Conditions: Aortic Valve Stenosis
Keywords: aortic stenosis, aortic valve replacement
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 5
Healthy volunteers: no

Brief summary:
The current trial is designed to assess the safety and efficacy of sustained oral administration of inorganic nitrate in patients with severe aortic stenosis and to assess the mechanisms by which inorganic nitrate enhances oxygen uptake and exercise capacity in this population.

Detailed description:
This is a randomized double-blind crossover clinical trial, in which 22 subjects who underwent transcatheter aortic valve replacement (TAVR) for severe aortic stenosis (AS) three or more months prior to enrollment will receive the following 2 interventions, in randomized order: (1) Potassium nitrate (KNO3), at a dose of 12-18 mmol/d by mouth for 4 weeks, or; (2) Potassium chloride (KCl), at a dose of 12-18 mmol/d by mouth for 4 weeks. A 1-week washout period will be introduced between the 2 interventions. The purpose of the trial is to test the safety of KNO3 and its efficacy on a number of clinical and physiologic endpoints in subjects who underwent TAVR for severe AS.

Arms:
arm 1: Capsules containing 18 mmoles of KNO3 per day, given as one capsule (6 mmoles) three times a day for 4 weeks. arm 2: Capsules containing 18 mmoles of KCl per day, given as one capsule (6 mmoles) three times a day for 4 weeks.

Interventions: intervention 1: Potassium Nitrate intervention 2: Potassium Chloride
Intervention descriptions: intervention 1: Potassium nitrate capsules will consist of potassium nitrate (KNO3-) crystals \[610 mg, corresponding to 6.03 mmoles of NO3-\] with 190mg of lactose monohydrate, spray dried, NF. The dose for this trial will be 18 mmoles of NO3-per day, given as one capsule (6 mmoles) three times a day. intervention 2: Potassium chloride capsules will consist of potassium chloride (KCl), granular, USP (450mg) plus lactose monohydrate, spray dried, NF (300mg). The dose for this trial will be 18 mmoles of KCl per day, given as one capsule (6 mmoles) three times a day.

Protocol document:
Prot_SAP_000.pdf: 
NCT #: 
NCT03483051  
Title: 
Targeting Pulsatile Load to Increase Exercise Capacity and 
Quality of Life After Aortic Valve Replacement for Severe 
Aortic Stenosis (PULSE AS)  
Date: 
02-Aug 2018  


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CONFIDENTIAL 
 
CLINICAL TRIAL PROTOCOL 
 
Targeting Pulsatile Load to Increase Exercise Capacity and Quality of Life After Aortic Valve 
Replacement for Severe Aortic Stenosis (PULSE AS) 
 
Version:  V3.0 Date: 02-Aug 2018 
Sponsor: University of Pennsylvania 
Sponsor Representative:  Emma A. Meagher, M.D. 
Sponsor email: psom-ind-ide@pobox.upenn.edu 
 
Principal Investigator: 
Julio A. Chirinos, M.D., Ph.D. 
 
Sub-Investigators: 
Saif Anwaruddin, M.D. 
Howard C. Herrmann, M.D. 
Anupam A. Kumar, M.D. 
Frank E. Silvestry, M.D. 
 
 
 
 
 
 
 
 
 
 
 


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Table of Contents 
Table of Contents .................................................................................................................. 2 
1. SPECIFIC AIMS ................................................................................................................... 4 
2. TRIAL SUMMARY TABLE (OBJECTIVES AND ENDPOINTS) ..................................................... 6 
3. BACKGROUND AND SIGNIFICANCE ..................................................................................... 7 
4. STUDY DESIGN AND POPULATION ...................................................................................... 8 
4.1. Overview of study design .........................................................................................................8 
4.2 Study Sites ................................................................................................................................8 
4.3. Study Population .....................................................................................................................8 
4.3.1. Inclusion Criteria ......................................................................................................................... 8 
4.3.2. Exclusion Criteria ......................................................................................................................... 9 
4.3.3. Criteria that will prompt exclusion from the trial at the phase 1 visit ........................................ 9 
4.4. Randomized intervention ....................................................................................................... 10 
5. STUDY VISITS AND PROCEDURES ...................................................................................... 10 
5.1. Initial Visit ............................................................................................................................. 11 
5.2. Randomization procedure ...................................................................................................... 13 
5.3. Intervention Phase 1 .............................................................................................................. 13 
5.4. Washout period ..................................................................................................................... 14 
5.5. Intervention Phase 2 .............................................................................................................. 14 
5.6. Adverse Event Reporting ........................................................................................................ 16 
5.7. Subject withdrawal / Early termination .................................................................................. 16 
5.8. Concomitant Medication ........................................................................................................ 16 
6. DRUG DISPENSING, ACCOUNTABILITY AND DESTRUCTION ............................................... 16 
6.1. Rules for un-blinding .............................................................................................................. 17 
7. ADVERSE EVENTS ............................................................................................................. 17 
7.1. Key definitions ....................................................................................................................... 17 
7.2. Classification of AE/ADEs regarding causal relationship to study interventions and severity .... 18 
7.3. Recording and Reporting of Adverse Events ............................................................................ 19 
7.4. Follow-up .............................................................................................................................. 20 
7.5. Management of Suspected Unexpected Serious Adverse Reaction .......................................... 20 
7.6. Pregnancy ............................................................................................................................. 20 
8. STUDY MEASUREMENTS AND DATA COLLECTION ............................................................. 21 
8.1. Assessment of exercise capacity ............................................................................................. 21 
8.2. Quality of life ......................................................................................................................... 21 
8.3. Doppler Echocardiography ..................................................................................................... 21 
8.4. Measurement of late systolic load and arterial wave reflections: ............................................ 22 
8.5. Blood nitrate/nitrite level measurements ............................................................................... 22 
8.6. Flow-mediated brachial artery dilation ................................................................................... 23 
8.7. Other assessments ................................................................................................................. 23 
9. STUDY CENTER MANAGEMENT PLAN ............................................................................... 23 


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9.1. Principal Investigator ............................................................................................................. 23 
9.2. Investigators Committee ........................................................................................................ 24 
9.3. Operational Affairs Committee .............................................................................................. 24 
9.4. Data Management ................................................................................................................. 24 
9.5. Quantification Core Laboratories............................................................................................ 25 
10. STATISTICAL CONSIDERATIONS ...................................................................................... 26 
10.1. Power calculations ............................................................................................................... 26 
10.2. Data Analysis Plan................................................................................................................ 26 
11. PROTECTION OF HUMAN SUBJECTS ................................................................................ 28 
11.1. Potential benefits of the proposed research to the study subjects and importance of the 
knowledge to be gained, and risk/benefit ratio ............................................................................. 28 
11.2. Risks to study subjects  ....................................................................................................... 28 
11.2.1. Potential Risks of study intervention (potassium nitrate) ...................................................... 29 
11.2.2. Potential Risks of study procedures ........................................................................................ 30 
11.3. Adequacy of Protection Against Risks  ................................................................................. 31 
11.3.1. Recruitment and Informed Consent ....................................................................................... 31 
11.3.2. Protection against risks associated with cardiopulmonary exercise tests ............................. 32 
11.3.3. Protection against risks of potassium nitrate administration: ............................................... 32 
11.3.4. Measures to minimize the risk of breach in confidentiality: .................................................. 33 
11.3.6 Other measures to minimize risk: ............................................................................................ 33 
12. REGULATORY STANDARDS ............................................................................................. 33 
12.1. Informed consent ................................................................................................................ 33 
12.2. Institutional Review Board (IRB) ........................................................................................... 33 
12.3. Source document handling and archiving ............................................................................. 34 
13. DATA AND SAFETY MONITORING PLAN: ......................................................................... 34 
14. SUBJECT STIPEND ........................................................................................................... 34 
15. REFERENCES .................................................................................................................. 35 
APPENDIX 1. SCHEDULE OF EVENTS ..................................................................................... 43 
 
 
 
 


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1. SPECIFIC AIMS  
Moderate or severe aortic stenosis (AS) affects 2.8% of people aged ≥ 75 years and is one of the 
most common valvular disorders worldwide(1). Without intervention, severe AS carries an 
extremely poor prognosis with a 1 and 5 year survival of ~60% and ~30%, respectively(2,3). 
Surgical and transcatheter aortic valve replacement (AVR) are to-date the only interventions that 
have been shown to improve morbidity and mortality in this population(4,5). However, there is 
heterogeneity in clinical course after AVR; even after a successful procedure, some patients show 
minimal symptomatic or hemodynamic improvement(6).   
Symptoms in aortic stenosis result from an inability of the left ventricle (LV) to overcome the 
systolic load caused by a fixed outflow obstruction. After correction of the stenotic valve, residual 
LV afterload results primarily from arterial load and is associated with poor outcomes. Indeed, 
higher pulsatile arterial load after AVR is significantly associated with mortality(7). An important 
component of pulsatile load is the magnitude of wave reflections in the arterial tree(8,9). Wave 
reflections arise in peripheral arterial sites and travel back to the heart, arriving while the LV is still 
ejecting blood, increasing the late systolic workload of the LV. Wave reflections are highly 
sensitive to nitric oxide (NO).  
The increased LV afterload during systole results in myocyte hypertrophy and increased wall 
thickness that is accompanied by a diffuse interstitial fibrosis in the myocardium(10). In fact, the 
amount of myocardial fibrosis, as determined by either histology or cardiac MRI, is an independent 
predictor of LV function improvement and late all-cause mortality after aortic valve replacement 
(6). However, myocardial fibrosis does not appear to regress significantly after aortic valve 
replacement(11,12). This suggests that arterial factors play a role in the pathogenesis and 
maintenance of fibrosis even after AVR. This additionally indicates that persistent myocardial 
fibrosis may contribute to poor quality of life after AVR. Thus, reducing fibrosis, by reducing wave 
reflections and arterial load, may be an effective strategy towards improving quality of life and 
exercise capacity in patients after aortic valve replacement for severe aortic stenosis.  
Prior data demonstrates that a single oral dose of inorganic nitrate reduces late systolic load in 
other populations. Dietary nitrate increases plasma nitrite, an endothelium-independent source of 
the potent vasodilator nitric oxide (NO). Thus, augmentation of the nitrate-nitrite-NO pathway has 
the potential for chronic “disease-modifying” benefits in aortic stenosis. Therefore, nitrate 
supplementation represents a logical intervention to improve exercise capacity and quality of life 
in aortic stenosis. 
The current trial is designed to assess the safety and efficacy of sustained oral administration of 
inorganic nitrate in patients with severe aortic stenosis and to assess the mechanisms by which 
inorganic nitrate enhances oxygen uptake and exercise capacity in this population. This is a 
randomized double-blind crossover clinical trial, in which 22 subjects who underwent surgical or 
transcatheter AVR for severe AS three or more months prior to enrollment will receive the 
following 2 interventions, in randomized order: (1) Potassium nitrate (KNO3), at a dose of 12-18 
mmol/d by mouth for 4 ± 1 weeks, or; (2) Potassium chloride (KCl), at a dose of 12-18 mmol/d by 
mouth for 4 ± 1 weeks. A 1-week ± 3 day washout period will be introduced between the 2 
interventions. The purpose of the trial is to test the safety of KNO3 and its efficacy on a number 
of clinical and physiologic endpoints in subjects who underwent AVR for severe AS.  


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Our specific aims are: 
Specific Aim 1: To assess whether KNO3 therapy improves endpoints with direct clinical 
relevance: aerobic capacity and quality of life (QOL)  
Hypothesis 1a: KNO3 will improve exercise capacity, quantified as: (1) Total work 
performed during a maximal-effort exercise test; (2) Peak oxygen consumption (VO2) 
during a symptom-limited maximal effort exercise test. These will be the co-primary 
endpoints of the trial.  
 
Hypothesis 1b: KNO3 will improve QOL, assessed using the Kansas City Cardiomyopathy 
Questionnaire.  
 
Specific Aim 2: To dissect the effects of KNO3 on specific physiologic adaptations to exercise  
Hypothesis 2a: KNO3 improves the systemic vasodilator response to exercise, measured 
as the change in systemic vascular resistance during a symptom-limited maximal effort 
exercise test.  
 
Hypotheses 2b: Since nitrates may exert venodilating effects and/or myocardial effects, 
we will test the hypotheses that KNO3 impacts LV diastolic filling parameters (measured 
with echocardiography at rest and after peak exercise) and improves myocardial strain 
(assessed with speckle-tracking echocardiography). 
 
Specific Aim 3: To assess whether KNO3 reduces late systolic LV load from wave reflections 
(assessed via comprehensive aortic pressure-flow relations, using arterial tonometry and Doppler 
echocardiography).  
This trial has the potential to establish a novel, inexpensive, and readily implementable 
therapeutic paradigm for post-AVR patients and to characterize specific mechanisms involved, 
enhancing our understanding of this disease. 
 


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2. TRIAL SUMMARY TABLE (OBJECTIVES AND ENDPOINTS) 
Trial Title / Acronym Targeting Pulsatile Load to Increase Exercise Capacity and Quality of 
Life After Aortic Valve Repair for Severe Aortic Stenosis (PULSE AS) 
Phase 
Phase IIb Randomized Clinical Trial (RCT) 
Number of Subjects 22 
Patient Population 
Ages 50-90; 3 or more months after AVR for severe AS 
Study Site 
University of Pennsylvania 
Study Design 
Randomized, double-blinded crossover study 
Randomized 
Intervention 
Potassium Nitrate (KNO3) capsules, at a dose of 6 mEq (1 capsule) 
three times daily for  4 ± 1 weeks, versus, potassium chloride (KCl) 
capsules administered at a dose of 6 mEq (1 capsule) three times daily 
for 4 ± 1 weeks 
Primary Aim 
To assess whether KNO3 therapy improves exercise capacity, quantified 
as: (1) Total work performed during a maximal-effort exercise test; and 
(2) Peak oxygen consumption (VO2) during a symptom-limited maximal 
effort exercise test. These will be the co-primary endpoints of the trial. 
Secondary Aims 
To assess whether KNO3:  
• 
improves quality of life 
• 
improves the systemic vasodilator response to exercise 
• 
improves LV diastolic function and myocardial systolic strain 
• 
reduces late systolic LV load from wave reflections 
Exploratory Aims 
To assess whether KNO3 improves: 
 
• 
Ventilatory threshold, VO2 kinetics 
• 
Natriuretic peptide levels 
• 
Physical activity  
 
To assess the relationship between nitrate and nitrite levels achieved 
and the therapeutic response achieved in each subject (e.g., 
improvement in endpoints in response to KNO3 vs control). To assess 
the effect on RNA expression. 
Primary Endpoints 
Peak oxygen uptake (VO2) during a symptom-limited maximal effort 
exercise test after 4 ± 1 weeks of KNO3 vs KCl 
Quality of life score, assessed using the Kansas City Cardiomyopathy 
Questionnaire 
Secondary 
Endpoints 
Measures of LV diastolic function (E/e’, left atrial volume index),and 
peak global systolic myocardial longitudinal and circumferential strain 
 
