Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age
Phase: PHASE2
Conditions: Aging, Sedentary Lifestyle, Frailty
Sponsor: Gladwin, Mark, MD

 
 
 
Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old 
Age 
 
 
 
 
 
 
 
 
 
Daniel E. Forman, MD 
Principal Investigator 
 
 
 
 
 
 
Professor of Medicine 
University of Pittsburgh Medical Center 
University of Pittsburgh School of Medicine 
Chair, Geriatric Cardiology Section 
 
Version 9 
 
August 28, 2024 
 
 
 
 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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TABLE OF CONTENTS 
Protocol Synopsis 
4-7 
1. Study Objective, Specific Aims, Background and Significance 
7-10 
1.1 Study Objectives 
7 
1.2 Specific Aims 
8 
1.3 Background and Rationale 
8 
1.4 Significance 
9 
2. Research Design and Methods 
10-25 
2.1 Classification and Methodological Design 
10 
2.2 Detailed Description of the Study 
10 
2.2.1 Study Visits and Assessments 
10 
2.2.2 Study Timetable 
18 
2.3 Study Drug 
20 
2.4 Statistical Analysis 
22 
3. Human Subjects 
25-26 
3.1 Participant Population 
25 
3.2 Inclusion Criteria 
25 
3.3 Exclusion Criteria 
25 
4. IRB Approval and FDA Amendments 
26-27 
5. Recruitment and Informed Consent Procedures 
27-28 
5.1 Recruitment Methods 
27 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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5.2 Informed Consent Procedure 
28 
6. Potential Risks and Benefits 
29-34 
6.1 Potential Risks 
29 
6.2 Alternative Treatments 
34 
6.3 Potential Benefits 
34 
7. Risk Management Procedures 
34-35 
7.1 Protection Against Risks 
34 
7.2 Protection Against Potential Risks of Experimental Inter-
vention 
35 
8. Reportable Events 
36-37 
8.1 Reportable Adverse Events 
36 
8.2 Adverse Events Reporting Timelines 
37 
9. Data Safety Monitoring 
37-38 
9.1 Data and Safety Monitoring Board 
37 
9.2 Data and Safety Monitoring Plan 
38 
9.3 Parameters to be Monitored 
38 
9.4 Frequency of Monitoring 
38 
10. Withdrawal of Participants and Stopping Criteria 
39-40 
11. Costs and Payments 
40-41 
11.1 Costs 
40 
11.2 Payments 
41 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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12. Qualifications and Source of Support 
41-43 
12.1 Qualifications of the Investigators 
41 
12.2 Source of Support 
43 
13. References 
44 
 
 
 

