................................................................................................................... 16 
K 
PUBLICATION PLAN ................................................................................................................ 16 
L 
ATTACHMENTS ........................................................................................................................ 16 
L1 
TABLES .................................................................................................................................. 17 
L2 
INFORMED CONSENT DOCUMENTS ........................................................................................... 17 
L3 
PATIENT EDUCATION BROCHURES ........................................................................................... 17 
L4 
SPECIAL PROCEDURES PROTOCOLS .......................................................................................... 17 
L5 
QUESTIONNAIRES OR SURVEYS ................................................................................................ 17 
M 
REFERENCES ............................................................................................................................. 17 
 
A Introduction 
 

 
 
 
 
 
 
5 
A1 Study Abstract 
This study has several arms.  The overall objective of the study is to evaluate the 
improvements in cardiac, skeletal muscle, and whole body performance after treatment 
with dietary nitrate (NO3-) derived from beet root juice (BRJ).  The rationale for this study 
is that it was fairly recently discovered that NO3- from the diet can be a source for nitric 
oxide (NO) production in the body through an ‘enterosalivary pathway.’  NO has 
pleiotropic effects in the body including relaxing smooth muscle (e.g., around arteries 
causing vasorelaxation) and, paradoxically, stimulating skeletal muscle contraction.  
Subjects with heart failure have reduced bioavailability of NO, so they may particularly 
benefit from increased NO production via the dietary pathway.  
Substudy #1 (V̇ O2peak study). The purpose of this study is to assess the effects of acute 
ingestion of a single dose of dietary nitrate vs. placebo on exercise performance (6 min 
walk test; V̇ O2peak test; short physical performance battery), skeletal muscle (energetics, 
blood flow and/or oxygen levels),  and cardiac muscle function (using magnetic 
resonance spectroscopy and imaging). Breath NO, plasma NO3-  and nitrite (NO2-) will 
also be measured before and after nitrate ingestion.  
 
Subjects with HF or controls (without HF) will be studied. The study design is a 
randomized, double-blind crossover study with at least a 7 d washout in between the 2 
study days (placebo [BRJ depleted of nitrate] and BRJ). 
Substudy #2 (Neuromuscular study).  The purpose of this study is to assess the 
effects of acute ingestion of a single dose of dietary nitrate on exercise performance (6 
min walk; muscle power and speed of contraction as measured using a Biodex machine; 
short physical performance battery). Breath NO, plasma NO3-  and nitrite (NO2-) will also 
be measured before and after nitrate ingestion. There is an optional muscle biopsy as a 
part of this substudy. The purpose of this is to evaluate the molecular changes after 
nitrate ingestion v. placebo.  
 
Subjects with heart failure or controls (without heart failure) will be studied.  The 
study design is a randomized, double-blind crossover study with at least a 7 d washout 
in between the 2 study days (placebo [BRJ depleted of nitrate] and BRJ).  
 
A2 Primary Hypothesis 
The primary hypothesis is that patients with heart failure will have improvements in 
exercise performance (primary endpoint is neuromuscular power, secondary is V̇ O2peak) 
after ingestion of BRJ containing dietary nitrate as compared to after ingestion with 
placebo.  We further hypothesize that breath NO, plasma nitrate and nitrite as well as 
cardiac or skeletal muscle nitrate/nitrite and cGMP will increase after ingestion of BRJ as 
compared with after placebo. 
A3 Purpose of the Study Protocol 
The primary purpose of this study protocol is to outline the procedures for each of the 
above-listed Substudies that comprise the overall dietary nitrate intervention study.   
 

 
 
 
 
 
 
6 
B Background 
 
B1 Prior Literature and Studies 
• Significance of Heart Failure (HF): HF is a major public health problem affecting 5.8 
million Americans. Moreover, adults over age 40 have a 1 in 5 chance of developing HF 
in their lifetime. Total HF costs are ~$39.1 billion. The human cost is great as well: 40% 
of patients die within 1 y of diagnosis.1 Moreover, the number affected by HF and the 
number of HF deaths continue to increase. In addition to being a major cause of death, 
HF is a significant cause of disability. Indeed, the New York Heart Association 
classification system of HF is based on the severity of patients’ inability to exercise and 
to perform normal activities of daily living. Patients with class II HF have mildly limited 
physical activity; those with Class III have markedly limited physical activity; and those 
with Class IV cannot carry out any physical activity without discomfort and have 
symptoms even at rest. Given the disabling nature of HF and its tremendous impact on 
quality of life, any improvement in exercise capacity or tolerance would be of enormous 
benefit to patients with this disease.  
 