Augmentation index, late systolic wall stress and aortic input impedance 


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3. BACKGROUND AND SIGNIFICANCE  
Calcific aortic stenosis (AS) occurs in 3-5% of people in the United States aged ≥ 75 years(13). 
Without intervention, severe AS carries an extremely poor prognosis with a 1 and 5 year survival 
of ~60% and ~30%, respectively(2,3). Surgical and transcatheter aortic valve replacement (AVR) 
are currently the only interventions that have been shown to improve morbidity and mortality in 
this population(4,5). Over the last decade, TAVR has been shown to result in a dramatic 
improvement of survival and quality of life for many patients who were deemed to be inoperable 
candidates for SAVR(4,5). However, there is heterogeneity in clinical course after AVR; even after 
a successful procedure, some patients show minimal symptomatic or hemodynamic 
improvement(6).  Among the extreme risk cohort of patients in the CoreValve US Pivotal trial, 39% 
of patients had a poor outcome 6 months after TAVR with 22% dying, 6% having poor quality of 
life by KCCQ and 1.4% with with a decline in their quality of life after the procedure(14). This was 
redemonstrated in reports from the PARTNER trial, in which 35% of patients had a poor outcome 
at 6 months post-procedure, with 19% subsequently dying and 16% with poor quality of life(15). 
This suggests an unmet need to identify and intervene upon physiologic abnormalities in patients 
who do not benefit from TAVR.  
There are a number of factors that determine a patient’s functional health status after AVR. Given 
the relative advanced age of patients who undergo TAVR, it should come as no surprise that 
presence of co-morbidities and overall frailty, in addition to age and gender, are critical. An 
analysis of patients from the PARTNER trial identified a number of significant pre-procedure risk 
factors that were predictive of poor outcomes after TAVR. Poor functional capacity, measured by 
Six-Minute Walk Test (6MWT) and lower mean aortic valve radients were the most predictive; 
other predictors were renal dysfunction, oxygen-dependent lung disease, and poor baseline 
cognitive function(16).  There are also clear cardiac and systemic hemodynamic characteristics 
that are associated with poor outcomes after AVR. Low-gradient AS and tricuspid valve 
regurgitation were shown to be associated with all-cause 6 month post-procedure mortality; 
severe mitral regurgitation and moderate/severe postprocedural aortic regurgitation appear to be 
independent predictors of poor response to treatment(17). Higher pulsatile arterial load after aortic 
valve repair is significantly associated with mortality(7).  
Symptoms in aortic stenosis result from an inability of the left ventricle to overcome the systolic 
load caused by a fixed outflow obstruction. Left ventricle (LV) afterload in patients with severe AS 
can be conceptualized as a series circuit with additive contributions by both the aortic valve and 
systemic arterial tree(8,9,18). After correction of the stenotic valve, residual LV afterload results 
primarily from arterial load and is associated with poor outcomes. Indeed, higher pulsatile arterial 
load after AVR is significantly associated with mortality(7). An important component of pulsatile 
load is the magnitude of wave reflections in the arterial tree(8,9). Wave reflections arise in 
peripheral arterial sites and travel back to the heart, arriving while the LV is still ejecting blood, 
increasing the late systolic workload of the LV. Available evidence causally link late systolic load 
with LV remodeling, diastolic dysfunction, and HF risk. Wave reflections are highly sensitive to 
NO.  
The increased LV afterload during systole results in myocyte hypertrophy and increased wall 


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thickness, which allows the ejection fraction to be maintained by decreasing LV wall stress(19). 
This myocyte growth is accompanied by a diffuse interstitial fibrosis in the myocardium, as 
fibroblasts increase collagen synthesis in response to the elevated intraventricular pressures(10). 
In fact, the amount of myocardial fibrosis, as determined by either histology or cardiac MRI, is an 
independent predictor of LV function improvement and late all-cause mortality after aortic valve 
replacement(6). However, myocardial fibrosis does not appear to regress significantly after aortic 
valve replacement(11,12). This  suggests that nonvalvular factors may play a role in the 
pathogenesis and maintenance of fibrosis even after AVR.  
Our trial will test a novel intervention (inorganic nitrate) to modify key physiologic abnormalities 
(arterial vasodilator reserve and late systolic LV load from arterial wave reflections), which have 
the potential for both immediate-term improvements in exercise tolerance and long-term “disease-
modifying” effects. The trial is designed to address the safety and potential clinical benefit of this 
approach in severe aortic stenosis using endpoints with direct clinical relevance, and also to 
characterize the specific physiologic mechanisms involved. This will potentially identify a new 
therapeutic paradigm and a readily implementable therapeutic intervention.  
4. STUDY DESIGN AND POPULATION 
4.1. Overview of study design 
In this phase IIb, double-blind, cross-over trial, we will assign 22 subjects who are post-AVR for 
severe AS to: (A) Potassium nitrate administered by mouth at a dose of 6 mEq three times daily 
for 4 ± 1 weeks, or (B) Potassium chloride (KCl) at identical doses. The order of the interventions 
(AB-BA design) will be randomized, with a 1-week ± 3 day washout period separating each 
intervention. A crossover design will enable each subject to receive both treatments, reducing 
inter-individual variability and maximizing our power to detect effects of potassium nitrate vs. 
control (KCl). The active drug (KNO3-) and control (KCl) will be prepared at the Investigational 
Drug Pharmacy at the University of Pennsylvania and dispensed by an investigational drug 
pharmacist, blinded to both the subjects and the investigators.  
4.2 Study Sites 
The site for this trial will be the Hospital of the University of Pennsylvania. 
4.3. Study Population 
Subjects will be identified from clinic visits at the University of Pennsylvania for post-TAVR follow 
up. We will enroll 22 subjects meeting the following criteria: 
4.3.1. Inclusion Criteria 
1. Adults aged 50-90 years of age 
2. Diagnosis of severe aortic stenosis prior to aortic valve repair 
3. Successful trancatheter aortic valve repair via transfemoral procedural approach or successful 
surgical aortic valve repair at least three months prior to enrollment  


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4. Stable medical therapy: no addition/removal/changes in antihypertensive medications, or 
beta-blockers in the preceding 30 days.  
 
4.3.2. Exclusion Criteria 
1. Supine systolic blood pressure (SBP) < 100 mmHg OR supine diastolic blood pressure (DBP) 
<60 mmHg 
2. Poorly controlled hypertension, as defined as SBP > 160 mmHg OR DBP > 100 mmHg 
3. Pregnancy. Women of childbearing potential will undergo a pregnancy test during the 
screening visit 
4. Atrial fibrillation within the prior 8 weeks before enrollment 
5. Inability/unwillingness to exercise 
6. Moderate or greater mitral regurgitation or aortic/perivalvular regurgitation, any degree of 
mitral stenosis, severe right-sided valvular disease, or presence of a mitral prosthetic valve. 
7. Moderate or severe patient prosthesis mismatch, as defined by Effective Orifice Area Index < 
0.85 cm2/m2 
8. Hypertrophic, infiltrative, or inflammatory cardiomyopathy 
9. Pericardial disease 
10. Current angina 
11. Acute coronary syndrome or coronary intervention within the past 2 months 
12. Primary pulmonary arteriopathy 
13. Clinically significant lung disease as defined by: Chronic Obstructive pulmonary disease 
meeting Stage III or greater GOLD criteria, treatment with oral steroids within the past 6 
months for an exacerbation of obstructive lung disease, or the use of daytime supplemental 
oxygen 
14. Ischemia on stress testing without subsequent revascularization (during the screening visit) 
15. Treatment with phosphodiesterase inhibitors that cannot be withheld 
16. Treatment with organic nitrates  
17. Significant liver disease impacting synthetic function or volume control (ALT/AST > 3x ULN, 
Albumin <3.0 g/dL) 
18. eGFR < 30 mL/min/1.73 m2  
19. G6PD deficiency. For males of African, Asian or Mediterranean decent, this will be evaluated 
prior to drug administration. A qualitative test positive for deficiency or a quantitative test with 
clinically significant G6PD deficiency (<60% of normal activity) will prompt  exclusion from the 
trial (prior to drug administration). 
20. History of methemoglobinemia or methemoglobin level >5% at baseline visit  
21. Serum K>5.0 mEq/L  
22. Severe right ventricular dysfunction. 
23. Any medical condition that, in the opinion of the investigator, will interfere with the safe 
completion of the study.  
4.3.3. Criteria that will prompt exclusion from the trial at the phase 1 visit  
1. Acute coronary syndrome or coronary intervention for unstable coronary disease after 
enrollment  
2. New onset ischemia on stress testing since the time of enrollment 
3. New treatment with organic nitrates or phosphodiesterase inhibitors that cannot be withheld. 


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4. Serum K>5.5 mEq/L. 
5. Any medical condition that, in the opinion of the investigator, will interfere with the safe 
completion of the study, or the validity of endpoint assessments. These ad-hoc 
discontinuations will be discussed with the PI of the trial and the medical director of the IND. 
In the following situations, subjects who meet the criteria below will not be immediately 
discontinued, but will be scheduled for an ad hoc visit for reassessment of values prior to drug 
initiation in phase 2: 
1. Supine systolic blood pressure < 90 mm Hg 
2. Acute kidney injury with eGFR < 30 mL/min/1.73m2  
3. Methemoglobinemia – baseline methemoglobin level > 5% 
 
If these criteria persist during the ad-hoc visit,  the subject will be discontinued from the study.  
4.4. Randomized intervention 
The randomized interventions will be the following (in random order, separated by a 1-week ± 3 
day washout period) 
1. Potassium nitrate:  Interventional drug capsules will consist of potassium nitrate (KNO3-) 
crystals [610 mg, corresponding to 6.03 mmoles of NO3-] with 190mg of lactose monohydrate, 
spray dried, NF. The dose for this trial will be 18 mmoles of NO3-per day, given as one capsule 
(6 mmoles) three times a day. 
 
2. Control: Capsules will consist of potassium chloride (KCl), granular, USP (450mg) plus lactose 
monohydrate, spray dried, NF (300mg). We chose potassium chloride as the control in order 
to isolate the effects of nitrate from any potential effect of potassium supplementation. The 
dose for this trial will be 18 mmoles of KCl per day, given as one capsule (6 mmoles) three 
times a day. 
 
5. STUDY VISITS AND PROCEDURES 
An overview of the study design and flow of study visits is presented in Figure 3. 
 
 
 
 
 
 
 
 


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Figure 3. Overview of the study design and procedures. 
 
 
 
The following investigational devices will be used during the study: 
Portalite: continuous wave near infrared spectroscopy (NIRS) system to measure oxygenation of 
muscle tissue 
Portamon: continuous wave near infrared spectroscopy (NIRS) system to measure oxygenation 
of muscle tissue 
5.1. Initial Visit  
 
During the baseline study visit, inclusion and exclusion criteria will be reviewed to ensure subject 
suitability. After eligibility has been confirmed, written informed consent will be obtained from the 
subject using Institutional Review Board (IRB)-approved documents. Informed consent will be 


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obtained before any study procedures. Subjects will be given the opportunity to have all questions 
regarding their participation answered in detail in a private setting before entering the study.  
Following informed consent, a physical examination, with measurement of orthostatic blood 
pressures, will be performed. A urine pregnancy test will be performed in women with child-
bearing potential. Blood will be collected for measurement of (a) comprehensive metabolic panel; 
(b) complete blood count;  (d) methemoglobin. A pregnancy test (for women of childbearing 
potential) and Glucose-6-phosphate dehydrogenase (G6PD) deficiency screening (for males of 
males of African, Asian or Mediterranean decent) will be performed as needed. Blood, urine, and 
saliva samples will be collected and frozen for nitrate/nitrite and biomarker level determination.  
The Kansas City Cardiomyopathy Questionnaire (KCCQ) will be administered. Subjects will also 
be given an informational brochure regarding foods that are high in nitrate/nitrite and thus should 
be avoided during the study.  
Echocardiography will then be performed using a standardized protocol. Images will be obtained 
from the parasternal long axis, short axis, apical 5-, 4-, 3-, and 2-chamber, subcostal, and 
suprasternal views for offline analysis of myocardial strain. Dedicated ventricular chamber images 
will be obtained in the 4- and 2-chamber apical positions for determination of left ventricular 
volumes. Mitral inflow velocities, including color M-mode interrogation, will be assessed in the 4-
chamber view. Tissue Doppler imaging will be performed at the mitral septal position, 
approximately 1-cm apical to the mitral valve plane. Additional images will be obtained in the 
parasternal short axis at the level of the papillary muscles, 2-chamber, and 4 chamber apical 
views for assessment of myocardial strain. Pulse-wave Doppler interrogation of the left ventricular 
outflow tract (LVOT) will be performed in the apical 5-chamber view. 
Concurrent arterial tonometry will be performed using a high-fidelity tonometer at the carotid, 
femoral, and radial arteries, using a Sphygmocor device. Waveforms will be calibrated using the 
brachial artery blood pressures, obtained using a validated oscillometric device. Waveforms will 
be digitally-stored for off-line analysis. Where available, we will also measure blood pressure 
using a BP+ device (Uscom, Sydney, Australia) , which is an FDA-approved device that measures 
brachial and central blood pressure using a standard brachial blood pressure cuff. Body surface 
measurements will be made to determine distance between the suprasternal notch to the carotid, 
radial, and femoral arteries. A 6-Minute Walk Test will also be performed during this visit. Patients 
who demonstrate significant desaturation during exercise ≤94% or a fall in Spo2 ≥5%) will be 
discontinued from the study at this timepoint.  
Initial visit procedures 
• 
Informed consent* 
• 
Eligibility assessment 
• 
Medical history, review of concomitant medications 
• 
Dietary counseling 
• 
KCCQ 
• 
Physical Exam (including orthostatic and anthropometric measurements) 
• 
ECG, 
laboratory 
tests 
(CBC, 
comprehensive 
metabolic 
panel, 
NTproBNP, 
methemoglobin; pregnancy test and G6PD deficiency screening as needed 
• 
Blood, urine, saliva collection 
• 
Echocardiogram, arterial tonometry 


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• 
6 minute walk test with oxygen saturation 
* Informed consent will be obtained before any study procedures  
5.2. Randomization procedure 
A blocked randomization will be performed. Each block will contain an equal number of allocations 
to KNO3 followed by KCl versus KCl followed by KNO3. The order of treatment sequences within 
each block will be randomized.  
5.3. Intervention Phase 1 
Subjects will be randomized to receive either active drug (KNO3) or control (KCl) as the “first” drug 
for phase A. The initial dose will be one 6 mmol capsule administered twice daily, to be taken with 
meals. We will provide subjects with a 14-day supply of study medication to account for potential 
delays in receiving the 2nd batch of medication (i.e., after the 1-week call).  
1-week call: Subjects will be called by telephone ~1 week later to assess if they are experiencing 
any side-effects. Subjects will receive reinforcement of the dietary restrictions imposed during the 
study. Subjects will be interrogated about headaches and dizziness. If these symptoms are not 
present, or are not worse compared to the subject’s baseline, the drug dose will be up titrated to 
18 mmol daily (One 6-mmol capsule taken 3 times per day). The presence of orthostatic 
symptoms (i.e. sustained lightheadedness upon standing) will prompt a visit to assess for 
orthostatic vital signs. The presence of symptomatic orthostatic hypotension (20 mmHg reduction 
in systolic blood pressure with associated symptoms such as dizziness) measured at this 
subsequent assessment visit will prompt exclusion of patients from the trial. 
 
For subjects who have a serum K at the initial visit between 4.7 and 5.0 mEq/L in the presence of 
either: (a) potassium-sparing diuretic use, or (b) Estimated glomerular filtration rate of 30-39 
mL/min/1.73m2, we will check a serum potassium 1 week after the implementation of the 18 
mmol/d dose as a safety measure. A serum potassium > 5.5 mEq/L will prompt exclusion at this 
point. 
 
At 3 weeks into each interventional phase, the Actigraph devices will be mailed to the subjects. 
The goal will be for the subjects to wear this device on their wrist during the final week of each 
interventional phase to look for differences with each therapy. The devices will be brought back 
by the subject at the endpoint visit. 
 