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PROTOCOL SYNOPSIS 
 
Protocol Title: 
Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and 
Physical Activity in Old Age  
Protocol Number: 
STUDY20110334 
NCT Number: 
NCT04405180 
Version # and Date: 
Version 9 / August 28, 2024 
Clinical Phase: 
Phase II Clinical Investigation 
Investigational Drugs: 
Nitrogen 14 Sodium Nitrite (14N Sodium Nitrite) 
Trial Site: 
University of Pittsburgh Single-Center Randomized Trial 
IND Sponsor: 
Mark T. Gladwin, MD 
University of Maryland 
655 West Baltimore Street, 14-029 
Baltimore, MD 21201-1509 
Principal Investigator: 
Daniel E. Forman, MD 
Professor of Medicine 
Chair, Geriatric Cardiology Section  
University of Pittsburgh 
3471 Fifth Avenue, Suite 500 
Pittsburgh, PA  15213 
Co-Investigators: 
Nancy Glynn, PhD 
Kara Hughan, MD, MHSc 
Theodore Huppert, PhD 
Anne Newman, MD, MPH 
Subashan Perera, PhD 
Frank Sciurba, MD 
Sruti Shiva, PhD  
Simon Watkins, PhD 
Study Monitor:               
Mark T, Gladwin, MD 
 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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Research Facilities: 
UPMC Montefiore Geriatric Research Center (GRC) 
3459 Fifth Ave. NE 547 
Pittsburgh, PA 15213 
UPMC Emphysema/COPD Research Center  
Division of Pulmonary and Critical Care Medicine  
University of Pittsburgh  
3471 Fifth Ave; Suite 1211 
Pittsburgh, PA 15213 
UPMC Montefiore Translational Research Center (TRC) 
3459 Fifth Ave. NW 823 
Pittsburgh, PA 15213 
UPMC Presbyterian Hospital Magnetic Resonance Research 
Center (MRRC) 
200 Lothrop St 
Pittsburgh, PA 15213 
University of Pittsburgh Pepper Center– Senior Mobility Ag-
ing Research Training (SMART) Center 
3471 Fifth Ave. Suite 1200 
Pittsburgh PA 15213 
Clinical Laboratories: 
UPMC Presbyterian Hospital 
3477 Euler Way 
Pittsburgh, PA  15213 
University of Pittsburgh-Jurczak Laboratory 
200 Lothrop Street 
Pittsburgh, PA 15213 
Study Rationale: 
Old age is associated with declining skeletal muscle mitochon-
drial bioenergetics with related decrements in cardiorespiratory 
fitness (CRF) and physical function1 that predispose to frailty, 
disability, and diminished quality of life.2 While exercise training 
may moderate and possibly even reverse declines3 in mitochon-
drial bioenergetics,4 and bring about clinical benefit, the potential 
for such improvement is typically confounded by exercise intoler-
ance with early fatigability that results from the same age-related 
mitochondrial declines.5 Consequently, sedentariness is en-
demic and insidious among the growing population of older 
adults.6 
The principal rationale for the trial is innovative as it is oriented 
to sedentariness and related fatigability, and a pharmacological 
means to moderate them. It targets a mechanistic underpinning 
to fatigability as a symptom related to limitations in mitochondrial 
bioenergetics and vascular function and explores the utility of ni-
trite supplements as a novel means to improve the underlying 
physiology and clinical correlates. 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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Study Objectives: 
To assess the impact of nitrite supplements on mitochondrial 
respiration, with associated focus on mitochondrial energetics 
and muscle perfusion, and to complete preliminary analysis on 
clinical correlates.    
Study Hypotheses: 
• We hypothesize that nitrite will increase skeletal muscle mito-
chondrial energetics.   
• We hypothesize that improved skeletal muscle mitochondrial 
respiration will relate to both direct enhancement of mito-
chondrial respiration as well as improved skeletal muscle per-
fusion. 
• We hypothesize that changes in skeletal muscle mitochon-
drial respiration will correspond to improved CRF, reduced fa-
tigability and improved PA.  
Study Aims: 
Aim 1: To show efficacy of 12-weeks of sodium nitrite treatment 
to improve mitochondrial energetics. 
Aim 2: To show efficacy of 12-weeks of sodium nitrite treatment 
to improve skeletal muscle perfusion. 
Aim 3: To explore the effects of nitrite-induced changes in mito-
chondrial energetics on CRF and metrics of clinical function.  
Overall, this protocol lays a mechanistically-oriented foundation 
for future studies to definitively assess the utility of nitrite treat-
ment as an important therapeutic option for the growing burden 
of sedentariness in old age 
Study Design: 
Prospective, randomized, placebo-controlled, double-blinded 
trial at 2 clinical sites to study the utility of a 12-week intervention 
(oral nitrite capsules vs. placebo) in older (age ≥70 years) 
healthy, sedentary adults (<1 hour of structured PA per week).  
Planned Sample Size:  
70 participants total  
Duration of Treatment: 
14 weeks (12 weeks of Intervention with 2 weeks of Post-inter-
vention testing) 
Major Inclusion Criteria:  • Age ≥70 years 
• Sedentary (<1 hour/week of volitional exercise activity) 
• Clinically stable (euvolemic; baseline HR <100 bpm) and with-
out hospitalization or invasive cardiac procedure for 6 weeks 
Major Exclusion Criteria: See complete list in section 3.3 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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Study Endpoints: 
Overall, study endpoints pre- and post- nitrites will include:  
1. 
Aim 1: Mitochondrial energetics: ex vivo assessments of 
permeabilized skeletal muscle fiber respiration with Oro-
boros (primary outcome) and in vivo assessments of skel-
etal muscle ATP production with phosphorus-magnetic 
resonance spectroscopy (31P-MRS).  
2. 
Aim 2: Skeletal muscle perfusion: near infrared spectros-
copy (NIRS) to oxygenation and perfusion.  
3. 
Aim 3: Exploratory assessment of clinical correlates. 
Symptom-limited (maximal) CPET will be used to assess 
CRF (measured as peak oxygen consumption [VO2]).7 
Submaximal CPET (resistance which corresponds to 
60%-80% peak VO2) to exhaustion to assess endurance. 
Steady-state walking test on a level treadmill (with gas ex-
change) to assess walking efficiency and fatigability.7,8 
Other functional measures include 400-m corridor walk, 
short physical performance battery (SPPB), handgrip 
strength, and daily physical activity (PA). 7,8 
 
 
 
 
1. STUDY OBJECTIVES, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
1. STUDY OBJECTIVES 
PRIMARY OBJECTIVE: To assess the impact of nitrite administration on mitochondrial 
respiration, with associated focus on mitochondrial bioenergetics and muscle perfusion. 
 