• Significance of Nutrition and Diet in the Management of HF: Compared to what is 
known about pharmaceutical treatments for HF, relatively little is known about how diet, 
nutrition, and the microbiota may improve HF. The main nutritional guidelines for HF 
recommend avoidance of high sodium and promoting ‘healthy’ diets. Guidelines on 
specific nutrients are mostly limited to advice to include adequate vitamin and mineral 
intake to avoid rare causes of HF (e.g., Beriberi and Keshan’s disease). Overall, there is 
a dearth of studies related to nutrition in HF patients. Our proposed study would help fill 
this gap in our knowledge.  
 
• Significance of Dietary NO3- for Improving Exercise Tolerance in Patients with 
HF: Pharmaceutical nitrates (in combination with hydralazine) improve quality of life and 
survival2 in HF patients. By investigating the efficacy of dietary NO3- on exercise 
tolerance in a well-designed and controlled study, our study would not only help improve 
our knowledge of nutrition in HF, but also potentially point towards a novel, relatively 
inexpensive “nutraceutical” that could be widely applied to HF treatment. There is 
already a burgeoning literature demonstrating the effectiveness of dietary NO3-, from 
beet root juice (BRJ) (from a standardized BRJ drink from James White Drinks Inc.), in 
enhancing exercise tolerance in normal subjects,3-6 in athletes,7 and in patients with 
peripheral arterial disease.8 Peak power during an incremental exercise test4, time to 
fatigue during constant intensity exercise in normoxia3,5 or hypoxia,6 average power 
during a simulated time trial7, and distance walked before the onset of claudication8 all 
improve after BRJ supplementation. If these findings hold true for patients with HF, it 
would demonstrate that BRJ supplementation improves exercise capacity and tolerance 
thereby likely improving quality of life and decreasing disability and cost of HF. 
Documenting an improvement in submaximal performance such as is demonstrated in a 
6 minute walk test (in addition to peak performance) would be important since most 
activities of daily living and work do not require a maximal exercise capacity but rather 
the ability to tolerate sustained exercise. 
 
• Significance of Dietary NO3- as a Substrate for the Alternative NO Production 
Pathway: One of the most powerful efficiency-enhancing molecules is nitric oxide (NO). 

 
 
 
 
 
 
7 
However, the classical pathway of endothelial NO production from L-arginine, mediated 
by endothelial NO synthase, is dependent upon the presence of oxygen (O2).9 In 
contrast, in the alternative pathway for NO production (i.e., NO3- → NO2- → NO) dietary 
NO3- is first reduced to NO2- by oral facultative anaerobic bacteria. After NO2- (and 
residual NO3-) are swallowed, stomach acid, and/or ischemic/deoxygenated conditions 
further reduce these compounds to NO. This process is O2 independent. Details of this 
enterosalivary pathway for NO2- and NO production are expanded upon in recent 
reviews.9,10 This alternative pathway is not only a parallel system but an important back-
up providing NO in conditions when the classical pathway cannot. These conditions 
include local ischemic and acidic conditions, which in HF patients can result from 
increased left ventricular (LV) wall stress, coronary artery disease, or rigorous exertion. 
Importantly, as listed in Figure 1, tolerance does not appear to develop following 
repeated stimulation of this alternative pathway, in contrast to treatment with 
pharmacologic nitrates (e.g., nitroglycerin).9 In sum, this is a heretofore under-
appreciated NO production system that may be exploited through the use of dietary 
NO3- to improve energy efficiency and, hence, function in patients with HF.  
 
• Significance of Oxygen Uptake Inefficiency in HF: Inefficiency (a poor cardiac 
work:O2 use ratio) is a hallmark of HF.11 In HF, both the heart and skeletal muscle are 
inefficient.11,12  It is an unfortunate irony that in HF, a condition in which it is especially 
important to get the most work from a given amount of O2, the normally tight coupling 
between the two is impaired. This inefficiency feeds into a vicious cycle in HF: 
decreased function leads to inefficiency, which further impairs function.11 Clearly, 
inefficiency in HF is an attractive target for the development of novel therapies. If this 
important input that contributes to continuing progression of HF can be improved, more 
energy should be available for work for the heart, skeletal muscle, and potentially other 
organs.  
 