Phase 1 endpoint assessment: Following 4 ± 1 weeks of therapy with KNO3/KCl, subjects will 
return for endpoint assessments. Physical examination with measurement of orthostatic blood 
pressure, KCCQ, dietary counseling, and side effect assessments will be performed. A urine 
pregnancy test will be performed in women with child-bearing potential. An intravenous catheter 
will be placed in the antecubital vein and securely fixed in place. Blood, urine and saliva samples 
will be collected prior to, and approximately 2 hours after the morning dose administration, given 
with a standardized low-nitrate breakfast. Blood RNA samples will be collected prior to the 


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14 
morning dose administration using Paxgene tubes (BD Biosciences, Franklin Lakes, NJ). 
Subjects will then undergo repeat echocardiography, arterial tonometry and blood pressure 
measurements (including BP+ central blood pressure measurements). Subjects will then perform 
a maximal-effort peak oxygen consumption (VO2) test using a supine bicycle exercise test with 
expired gas analysis. Cardiac output at rest will be measured immediately prior to exercise using 
pulsed wave Doppler echocardiography. Flow-mediated dilation of the brachial artery to assess 
endothelial function will be performed (Unex Co. Ltd., Nagoya, Japan). An additional blood sample 
will be obtained at peak exercise. The Kansas City Cardiomyopathy Questionnaire (KCCQ) will 
be administered.  Download of actigraph device data will also be done.  
Phase 1 visit 
• 
Interim medical history, review of concomitant medications 
• 
Physical Exam (including orthostatic vital signs) 
• 
Blood, urine, saliva collection 
• 
Blinded drug administration 
• 
ECG, laboratory tests (CBC, comprehensive metabolic panel, methemoglobin; 
pregnancy test as needed, BD PAXgene RNA samples 
• 
KCCQ 
• 
Blood, urine and saliva collection approximately 2 hours post-administration 
• 
Echocardiogram, arterial tonometry 
• 
Cardiopulmonary test with resting cardiac output measurements, blood draw at peak 
exercise 
• 
Download of Actigraph data and continuation of outpatient physical activity 
• 
Endothelial function assessment with flow-mediated brachial artery dilation 
5.4. Washout period 
Following the endpoint assessment, subjects will enter a 1-week ± 3 day washout period during 
which they will not receive any study medications.  
At 3 weeks into each interventional phase, the Actigraph device will be mailed to the subjects. 
The goal will be for the subjects to wear this devices on their wrist during the final week of each 
interventional phase to look for differences with each therapy. The device will be returned by the 
subject at the endpoint assessment. 
5.5. Intervention Phase 2 
Following the washout period, subjects will receive either active drug (KNO3) or control (KCl) 
during phase 2 of the trial. Subjects will receive the intervention (KNO3 or KCl) that was not 
administered to them in phase 1, such that each subject will receive both study interventions in 
this cross-over design. The ~1-week call and the ~4-week study visit will be repeated as above. 
We will provide subjects with a 14-day supply of study medication to account for potential delays 
in receipt of the 2nd batch of medication (i.e., after the 1-week call).  
  


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15 
1-week call: Subjects will be called by telephone ~1 week later to assess if they are 
experiencingside-effects. Subjects will receive reinforcement of the dietary restrictions imposed 
during the study. Subjects will be asked about headaches and dizziness. If these symptoms are 
not present, or are not different than the subject’s baseline, the drug dose will be up-titrated to 18 
mmol daily (One 6-mmol capsule taken 3 times per day). The presence of orthostatic symptoms 
will prompt a visit to assess for orthostatic vital signs. The presence of symptomatic orthostatic 
hypotension (20 mmHg reduction in systolic blood pressure with associated symptoms such as 
dizziness) at the follow up visit will prompt exclusion of patients from the trial. 
 
Phase 2 endpoint assessment: Following 4 ± 1-weeks of therapy with KNO3  or KCl, subjects 
will return for a repeat endpoint assessment. Physical examination with measurement of 
orthostatic blood pressure, KCCQ, dietary counseling, and side effect assessments will be 
performed. A urine pregnancy test will be performed in women with childbearing potential. An 
intravenous catheter will be placed in the antecubital vein and securely fixed in place.  Blood, 
urine and saliva samples will be collected prior to, and approximately 2 hours after, morning dose 
administration, given with a standardized low-nitrate breakfast. Blood RNA samples will be 
collected prior to the morning dose administration using Paxgene tubes (BD Biosciences, Franklin 
Lakes, NJ). Subjects will then undergo repeat echocardiography, arterial tonometry and blood 
pressure measurements (including BP+ central blood pressure measurements). Subjects will then 
perform a maximal-effort peak oxygen consumption (VO2) test using a supine bicycle exercise 
test with expired gas analysis. Cardiac output at rest will be measured immediately prior to 
exercise using pulsed wave Doppler echocardiography. An additional blood sample will be 
obtained at peak exercise. Flow-mediated dilation of the brachial artery to assess endothelial 
function will be performed (Unex Co. Ltd., Nagoya, Japan). The Kansas City Cardiomyopathy 
Questionnaire (KCCQ) will be administered. Download of actigraph device data will also be done. 
Following the final endpoint assessment, subjects’ participation in the study will be over.   
 
Phase 2 visit (final visit) 
• 
Interim medical history, review of concomitant medications 
• 
Physical Exam (including orthostatic vital signs) 
• 
Blood, urine, saliva collection 
• 
Blinded drug administration 
• 
ECG, laboratory tests (CBC, comprehensive metabolic panel, methemoglobin; 
pregnancy test as needed, BD PAXgene RNA samples 
• 
KCCQ 
• 
Blood, urine and saliva collection approximately 2 hours post-administration 
• 
Echocardiogram, arterial tonometry 
• 
Cardiopulmonary test with resting cardiac output measurements, blood draw at peak 
exercise 
• 
Download of Actigraph data and continuation of outpatient physical activity 
• 
Endothelial function assessment with flow-mediated brachial artery dilation 


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16 
5.6. Adverse Event Reporting 
All adverse events and adverse device effects will be reported following FDA guidelines. The 
research team will keep a log of all adverse events that occur in the trial, and any reportable 
events will be reported as described in Section 7 of this protocol. The study team in charge of the 
conduct of the trial is up to date on all trainings pertaining to safety guidelines and adverse event 
reporting. Adverse Events will be reported to the site IRB, the Sponsor and the NIH Program 
Office in a timely fashion, as specified in section 7.  
5.7. Subject withdrawal / Early termination 
 
Subjects may voluntarily withdraw from the study at any time and for any reason, or this may be 
at the investigator’s discretion. The investigator may withdraw a patient from the study due to 
protocol non-compliance, incorrect enrollment or randomization, or for any other reasons related 
to subject safety. The reason for study discontinuation will be recorded on the source documents 
and all such subjects will be asked to complete an early termination visit. 
During this visit, we will document: (1) vital signs; (2) compliance with the medications (3) Adverse 
effects. (4) Specific reason for withdrawal. 
 
Subject Early Termination Visit Procedures 
• Vital Signs and Physical Exam 
• Medication adherence log 
• Safety Labs 
• Adverse event assessment 
• Documentation of reason for withdrawal 
5.8. Concomitant Medication 
Subjects should be treated with standard of care medications for AS or associated comorbidities. 
As per inclusion criteria, subjects should be on a stable medical regimen prior to entry. Further 
adjustment of blood pressure medications during the study period is discouraged and should only 
be performed according to new and clinically compelling worsening of clinical status. Therapy with 
organic nitrates or phosphodiesterase-5 inhibitors is contraindicated during the study period.  
 
6. DRUG DISPENSING, ACCOUNTABILITY AND DESTRUCTION 
KNO3 and KCl capsules will be prepared at the University of Pennsylvania Investigational Drug 
Service [3600 Spruce Street, Ground floor Maloney Building, Philadelphia, PA 19104]. Drug 
bottling, labeling and dispensing will also be managed by the Penn Investigational Drug 
Pharmacy. Prior to initiation of each phase, participants will receive a sufficient supply of KNO3 
or control capsules to last until the end of each treatment phase, allowing for 100% compliance 
with the regimen for the entire treatment phase, including a window of ±7 days for the 1-week up-


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17 
titration  (to ensure an adequate supply of medication before additional medications are received 
by the subject) and ±1 week for the endpoint assessment visit.  
Subjects will be instructed to take the medication as required by the protocol, and compliance will 
be assessed via pill count performed by IDS.   
Subjects will be instructed to return unused capsules at the end of each treatment phase. A pill 
count will be performed by IDS prior to destruction of the drug. Returned trial capsules will be 
stored separately from the non-allocated trial capsules until returned trial capsules are disposed 
of. The IDS personnel will keep track of all received, used, partly used and unused trial products. 
Used and unused study drug will be destroyed at the instruction of the sponsor. A copy of the 
drug destruction SOP should be maintained in the pharmacy section of the Regulatory Binder.  
Study drug should not be destroyed until drug accountability has been completed.   
6.1. Rules for un-blinding 
Randomization data will be kept strictly confidential, accessible only to authorized IDS personnel, 
until the time of un-blinding.  The investigators will be given access to the treatment code for their 
patients for emergency un-blinding by calling the IDS. This is considered to be a very unlikely 
occurrence. Any suspected study drug-related events will be treated as though the patient 
received active (KNO3). Nevertheless, in the rare event of necessary un-blinding, the site PI and 
the sponsor must be notified.   
 
7. ADVERSE EVENTS 
7.1. Key definitions 
Adverse Event: An adverse event (AE) is any untoward medical occurrence associated with the 
use of a drug in a subject whether or not considered drug or biologic related. An AE can therefore 
be any unfavorable and unintended sign, symptom or disease temporally associated with the use 
of the pharmaceutical product. 
Suspected Adverse Reaction: A suspected adverse reaction (SAR) is any adverse event for which 
there is a reasonable possibility that the drug caused the event. “Reasonable possibility” suggests 
there is a causal relationship between the drug and the adverse event. “Suspected adverse 
reaction” implies a lesser degree of certainty about causality than adverse reaction, which means 
any adverse event caused by a drug 
Serious Adverse Events (SAE): An adverse event or suspected adverse reaction is considered 
serious if the investigator or sponsor believes any of the following outcomes may occur: 
• 
Death 
• 
Life-threatening AE:  Places the subject at immediate risk of death at the time of the event 
as it occurred. It does not include an AE that, had it occurred in a more severe form, might 
have caused death. 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions. 


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• 
Inpatient hospitalization or prolongation of hospitalization.  
• 
Congenital anomaly or birth defect.  
• 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon appropriate 
medical judgment, they may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition above. 
This determination is based on the opinion of either the investigator or sponsor (e.g., if either 
believes it is serious, it must be considered serious). 
Unanticipated Adverse Device Effect (ADE): Any serious adverse effect on health or safety, 
any life-threatening problem or death caused by, or associated with a device, if that effect, 
problem, or death was not previously identified in nature, severity, or degree of incidence in the 
application; or any other unanticipated serious problem associated with a device that relates to 
the rights, safety, or welfare of subjects.  
7.2. Classification of AE/ADEs regarding causal relationship to study interventions and 
severity 
A medically-qualified investigator must assess all AEs in terms of causal relationship to 
intervention, severity, and “expectedness” using the following guidelines: 
Classification of Adverse Events for Causal Relationship to Study Interventions 
Not related 
There is not a reasonable causal relationship to the investigational 
product and the adverse event 
Unlikely related 
No temporal association or the cause of the event has been identified, or 
the drug or device cannot be implicated. 
Possibly related:  
There is reasonable evidence to suggest a causal relationship between 
the drug and adverse event. 
Related 
 
There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. 
 
Classification of Adverse Events Regarding Severity Scale 
1 
Mild AE. Awareness of sign, symptom, or event, but easily tolerated; no treatment 
required 
2  
Moderate AE. Discomfort enough to cause interference with usual activity and may 
warrant intervention. In the latter scenario, AE responds to treatment 
3 
Severe AE. Incapacitating, limiting usual/normal activities or significantly affects 
clinical status requiring hospitalization or prolongation of hospitalization.  
4 
Life-threatening or disabling 
5 
Fatal AE 


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19 
 
Expectedness:  The expectedness of an AE/ADE or SAR shall be determined according to the 
specified reference document containing safety information (e.g., most current investigator’s 
brochure or product label). Any AE/ADE that is not identified in nature, severity, or specificity in 
the current study reference document(s) (e.g. protocol or investigator’s brochure) is considered 
unexpected. Events that are mentioned in the investigator's brochure as occurring with a class of 
drugs or as anticipated from the pharmacological properties of the drug, but not specifically 
mentioned as occurring with the particular drug under investigation are considered unexpected. 
The following AEs are expected, disease-related events in patients post-transcatheter or surgical 
AVR (84).  
• 
Unplanned hospitalization, ER visit or clinic visit 
• 
Arrhythmias 
• 
Permanent pacemaker implantation 
• 
Sudden cardiac death 
• 
Acute coronary syndrome 
• 
Cerebrovascular event 
• 
Paravalvular aortic regurgitation 
• 
Wound dehiscence 
• 
Infective endocarditis 
• 
Aortic dissection 
• 
Sternal infection 
• 
Thromboembolism 
 
The following are potential expected side effects of KNO3: 
• 
Stomach discomfort  
• 
Slight headache 
• 
Dizziness 
• 
Lightheadedness 
• 
Low blood pressure 
• 
Stomach Ache, diarrhea, nausea, or vomiting 
• 
Shortness of breath 
• 
Flushing 
• 
Rash 
• 
Mild orthostatic hypotension 
• 
Clinically significant methemoglobinemia or significant symptomatic orthostatic 
hypotension (20 mmHg or greater reduction in systolic blood pressure with 
associated symptom such as dizziness) are potential side effects, although we will 
consider them unexpected given prior data in this patient population with this 
particular drug and dosing scheme.  
7.3. Recording and Reporting of Adverse Events 
The site PI will continuously supervise all aspects of the trial and review the records of the study 


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20 
subjects following each visit and at the end of their participation. The site PI will be responsible 
for ensuring that all adverse events are noted, followed and reported to the IRB, Sponsor (at 
psom-ind-ide@psom.upenn.edu) and IND Medical Director as appropriate.  
SAEs occurring from the time of signed informed consent to the final study visit will be captured 
on the SAE eCRF. AEs will be classified according to the guidelines/definitions specified in section 
7 of the protocol. Any AE rated ≥ 3 in severity (i.e., SAE) must be reported by the site investigator 
or qualified designee within 1 working day of first becoming aware of the event, to the sponsor via 
email and  regulatory medical director. The IRB should be notified as per institutional guidelines. 
The medical director and site IRB, as appropriate, will make an immediate determination about 
the necessity to modify the protocol, include additional information in the consent form, inform 
previous participants, temporarily hold enrollment of patients, or terminate the study. The study 
will proceed only if the medical director and site IRB all agree on this course of action.  
The investigator or qualified designee will enter the required information regarding the SAE into 
the appropriate module of the eCRF, All study procedures and cumulative adverse events are 
subject to full IRB review at least yearly. 
Events significant enough to necessitate modification of study drug dosing will be captured on an 
appropriate eCRF module (“Study Drug Dosing” page). Should the PI determine that modification 
to the study drug is appropriate, the PI must notify the sponsor.  
7.4. Follow-up 
The Investigator will record safety follow-up information according to the same process used for 
reporting the initial event as described above. The Investigator will follow all reportable events 
until resolution, stabilization or the event is otherwise explained.  
7.5. Management of Suspected Unexpected Serious Adverse Reaction 
AEs that meet the criteria of serious, related to study intervention, and unexpected for the study 
intervention, qualify for expedited reporting by the sponsor to the regulatory authorities. The Site 
Investigator will assess all SAEs and evaluate for “unexpectedness” and relationship to study 
drug. The Site Investigator is required to complete a report for any events identified as serious, 
study drug related and unexpected, using the CIOMS format.  The sponsor will submit a voluntary 
CIOMS form. See Section 7.4 for reporting procedures. 
A copy of this report should be kept at the site and also forwarded to the sponsor regulatory lead 
within the same timeline used for reporting to regulatory authorities.  
7.6. Pregnancy 
Pregnancy is a contraindication to enrollment in the study. Pregnancy occurring during the study 
period, although not considered an SAE, must be reported to the NIH, local IRB and Sponsor 
within the same timelines as an SAE. The pregnancy will be recorded on the appropriate case 
report form. The drug will be discontinued immediately and subject discontinued from the trial, but 
the pregnancy will be followed until final outcome. Any associated AEs or SAEs that occur to the 


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21 
mother or fetus/child will be recorded in the AE case report form or CIOMS form for SAEs.  
 