1.2 SPECIFIC AIMS 
Two-site randomized controlled trial to clarify the impact of a 12-week course of nitrite sup-
plements versus placebo on mitochondrial energetics in older sedentary adults. We will 
take an integrative physiology approach to determine the effect of nitrite therapy on a com-
prehensive assessment of mitochondrial energetics, skeletal muscle vascular function, and 
whole-body physical function (cardiorespiratory fitness [CRF], exercise endurance, 
strength, balance, and daily physical activity [PA]) and fatigability. 
Aim 1: To show efficacy of 12 weeks of sodium nitrite treatment to improve mitochondrial 
energetics. 
• We hypothesize that compared to placebo, the participants receiving nitrite supplements 
will show an improvement in: 
H1a: Skeletal muscle mitochondrial respiration, efficiency (high resolution respirometry 
[primary outcome]) and adenosine triphosphate (ATP) production (phosphorus-
magnetic resonance spectroscopy) 
H1b. Morphology and molecular underpinnings of muscle energetics (Direct Immunofluo-
rescence and light microscopy of mitochondrial structure, RNA-Seq and Western 
Blot) 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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H1c: Platelet mitochondrial respiration (Seahorse), i.e., an analysis to clarify whether 
platelets can be used as a surrogate to skeletal muscle to assess equivalent 
changes in mitochondrial bioenergetics 
Aim 2: To show efficacy of 12 weeks of sodium nitrite supplements to improve skeletal 
muscle perfusion. 
• We hypothesize that, compared to placebo, the nitrite group will show a greater improve-
ment in: 
H2a: Tissue oxygenation, as measured using near infrared spectroscopy (NIRS) during 
exercise. 
Aim 3:  To explore the effects of nitrite-induced changes in mitochondrial energetics on 
CRF and metrics of clinical function.  
• We hypothesize that, compared to placebo, the participants receiving nitrite supplements 
will show a greater improvement in: 
H3a: Increased CRF (peak oxygen uptake [VO2] achieved during progressive resistance 
on a cycle ergometer), increased endurance (duration of exercise at fixed re-
sistance 60%-80% peak VO2 intensity), increased efficiency of walking (decreased 
VO2 at steady-state [1.5 mph] and self-paced walking), increased 400-meter corri-
dor walk, increased SPPB score; increased handgrip strength, increased PA (ac-
celerometry), and decreased fatigability (during steady-state walking and 400-me-
ter corridor walk respectively).  
H3b: Changes in mitochondrial energetics and skeletal muscle perfusion induced by ni-
trite will be associated with changes in daily activity (measured by accelerometry), 
and fatigability (measured by rate of perceived exertion during steady-state [1.5 
mph] walking).  
 
1.3. BACKGROUND AND RATIONALE 
Our multi-disciplinary team has published seminal work indicating that mitochondrial bioen-
ergetics and CRF are significant determinants of physical function in older adults.9 In paral-
lel efforts, we showed efficacy of chronic nitrite supplements to improve mitochondrial bio-
energetics in older sedentary adults. Only one month of nitrite supplements significantly im-
proved ex vivo assessments of mitochondrial energetics in skeletal muscle biopsies, con-
comitant with increased skeletal muscle sirtuin-3 expression, a nicotinamide adenine dinu-
cleotide (NAD) dependent lysine deacetylase and key regulator of mitochondrial metabo-
lism. These key data reinforce the premise that nitrite enhances vital mitochondrial metabo-
lism in older adults. Moreover, improvement in muscle energetics in nitrite-treated older 
adults was linked with increased exercise efficiency as evidenced by reduced VO2 during 
submaximal steady-state walking. These data support our overarching hypothesis that ni-
trite supplements will make physical function easier such that PA will increase. 
Older adults represent the fastest growing demographic in the population of the United 
States and constitute a unique and growing healthcare challenge.10 Aging is associated 
with an inexorable decline in CRF11 and decreased PA12 with harmful implications13 that in-
clude increased vulnerabilities to disease, frailty, reduced independence, diminished quality 
of life as well as escalating healthcare costs. Yet despite typical age-related declines, many 
older adults are able to mitigate the rate of decline in fitness and activity, and to thereby 
better preserve healthfulness and well-being.14 While behavioral and community interven-
tions are often pursued as vital strategies to optimize activity and health, for many older 
adults such good intentions are stymied by intrinsic age-related exercise intolerance and 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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early fatigability15 that undermine goals to be active. Our protocol responds to such chal-
lenges by focusing on the utility of nitrite supplementation to enhance skeletal muscle mito-
chondrial energetics in older sedentary adults. We will build on our exciting preliminary data 
to clarify if mitochondrial benefits extend to improved physical function, including CRF, en-
durance, strength, balance, and PA.  
 