• Significance of Dietary NO3- as a Treatment of Inefficiency in HF: There are limited 
therapeutic options for improving efficiency in HF. A few medications exist that may 
improve efficiency by altering substrate metabolism preference (e.g., trimetazidine and 
perhexiline), but they are not available in the U.S., and the latter an unattractive side 
effects. However, studies of dietary NO3- supplementation show that it reduces the O2 
cost of exercise, i.e., after dietary NO3- supplementation peak exercise time and output 
increase, but V̇ O2 at a given power decreases.3-5,13-15  An in vitro study of mitochondria 
from healthy volunteers who were supplemented with BRJ demonstrated improved 
oxidative phosphorylation efficiency (i.e., an improved P:O ratio) and a decrease in state 
4 respiration.15 The improved P:O ratio correlated with a decrease in the O2 cost of 
whole-body exercise. Thus, thermodynamic efficiency and coupling of muscle 
mitochondria was improved by BRJ in normals. Whether BRJ-derived NO can also 
improve the whole body inefficiency of HF is unknown. Our study will help fill this gap in 
our knowledge.  
 
 
B2 Rationale for this Study 
The rationale for this study is that it was fairly recently discovered that NO3- from the diet 
can be a source for nitric oxide (NO) production in the body through an ‘enterosalivary 
pathway.’  NO has pleiotropic effects in the body including relaxing smooth muscle (e.g., 
around arteries causing vasorelaxation) and, paradoxically, stimulating skeletal muscle 

 
 
 
 
 
 
8 
contraction.  Subjects with heart failure have reduced bioavailability of NO, so they may 
particularly benefit from increased NO production via the dietary pathway. This increase 
in NO bioavailability should translate into improved skeletal and cardiac muscle and 
blood vessel function.  These improvements in organ function should, in turn, result in 
improved exercise performance, especially in patients with heart failure. 
 
 
C Study Objectives 
 
C1 Primary Aim 
The primary aim of this study is to determine if dietary nitrate (NO3-), in this case from 
BRJ, is efficacious in improving exercise performance (V̇ O2peak and muscle power and 
speed) in patients with heart failure. 
Rationale for the Selection of Outcome Measures 
The primary outcome measures are the V̇ O2peak and peak leg muscle power and speed.  
These were chosen because they have been correlated with survival in patients with 
heart failure.  Furthermore, these endpoints directly impact a patient’s ability to perform 
activities of daily living. 
Note: there are other Tertiary Aims including assessment of the mechanisms by which 
dietary nitrate may improve exercise performance. To this end, the Tertiary Aims are to 
determine the effects of BRJ on plasma nitrate and nitrite levels, breath NO. 
D Investigational Agent  
 
D1 Preclinical Data 
In 2004, Webb et al. (Proc Natl Acad Sci USA 2004;101:13683–13688.) reported 
protective effects of nitrite in isolated perfused heart preparations subjected to IR 
injury. Under ischaemic conditions, both rat and human myocardium generated 
NO from nitrite. Although NO was produced in a dose-dependent manner (10–
100 mM), the degree of protection did not differ, suggesting that the beneficial 
effect of nitrite treatment was reached already at low concentrations. 
Furthermore, the authors showed that the conversion to NO was dependent on 
xanthine oxidoreductase (XOR), since co-administration of allopurinol or BOF-
4272 attenuated nitrite-dependent NO formation. XOR is generally thought to 
contribute to IR injuries via production of reactive oxygen species (ROS), 
including superoxide (O − 2). However, the findings by Webb et al. suggest that 
during hypoxic conditions, nitrite supplementation may partly shift the activity of 
XOR from generation of damaging 
O −2 to protective NO. 
 
Soon after, Duranski et al. (J Clin Invest 2005;115:1232–1240.) 
demonstrated potent cytoprotective effects of low dose nitrite using in vivo mouse 
models of myocardial infarction and liver ischaemia. The nitrite-mediated effects 

 
 
 
 
 
 
9 
were independent of NOS and abolished by co-administration of the NO 
scavenger carboxy-PTIO (cPTIO), suggesting NO as an active mediator. 
D2 Clinical Data to Date  
Based on the results from this study, we have shown that dietary nitrate (in the 
form of BRJ) has improved: 1) muscle power, 2) speed of muscle contraction, 3) 
nitrate, nitrite, and NO levels, 4) V̇ O2peak.  The improvements in muscle power 
and speed and nitrate/nitrite levels was shown in patients with HF and in 
controls. Our preliminary data also suggest that muscle blood flow and oxygen 
levels are increased in patients with heart failure. 
D3 Dose Rationale and Risk/Benefits  
Initially, we had started off with giving one bottle of BRJ with ~5-6mmol of dietary 
nitrate.  However, we were not seeing any differences between the BRJ studies 
and the placebo studies.  After increasing the dose to ~ 11.2 mmol, we showed a 
difference between the BRJ and placebo studies.  Despite this increase in dose, 
we have had no episodes of increased side effects including hypotension. 
E Study Design  
 