8. STUDY MEASUREMENTS AND DATA COLLECTION 
8.1. Assessment of exercise capacity 
We will use a supine bicycle exercise protocol in conjunction with expired gas analysis to assess 
oxygen consumption (VO2) during exercise. Subjects will perform a maximal exertion-limited 
exercise test using a graded-exercise protocol. We will use a supine cycle ergometer designed 
for stress echocardiography (Stress Echo Ergometer 1505, Medical Positioning, Inc, Kansas City, 
MO). Subjects will undergo expired gas analysis with a Parvo Medics True One 2400 device 
(Parvo Medics, Sandy, UT), an Innocor device (Innovision Inc) or equivalent. Resistance began 
at 15 W for 3 minutes, increasing to 25 W for 3 minutes, and then increasing by 25 W every 3 
minutes thereafter. Breath-by-breath information will be recorded. We will use custom-designed 
software already developed in Matlab (MathWorks, Natick, MA) at our lab for offline processing 
and quantification of all exercise data.(85) All data quantification will be blinded to treatment. Total 
work performed will be computed and exercise efficiency will be defined as (total work/total 
oxygen consumed). Exercise efficiency is not a formal endpoint of the trial, but will be used to 
interpret changes in the co-primary outcomes. After these exercises, we will use a device that 
uses Near Infrared Spectroscopy [NIRS] to measure the oxygen levels in the left calf and forearm 
muscles (Portamon and Portalite devices, Artemis, Netherlands). 
8.2. Quality of life 
Quality of life will be assessed with the Kansas City Cardiomyopathy Questionnaire (KCCQ).(86) 
We will administer the KCCQ to subjects prior to randomization and at the end of each intervention 
phase (6-week time point in both phases). This is a validated 23-item questionnaire that assess 
physical function, symptoms, social function, self-efficacy and knowledge, and quality of life. It 
has been used extensively in heart failure studies (http://cvoutcomes.org/pages/3214) 
8.3. Doppler Echocardiography 
This procedure is similar to clinical cardiac echocardiograms. An ultrasound probe will be placed 
on the surface of the skin to obtain images of the heart. This takes approximately 20 minutes. 
This will occur at all three visits. 
Measurement of LV filling and myocardial strain: given that inorganic nitrate may also induce 
venodilation and myocardial effects, we will also assess the effects of potassium nitrate on (1) 
Early mitral annular early diastolic tissue velocity; (2) The ratio of early diastolic mitral inflow 
velocity to mitral annular tissue velocity (a surrogate of LV filling pressures); (3) Peak early 
diastolic intraventricular pressure gradient, a marker of ventricular relaxation, assessed with color 
M-mode interrogations of mitral inflow.(87,88) We note that the latter is not simply based on the 
inflow propagation velocity, but rather on solving Euler momentum equation.(87) This method is 
able to accurately assess LV relaxation(87). Systolic function will be assessed via systolic 


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22 
myocardial strain (using speckle tracking echocardiography), which we have successfully applied 
in previous studies.(66,69,89) 
8.4. Measurement of late systolic load and arterial wave reflections:  
We will use a high-fidelity Millar applanation tonometer(54) to record carotid pressure waveforms, 
which will be calibrated using brachial artery pressures. Central arterial tonometric recordings and 
Doppler flow velocity files will be processed off-line using custom-designed software written in 
Matlab (The Mathworks, Natick, MA) as previously described.(66,90) We have successfully 
implemented this method in multiple previous studies(8,57,66,67,91) and have published a tutorial 
detailing our analysis methods.(8,53) After signal-averaging of pressure and flow, time alignment of 
carotid pressure and LV outflow curves will be performed to maximize concordance of the rapid 
systolic upstroke of pressure and flow, concordance of the dicrotic notch and cessation of flow, 
zero value of the phase angle of higher-frequency harmonics (7th to 10th) of input impedance, and 
linearity of the early systolic pressure-flow relationship.(90) After computation of aortic input 
impedance, proximal aortic characteristic impedance (Zc) will be computed in the frequency 
domain as previously described.(90) Pressure and flow harmonics were separated into forward and 
backward components using standard wave separation analysis.(8,53,90,92) The sum of forward and 
backward pressure harmonics yields the forward and backward waves, respectively. We will 
assess the reflection coefficient in the first 3 harmonics. A reflection coefficient (Γ ) is derived from 
the ratio of two sine waves, it is a complex number with an amplitude and phase-angle, which can 
correspond to different degrees of destructive or constructive interference between forward and 
backward waves. Therefore, the net-effect of reflections will be expressed as the real part of Γ, 
which becomes increasingly positive as pressure from wave reflections increases (constructive 
interference), and negative when destructive interference leads to a net decrease in pressure by 
wave reflections at a given harmonic.(93) Since all harmonics of wave reflection contribute variably 
to systolic LV load, our primary measure of late systolic load will be the net pressure related to 
wave reflections during ejection in the time domain, which better represents the impact of 
reflections on LV afterload. We will first compute the product of flow and aortic Zc (QZc product), 
which represents the pressure resulting from the interaction of blood flow with aortic root Zc.(8,54) 
The relation between QZc and measured pressure reveals the direct effect of wave reflections on 
the arterial system.(94) We will therefore quantify the additional ejection-phase pressure load from 
wave reflections arising distal to the root (i.e., reflection-related pressure time integral during 
ejection, as the difference between measured pressure and the QZc product). Carotid-femoral 
pulse wave velocity (PWV), an index of large artery stiffness,(95,96) will also be measured(54,97) using 
a Sphygmocor device (Atcor Medical).(95) We will also measure carotid-radial PWV using the 
Sphygmocor device. PWV is not a formal endpoint of this trial, since our pilot trial did not 
demonstrate an effect of nitrate-rich beetroot juice on this endpoint (unpublished data). However, 
it may aid in interpreting changes in pulsatile hemodynamics in the trial. Similarly, when available, 
arterial tonometry may be performed during and immediately after exercise for exploratory 
purposes.  
8.5. Blood nitrate/nitrite level measurements 
We will document an intervention-related change in blood nitrate and nitrite. Nitrate and nitrite 


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23 
levels will be sampled before and approximately 2 hours after the last oral dose on the morning 
of the exercise test, after the patient has been on each intervention for 4 ± 1 weeks. 
Measurements will be performed at Dr. Harry Ischiropoulos’ lab at the University of Pennsylvania. 
Venous blood samples will be drawn into lithium-heparin tubes (which have very low levels of 
nitrate/nitrite) and centrifuged at 4,000 rpm for 10 min within 3 min of collection and frozen at -
80°C for later analysis. Blood will also be obtained at peak exercise. After thawing at room 
temperature, blood samples will be deproteinized using cold ethanol precipitation as previously 
described.(98) The nitrate and nitrite content of deproteinized blood will be determined using a 
modified detection chemiluminescence technique using a Ionics/Sievers nitric oxide analyzer 
(NOA 280), as first described by Dr. Ischiropoulos’ lab(99) and later adapted by Allen et al for 
human blood.(100)  
 
8.6. Flow-mediated brachial artery dilation 
Endothelial function will be assessed using flow-mediated dilation of the brachial artery (FMD) 
according to published guidelines.(101) The protocol is described in greater detail elsewhere.(102-
104) Briefly, brachial artery diameter will be measured using a 10-Mhz ultrasound transducer (Unex 
Co. Ltd., Nagoya, Japan) at baseline after 5 minutes of supine rest. A stereotactic arm will be 
used for optimal transducer position. A pneumatic cuff will be inflated over the right forearm to 50 
mmHg above the systolic pressure and deflated after 5 minutes.  The diastolic brachial artery 
diameter will be measured continuously using the Unex device beginning at 30 seconds prior to 
cuff inflation and ending 2 minutes after cuff release. Maximal arterial vasodilation will be 
evaluated by assessment of change in diameter following cuff release.  
8.7. Other assessments 
Weight and Height: Weight will be measured to the nearest 0.1 kg with the use of calibrated 
scales, while the subjects are wearing light clothing and no shoes. Height will be measured at 
baseline to the nearest 0.1 cm using a wall-mounted stadiometer.  
Genetic Expression Analysis: Blood RNA samples will be collected using BD PAXgene blood 
RNA tubes (BD Biosciences) in order to explore changes in genetic expression in response to the 
study medication (potassium nitrate) and whether patterns of genetic expression predict response 
to the study medication. Two 2.5 mL samples (total of 5 mL of blood) will be collected at each 6-
week endpoint assessment visit.  
 
9. STUDY CENTER MANAGEMENT PLAN 
9.1. Principal Investigator 
Dr. Chirinos will serve as the Principal Investigator for this protocol. In this role, he will lead 
operations at enrollment site and will be responsible for various aspects of the study, including 
data collection, adherence to all policies and procedures, and maintenance of study protocol to 
ensure that the specific objectives are being met.  The PI will oversee the safety of study 


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24 
participants. The PI  will also interact with the data management team and the IND sponsor as 
needed. He will also establish subcommittees and working groups to complete specific activities, 
monitor study implementation, and hold regular investigator calls/meetings. Dr. Chirinos will 
supervise the investigative team to develop and coordinate procedures  and generate reports and 
presentations. Other roles of Dr. Chirinos will include directing the echocardiography and arterial 
hemodynamics core laboratory for this trial. 
9.2. Investigators Committee 
An Investigators Committee will hold a monthly call and discuss aspects of the study, including 
enrollment, retention, data management, quality assurance, and other issues as they arise.  Dr. 
Chirinos will chair the committee, which will include the following members: Julio Chirinos, 
Anupam Kumar, Howard Hermann, Frank Silvestry, Saif Anwaruddin. Minutes of these meetings 
will be distributed and kept on file. The PI will have authority to make decisions that require 
immediate attention when the committee is unavailable to meet. The PI will adjudicate 
disagreements within the committee. The sponsor has the final responsibility for all study 
decisions.  
9.3. Operational Affairs Committee 
This committee will be responsible for oversight and coordination of the day-to-day activities of 
the project.  Committee members will include the site PI, the study coordinators, and the 
echocardiography/tonometry quantification technician. This group, along with all of the members 
of the Investigators Committee, will hold an in-person meeting at the beginning of the project to 
review the study protocol and the policies and procedures for its implementation. Members of the 
Operational Affairs Committee will participate in the Investigators Committee, but may hold 
additional meetings/calls throughout the project to review subject recruitment, retention, data 
transfer, quality of data, etc. Minutes of these meetings will be distributed and kept on file.  
9.4. Data Management 
The data for this trial will be collected in ad hoc source documents by the research staff during 
each study visit, using trial-specific and visit-specific data collection forms. All source documents 
collected in this trial will be housed inside of a locked cabinet in the offices of the research staff. 
Data capture and storage will be accomplished within the framework of the Research Electronic 
Data Capture (REDCap) project. REDCap is a secure, web-based application designed 
exclusively to support data capture for research studies. It provides an intuitive interface for data 
entry with data validation, audit trails for tracking data manipulation and export procedures, 
automated export procedures for seamless data downloads to common statistical packages, 
including SAS, and procedures for importing data from external sources. As of March 2015, 
REDCap was in production use or prototype build-status for more than 99,000 studies spanning 
numerous research focus areas across a consortium of 940 active institutional partners, including 
the University of Pennsylvania.  Once the REDCap database is launched into production mode, 
the study coordinators will be responsible for transmitting the participant coded electronic data 
using standardized data collection instruments to the database manager who will check the data 


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25 
for accuracy and completeness and follow-up with the research assistants as needed. The 
research coordinators will perform manual data entry from source documents and self-
administered participant questionnaires (such as quality of life questionnaires).  
9.5. Quantification Core Laboratories 
Various physiologic core laboratory units will be in charge of analyzing physiologic data from the 
trial.  
Echocardiography Core Laboratory: The echocardiography core lab, directed by Dr. Chirinos, 
has extensive experience in analyzing echocardiographic data for multicenter trials. The lab 
functions as the core lab for 2 ongoing international multicenter trials in heart failure (CVRx 
HOPE4HF and CVRx NeoHF) and one US-based multicenter study focused on HFPEF and 
HFREF (BMS CV002004). The core lab has all the capabilities required for echocardiographic 
data management and quantification in this study. The core lab has well-established standard 
procedures for receiving and logging imaging data, quantitative analyses, quality control, audit 
trails, and communication with study sites (queries about individual issues, feedback for quality 
control). The core lab will be responsible for generating and updating manuals for 
echocardiographic data acquisition, which will standardize procedures across the sites. The core 
lab will certify individual technicians performing measurements for the study after reviewing of 
sample scans (from normal volunteers) across the 2 sites, and will review and update certification 
as needed. The core lab will have the capacity to veto individual technicians from performing 
studies for the trial, based on data quality. The echocardiographic core lab will hold monthly calls 
with a representative of the data management core to discuss issues regarding core lab data 
management. 
 
Arterial hemodynamics and exercise physiology core laboratory: The arterial hemodynamics 
core lab, directed by Dr. Chirinos, has experience in analyzing hemodynamic data for various 
large cohort studies (MESA, Asklepios)(57,67,69,71,106-109) and currently functions as the core 
lab for physiologic data for several ongoing multicenter studies (ACRIN 4008, BMS CV002004, 
iCAP study, CREST study). The core lab has specific experience in assessing specific exercise-
related phenotypes relevant to inorganic nitrate effects(82) and has adequate capabilities required 
for physiologic data management and quantification in this study. The physiology core lab will 
hold monthly calls with a representative of the data management core to discuss issues regarding 
core lab data management. 
  
Imaging data management: All imaging electronic data will be stored within a secure HIPAA-
compliant network-attached storage server, and is accessible only to those who are specifically 
given access by the core lab director. The secure server has mechanisms for redundant storage 
and data backup at different physical locations, to prevent data loss from disasters (such as a 
fire).  