1.4 SIGNIFICANCE 
The protocol is conceptually innovative in multiple aspects: 
• 
The principal rationale for the trial is innovative as it targets mechanistic underpinnings, 
i.e. mitochondrial energetics and vascular function, as integral means to foster physical 
function, PA, and to diminish fatigability.   
• 
The application of oral nitrite supplementation is innovative. While seminal literature re-
garding nitrite focuses predominantly on vascular effects,30 our pilot data raise im-
portant perspectives regarding its intrinsic effects on skeletal muscle mitochondria, 
which are the source of over 90% of adenosine triphosphate (ATP) needed for move-
ment. Age-related changes in skeletal muscle mitochondrial function likely contribute to 
functional decline.1,22,31-34 
• 
We will use high-resolution respirometry to examine the activity of electron transport 
system (ETS) in permeabilized intact muscle fibers from freshly collected muscle speci-
mens. This allows us to assay ETS function in a highly controlled ex vivo experiment at 
the myocellular level, removed from other potentially limiting physiological factors in-
cluding supplies of substrates and oxygen. Specifically, we will use an Oroboros Ox-
ygraph 2k35,36 to interrogate mitochondrial ETS function when reducing equivalents are 
channeled via complex I, complex II or electron transfer flavoprotein (fatty acids). This 
method preserves mitochondrial reticulum structure and function, which avoids errone-
ous mitochondrial dysfunction observed in isolated mitochondria37,38 and will comple-
ment in vivo assessment of mitochondria. 
• 
Mitochondrial energetic capacity (ATPmax) will be determined by (phosphorus-mag-
netic resonance spectroscopy [31P-MRS]) of the non-dominant quadriceps muscle. 
ATPmax incorporates all aspects of mitochondrial content and function including oxy-
gen consumption, efficiency, and Ca2+ handling, all working together to produce ATP.39 
• 
The molecular underpinnings of improved skeletal muscle mitochondrial energetics will 
be determined via transcriptional RNA transcriptionomic profiling (RNA-seq), expression 
of key proteins (Western blots), and immunohistology (fiber type, capillary density). 
• 
The skeletal muscle mitochondrial assessments will be coupled with an analysis of 
platelet mitochondrial energetics (Seahorse) to determine the utility of platelet measure-
ments to be applied as (1) a novel and convenient biomarker of mitochondrial energet-
ics.  
• 
NIRS will be used to assess: (1) skeletal muscle mitochondrial metabolic performance 
profile during the hypoxic stress40 by gauging perfusion changes pre- and 12-week post-
nitrite versus placebo. Clinical and Physiological assessments:  
Fitness and Efficiency (1) CRF (peak VO2) on a progressive intensity cycle ergom-
eter will clarify how changes in mitochondrial respirometry translate into systemic 
oxygen utilization, highlighting physiological linkage between cellular respiration 
and whole-body oxygen utilization and putative benefits of nitrite to improve these 
relationships; (2) VO2 at 60%-80% exercise resistance (watts) on a cycle ergome-
ter until exhaustion will assess nitrite-medicated changes in endurance41,42; (3) VO2 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
11 
at a steady-state walking (1.5 mph) will assess nitrite-mediated changes in the effi-
ciency of walking and associated perceived fatigability (changes in rated of per-
ceived exertion); (4) 400-meter corridor walk (with portable gas exchange) will be 
used to assess overall capacity (disability), physical fatigability (slowing of walking 
speed), as well as the energy of walking.  
Additional functional measures: (1) short physical performance battery (SPPB) to 
assess strength, balance, and an index of frail7; (2) handgrip to assess strength; 
(3) daily activity (accelerometry).  
Fatigability: Fatigability assessments are novel, and include both “perceived” (rate 
of perceived exertion [RPE])43 during steady-state walking8, as well as “perfor-
mance” (energy consumed during a 400-meter corridor walk)44 subtypes. 
 