E1 Overview or Design Summary 
Please see above for specifics of the designs of each of the studies.  All studies are 
prospective and interventional. All substudies are of a cross-over design. 
E2 Subject Selection and Withdrawal  
Subjects will include patients with heart failure with reduced ejection fraction. 
Inclusion Criteria  
HF Subjects: 
• Men and women with a diagnosis of heart failure with reduced ejection fraction 
• Age > or = 18 y 
 
2.a 
Exclusion Criteria  
HF and Control Subjects: 
• Age <18 y 
• Those taking phosphodiesterase inhibitors (e.g., Viagra) will be excluded, as 
these can potentiate NO effects 
• Those taking prescription hormones (e.g., birth control pills, HRT), as these may 
block conversion of nitrate to nitric oxide 
• Women who are pregnant or intend to become pregnant during the study 
• Individuals who are not able to perform the exercise tests 
• Individuals who can not give informed consent 

 
 
 
 
 
 
10 
2.b 
Ethical Considerations  
Vulnerable populations, such as children and prisoners, will not be enrolled. All study 
procedures will be conducted with the highest ethical standards, in accordance with IRB 
and HIPAA regulations.  
2.c 
Subject Recruitment Plans and Consent Process 
 
We will recruit subjects from the following resources:  
• 
the Washington University Volunteer for Health database 
• 
Dr. Peterson's V̇ O2max database (which contains subjects who have already 
signed an informed consent document to be contacted about future studies) 
• 
Dr. Mann's heart failure registry 
• 
the Washington University Heart Failure and Cardiology clinics 
• 
Dr. Peterson's previously approved study #201107366 (Heart failure and 
myocardial metabolism) who have agreed (in their signed consent form) to allow 
us to contact them for future studies, if they meet the entry criteria 
2.d 
Randomization Method and Blinding 
Subjects are randomized by the dietician in the Clinical Research Unit (Suzanne Waller). 
It is she who hands out the BRJ and the placebo.  The BRJ and placebo are packaged 
the same and have similar taste, texture, and color. 
2.e 
Risks and Benefits 
See this description in the HRPO website for this study. 
2.f 
Early Withdrawal of Subjects  
Subjects may withdraw from this study at their discretion or the discretion of the PIs. 
2.g 
When and How to Withdraw Subjects  
Subjects are withdrawn from the study at the discretion of the PIs based on patient 
safety considerations and/or compliance.  
2.h 
Data Collection and Follow-up for Withdrawn Subjects  
If subjects are withdrawn, this is reported in publications according to the Study flow 
charts. 
 
E3 Study Drug  
 
3.a 
Description  
Beet IT Sport shot and Beet IT organic stamina shot. 
3.b 
Treatment Regimen  
See above for the particular treatment regimen for each substudy. 

 
 
 
 
 
 
11 
3.c 
Method for Assigning Subjects to Treatment Groups  
For all substudies that are cross-over, Suzanne Waller randomly assigns the treatment 
order.  For the substudies in which the BRJ or placebo are randomly assigned, Suzanne 
performs the randomization and assigns subjects to a treatment group. 
3.d 
Preparation and Administration of Study Drug  
The BRJ or placebo are placed in a cup and the patient is to ingest it orally. 
3.e 
Subject Compliance Monitoring  
We watch the subjects ingest the BRJ/placebo on the study days.  For the longer-term 
studies, we ask the subjects about compliance. 
3.f 
Prior and Concomitant Therapy  
Subjects may stay on all allowed previously prescribed medications. 
3.g 
Packaging  
Per James White Drinks, LTD (Ipswitch, UK) 
3.h 
Blinding of Study Drug  
Per Suzanne Waller of the CRU 
3.i 
Receiving, Storage, Dispensing and Return  
Study BRJ and placebo are shipped to Dr. Peterson and may be stored at room temp. or 
at refrigerated temperatures.  BRJ/placebo are dispensed by Suzanne Waller.  No 
BRJ/placebo is returned due to high costs of shipment to the UK. 
F Study Procedures  
 
F1 Screening for Eligibility 
Subjects’ medical chart will be reviewed after they sign consent to assess for eligibility.  
F2 Schedule of Measurements 
For all studies and sub-studies: 
• Subjects will be consented. 
• Subjects will give permission for the investigators to review their medical records. 
• Subjects for the main VO2 study, and the neuromuscular function testing will be HF 
patients.   
• I. BRJ main study (1-2 shot BRJ intervention): 
1) After consenting to participate, subjects will be instructed to refrain from spitting or 
the use of an antibacterial mouthwash, antacids, proton pump inhibitors, or chewing 
gum during the study. 
 