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10. STATISTICAL CONSIDERATIONS 
10.1. Power calculations 
We will randomize 22 subjects to one of 2 sequences (i), each of which consists of 2 periods 
(AB/BA design). We now have pilot data regarding the distribution of the changes in peak VO2 
(the primary endpoint of the study) as well as various secondary endpoints, among patients with 
HFpEF. We considered an increase in peak VO2 of ~0.6 ml/kg/min to be the minimum clinically-
significant change. This was based on the fact that even “modest” changes of comparable 
magnitude in peak VO2 have been associated with improved outcomes.(110) The standard 
deviation of the change in peak VO2 in our pilot trial was 1.23 ml/kg/min. Therefore, the 
standardized effect size is 0.49 or greater. Assuming a 90% retention rate, enrolling 22 subjects 
in this cross-over trial will have 80% power to detect an effect size as low as 0.66 in the 
intervention-induced change of our study endpoints, with a two-sided α=0.05.  
Based on the distribution of the changes in study endpoints from repeated measurements of the 
Kansas City Cardiomyopathy Questionnaire in AS patients, we estimated that this trial study is 
powered (80%) to detect the following changes in other endpoints: 4.6 points for the Kansas City 
Cardiomyopathy Questionnaire, 8.2% for exercise vasodilatory reserve, 19% for cardiac output 
reserve and 4.2% change in reflection magnitude.  We note that these differences are well below 
what is considered a clinically significant change in KCCQ (10 points or more, 
http://cvoutcomes.org/pages/3217) or the effect size that has been observed for these endpoints 
in our pilot  trial.(85) Therefore, we are confident that we will achieve adequate power not only for 
our clinical endpoints, but also for our physiologic measures. PASS11(111) was used to perform 
power analyses.  
10.2. Data Analysis Plan  
The co-primary outcome variables will be peak VO2 and KCCQ score. All secondary outcome 
measures are continuous variables. The predictor of interest for all aims will be intervention (KNO3 
therapy vs. control), with analyses based upon the total number of subjects randomized. Initial 
descriptive estimates of all measures will be generated for study participants at each time point 
by treatment group. Statistics will include estimates of central tendency, measures of variability, 
and derived moments of skewness and kurtosis. Analyses of distributional properties will be 
performed to determine if variance stabilizing or normalizing transformations should be applied. 
Outliers will be assessed via visual inspection of distributions and checked for accuracy.  Aim 1 
will assess the effects of potassium nitrate therapy on peak O2 / work performed and quality of 
life score (co-primary outcomes).  An initial assessment of the treatment effect will be performed 
using the paired t-test and the non-parametric Wilcoxon sign-rank test on the difference between 
the paired within subject outcome measures. This will be followed by a more comprehensive linear 
mixed-effects model analysis(112) allowing for assessments of the treatment effect on each 
continuous outcome of interest while controlling for effects of other covariates such as period, 
sequence, and a random subject effect nested within sequence. For non-normal distributed 
outcomes, we will utilize non-parametric methods or consider distribution-stabilizing 
transformations. The intervention groups will initially be compared within each period (time 
invariant covariates will only be compared for period 1) according to continuous covariates 


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described above using parametric or non-parametric one-way ANOVA models, depending upon 
whether or not normality appears to be in question. Levine’s tests will be used to assess 
homogeneity of variance. Additionally, the intervention groups will be compared within period 
according to categorical covariates using Fisher’s Exact tests. Significant differences between 
groups on these variables will result in their use as control variables in the modeling of outcome.  
Separate models will be generated for each of the two outcome measures, with each outcome 
measure regressed on intervention group assignment, along with baseline outcome and any other 
covariates deemed prognostic in preliminary analyses. The linear mixed-effects models will 
incorporate adjustments for any period effect and include data from dropouts.(113-115) The model 
will include subject-specific intercepts as random effects, and assumes independent and 
identically distributed random errors within subject. Restricted maximum likelihood estimation will 
be used, and an appropriate covariance matrix will be specified.  Model assumptions will be 
examined (e.g., QQ plots to assess  normally distributed residuals for valid Wald tests). We will 
make every possible effort to minimize missing data and ensure final assessments for participants 
opting to discontinue study participation.  Missing data, however, is an inevitable problem in a 
longitudinal study. The mechanism for missingness-missing completely at random (MCAR), 
missing at random (MAR), nonignorable or not missing at random (NMAR)-will be evaluated prior 
to implementing methodology intended to minimize bias from missing data.(116) We anticipate that 
~10% of randomized subjects will not complete the study.  
 The intent-to-treat principle of including all randomized participants in the outcome models will 
be followed. SAS software procedures will be used for creating and analyzing multiple imputed 
data sets for incomplete multivariate data. Instead of filling in a single value for each missing 
value, Rubin's(117) multiple imputation procedure replaces each missing value with a set of 
plausible values that represent the uncertainty about the correct value to impute. These multiple 
imputed data sets are then analyzed using standard procedures for complete data and combining 
the results from this analysis. Assuming monotone missing data patterns emerge, either a 
parametric regression method that assumes multivariate normality or a nonparametric method 
that uses propensity scores will be implemented, depending upon distributional patterns.   
Analytic methods described above will take advantage of all available data. The possibility of 
systematic bias in the outcomes for those who withdraw exists. Baseline characteristics will be 
compared among subjects with and without complete follow-up, recognizing that statistical power 
associated with finding true statistical differences may be limited. To assess potential biases, a 
comparison of withdrawal rates and or/time to withdrawal will be included. If the number of 
subjects lost to follow-up is small and the missing observations can be documented as being MAR 
or MCAR, then the primary hypotheses will be tested using the complete observed data. If missing 
observations cannot be assumed to be MAR or MCAR, more complex approaches will be 
considered.  Sensitivity analyses for these models will be performed should they be 
implemented.(118)  
Our secondary aims involve exploratory analyses investigating mechanisms of action. In addition 
to assessing the effect of our randomized intervention on each mechanistic endpoint, exploratory 
structural equation modeling will be used to evaluate associations between outcomes and biologic 
mechanistic pathways. The modeling will be carried out in three sequential steps: (1) exploratory 
factor analysis, (2) confirmatory factor analysis, and (3) structural equation modeling. The 
exploratory factor analysis will be based on principal axis factoring to decrease the number of 


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variables.  For both theoretical and empirical reasons, it will be assumed that retained factors are 
correlated and thus an oblique rotation method will be used. Measured and latent variables will 
be examined for co-linearity, and related variables will be combined into single factors with 
individual factor loadings. To determine the number of factors, the model will be evaluated against 
the following four rules: (1) eigenvalues greater than 1.0(119); (2) Glorfeld’s(120) extension of parallel 
analysis, where a large number of random correlation matrices are generated  to compare the 
number of eigenvalues that are significant by chance(121); (3) high internal consistency (an alpha 
coefficient of ≥ 0.70) for unit-weighted factors,(122) and (4) interpretability.(123) The heaviest weight 
will be placed on the Minimum Average Partial and parallel analysis methods, with the scree test 
as a visual adjunct.(124) The next step involves incorporating the factors into a model using 
confirmatory factor analysis.  The model will be tested using goodness of fit tests to assess the 
overall fit of the model to the data.  Various models will be tested and compared prior to arriving 
at the best fitting model. And finally, the best fitting model obtained from confirmatory factor 
analysis will incorporate a structural equation model designed to examine the links between the 
randomized intervention, the mechanistic variables and the clinical variables assessed in the trial.    
 
11. PROTECTION OF HUMAN SUBJECTS 
11.1. Potential benefits of the proposed research to the study subjects and importance of 
the knowledge to be gained, and risk/benefit ratio 
Potential benefits: There are no anticipated direct benefits to the subjects as a result of their 
participation in this study nor will this be implied when obtaining consent. However, if our 
hypothesis is correct, subjects in the active medication groups may experience improvements in 
their functional class and quality of life, although this will not be implied in any way during informed 
consent or enrollment.  
Importance of the knowledge to be gained: If our hypothesis is correct, this study may identify an 
effective specific intervention to treat mechanistic abnormalities in AS patients. Furthermore, if 
improving these abnormalities leads to an enhanced exercise capacity in this patient population, 
this would lead to a new paradigm in the field and a better understanding of the mechanisms that 
lead or contribute to AS, which will accelerate the discovery of new treatments for this condition. 
In addition, if potassium nitrate proves effective in enhancing exercise capacity and/or quality of 
life in this trial, this would identify a readily implementable, inexpensive therapy for this condition. 
Risk/benefit ratio: the results of this study may ultimately lead to an effective treatment for 
persistent symptoms of AS after AV. Since there is minimal risk and potential benefits to medical 
knowledge and society, the risk / benefit ratio is acceptable.  
11.2. Risks to study subjects  
The conduct of these studies will involve 22 human subjects, each exposed to both the active 
drug (potassium nitrate) and control (potassium chloride) intervention (cross-over design), and 
therefore studied as their own controls. All subjects will be adults able to give informed consent. 
Participants will be recruited from the Hospital of the University of Pennsylvania. The ethnic 
distribution of the population in our center will favor adequate representation of minorities 
(particularly, African-Americans) in the sample.  


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The study involves various tests (arterial tonometry, Doppler echocardiography, a 
cardiopulmonary exercise stress, blood draws) and the administration of randomized therapy 
(KNO3 vs. control). 
 
11.2.1. Potential Risks of study intervention (potassium nitrate) 
The main potential risks of nitrate administration are related to its potential effect on (1) blood 
pressure; and (2) methemoglobin levels.  
Effects on blood pressure: Regarding the blood pressure reduction, as described above, many 
studies have demonstrated a reduction in blood pressure in both hypertensive and normal 
subjects following nitrate supplementation. This was summarized in a recent meta-analysis, 
demonstrating an approximate 4 mm Hg reduction in systolic blood pressure and 1 mm Hg 
reduction in diastolic blood pressure.(43) Importantly, in a study by our group of a single dose of 
inorganic nitrate in subjects with HFpEF, we did not observe any change in blood pressure 
following nitrate ingestion. Importantly, the lack of a blood pressure response to nitrate has been 
shown in both elderly(125) and diabetic individuals,(126) suggesting that there may be important 
differences in nitrate effect by subgroups. In our pharmacokinetics study, we found a mild (~12 
mm Hg) asymptomatic reduction in blood pressure recorded at the time of our study visits during 
2-week drug administration, which occurred in the absence of any hypotensive symptoms.(127) We 
did not find a reduction in ambulatory blood pressure. Therefore, although we do not anticipate 
major changes in BP or significant hypotension with this dose, we will implement exclusion criteria 
and safety blood pressure checks throughout the conduct of the study to ensure subject safety 
(see next section). 
Impact of NO3- on methemoglobin levels: Nitrate supplementation increases serum nitrite 
levels through the actions of an enterosalivary circuit and reduction of nitrate to nitrite by anaerobic 
bacteria in the oral cavity.(20,25-28) Methemoglobin is generated by the reaction of nitrite with 
hemoglobin. According to Toxnet, doses between 2-9 grams of NO3- have been associated with 
methemoglobinemia. The dose we will use in our study (18 mmol KNO3-) will provide only 1.11 
grams (18 mmol NO3- x 62 grams/mol NO3- x 1mol/1000 mmol), which is well below this threshold. 
Moreover, this level of nitrate supplementation provides less inorganic nitrate than that found in a 
traditional Japanese diet, which is rich in vegetables (traditional Japanese diet provides 18.8 
mg/kg of nitrate per day x 70 kg individual = 1.3 grams of nitrate).(128) Moreover, not a single cases 
of methemoglobinemia was observed in our pharmacokinetics trial , at the same doses used in 
this trial and in the same patient population. In the pharmacokinetics study we measured 
methemoglobin every half hour after the first administration as well as during steady-state 
continued oral administration at doses of both 12 mmol/d and 18 mmol/d and did not find any 
clinically-significant elevations in methemoglobin. We don’t anticipate any significant issues with 
methemoglobinemia in this study since this is a problem that has been reported with nitrite, but 
not with nitrate at the doses used in this study.  
Safety data:  
In a double-blind randomized controlled trial in HFPEF, in which 12.9 mmol of inorganic were 
administered to patients with HFpEF, efficacy was demonstrated for the study endpoint (please 
refer to research plan). In this pilot study, no side effects were noted in any subject, except for 
pink urine (which was not different between the nitrate-rich and nitrate-poor beetroot juice, 


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because this is a consequence of other components of beetroot juice).  As the body is able to 
reduce oxidized Fe3+ in methemoglobin back to Fe2+ at a rate of approximately 15% per hour,(129) 
the gradual increase in nitrite that occurs after nitrate administration (and the consequent 
formation of methemoglobin), should occur slowly enough to prevent significant rises in 
methemoglobin. To the best of our knowledge, methemoglobinemia has not been reported with 
oral nitrate administration. In our pharmacokinetic study, we did not observe any clinically-relevant 
elevations in methemoglobin levels(127).  
Other potential safety considerations:  
There has been concern regarding whether a high nitrate diet may predispose to gastric cancer 
in humans through conversion to nitrite in the stomach or through increased urinary excretion of 
nitrate metabolites. This is a controversial issue derived from animal studies(130), although the 
relationship between nitrate intake and cancer has not been demonstrated in humans.(23,131,132) In 
fact, many studies show either no relationship or even an inverse relationship between a high 
intake of nitrate and the occurrence of gastric cancer.(133,134) (135,136) (137,138) The Joint FAO/WHO 
Expert Committee on Food reviewed all the available evidence, but failed to establish a definite 
link between nitrate intake and risk of developing cancer.(139,140) Furthermore, The World Cancer 
Research Fund/American Institute of Cancer Research found no evidence linking ingestion of 
vegetables which are known to be high in nitrate with the development of cancer(141). We note that 
the highly controversial concern regarding cancer risk associated with nitrate intake is related to 
long-term intake and not a significant issue with our 6-week administration scheme of potassium 
nitrate. 
11.2.2. Potential Risks of study procedures 
Potential risks are associated with the study tests, the study interventions (potassium nitrate) and 
potential breaches in confidentiality.  
 
Cardiopulmonary stress test 
This test is used extensively for research purposes with minimal risk to subjects. The most 
significant risks of the test are dysrhythmias or other cardiovascular complications, which are 
extremely rare. These procedures will be performed by qualified personnel according to 
established American Heart Association Guidelines.(142,143) Non-revascularized myocardial 
ischemia, which may increase the risk of complications during exercise testing, is an exclusion 
criterion for the study. We note that prior studies in HFpEF also enrolled subjects with atrial 
fibrillation(144) and that rate-controlled atrial fibrillation is not a contraindication to exercise testing.  
Subjects may feel uncomfortable as a result of pushing themselves during the maximal effort 
exercise test. Subjects will likely feel short of breath and fatigued as a result of the exercise test. 
Various other complaints, such as nausea, lightheadedness, and other aches and pains are also 
possible as a result of the maximal effort exercise study. Although exercise testing may result in 
exhaustion, rarely do people develop abnormal HR or heart complications during exercise tests. 
The risk of this happening is the same as if the participant would exert themselves during stressful 
situations or during exercise elsewhere. 
We will perform EKG, HR, and blood pressures monitoring during our exercise test. In addition to 
the blood pressure (generally increases) and heart rate (generally increases) changes during 


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exercise, we will also monitor arterial saturation. This will be done non-invasively using a pulse 
oximeter. Of note, oxygen levels can decrease with exercise, even in individuals without 
significant cardiopulmonary disease.(145,146) If the arterial saturation falls to below 88% (“severe 
exercise induced hypoxemia”(146)), we will alert the care provider as this may prompt consideration 
for additional/alternative causes for arterial hypoxemia.  
Venipuncture and IV placement 
According to the 2010 WHO guidelines on phlebotomy, major risks associated with blood 
donations include hematoma at the site of venipuncture in 2-3%, and vasovagal reactions and 
fainting in 1%. The placement of an intravenous catheter would be anticipated to increase the risk 
of hematoma and discomfort slightly. Given that the catheter will be in place for a short-period of 
time, infection is an unlikely complication. Of note, the amount of blood to be drawn in the study 
is less than the amount of blood drawn at routine blood donation where the risk of syncope of 1% 
was defined.  
Arterial tonometry and assessments of oscillometric arterial pressure waveforms are non-invasive 
procedures and do not have any known risks.  
During various procedures (echocardiography, arterial tonometry), we will use adhesive 
electrodes attached to the participant’s skin to record the electrical signal from the heart. These 
may occasionally cause skin itching and irritation. 
Pregnancy Risks 
We will not be enrolling subjects who are pregnant in this study. Right now, there is not enough 
research on the potassium nitrate to determine the safety of the mother or unborn child. A 
pregnancy test will be given to women of child-bearing potential prior to enrollment in the study 
and administration of the supplement. The pregnancy test will be repeated at the 6-week visit 
(prior to initiation of the second treatment period). If a woman is enrolled of child-bearing potential, 
we will ask that they use a medically accepted method of birth control (such as an IUD, birth 
control combination pill, patch, ring, progestin-only pills, Depo Provera Shot, Implanon, complete 
abstinence, or condoms) while they participate in the study. In addition, we will test for pregnancy 
at each study visit and discontinue subjects from the study immediately if she becomes pregnant. 
As subjects with AS are generally older (>55 years old) we do not anticipate this concern to occur 
with our study population. We will not enroll pregnant women or women who are lactating. 
As with any clinical research study, there is a potential for breach of confidentiality. Adequate 
measures will be taken to minimize this risk (below). 
11.3. Adequacy of Protection Against Risks  
11.3.1. Recruitment and Informed Consent 
Written informed consent will be obtained from the subjects by the investigators prior to entry into 
the research study. This will be performed in accordance with the guidelines and under the 
supervision of the University of Pennsylvania Institutional Review Boards. The study procedures 
and interventions and the associated risks will be explained to the subjects during the informed 
consent process. Only IRB-approved consent forms and related materials will be used. 