2.   RESEARCH DESIGN AND METHODS 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
We will conduct a prospective, randomized, placebo-controlled, double-blinded trial to study 
the utility of a 12-week intervention (oral nitrite capsules vs. placebo) in older (age ≥70 
years) healthy, sedentary adults (<1 hour of structured PA per week). The primary aims of 
this protocol are to demonstrate the utility of chronic nitrite treatment to enhance (1) mito-
chondrial energetics, (2) vascular perfusion and (3) to explore whether physiological im-
provements translate to clinical enhancements (physical function, physical activity, fatigabil-
ity). 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN  
Study Procedures and Visits:  
Study Visits and procedures will take place in the UPMC Magnetic Resonance Research 
Center (MRRC), UPMC Montefiore Translational Research Center (TRC), University of 
Pittsburgh Geriatric Research Center (GRC), or UPMC Kaufmann at the University of Pitts-
burgh Pepper Center– Senior Mobility Aging Research Training (SMART) Center and 
UPMC Emphysema/COPD Research Center.  
Water and snacks will be available to participants throughout non-fasting visits. Participants 
will be provided a meal during fasting visits 
Reminder Calls: 
Study staff will call participants to remind them of study visit details, and of medications 
hold plans. The study staff will remind participants to hold medications per their anti-platelet 
and anti-coagulation medications hold plan at reminder calls for Visit 4 and Visit 9.  Partici-
pants with diabetes who are on hypoglycemic medications will be advised if they need ad-
justments to their usual regimen for fasting and functional testing visits. Participants with di-
abetes will be asked to bring their own glucometer to assess glucose as needed. If a partic-
ipant does not provide their own glucometer, a study glucometer will be available. 
 
2.2.1 Study Procedures and Assessments 
 
Consent Visit 
1.  Obtain written informed consent using standardized procedures.  

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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2.  Medical histories will be obtained prior to the visit. At the study visit, details about 
the participants’ medical histories will be reviewed. PI or a study clinician will evalu-
ate the participants. Participants will be instructed to notify the study staff if there 
are any health or medication changes during their participation in the study. The 
Charlson Comorbidity Index and the Pittsburgh Fatigability Scale  will be adminis-
tered. 
3.  Weight, height, and vital signs (temperature, blood pressure (BP), heart rate (HR), 
respiratory rate (RR), SpO2). BP will be re-measured if it is initially outside of the in-
clusion range. This allows for common clinical variations attributable to anxiety, de-
hydration, timing of medications, and other factors.   
4.   Screening blood work by trained study staff will be completed be completed after 
consent and before randomization. See Study Timeline 2.2.2 
 
5    7-Day Home Activity Monitoring: Participants will be issued an accelerometer 
(ActiGraph) non-invasive wrist monitoring device to be worn for a full 7 days following 
visit to assess changes in daily activity, including level/intensity of activity and sleep. 
Serial assessments by ActiGraph over the weeks of the protocol provide key data to 
gauge changes in physical activity that may result from nitrite supplements over time.  
Participants will receive instruction in the use of the ActiGraphs and asked to main-
tain an ActiGraph physical activity/sleep diary for the days that they are wearing the 
device. Participants will be reminded to return their ActiGraph at the visit following 
their 7-day wear. 
 
 
 
Pre-drug baseline study visits (Visits 1- 4) 
Visit 1  
Participants will be encouraged to have a light meal prior to arrival. 
1. Interval history, vital signs, and a brief physical exam. 
2. Exercise testing: 
a. Cardiopulmonary Exercise Testing (CPET)46: A symptom-limited (maximal) 
graded cycle ergometer exercise test in association with air-gas-exchange, 
an optimal gauge of aerobic capacity will be conducted by trained research 
exercise physiologists. A study clinician will be immediately available if 
needed. A computerized cycle ergometer will be used to generate a symp-
tom-limited exercise stimulus. ACSM criteria for starting and stopping will 
also be utilized. A face mask or mouthpiece will be positioned over the sub-
ject’s mouth and nose during the exercise for gas exchange assessments. 
VO2, minute ventilation (VE)/exhaled carbon dioxide (VCO2) slope, and res-
piratory exchange ratio (RER) will be measured as well as hemodynamics 
(max HR and BP), time, and ECG waveforms). Any unexpected abnormali-
ties will be reported to the participant’s primary provider(s); continued partici-
pation in the study will require the primary provider’s clearance. The rate of 
perceived exertion (RPE) will be completed during the CPET. 
b. 400-meter corridor walk8: Participants will walk over a 400-meter course. 
During the 400-meter corridor walk, average VO2 per lap during steady-state 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
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(2 minutes of plateaued VO2) will be used to calculate the energy cost of 
walking by normalizing the VO2 to lap gait speed (ml/kg/m).  
c. For participants with diabetes, they will be asked to bring their own glucome-
ter to assess glucose before and after the CPET.  
d. NIRS: NIRS will be worn during the CPET and 400-meter corridor walk. Sen-
sors will be placed on the skin of one leg to allow the non-ionizing, LED light 
source to assess tissue oxygenation and perfusion via measurements of the 
optical absorption of hemoglobin.   
 
e. Accelerometry: An ActiGraph wrist accelerometer will be worn during the 
400-meter corridor walk to establish baseline activity parameters and during 
the CPET.  
3. A battery of questionnaires will be administered. 
a. The following questionnaires will be administered:   
b. Community Healthy Activities Program for Seniors (CHAMPS)48  
c. Fried Frailty Index50 
d. EuroQol49 
e. Montreal Cognitive Assessment51,22 
f. Patient Health Questionnaire (PHQ-2)52  
g. PROMIS-Sleep Disturbance Short Form                                                                                  
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261577/) 
h. Self-efficacy: Sullivan54 
 
4. Participants who fail to meet applicable inclusion/exclusion criteria based upon the 
results of the screening assessment will be excluded from further study participa-
tion. Such participants will be paid for the visit. 
5. Prior to departing, participants will be reminded to resume their usual medication 
regimen and clarify any modifications related to the study if indicated.  
 