 
 
 
 
 
 
12 
2) Subjects will be asked to answer questionnaires regarding their medical health (basic 
health questionnaire, Minnesota Living with Heart Failure questionnaire, a combined 
fatigue questionnaire, a magnetic resonance safety questionnaire -i.e., questions 
regarding implanted pacemakers, etc.). Subjects will have a physical examination. 
 
3) Subjects will be instructed by study personnel to minimize dietary intake of foods that 
are high in dietary nitrates the day before each study visit.  
 
4) During study visit #1, subjects will be randomized to receive either one to two "shots" 
of BRJ (James White Drinks) or placebo (BRJ without nitrates).   
 
5) Before and at 3 time points after receiving the BRJ or placebo, subjects will undergo 
phlebotomy for plasma nutrient/hormone levels and genetic testing and will have 
their blood pressure checked and will blow into a tube connected to a small machine 
that will measure the amount of nitric oxide in their breath. 
 
6) Subjects will undergo transthoracic echocardiographic imaging at rest. They will also 
have their body composition determined via DXA. 
 
7) ~2 h after ingestion, subjects will blow into another machine to measure their 
respiratory muscle function.  
 
8) Subjects will pedal a cycle ergometer for 6 min at ~50, ~60, and ~70% of V̇ O2peak 
while V̇ O2 is measured using a ParvoMedics TrueOne metabolic cart. The average 
V̇ O2 during the last 2 min of each stage will be used to calculate gross and delta 
efficiency using stoichiometric equations as previously described. Following a 10 min 
rest period, peak power and V̇ O2peak will determined during a continuous exercise 
test performed using a 10 W/min ramp protocol.  Subjects will have their blood 
pressure, heart rate and rhythm monitored during this study. They will also have their 
muscle oxygen levels measured using small sensors taped to their thighs. 
 
11) Subjects will undergo a 7 d washout period. 
 
12) Subjects will come in for study day 2 in which they will receive whichever treatment 
(BRJ or placebo) they did not receive at first and then repeat the studies listed above 
5)-11). 
 
 
II. BRJ neuromuscular function testing sub-study: 
1) Subjects will be asked to undergo steps 1-7 of the main study described above. 
 
2) Subjects will undergo a test of neuromuscular function using an isokinetic 
dynamometer (a device that measures voluntary muscle force production while 
controlling the speed of movement).   
 
3) Subjects will undergo a 7 d washout period. 
 
4) Subjects will come in for Study day 2 in which they will receive whichever treatment 
(BRJ or placebo) they did not receive at first and then repeat the studies listed above 
1-5. 

 
 
 
 
 
 
13 
 
 
 
F3 Safety and Adverse Events  
 
3.a 
Safety and Compliance Monitoring 
 
We have a Data and safety monitoring board meeting at least yearly and should any 
AEs or SAEs arise, we may have them more frequently. 
 
 
3.b 
Medical Monitoring  
 
i 
Investigator only 
 
ii 
Independent expert to monitor 
 
iii 
Institutional Data and Safety Monitoring Board 
Dr. Geltman is the head of the Data and Safety Monitoring Board. 
iv 
Independent Data and Safety Monitoring Board 
 
3.c 
Definitions of Adverse Events 
Any adverse event that occurs during the study.  This includes patient complaints or 
medically-recognized events, e.g., hypotension. 
3.d 
Classification of Events 
 
i 
Relationship 
We have only had on AE during the study. The only AE we had was one person had 
abdominal discomfort and urgency after drinking the BRJ.  This was added to the 
expected risks in the consent forms. 
ii 
Severity 
We have had no SAEs. 
iii 
Expectedness 
Our consent forms include all of our expected risks and their likelihood of occurring.  We 
have had no SAEs.  The only AE we had was one person had abdominal discomfort and 

 
 
 
 
 
 
14 
urgency after drinking the BRJ.  This was added to the expected risks in the consent 
forms. 
3.e 
Data Collection Procedures for Adverse Events 
We will follow the HRPO-outlined procedures for collecting and reporting adverse 
events. 
3.f 
Reporting Procedures 
We will follow the HRPO-outlined procedures for collecting and reporting adverse 
events. 
 