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11.3.2. Protection against risks associated with cardiopulmonary exercise tests 
These tests will be performed by qualified personnel according to established American Heart 
Association Guidelines(142), under ECG monitoring. Personnel with adequate cardiopulmonary 
resuscitation training and resuscitation equipment (crash cart) will be available during these tests. 
Similarly, these tests will be performed in a hospital setting where a full code team can be 
deployed immediately should complications occur. 
11.3.3. Protection against risks of potassium nitrate administration: 
Subjects will be thoroughly advised regarding the potential risks of the study medication and 
precautions needed during its administration. We will exclude individuals who are taking sildenafil 
(Viagra®), tadalafil (Cialis®), or vardenafil (Levitra®) and we will instruct them not to take these 
medications while participating in the study, since it is likely that these medications may 
exaggerate the vasoactive effects of potassium nitrate. Similarly, the use of organic nitrate will be 
contraindicated during participation in this trial. 
Supine and orthostatic blood pressure measurements: Supine vital signs will be measured at the 
screening visit. The subject will rest in a supine position for a minimum of 3 minutes prior to 
obtaining vital sign measurements. Subjects with a supine systolic blood pressure >110 will be 
enrolled in the study. The subject will then assume a standing position for 3-5 minutes. Vital signs 
(BP and pulse rate) will then be measured while the subject is standing. A reduction in systolic 
blood pressure >20 mmHg will be considered an exclusion criterion for the study. Blood pressure 
measurements will be repeated after 4  ± 1 weeks of randomized therapy. Measurements will also 
be prompted by reports of any report of orthostatic symptom by study subjects at any point during 
the trial. The presence of symptomatic orthostatic hypotension (20 mmHg reduction in systolic 
blood pressure with associated symptom such as dizziness) will prompt exclusion of patients from 
the trial. 
 
Safety Clinical Laboratory Tests:  Laboratory evaluations will be collected prior to study drug 
initiation.  Among women who are not surgically sterilized or post-menopausal, a urine pregnancy 
test will be performed at screening (prior to all baseline studies and drug initiation), at the end of 
the first treatment phase  (prior to the initiation of the second treatment phase). Methemoglobin 
levels will be measured before initiation of randomized therapy and 4  ± 1 weeks after initiation of 
randomized therapy in both treatment phases (1 and 2). Methemoglobinemia (methemoglobin 
levels >10%) will prompt discontinuation of patients from the trial. Other tests, including a 
complete blood count, liver enzymes, a basic metabolic panel (including serum creatinine for 
assessments of renal function) will also be performed prior to randomization and 4 ± 1 weeks 
after initiation of randomized therapy.   
Significant methemoglobinemia was not observed in our pharmacokinetics trial thus far, at the 
same doses used in this trial, in a similar patient population. In this study we are measuring 
methemoglobin every half hour after the first administration as well as during steady-state 
continued oral administration at doses of both 12 mmol/d and 18 mmol/d. We don’t anticipate any 
significant issues with methemoglobinemia since this is a problem that has been reported with 
nitrite, but not with nitrate at the doses used in this study. However, a more strict monitoring 
approach for methemoglobinemia will be implementing should the results of our ongoing 
pharmacokinetics/safety study raise any concerns.  


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Side Effect management Plan:  No side effects have been observed in our single-dose study or 
our pharmacokinetics study (in particular, no hypotension or methemoglobinemia). However, a 
plan for management of side effects will be in place. The use of acetaminophen may be used for 
headaches during the study. Although hypotension is not expected, it should be managed as per 
standard clinical practice.  In the unlikely event that methemoglobinemia does occur during the 
trial, we will manage according to best clinical standards.(147) As the endogenous rate of 
reconversion from methemoglobin to hemoglobin is approximately 15% per hour, most 
asymptomatic patients can be managed conservatively. Generally, methemoglobin levels >20% 
with symptoms, or >30% without symptoms, warrant therapy; any patient with levels that are 
elevated to this range will be referred to the emergency room for management.(147)  
11.3.4. Measures to minimize the risk of breach in confidentiality:  
All records will be treated with strict confidentiality according to HIPAA guidelines (all study 
personnel are trained on HIPAA regulations). Blood samples obtained from subjects will be used 
only for research purposes. Records will be treated with strict confidentiality and stored in a 
secured, limited access area. A randomly assigned number rather than name will identify all 
collected samples.  A secure database of patient information will be maintained. The investigators 
and the sponsor will have access to research information and will follow IRB and institutional 
HIPAA guidelines. Paper files will be saved under lock in a secure IRB-approved area.  
 
11.3.6 Other measures to minimize risk:  
Phlebotomy, arterial tonometry, and Doppler echocardiographic examinations will only be 
performed by appropriately trained personnel as per our institutional standards.  
12. REGULATORY STANDARDS 
12.1. Informed consent 
The site investigator, or a person designated by the site investigator, will fully inform the subject 
of all pertinent aspects of the clinical trial including the review of the informed consent form 
approved by an Institutional Review Board (IRB).  Prior to a subject's participation in the clinical 
trial, the Informed Consent Form will be signed and personally dated by the subject or by the 
subject’s legally acceptable representative.  All subjects will receive a copy of the informed 
consent form.  
12.2. Institutional Review Board (IRB) 
The site Principal Investigator will submit this protocol to the Penn IRB. The study (study number, 
protocol title and version number), the document reviewed (protocol, Informed Consent Form, 
etc.) and the date of the review will be clearly stated on the written IRB approval opinion.  During 
the study, any amendment or modification to the protocol will be sent to the IRB. It will also be 
informed of any event likely to affect the safety of subjects or the continued conduct of the study, 
in particular any change in safety and all updates to the protocol will be sent to IRB.  


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12.3. Source document handling and archiving 
Hard copies of subject’s study records, including signed informed consent forms, HIPPA forms, 
source documents, and other study related materials will be stored in the subject binders, in a 
locked file cabinet in the research coordinator’s study office until archived. Research records will 
be retained for at least 2 years after the completion of this study. All study electronic files will be 
kept for at least 6 years after IRB acknowledgement of study termination. Files are not be 
destroyed or deleted without Sponsor approval. Data will be made available to the study sponsor 
National Institute of Health (NIH) members of the Institutional Review Board, and the Food and 
Drug administration, if requested by any of these entities.     
13. DATA AND SAFETY MONITORING PLAN:  
The data and safety monitoring plan is contained in Appendix 3. 
14. SUBJECT STIPEND 
Subjects will receive financial compensation for participation in this study. We will follow the 
following reimbursement scheme: 
• 
Completion of Visit #1: $100 
• 
Completion of Visit #2: $150 
• 
Completion of Visit #3: $150 
 
The maximum total amount (if all study visits are completed) will be $400.  
 
 
 


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15. REFERENCES  
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Benjamin EJ, Blaha MJ, Chiuve SE et al. Heart Disease and Stroke Statistics-2017 
Update: A Report From the American Heart Association. Circulation 2017;135:e146-e603. 
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Varadarajan P, Kapoor N, Bansal RC, Pai RG. Clinical profile and natural history of 453 
nonsurgically managed patients with severe aortic stenosis. Ann Thorac Surg 
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Leon MB, Smith CR, Mack M et al. Transcatheter aortic-valve implantation for aortic 
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Smith CR, Leon MB, Mack MJ et al. Transcatheter versus surgical aortic-valve 
replacement in high-risk patients. N Engl J Med 2011;364:2187-98. 
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Azevedo CF, Nigri M, Higuchi ML et al. Prognostic significance of myocardial fibrosis 
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Lindman BR, Otto CM, Douglas PS et al. Blood Pressure and Arterial Load After 
Transcatheter Aortic Valve Replacement for Aortic Stenosis. Circ Cardiovasc Imaging 
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Chirinos JA, Segers P. Noninvasive evaluation of left ventricular afterload: part 2: arterial 
pressure-flow and pressure-volume relations in humans. Hypertension 2010;56:563-70. 
9. 
Nichols WW ORM, Vlachopoulos C. McDonald's Blood Flow in Arteries: Theoretical, 
Experimental and Clinical Principles, 6th Edition. Hodder Arnold 2011. 
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Confidential and Proprietary Information of the Trustees of the University of Pennsylvania 
43 
APPENDIX 1. SCHEDULE OF EVENTS 
Schedule of Events 
Baseline 
assessment 
Phase 1 
1-week 
washout 
Phase 2 
1 week 
4 ± 1 
week 
1 week 
4 ± 1 
week 
Informed consent 
X 
 
 
 
 
 
Eligibility assessment 
X 
 
 
 
 
 
Medical History  
X 
 
X 
 
 
X 
Concomitant medications 
X 
 
X 
 
 
X 
Randomization 
X* 
 
 
 
 
 
Dietary counseling 
X 
 
X 
 
 
X 
KCCQ 
X 
 
X 
 
 
X 
Physical Exam 
X 
 
X 
 
 
X 
12-lead ECG 
X 
 
X 
 
 
X 
Laboratory tests 
X 
 
X 
 
 
X 
RNA collection with PAXgene tubes 
 
 
X 
 
 
X 
G6PD test as needed 
X 
 
X 
 
 
X 
6 minute walk test 
X 
 
 
 
 
 
Blood, urine, saliva collection and freezing 
X 
 
X 
 
 
X 
2D Echocardiogram 
X 
 
X 
 
 
X 
Arterial tonometry 
X 
 
X 
 
 
X 
Drug accountability 
 
 
X 
 
 
X 
Maximal effort exercise test with expired 
gas 
 
 
X 
 
 
X 
Retrieval of actigraph 
 
 
X 
 
 
X 
Arterial dilation (FMD) 
 
 
X 
 
 
X 
Assessment of adverse events 
 
X 
X 
 
X 
X 
*performed after baseline screening lab results are available 
 
 
 
 

ICF_001.pdf: 
NCT #: 
NCT03483051  
Title: 
Targeting Pulsatile Load to Increase Exercise Capacity and 
Quality of Life After Aortic Valve Replacement for Severe 
Aortic Stenosis (PULSE AS)  
Date: 
02-Aug 2018  


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UNIVERSITY OF PENNSYLVANIA 
RESEARCH SUBJECT COMBINED 
INFORMED CONSENT AND HIPAA 
AUTHORIZATION FORM 
 
 
Protocol Title:   
Targeting Pulsatile Load to Increase Exercise 
Capacity and Quality of Life After Aortic Valve 
Replacement for Severe Aortic Stenosis (PULSE 
AS) 
 
Principal 
Investigator: 
Julio Chirinos, MD, PhD 
Hospital of the University of Pennsylvania 
11 South PCAM Pavilion  
3400 S. Civic Center Boulevard  
Philadelphia, PA 19104 
(215) 220-9580 
Emergency 
Contact: 
24-Hour Emergency Number: (215) 662-4000 
(Ask for Cardiology Fellow on Call) 
  
 
Why am I being asked to volunteer? 
You are being invited to participate in a research study because you recently had an 
aortic valve replacement for aortic stenosis. Our goal is to look at the effects of 
potassium nitrate on your health. Your participation is voluntary which means you can 
choose whether or not you want to participate. If you choose not to participate, there will 
be no loss of benefits to which you are otherwise entitled. Before you can make your 
decision, you will need to know what the study is about, the possible risks and benefits 
of being in this study, and what you will have to do in this study. The research team is 
going to talk to you about the research study, and they will give you this consent form to 
read. You may also decide to discuss it with your family, friends, or family doctor. You 
may find some of the medical language difficult to understand. Please ask the study 
doctor and/or the research team about this form. If you decide to participate, you will be 
asked to sign this form. 


PULSE AS 
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2 
 
What is the purpose of this research study? 
 
The purpose of this study is to test whether potassium nitrate (KNO3) will improve exercise 
and quality of life in people who recently had an aortic valve replacement for aortic 
stenosis. After aortic valve replacement, some patients continue to be limited in their 
ability to do all the things they want to do, and exercise as much as they would like, due 
to becoming tired and short of breath early. We do not know exactly why these limitations 
occur. 
 
There is some evidence that in addition to problems with the heart, patients who have 
aortic stenosis develop problems with their arteries that affect their ability to exercise. 
potassium nitrate has been shown to improve blood flow in the body in healthy individuals. 
We would like to now study these pills in patients in who recently had an aortic valve 
replacement for aortic stenosis.  
 
The use of potassium nitrate in this study is experimental. Potassium nitrate has not been 
approved by the Food and Drug Administration (FDA) for the use being evaluated in this 
study.  
 
How long will I be in the study? How many other people will be in the 
study?  
 
You will be involved with this study for approximately 9 weeks. The study involves 3 
visits to our research center. The details of the visits are outlined below. We will enroll 
approximately 22 individuals in this study.  
 
What am I being asked to do? 
 
If you participate in this study, you will be asked to come to the research center for a total 
of 3 scheduled visits; there may be additional visits if you experience any side effects. 
During each visit, you will be asked to perform several tests related to exercise, heart 
function, and muscle function, which will be discussed below. During the first visit, we will 
perform tests to make sure you fit the criteria for our study. We will then ask you to start 
study medications for approximately 4 weeks (Intervention Phase 1). At the end of that 
period, we will ask that you come back to our research center for testing, including an 
exercise test. After this visit, you will not take any study medications for approximately 1 
week to allow the drugs to wash out of your system (Washout Period). We will then start 
you on a second study medication (Intervention Phase 2) for another period of 
approximately 4 weeks. At the end of this second period, you will be asked to come in 
and repeat the tests for a 3rd and final time, which will include your final exercise test. 
Your participation in the study will be over at the end of this endpoint assessment.  
 
This study is a crossover study, which means that over the course of the study, all 
participants will receive both the “active” study medication, KNO3, and the “control,” which 
is a study medication that has no known actions on your blood vessels. The control that 


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3 
we will use will be potassium chloride (KCl). Neither you, nor the study investigators, will 
know which drug you are getting at which time. However, we can always get this 
information from the research pharmacy if we need to for an emergency. 
 
If at any point, you are experiencing intolerable side effects, or your blood tests indicate 
that it will not be safe, we will ask you to stop the study medication, and your participation 
in the study will be over. You can also request to leave the study at any time.  
 
Procedures and Visit Schedule 
 
Visit 1: In person visit at our research center. This visit will take one-half of a day. 
 
On the first day, we will perform tests and studies to make sure you are a good candidate 
for our study (as discussed below). At the end of this visit, you will be randomized to study 
medications for the initial period (Intervention Phase 1).  
 
During this visit, we will give you a questionnaire about your symptoms. We will also 
perform a physician exam, and obtain blood, urine and saliva (spit) samples from you.  
 