Visit 2 
Participants will be encouraged to eat a light breakfast prior to arrival.  
1. Interval history, vital signs, and a brief physical exam. 
2. Steady-State Walking Test: A steady-state walking (1.5 mph) test will be completed 
on a treadmill. Participants will wear a portable CPET device during the steady-state 
walk. The rate of perceived exertion (RPE) will be completed during the steady-state 
walking test. 
3. Submaximal CPET: the participant will pedal on a cycle ergometer with a fixed re-
sistance (the resistance associated with 60%-80% peak VO2 achieved during the 
initial symptom-limited CPET on visit one). The rate of perceived exertion (RPE) will 
be completed during the submaximal CPET. 
      4.  Handgrip Strength Assessment       
      5.  Short Physical Performance Battery (SPPB) will be completed:             
a.  Sitting and standing out of a chair 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
14 
b.  Walking over a 4-meter course (to measure walking speed) 
c.  Standing with their feet in different positions to measure their balance 
6. NIRS will be worn during the submaximal CPET. NIRS and ActiGraph will be worn 
during the steady-state walk.   
7.   Standard breath-by-breath CPET ventilatory indices VO2, VE/VCO2, and RER will 
be collected and used to characterize the performance during the submaximal 
CPET as well as the steady-state treadmill walk completed in this study visit. 
8.   Three-day food record (3DFR): participants will be provided forms to complete a 
3DFR which will be analyzed for plant-based dietary nitrite and nitrate. Participants 
will be assigned dates for these self-assessments, including one weekend day. Par-
ticipants will be asked to maintain a diet that is consistent throughout the trial. Major 
dietary modifications during the 16-week trial will be discouraged. 
9.   Prior to departing, we will remind diabetic participants to resume their usual medica-
tion regimen and clarify any modifications related to the study if indicated. 
 
 
Visit 3  
Participants will be encouraged to eat a light breakfast prior to arrival.   
1.  Vital signs will be completed by study or MRRC staff.  Any concern for medical in-
stability after collecting the vital signs and the interval history will prompt contact 
with a study clinician, and possible physical exam before proceeding.  
2.   Magnetic Resonance Spectroscopy (MRS): 
MRS is an in-vivo test to measure maximal mitochondrial capacity to generate ATP. 
It will be performed on the subject’s leg as they complete a flexion-extension exer-
cise within the bore of the magnet as described below.   
Prior to the exercise, the technician will collect a fully relaxed, high-resolution 31P 
spectrum image of the resting muscle.  
The exercise protocol will then be performed: Participants lay supine with the non-
dominant knee (unless contraindicated) elevated at ~30º and straps are placed over 
the legs. Participants perform voluntary isometric contractions (kicking) repeatedly 
as hard and as fast as they can for two bouts each followed by a rest period. The 
protocol is designed to deplete PCR stores by 33-66% to ensure high signal to 
noise defining PCR recovery without inducing acidosis (pH <6.8), which inhibits oxi-
dative phosphorylation. 
3.  Participant may complete any remaining functional testing from Visit(s) 1 or 2. Visit 3 
and Visit 8 will not occur if the Subject is ineligible for the MRS. 
 
Visit 4 
Participants will be asked to fast for at least 8 hours prior to arrival. 
Participants will be asked to hold their medications per the Anti-coagulation Medications 
Hold Plan.  
1. Interval history, vital signs, and a brief physical exam  

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
15 
2. Staff will obtain verbal confirmation from participants of fasting for at least 8 hours 
prior to arrival and that Anti-coagulation Medications Hold Plan was followed. 
3. An IV line will be placed  
4. Skeletal Muscle Biopsy #1: Participants will lie comfortably in the supine position for 
biopsy of the vastus lateralis muscle of the non-dominant leg. Biopsy sites will be 
disinfected with chlorhexidine, lidocaine will then be administered to biopsy site.  
The physician investigator or study clinician will then make a small incision in the 
skin and insert a needle into the vastus lateralis to remove approximately 150 mg of 
muscle.  Additional passes of the needle may be necessary for an adequate muscle 
sample. The wound site will be closed with Steri-strips™ (or a suture if subject is al-
lergic to Steri-strips™) and a sterile pressure bandage.  
a. Muscle specimens will be immediately processed and stored. Muscle speci-
mens will be divided into portions for specific analyses. Any extra tissue will 
be stored for future analyses.       
b. The biopsy wound will be compressed and dressed. 
5. Randomization: Subject will be randomized following review of the participant safety 
status by PI. 
 