3.g 
Adverse Event Reporting Period 
Per the HRPO suggestions, AEs should be reported within 24h 
3.h 
Post-study Adverse Event 
There is no long-term follow-up of these very short-term, low morbidity studies.  We have 
also not heard of any post-study adverse events. 
F4 Study Outcome Measurements and Ascertainment 
 
G Statistical Plan  
 
G1  Sample Size Determination and Power 
These are first-in-human, exploratory proof of concept studies.  Thus a formal power 
analysis was not performed.  
G2  Interim Monitoring and Early Stopping 
NA – if the DSMB determines there is reason for early stopping, any of the Substudies 
may be stopped. 
G3  Analysis Plan 
See our publications for the specific statistical methods used for each particular 
substudy.   
G4  Statistical Methods  
Please see our publications for the specific statistical methods used for each particular 
substudy.   
G5  Missing Outcome Data 
This is reported in all publications. 

 
 
 
 
 
 
15 
G6  Unblinding Procedures  
After a particular substudy is complete, we unblind the data. 
 
H Data Handling and Record Keeping  
 
H1 Confidentiality and Security 
Per HRPO guidelines. 
H2 Training  
NA 
H3 Case Report Forms and Source Documents 
The data collection sheets are kept in the subjects’ hardcopy folders, which are kept in a 
secure location. 
H4 Records Retention 
All records are retained in compliance with the HRPO guidelines under double-lock and 
password protected, encrypted computers. 
H5 Performance Monitoring  
NA  
 
I Study Monitoring, Auditing, and Inspecting  
 
I1 
Study Monitoring Plan  
The study is monitored by the PI, the study coordinators, and the DSMB. 
I2 
Auditing and Inspecting  
NA for this single center, small study 
J Study Administration 
 
J1 Organization and Participating Centers 
WUSM only 
J2 Funding Source and Conflicts of Interest 
CCIR JIT funding is current.  Dr. Peterson also has funds to continue these studies.  
These funds are separate from a grant. There are no conflicts of interest 

 
 
 
 
 
 
16 
J3 Committees 
NA 
J4 Subject Stipends or Payments  
Subjects are reimbursed for their time for each Substudy as outlined in the consent 
forms for that particular Substudy. 
J5  Study Timetable 
The study is ongoing. It is not limited by  
 
K Publication Plan  
We already have several publications from this study: 
1. 
Coggan AR, Leibowitz JL, Kadkhodayan A, Thomas DT, Ramamurthy S, Anderson-Spearie 
C, Waller S, Farmer M, Peterson LR. Effect of acute dietary nitrate intake on maximal knee 
extensor speed and power in healthy men and women. Nitric Oxide: Biol and Chem 
2015;48:16-21. PMCID: PMC4362985 (http://dx.doi.org/doi:10.1016/j.niox.2014.08.014). 
 
2. 
Coggan AR, Leibowitz JL, Anderson Spearie C, Kadkhodayan A, Thomas DP, 
Ramamurthy S, Mahmood K, Park S, Waller S, Farmer M, Peterson LR. Acute dietary 
nitrate intake improves muscle contractile function in patients with heart failure: a double-
blind, placebo-controlled, randomized trial. Circ Heart Fail 2015;8:914-20. (the most cited 
article from this journal in the lay press per Altmetrics). PMCID: PMC4573847 
http://circheartfailure.ahajournals.org/content/early/2015/07/15/CIRCHEARTFAILURE.115.
002141.full.pdf?ijkey=81kIxpBz5FebFXK&keytype=ref 
 
3. 
Rimer EG, Peterson LR, Coggan AR, Martin JC.  Acute dietary nitrate supplementation 
increases maximal cycling power of trained athletes. Int J Sports Physiol Perform 2016; 
11:715-20. PMCID: PMC4889556 
 
4. 
Coggan AR, Broadstreet SR, Mahmood K, Mikhalkova D, Madigan M, Bole I, Park S, 
Leibowitz J, Kadkhodayan A, Thomas D, Thies D, Peterson LR. Dietary nitrate increases 
VO2 peak and performance but does not alter ventilation or efficiency in patients with heart 
failure with reduced ejection fraction. J Card Fail  (in press). 
 
5. 
Coggan AR, Broadstreet S, Mikhalkova D, Bole I, Leibowitz J, Kadkhodayan A, Park S, 
Thomas D, Thies D, Peterson LR.  Dietary nitrate-induced increases in human muscle 
power: high vs. low responders. Physiol Rev (in review). 
 
6. 
Coggan AR, Peterson LR. Dietary nitrate and skeletal muscle contractile function in heart 
failure. Curr Heart Fail Rep 2016;13:158-165. PMCID: PMC Journal PMID: 27271563. 
 
We will continue to publish our findings as they become available. 
 