 
Here is a list of the events for Visit #1: 
• Review and sign this informed consent form.  
• Vital Signs and brief Physical Exam – We will check your blood pressure and heart 
rate, and a qualified member of our staff will perform a brief physical exam. We will 
also perform orthostatic blood pressure measurements, where your blood 
pressure will be assessed both while lying down and with standing.  
• Blood Draw - We will draw blood from you. Blood will be drawn by inserting a 
needle into a vein in your arm. We will measure various substances in the blood 
that tell us about the pressures in your heart, your nitrate levels, and your 
methemoglobin levels. We will discuss methemoglobin more below, but in brief, 
this is a compound that can accumulate in the blood and make it more difficult for 
your blood to carry oxygen to the body. Some drugs, and possibly potassium 
nitrate, may increase your methemoglobin levels, so we want to track this carefully. 
We will draw 4 tablespoons of blood during the blood draw. We will also collect a 
saliva (spit) and a urine sample. 
• Questionnaires –We will give you a questionnaire that asks you questions about 
your symptoms and how you feel you are functioning in your day-to-day life 
(Kansas City Cardiomyopathy Questionnaire)  
• Arterial Tonometry - This is a simple non-invasive test in which we will examine 
the pressure in your blood vessels. This test will allow us to determine how stiff 
your arteries are and to measure the pressure the heart is pumping against. This 
test will be done by a trained member of our team over approximately 30 minutes. 
Sticker-like electrodes will be placed over your skin on the chest to measure the 
electrical current generated by your heart. Next, the investigator will gently put the 


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4 
tonometer, which looks like a pencil, against your wrist, over the side of your neck 
and finally in your groin to measure the pulse. 
• Doppler Echocardiogram - This procedure is similar to previous echocardiograms 
that you may have had. In this procedure we put some gel on the skin on the left 
side of your chest and use a probe to take pictures of the heart and blood vessels. 
This takes approximately 20 minutes. 
• Electrocardiogram – During this procedure, we will place electrodes (stickers) on 
your chest to capture the electrical signals from the heart.  
• 6-minute walk test: you will be asked to do a six-minute walking test. The objective 
of this test is to walk as quickly as you can for six minutes in a hallway track (up 
and down the corridor) so that you cover as much ground as possible. You may 
slow down if necessary. If you stop, we ask that you to continue to walk again as 
soon as possible. You will be kept informed of the time and you will be encouraged 
to do your best. Your goal is to walk as far as possible in six minutes. We do this 
test because we want to make sure that your oxygen levels do not drop too much 
during exercise, which would make you ineligible to continue in the study. 
• Activity Monitor (Actigraph device)- this device will track the number of steps you 
take in a day and how active you are. We would like you to wear this device on 
your wrist as much as possible for one week prior to your return visit We will ask 
you to return the device to the research team when you return for your following 
visit. The device is not waterproof, so you will need temporarily to take it off to take 
baths or showers.   
• Randomization - After baseline testing is complete, you will be randomly assigned 
to receive either potassium nitrate or control first. You will not be told which 
medication you are getting.  
 
 
At the end of this visit, you will be given study medications to be taken twice daily during 
Intervention Phase 1. 
 
~1 week phone call: We will call you after approximately 1 week to see how you are 
doing. If you are feeling well, we will then increase the dose to three times a day. 
Medication should be taken with food. If you are having side effects, we may ask you 
to come into our research center for us to see you. We will ask you to take study 
medications for approximately 4 weeks.  
 
Visit 2: This visit will occur approximately 4 weeks after your first visit. Please plan on 
this visit to last from 8 am – 5 pm.  
 
Please arrive in the morning before taking your morning dose of study medication. We 
will place an IV in your arm and check the levels in your blood. We will also collect another 
saliva and urine sample. We will then give you a light breakfast with your morning dose 
of study medication. We will then ask you about side effects. We will then ask you to 
perform some of the study procedures. About two hours after you have taken the study 


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medication, we will check your blood levels from your IV and collect another spit sample. 
We will then ask you to perform the rest of the study procedures, as listed below.  
 
List of Visit #2 Procedures: 
• Brief interval history, review of all medications you are taking 
• Vital Signs (including orthostatic blood pressure measurement) and brief Physical 
Exam 
• Light breakfast with study medication 
• Questionnaires – We will ask you about any potential side effects you may be 
experiencing from the study medication. We will also administer the questionnaire 
asking about your symptoms (Kansas City Cardiomyopathy Questionnaire). 
• Blood Draws from IV and saliva and urine samples 
• Arterial Tonometry 
• Doppler Echocardiogram 
• Bicycle Exercise Test with peak exercise blood sample 
• Arterial dilation assessment 
 
Bicycle Exercise Test: We will ask you to perform a bicycle exercise test where you will 
be lying on your back. This test will be used to assess your overall exercise capacity. 
The test will begin with a low level of resistance that will then increase every 3 minutes. 
We would like you to exercise for as long as you possibly can, as the goal is to push 
yourself to your peak. We will be monitoring you throughout this period and will stop you 
if we see anything unsafe. When you stop exercising, we will ask you to remain lying in 
the same position for about 6 minutes while we continue to collect information about 
your recovery. The total length of this bicycle exercise test will depend on you.   
 
During bicycle exercise testing you will breathe through a mouthpiece that is connected 
to a machine that monitors that air that you breathe in and out. Specifically, the machine 
tells us about how much oxygen you are using, and how much carbon dioxide your 
body is making during exercise. We will also continuously monitor your heart rate and 
rhythm, and we will be checking your blood pressure frequently during the study. This 
information will allow us to determine how much oxygen your body is using during 
exercise, the electrical conduction of your heart, how fast your heart is beating during 
exercise, and how much pressure there is in your arteries during exercise.  
 
The length of the exercise test will vary based on how long you can exercise for, but we 
want you to exercise for as long as you safely can while we monitor you the entire 
time.  We will draw a blood sample from your IV immediately when you stop exercising. 
 
Arterial dilation assessment: We will take pictures of the artery in your arm using 
ultrasound. We will then inflate a blood pressure cuff wrapped around your forearm for 5 
minutes. This may make your hand feel “pins and needles” or heavy. This may be 
slightly uncomfortable for you, but there is no serious harm to this. We will then deflate 


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the blood pressure cuff and measure the increase in blood flow as blood rushes into 
your forearm and hand. We will measure how your artery responds (dilates) in response 
to this inflation, using ultrasound. None of these devices are associated with any known 
side effects. You may, however, notice slight bruising on your arm from the blood 
pressure cuff, though this will not last long. 
 
Other tests (in an identical fashion to visit 1): 
• Vital Signs (including orthostatic blood pressure measurement) 
• Questionnaires – We will ask you about any potential side effects you may be 
experiencing from the study medication. We will also administer the questionnaire 
asking about your symptoms (Kansas City Cardiomyopathy Questionnaire). 
• Blood Draws from IV and saliva and urine samples 
• Arterial Tonometry 
• Doppler Echocardiogram 
 
Washout period: following the completion of the studies during your second visit, we 
will ask that stop taking any of our study medication for 1 week ± 3 days to allow the 
drug to clear out of your body. You should continue all of your other regularly-prescribed 
medications. 
 
Intervention Phase 2: At the end of the washout period, we will give you the second 
study medication to be taken twice a day. This will be either KNO3 or control, and will 
be the opposite of what you got during Intervention Phase 1. In this way, every subject 
in the study will receive both KNO3 and control, though the subjects may receive these 
medications in a different order. 
 
~1 week phone call: We call you after about one week of taking the Intervention 
Phase 2 study medications twice a day. We will discuss how you are feeling and any 
side effects. If you are having side-effects, we may ask you to come in to our research 
center to be evaluated. If you are doing well, we will increase the dose of the 
Intervention Phase 2 study medications to three times a day with a meal.  
 
Visit 3: This visit will occur after you have been on Intervention Phase 2 study 
medications for approximately 4 weeks. Please plan on this visit to last from 8 am – 5 
pm.  
 
Please arrive in the morning before taking your morning dose of study medication. We 
will place an IV in your arm and check the levels in your blood. We will also collect another 
saliva and urine sample. We will then give you a light breakfast with your morning dose 
of study medication. We will then ask you about side effects. We will then ask you to 
perform some of the study procedures. About two hours after you have taken the study 
medication, we will check your blood levels from your IV and collect another spit sample. 
We will then ask you to perform the rest of the study procedures, as listed below.  
 
 
List of Visit #3 Procedures: this visit will be exactly the same as Visit #2 


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7 
• Brief interval history, review of all medications you are taking 
• Vital Signs (including orthostatic blood pressure measurement) and brief Physical 
Exam 
• Light breakfast with study medication 
• Questionnaires – We will ask you about any potential side effects you may be 
experiencing from the study medication. We will also administer the questionnaire 
asking about your heart failure symptoms (Kansas City Cardiomyopathy 
Questionnaire). 
• Blood Draws from IV, urine and saliva samples 
• Arterial Tonometry 
• Doppler Echocardiogram 
• Bicycle Exercise Test with blood sample at peak exercise 
• Arterial dilation assessment 
 
Your participation is complete after Visit 3. We will compare your results from Visit 2 
and Visit 3. 
 
 
 


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8 
The study chart below explains what is expected of you at each study visit: 
 
Visit 1 
• Read and sign this informed consent form (ICF). 
• If you are a pre-menopausal woman, we will confirm 
that you are not pregnant using a urine pregnancy 
test. 
• Brief physical exam with EKG and vital signs.  
• Complete a questionnaire on your symptoms 
• Have your blood drawn, an IV placed, and give a 
saliva and urine sample.  
• The Arterial Tonometry test. 
• Have a Doppler Echocardiogram. 
• Have a 6-minute walk test 
 
If you meet all of our criteria, we will mail the 
medications to you after reviewing your 
laboratory studies (Intervention Phase 1). We will 
also mail you the activity monitor to wear for the 
one-week immediately prior to your return visit.  
 
Visit 2 
(after ~4 
weeks of 
study 
medications) 
• Record Vital Signs. 
• Retrieve the activity monitor to download the data 
• Have your blood drawn and a new IV placed. 
• Give a saliva and urine sample. 
• If you are a pre-menopausal woman, we will confirm 
that you are not pregnant using a urine pregnancy 
test. 
• Have a light breakfast with your morning dose of 
study medication. 
• Complete a questionnaire on your symptoms. 
• Discuss potential side effects. 
• Arterial dilation assessment 
~ After 2 hours~ 
• Blood draw from your IV and give a saliva and urine 
sample. 
• The Arterial Tonometry test. 
• Have a Doppler Echocardiogram. 
• Complete the bike exercise tests with blood 
sample at peak exercise 
 


PULSE AS 
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9 
After visit 2, you will start the ~ 1-week washout period 
during which you will NOT take any study medication. 
Please continue your other medications. 
 
After this washout period, you will start Intervention 
Phase 2 study medications which you will receive by 
mail. We will also mail you the activity monitor to wear 
for the one-week immediately prior to your return visit. 
 
Visit 3 
(after ~4 
weeks of 
Intervention 
Phase 2 
study 
medications) 
• Record Vital Signs. 
• Have your blood drawn and an IV placed. 
• Give a saliva and urine sample. 
• If you are a pre-menopausal woman, we will confirm 
that you are not pregnant using a urine pregnancy 
test. 
• Have a light breakfast with your morning dose of 
study medication. 
• Complete a questionnaire on your symptoms. 
• Discuss potential side effects. 
• Arterial dilation assessment 
 
~ After 2 hours~ 
• Blood draw from your IV and give a saliva and urine 
sample. 
• The Arterial Tonometry test. 
• Have a Doppler Echocardiogram. 
• Complete the bike exercise tests with blood draw 
at peak exercise. 
• Return your activity monitor  
 
Your participation in the study is now over. 
 
 
 
 


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What are the possible risks or discomforts?  
 
Study Drug - Potassium nitrate (KNO3) 
Currently, we do not have much information regarding possible side effects of potassium 
nitrate in patients who recently had an aortic valve replacement for aortic stenosis. In 
other studies of generally healthy individuals, nitrate given either as a juice or in capsule 
form was generally well tolerated. If taken without food, the most common side effect 
people experienced was stomach discomfort. This was prevented by taking the study 
drug with a meal. Other possible side effects include: 
• Slight headache 
• Dizziness 
• Lightheadedness 
• Low blood pressure 
• Stomach ache, diarrhea, nausea, or vomiting 
• Shortness of breath 
• Flushing 
• Rash 
• Fatigue 
• Changes in blood pressure when standing up 
• Orthostatic hypotension – this is a large change in blood pressure that occurs from 
moving from sitting to standing and can cause people to get very light headed and 
even pass out. 
• Methemoglobinemia - One of the main functions of blood is to carry oxygen from 
the lungs to the rest of the body. This is accomplished by something called 
“hemoglobin” that is found in blood. Hemoglobin exists in many forms, one of which 
is methemoglobin. This version is not active and cannot participate in carrying 
oxygen. In healthy individuals, methemoglobin makes up less than 3% of 
circulating hemoglobin. Some genetic conditions, and certain drugs, can increase 
methemoglobin levels. Nitrates, and drugs in this family, are in this group. In our 
preliminary studies, we found that potassium nitrate does not significantly increase 
the levels of methemoglobin. However, we will measure your methemoglobin 
levels during this study and if you are a male of African, Asian or African, Asian or 
Mediterranean decent, we will make sure that you don’t have Glucose-6-phosphate 
dehydrogenase (G6PD) deficiency, which would make you more susceptible to 
methemoglobinemia. 
 
We did not see any serious side effects from potassium nitrate in previous studies. If you 
experience any side effects you find too uncomfortable, you may withdraw from the study 
at any time. The study team will specifically assess you for the presence of symptoms 
after the medication is initiated or after the dose in increased, in order to determine 
whether it is safe for you to continue taking these medications. 
 
 


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Potential Drug Interactions and Instructions During the Study 
 
There is not much information on potassium nitrate in subjects who recently had an aortic 
valve replacement for aortic stenosis. You are not eligible for this study if you are on other 
nitrate-containing medications such as isosorbide mononitrate (Imdur) or isosorbide 
dinitrate (Isordil). You are also not eligible for this study if you are taking allopurinol 
(Zyloprim) or febuxostat (Uloric, Adenuric) for gout, as these drugs may interfere with 
activation of the study medication.  
 
If you take medications for erectile dysfunction, such as sildenafil (Viagra), tadalafil 
(Cialis), or vardenafil (Levitra) we will ask you to stop taking this medication for 7 days 
before starting the study and for 7 days after study completion. This is due to a chance 
that taking both nitrates and these medications could lead to a dangerous drop in your 
blood pressure.  
 
Potassium nitrate (KNO3) and potassium chloride (KCl) contains potassium, which can 
be dangerous at high levels. We will monitor your potassium level at each study visit to 
minimize the chance of harm.  
 
A major source of nitrates is your diet. We will discuss your diet and foods to avoid during 
the study. 
 
Finally, we will ask you to not use mouthwash during the study. While brushing your 
teeth and flossing are fine, mouthwash kills the bacteria in your mouth. These bacteria 
are needed for activation of the study medication. Please let us know if you are put on 
antibiotics during the study. 
 
Drug Interaction and Instructions Summary: 
• You are not eligible for this study if you take any form of nitrates (nitroglycerin, 
isosorbide mononitrate (Imdur), isosorbide dinitrate (Isordil, Dilatrate). 
• You must not take medications for erectile dysfunction, such as sildenafil (Viagra), 
tadalafil (Cialis), or vardenafil (Levitra) for 7 days before starting the study and for 
7 days after the study is complete. This is to avoid any a dangerous drop in your 
blood pressure. 
• Do not use mouthwash during the study. 
• We will go over information regarding which foods to avoid during the study. 
• Please let us know if you are started on antibiotics for any reason during the study. 
 