6. Pharmacokinetics (pK) 
a. Nursing staff will measure BP, RR, HR, SpO2, and MetHb to ensure subject 
is at a steady-state before administering the study drug. Baseline assess-
ments of nitrite/nitrate will be collected. Participants will receive their first 
dose of study drug (20 mg Nitrite vs. Placebo). Then, the following will be 
assessed:  BP(orthostatic hypotension assessment), HR and MetHb, RR 
and SpO2 every 15 minutes for the 2 hours post-drug administration period 
per standard operating procedure. If high MetHb level or if subject is hypo-
tensive or symptomatic, after the study drug is administered, the PI or study 
clinician will be informed and advise on plan for management. 
7. Blood assessments: 
a. Nitrite/Nitrate Serum Levels and other Research Bloods: baseline (before 
study drug), 30 minutes, 60 minutes 
b. Platelet Mitochondrial Respirometry: baseline (before study drug), 30 
minutes 
c. Research Inflammatory Indices: baseline (before study drug) 
d. Heparinized blood will be immediately centrifuged at bedside to separate 
plasma and red cells and then flash frozen for future analyses. Total volume 
of blood drawn for 3 samples will be about 80 ml (about 5.4 tablespoons).   
8. Skeletal Muscle Biopsy #2 immediately after the 30-minute blood sample is drawn.  
9. A breakfast will be provided after the skeletal muscle biopsy #2. 
10. If the study drug is well tolerated, the PI will be contacted and IDS orders for 
enough study drug/placebo will be ordered to sufficiently cover dosing for partici-
pants as described in drug distribution section. 
11. Drug supply dispersal 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
16 
a. Participants will receive sufficient study drug/placebo supply. We will instruct 
participants to return all of the study drug packaging, including unused study 
drug and empty packaging at each study visit and/or at the time of discontin-
uation from treatment. 
12. Study staff will review medication administration and use of the drug diary card with 
all participants. Participants will be asked to record the time they take the drug each 
day and any symptoms with severity rating and/or health events during the dosing 
period. 
13. The study staff will review biopsy site care with participants and if indicated, remind 
participants to resume their normal anti-coagulation/anti-platelet/anti-diabetic medi-
cations.  
14. Participants will be called 2 days after biopsy for assessment of biopsy healing.  All 
participants will be given the option of returning for an Interim Visit for direct assess-
ment if symptomatic.  
15. Daily activity: Participants will be encouraged to begin or extend a daily walking rou-
tine with specific instructions and encouragement to progress in time and length as 
tolerated.  
In order to track these changes over the course of the 12-week intervention, participants 
will be asked general questions about activity level, exertion level, and barriers to activity. 
Furthermore, the study nurse will ask about any progress as part of the interim study on-
site and phone-based assessments.  
The Daily Activity questions are not a study endpoint, but rather they are pragmatic tools to 
encourage motivation and behaviors of activity. Only the ActiGraph data are study end-
points. 
 
Midway Drug Intervention Assessment (Visit 5) 
Visit 5 (Midway): On-site assessment to assess safety and abbreviated functional assess-
ment during the 12-week intervention phase. Participants will be asked to fast for at least 8 
hours prior to arrival. 
The following procedures and assessments will be completed:  
1. Interval history, vital signs, and a brief physical exam.  
2. IV line will be placed 
3. Dose Monitoring: 
a. Subject will take morning dose of study drug 
b. Research Blood assessments at baseline (before study drug), 30 
minutes, 60 minutes: 
i. Nitrite/Nitrate Serum Levels and other Research Bloods: base-
line, 30 minutes, 60 minutes 
ii. Platelet Mitochondrial Respirometry: baseline (before study 
drug), 30 minutes 
iii. Research Inflammatory Indices: baseline (before study drug) 
iv. CBC (for safety assessment) 
4. pK assessments as in Visit 4 with orthostatic hypotension assessment Blood 
Pressures. 
5. Functional evaluation: 
a. Participants will complete the following: 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
17 
i. Steady-state walk, 400-meter corridor walk (with VO2 assess-
ment), SPPB, Handgrip Strength 
ii. Participants will complete the Steady-state walk test 
30minutes(+/-10 minutes from the 30 minute blood collection) 
6. Additional study drug capsules distributed if needed 
7. A breakfast will be provided after the 60-minute blood draw 
8. 7-Day Home Activity Monitoring 
 
Post-Drug Assessments (Visits 6-9)  
The procedures and assessments are generally similar to those performed Pre-drug base-
line study visits. 
 