 
 
 
L  Attachments  
 

 
 
 
 
 
 
17 
L1  Tables  
 
L2  Informed consent documents 
See the consents attached to the HRPO website for this study.  
L3  Patient education brochures 
NA 
L4  Special procedures protocols  
NA 
L5  Questionnaires or surveys 
See questionnaires that are attached to the HRPO website for this study. 
 
M References 
1. 
Bleumink GS, Knetsch AM, Sturkenboom MC, Straus SM, Hofman A, Deckers JW, 
Witteman JC, Stricker BH. Quantifying the heart failure epidemic: prevalence, 
incidence rate, lifetime risk and prognosis of heart failure The Rotterdam Study. 
Eur Heart J 2004;25:1614-1619. 
2. 
Taylor AL, Ziesche S, Yancy C, Carson P, D'Agostino RJ, Ferdinand K, Taylor M, 
Adams K, Sabolinski M, Worcel M, Cohn JN. Combination of isosorbide dinitrate 
and hydralazine in blacks with heart failure. N Engl J Med 2004;351:2049-2057. 
3. 
Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr 
J, Benjamin N, Jones AM. Dietary nitrate supplementation reduces the O2 cost of 
low-intensity exercise and enhances tolerance to high-intensity exercise in 
humans. J Appl Physiol 2009;107:1144-1155. 
4. 
Bailey SJ, Fulford J, Vanhatalo A, Winyard PG, Blackwell JR, DiMenna FJ, 
Wilkerson DP, Benjamin N, Jones AM. Dietary nitrate supplementation enhances 
muscle contractile efficiency during knee-extensor exercise in humans. J Appl 
Physiol 2010;109:135-148. 
5. 
Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Blackwell JR, 
DiMenna FJ, Gilchrist M, Benjamin N, Jones AM. Dietary nitrate supplementation 
reduces the O2 cost of walking and running: a placebo-controlled study. J Appl 
Physiol 2011;110:591-600. 
6. 
Vanhatalo A, Fulford J, Bailey SJ, Blackwell JR, Winyard PG, Jones AM. Dietary 
nitrate reduces muscle metabolic perturbation and improves exercise tolerance in 
hypoxia. J Physiol 2011 Sep 12. [Epub ahead of print]. 
7. 
Lansley KE, Winyard PG, Bailey SJ, Vanhatalo A, Wilkerson DP, Blackwell JR, 
Gilchrist M, Benjamin N, Jones AM. Acute dietary nitrate supplementation 
improves cycling time trial performance. Med Sci Sports Exerc 2011;43:1125-1131. 
8. 
Kenjale AA, Ham KL, Stabler T, Robbins JL, Johnson JL, Vanbruggen M, Privette 
G, Yim E, Kraus WE, Allen JD. Dietary nitrate supplementation enhances exercise 
performance in peripheral arterial disease. J Appl Physiol 2011;110:1582-1591. 
9. 
Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in 
physiology and therapeutics. Nat Rev Drug Discov 2008;7:156-167. 

 
 
 
 
 
 