 
 
 
Possible Risks or Discomforts of Study Procedures 
Arterial Tonometry 


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12 
• Minor discomfort may occur when the tonometer is placed against your neck and 
groin. This will feel like someone is taking your pulse in the neck or groin. 
• We will use adhesive electrodes attached to your skin. These may occasionally 
cause itching and irritation in your skin. 
 
Doppler Echocardiogram 
• We will use adhesive electrodes attached to your skin. These may occasionally 
cause itching and irritation in your skin. 
• Minor discomfort may occur when the probe is placed on your chest to obtain 
pictures of your heart.  
 
Bicycle Exercise Test  
• This exercise testing protocol is considered safe. Rarely, people have an adverse 
event during exercise. The risk of this happening is the same in our lab as it 
would be if you exerted yourself elsewhere. Individuals who are trained to 
respond to situations that might develop during exercise will closely monitor you 
throughout the exercise period. It is expected that you will become tired and short 
of breath during this exercise test, as we do ask that you push yourself to the 
most that you can possibly do. 
• You will likely become short of breath and tired during this test. This test may 
result in exhaustion or abnormal heart rates, however you will be monitored using 
EKGs to check for this. In rare cases, exercise may lead to a major heart 
complication (such as a heart attack, passing out, or death).  
• We will have staff on hand to monitor your heart throughout the process and will 
be equipped to handle emergencies in the event of any problems. 
• Importantly, exercise testing has been shown to be safe in the vast majority of 
stable patients with heart failure. 
 
Blood Draw  
• There may be minor discomfort or pain (sometimes accompanied by redness, 
swelling, and warmth to the area) from the needle used to place the IV line. 
Some bleeding or bruising may occur. Fainting and infection at the site of the IV 
are both possible, although these occurrences are unlikely. You may develop 
inflammation in the vein where the IV was placed (superficial phlebitis) or a clot 
of the vein. These problems generally resolve over time on their own after the IV 
has been removed.  
• Blood samples collected during testing will be stored confidentially in dedicated 
blood laboratory space. Your blood samples will be analyzed for research 
purposes. All collected blood samples will be stored with your unique coded 
identifier and only approved study personal will have access to the key to the 
code.  
Reproductive Risks   
Because of the effects of this drug, there could be serious harm to unborn children or 
children who are breast-feeding. These effects could also harm the mother. It is also 


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possible that harmful side effects that are not yet known could happen to both the mother 
and unborn or breast-feeding child. If you are currently pregnant, it is important that you 
inform the investigator because you will not be able participate in the study.   
 
If you are able to become pregnant, you will be given a pregnancy test before entry into 
the study. You will be asked to use a medically accepted method of birth control (such as 
an IUD, birth control combination pill, patch, or ring, progestin-only pills, Depo Provera 
Shot, Implanon, complete abstinence, or condoms) while you participate in the study. You 
should not become pregnant while you are in this study. If you do become pregnant, you 
must tell the study investigator and consult an obstetrician or maternal-fetal specialist. If 
you become pregnant during this study, you will be discontinued from the trial, but we will 
collect information about the pregnancy and its outcome via telephone call.  
 
Risks of Genetic Testing 
This research includes genetic testing. We will be measuring how your body changes its 
expression of different genes over the course of this study. Even without your name or 
other identifiers, your genetic information is unique to you. The researchers believe the 
chance that someone will identify you is very small, but the risk may change in the 
future as people come up with new ways of tracing information. 
 
There can be a risk in knowing genetic information. New health information about 
inherited traits that might affect you or your blood relatives could be found during a 
research study. Even though your genes are unique, you share some of the same 
genes with your blood relatives. Although we are not able to know all of the risks from 
taking part in research on inherited traits, we believe that the risks to you and your 
family are very low, because your samples will be coded. Research results will not be 
returned to you or your doctor.  
 
Very rarely health or genetic information could be misused by employers, insurance 
companies, and others. For example, it could make it harder for you to get or keep a job 
or insurance, or life insurance companies may charge a higher rate based on this 
information. We believe the chance these things will happen is very small, but we 
cannot make guarantees. 
 
A federal law (Genetic Information Non-Discrimination Act, GINA) helps reduce the risk 
from health insurance or employment discrimination. The law does not include other 
types of misuse by life insurance or long term care insurance. If you want to learn more 
about GINA, you can find information about it on the internet or ask the study staff. 
 
Other Risks 
Although a breach in confidentiality is a potential risk, we have implemented various 
measures to minimize the possibility of a breach in confidentiality. If you decide to 
participate in the study, you will be assigned a unique identifier number (example: ‘#023’) 
and all your data will be handled in a way that cannot be linked to your name or other 
personal information. Only the investigator and study staff will have access to the list that 
connects your name to your data.  


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If you are injured while participating in this study, you should inform your primary care 
physician that you are participating in this research study. Your primary care physician 
can also contact us regarding your participation in this study at any time. 
 
In addition to the possible risks detailed above, the research may involve risks that are 
currently unforeseeable.  
 
This is not a complete list of possible side effects, but the most significant are listed above. 
If you notice other effects or new symptoms not listed above, please contact the study 
team. You can contact Dr. Anupam Kumar at 267-293-9247. 
 
What if new information becomes available about the study? 
During the course of this study, we may find more information that could be important to 
you. This includes information that, once learned, might cause you to change your mind 
about being in the study. We will notify you as soon as possible if such information 
becomes available. 
 
What are the possible benefits of the study?  
You may not benefit from participating in this research study. However, your participation 
will help us understand how potassium nitrate affects the body’s response to exercise. 
 
What other choices do I have if I do not participate?  
There is no penalty if you choose not to join the research study.  
 
At this moment, we don’t have effective treatments for your condition; however, we don't 
yet know whether the therapy we are testing in this study is effective. 
As an alternative to participation, you may continue your standard care and therapy 
without joining the research study. You may also discuss alternate forms of therapy with 
your personal physician. 
 
Will I be paid for being in this study? 
You will receive financial compensation for your participation in this study in the form of 
a check or prepaid card (GreenPhire ClinCard). We will follow the following 
reimbursement scheme: 
• Completion of Visit #1: $100 
• Completion of Visit #2: $150 
• Completion of Visit #3: $150 
The maximum total amount (if you complete all study visits) will be $400. You will be paid 
for each visit that you complete. Your reimbursement will be mailed out as one check 
approximately 4-8 weeks after each visit is completed.  
 
Please note: In order to be compensated for your participation in this study, you must 
provide your Social Security Number. Additionally, please note that the University of 


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Pennsylvania is required to report to the IRS any cumulative payments for participation in 
research studies that exceed a total of $600 in a calendar year. 
 
Will I have to pay for anything? 
You will not have to pay for any research procedures or tests that result from participating 
in this study.  
 
You are still responsible for any deductibles or applicable co-pays for routine office visits, 
scans and blood work not related to this study. Please talk to your doctor and study team 
about putting you in touch with a financial counselor to determine exactly what the 
deductible and co-pay will be for you; this is highly variable depending on your type of 
insurance. 
 
What happens if I am injured from being in the study?  
We will offer you the care needed to treat injuries directly resulting from taking part in this 
research. We may bill your insurance company or other third parties, if appropriate, for 
the costs of the care you get for the injury, but you may also be responsible for some of 
them. 
 
There are no plans for the University of Pennsylvania to pay you or give you other 
compensation for the injury.  
 
You do not give up your legal rights by signing this form.   
 
If you think you have been injured as a result of taking part in this research study, tell the 
person in charge of the research study as soon as possible.  The researcher’s name and 
phone number are listed in the consent form. 
 
When is the Study over?  Can I leave the Study before it ends? 
This study is expected to end after all participants have completed all visits, and all 
information has been collected. This study may also be stopped at any time by your 
physician, the study Sponsor, or the Food and Drug Administration (FDA) without your 
consent because: 
• 
The Primary Investigator feels it is necessary for your health or safety.  Such an 
action would not require your consent, but you will be informed if such a decision 
is made and the reason for this decision. 
• 
You have not followed study instructions.  
• 
The Sponsor, the study Principal Investigator, or the Food and Drug 
Administration (FDA) has decided to stop the study. 
 
If you decide to participate, you are free to leave the study at any time. Withdrawal will 
not interfere with your future care. If you choose to withdraw from the study, you will be 
asked to attend an Early Termination visit in which we will take your vital signs, check 
your blood levels, complete a medical history and physical, and get any information 
regarding side effects you may be experiencing at that time.  


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Who can see or use my information?  How will my personal 
information be protected?   
We will do our best to make sure that the personal information obtained during the course 
of this research study will be kept private. However, we cannot guarantee total privacy. 
Your personal information may be given out if required by law.   
 
If information from this study is published or presented at scientific meetings, your name 
and other personal information will not be used. If this study is being overseen by the 
Food and Drug Administration (FDA), they may review your research records.   
 
The research team will make every effort to keep all the information you tell us during the 
study strictly confidential, as required by law. Any documents you sign, where you can be 
identified by name will be kept in a locked file cabinet in Dr. Kumar’s research office. All 
of your electronic information will be kept in a secure server and the file that contains your 
health information will not be the same as the file that contains your name. Only the 
investigators will have access to the codes that link your health information with your 
personal data. These documents will be kept confidential.   
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as 
required by U.S. law. This website will not include information that can identify you. At 
most, the website will include a summary of the results. You can search this website at 
any time. 
 
Electronic Medical Records and Research Results  
 
What is an Electronic Medical Record and/or a Clinical Trial Management  
System?  
 
An Electronic Medical Record (EMR) is an electronic version of the record of your care 
within a health system. An EMR is simply a computerized version of a paper medical 
record.  
 
A clinical trial management system (CTMS) is used to register your information as a 
participant in a study and to allow for your research data to be entered/stored for the 
purposes of data analysis and any other required activity for the purpose of the conduct 
of the research.  
 
If you are receiving care or have received care within the University of Pennsylvania 
Health System (UPHS) (outpatient or inpatient) and are participating in a University of 
Pennsylvania research study,  information related to your participation in the research 
(i.e. laboratory tests, imaging studies and clinical procedures) may be placed in your 
existing EMR maintained by UPHS. Information related to your participation in clinical 
research will also be contained in the CTMS. 
 


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If you have never received care within UPHS and are participating in a University of 
Pennsylvania research study that uses UPHS services, an EMR will be created for you 
for the purpose of maintaining any information produced from your participation in this 
research study.  The creation of this EMR is required for your participation in this study. 
In order to create your EMR, the study team will need to obtain basic information about 
you that would be similar to the information you would provide the first time you visit a 
hospital or medical facility (i.e. your name, the name of your primary doctor, the type of 
insurance you have). Information related to your participation in the study (i.e. laboratory 
tests, imaging studies and clinical procedures) may be placed in this EMR.  
Once placed in your EMR or in the CTMS, your information may be accessible to 
appropriate UPHS workforce members that are not part of the research team.  
Information within your EMR may also be shared with others who are determined by 
UPHS to be appropriate to have access to your EMR (e.g. health insurance company, 
disability provider, etc.). 
 
What information about me may be collected, used or shared with 
others?   
• Name 
• Address 
• Telephone number 
• Date of birth 
• Social Security number (for payment purposes) 
• Personal and family medical history  
• Results from physical examinations, tests or procedures 
• Dates of tests or procedures 
• Medical record numbers 
• Echocardiogram images obtained during the study 
• Blood, urine and saliva samples 
• Medication history 
• Several questionnaires 
• Email address 
 
 
 


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Why is my information being used? 
Your contact information is used by the research team to get in touch with you during the 
study. Your information and results of tests, procedures, and questionnaires are used to: 
• Complete the research. 
• Oversee the research. 
• Ensure that the research was done right.   
 
Who may use and share information about me?   
The following individuals may use or share your information for this research study: 
• All research staff directly associated with this trial 
• University of Pennsylvania Institutional Review Board (IRB) 
• University of Pennsylvania Office of Clinical Research (OCR) 
• Other research personnel with access to the databases for research and/or study 
coordination and as otherwise approved by the IRB  
 
Who, outside of the School of Medicine, might receive my 
information?  
• University of Pennsylvania Institutional Review Board (IRB) 
Oversight organizations 
• The Food and Drug Administration (FDA) 
• The Office of Human Research Protections (OHRP) 
 
Once your personal health information is disclosed to others outside the School of 
Medicine, it may no longer be covered by federal privacy protection regulations.   
 
The Principal Investigator or study staff will inform you if there are any additions to the list 
above during your active participation in the study. Any additions will be subject to 
University of Pennsylvania procedures developed to protect your privacy. 
 
How long may the School of Medicine use or disclose my personal 
health information?   
Your authorization for use of your personal health information for this specific study does 
not expire.  
 
 
 


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Your information may be held in a research database.  However, the School of Medicine 
may not re-use or re-disclose information collected in this study for a purpose other than 
this study unless: 
• You have given written authorization. 
• The University of Pennsylvania’s Institutional Review Board grants permission. 
• As permitted by law. 
 
Can I change my mind about giving permission for use of my 
information? 
Yes. You may withdraw or take away your permission to use and disclose your health 
information at any time.  You do this by sending written notice to the investigator for the 
study.  If you withdraw your permission, you will not be able to stay in this study. 
 
What if I decide not to give permission to use and give out my health 
information? 
Then you will not be able to be in this research study. 
 
You will be given a copy of this Research Subject HIPAA Authorization describing your 
confidentiality and privacy rights for this study.  
 
By signing this document you are permitting the School of Medicine to use and disclose 
personal health information collected about you for research purposes as described 
above. 
 
The study doctors believe that there may be opportunities in the future with other studies 
in aortic stenosis. We would like your permission to contact you in the future. Please initial 
below if you agree to being contacted by telephone in the future (for up to 3 years after 
your final visit) about additional studies.  
 
______:   I agree to being contacted in the future about studies related to findings in this 
study. 
______:  I do not agree to be contacted in the future about studies related to findings in 
this study. 
 
If you should change your mind about someone contacting you in the future, you will need 
to send a written letter to the study team at the address on the first page of this consent 
form.  
 
Who can I call with questions, complaints or if I’m concerned about 
my rights as a research subject? 
If you have questions, concerns or complaints regarding your participation in this research 
study or if you have any questions about your rights as a research subject, you should 
speak with the Principal Investigator listed on page 1 of this form.  If a member of the 
research team cannot be reached or you want to talk to someone other than those 


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working on the study, you may contact the Office of Regulatory Affairs with any question, 
concerns or complaints at the University of Pennsylvania by calling (215) 898-2614. 
 
The person leading this medical research study, Dr. Chirinos, is named as an inventor 
on a patent application on the concept of using potassium nitrate for the treatment of 
heart failure with preserved ejection fraction but not for aortic stenosis. Therefore, the 
researcher cannot benefit financially from the results of this research study. If you would 
like more information, please ask the researchers or the study coordinator.  
 
When you sign this form, you are agreeing to take part in this research study. 
This means that you have read the consent form, your questions have been 
answered, and you have decided to volunteer.  Your signature also means that 
you are permitting the University of Pennsylvania to use your personal health 
information collected about you for research purposes within our institution. You 
are also allowing the University of Pennsylvania to disclose that personal health 
information to outside organizations or people involved with the operations of this 
study. 
A copy of this consent form will be given to you.  
 
 
________________________      _____________________________________ 
Name of Subject  
 
 Signature of Subject  
 
           Date 
  (Please Print) 
 
 
 
 
________________________ 
_____________________________________ 
Name of Person Obtaining 
 Signature                                  
 
Date 
Consent (Please Print)