18 
10. Gilchrist M, Winyard PG, Benjamin N. Dietary nitrate--good or bad? Nitric Oxide. 
2010;22:104-109. 
11. Opie LH. The metabolic vicious cycle in heart failure. Lancet 2004;364:1733-1734. 
12. Witte KK, Levy WC, Lindsay KA, Clark AL. Biomechanical efficiency is impaired in 
patients with chronic heart failure Eur J Heart Fail. 2007;9:834-838. 
13. Bescós PB, Rodríguez FA, Iglesias X, Ferrer MD, Iborra E, Pons A. Acute 
administration of inorganic nitrate reduces VO2peak iun endurance athletes. Med 
Sci Sports Exerc 2011;43:1979-1986. 
14. Larsen FJ, Weitzbeg E, Lundberg JO, Ekblom B. Dietary nitrate reduces maximal 
oxygen consumption while maintaining work performance in maximal exercise. 
Free Rad Biol Med 2010;48:342-347. 
15. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg 
E. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell 
Metab 2011;13:149-159. 
16. Peterson LR, Schechtman KB, Ewald GA, Geltman EM, de las Fuentes L, Meyer 
T, Krekeler P, Moore ML, Rogers JG. Timing of cardiac transplantation in patients 
with heart failure receiving beta-adrenergic blockers. J Heart Lung Transplant 
2003;22:1141-1148. 
17. Peterson LR, Waggoner AD, Schechtman KB, Meyer T, Gropler RJ, Barzilai B, 
Davila-Roman VG. Alterations in left ventricular structure and function in young 
healthy obese women: assessment by echocardiography and tissue Doppler 
imaging. J Am Coll Cardiol 2004;43:1399-1404. 
18. Peterson LR, Schechtman KB, Ewald GA, Geltman EM, Meyer T, Krekeler P, 
Rogers JG. The effect of beta-adrenergic blockers on the prognostic value of peak 
exercise oxygen uptake in patients with heart failure. J Heart Lung Transplant 
2003;22:70-77. 
19. Stolker JM, Heere B, Geltman EM, Schechtman KB, Peterson LR. Prospective 
comparison of ventilator equivalent versus peak oxygen consumption in predicting 
outcomes of patients with heart failure. Am J Cardiol 2006;97:1607-1610. 
20. Peterson LR, Rinder MR, Schechtman KB, Spina RJ, Glover KL, Villareal DT, 
Ehsani AA. Peak exercise stroke volume: associations with cardiac structure and 
diastolic function. J Appl Physiol 2003;94:1108- 1114. 
21. Spina RJ, Ogawa T, Coggan AR, Holloszy JO, Ehsani AA. Exercise training 
improves left ventricular contractile response to beta-adrenergic agonist. J Appl 
Physiol 1992;72:307-311. 
22. Spina RJ, Ogawa T, Martin WHr, Coggan AR, Holloszy JO, Ehsani AA. Exercise 
training prevents decline in stroke volume during exercise in young healthy 
subjects. J Appl Physiol 1992;72:2458-2462. 
23. Coggan AR, Habash DL, Mendenhall LA, Swanson SC, Kien CL. Isotopic 
estimation of CO2 production during exercise before and after endurance training. 
J Appl Physiol 1993;75:70-75. 
24. Ehsani AA, Spina RJ, Peterson LR, Rinder MR, Glover KL, Villareal DT, Binder EF, 
Holloszy JO. Attenuation of cardiovascular adaptations to exercise in frail 
octogenarians. J Appl Physiol 2003;95:1781-1788. 
25. Ruby BC, Coggan AR, Zderic TW. Gender differences in glucose kinetics and 
substrate oxidation during exercise near the lactate threshold. J Appl Physiol 
2002;92:1125-1132. 
26. Peterson LR, Courtois M, Peterson LF, Peterson MR, Davila-Roman VG, Spina 
RJ, Barzilai B. Estrogen increases hyperemic microvascular blood flow velocity in 
postmenopausal women. J Gerontol A Biol Sci Med Sci 2000;55:M174-9. 
27. O'Connor RD, Gropler RJ, Peterson L, Schaffer J, Ackerman JJ. Limits of a 

 
 
 
 
 
 
19 
localized magnetic resonance spectroscopy assay for ex vivo myocardial 
triacylglycerol. J Pharm Biomed Anal 2007;45:382-389. 
28. Frimel TN, Deivanayagam S, Bashir A, O'Connor R, Klein S. Assessment of 
intrahepatic triglyceride content using magnetic resonance spectroscopy. J 
Cardiometab Syndr 2007;2:136-138. 
29. Mancini DM, Coyle E, Coggan A, Beltz J, Ferraro N, Montain S, Wilson JR. 
Contribution of intrinsic skeletal muscle changes to 31P NMR skeletal muscle 
metabolic abnormalities in patients with chronic heart failure. Circulation 
1989;80:1338-1346. 
30. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, 
Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, 
Webb AJ, Ahluwalia A. Inorganic nitrate supplementation lowers blood pressure in 
humans: role for nitrite-derived NO. Hypertension 2010;56:274-281. 
31. Govoni M, Jansson EA, Weitzberg E, Lundberg JO. The increase in plasma nitrite 
after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. 
Nitric Oxide 2008;19:333-337. 
32. Kemp GJ, Radda GK. Quantitative interpretation of bioenergetic data from 31P and 
1H magnetic resonance spectroscopic studies of skeletal muscle: an analytical 
review. Magn Reson Q 1994;10:43-63. 
33. Moseley L, Jeukendrup AE. The reliability of cycling efficiency. Med Sci Sports 
Exerc 2001;33:621-627. 
34. Recommendations for exercise testing in chronic heart failure patients. Eur Heart J 
2001;22:37-45. 
35. Stamler J, Meissner G. Physiology of Nitric Oxide in Skeletal Muscle. Physiol Rev 
2001;81:209-237. 
36. van Heerebeek L, Hamdani N, Falcao-Pires I, Leite-Moreira AF, Begieneman MP, 
Bronzwaer JG, van der Velden J, Stienen GJ, Laarman GJ, Somsen A, Verheugt 
FW, Niessen HW, Paulus WJ. Low myocardial protein kinase G activity in heart 
failure with preserved ejection fraction. Circulation 2012;126:830-9.