Visit 6 
Participants will be encouraged to have a light meal before arrival.  Participants will be 
asked to hold their AM study drug prior to arrival. 
See Visit 1 for details. 
Study staff will update concomitant medication list and complete a symptom assess-
ment. The drug diary cards and empty drug boxes for completed weeks on study 
drug/placebo will be collected. Compliance of the study drug will be reviewed.  
 
1. Interval History, vital signs, and a brief physical exam. 
2. The participant will take their study drug 30 minutes before the CPET.   
3. Cycle Ergometer symptom-limited CPET 
4. 400-meter corridor walk 
5. Questionnaires  
6. Prior to departing, we will remind diabetic participants to resume their usual medica-
tion regimen and clarify any modifications related to the study. 
7. Blood work collected by trained study staff. (See Study Timeline 2.2.2) 
8. Prior to departing, we will remind diabetic participants to resume their usual medica-
tion regimen and clarify any modifications related to the study if indicated. 
 
Visit 7 
Participants will be encouraged to eat a light breakfast prior to arrival. The partici-
pants will be asked to take their study drug 30 minutes prior to the submaximal 
CPET.   
Interval history, vital signs, and a brief physical exam. 
 
See visit 2 for details. 
1. Steady-State Walking Test 
2. Submaximal CPET 
3. Handgrip Strength Assessment. 

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
18 
4. SPPB 
5. 3DFR will be provided to subject and collected at following visit. 
6. Prior to departing, we will remind diabetic participants to resume their usual medica-
tion regimen and clarify any modifications related to the study if indicated. 
 
Visit 8 
Participants will be encouraged to eat a light breakfast prior to arrival. The participant 
will be asked to hold the AM dose of the study drug prior to arrival. The participant will 
take the study drug ~30 minutes prior to start of the MRS.  
See Visit 3 details.   
1. MRRC Staff will collect vital signs. Any concern for medical instability after collecting 
the vital signs and the interval history will prompt contact with a study clinician, and 
possible physical exam before proceeding. 
2.   MRS 
3.   Participant may complete any remaining functional testing from Visit(s) 6 or 7 
4. Participant will take the afternoon dose of study drug 30 minutes prior to completing 
any remaining functional testing from visit 7.   
 
Visit 9 
Participants will be asked to fast for at least 8 hours prior to arrival. Participants will be 
asked to hold their AM dose of the study drug prior to arrival.   
See Visit 4 for details. 
1. Interval history, vital signs, and a brief physical exam. 
2. Staff will also confirm participant has been fasting for at least 8 hours prior to arrival 
and that any customary anti-platelet or anti-coagulation medications were held as 
instructed.  
3.   An IV line will be placed  
4. Skeletal Muscle Biopsy #1 
5. Participant will self-administer AM dose of study drug. 
6. Research Blood assessments at baseline (before study drug), 30 minutes, 60 
minutes, as per Pre drug biopsy visit. 
7. Skeletal Muscle Biopsy #2 immediately after the 30-minute blood sample is drawn. 
8. Pharmacokinetics (pK) with orthostatic hypotension assessment Blood Pressures 
every 15 minutes as noted in Visit 4. 
9. A breakfast will be provided after the second skeletal muscle biopsy. 
10. Remaining study drug/placebo, 3DFR, daily activity questions, drug diary cards, 
and/or Actigraphy and diary will be collected from subject prior to discharge. 
11. Participants will be called 2 days after biopsy for assessment of biopsy healing.  All 
participants will be given the option of returning for an Interim Visit if they prefer a 
direct assessment.  

Nitrites, Skeletal Muscle Mitochondrial Bioenergetics, and Physical Activity in Old Age               
19 
12. The study staff will review biopsy site care with participants and if indicated, remind 
participants to resume their normal anti-coagulation/anti-platelet/anti-diabetic medi-
cations 
13. Following safety monitoring the study staff will schedule final follow up phone call for 
1 week after visit 9.  
 
Weekly Telephone Calls    
Following Visit 4, the study staff will contact participants by phone each week to review any 
symptoms. Staff will continue to call weekly until 1 week after visit 9. Each call will last ap-
proximately 5-10 minutes. We will ask participants if they have missed any study drug 
doses and if there have been any changes in their medications or medical history, including 
emergency room visits or hospitalizations since their study visit. We will ask participants to 
review their drug diary card to report what they wrote down for the week to help them re-
member.    
We will also remind participants to contact the study staff or doctor anytime their health 
care team suggests a change in medications or if they have any symptoms that cause con-
cern. There will be study personnel available to address participant’s questions or concerns 
24 hours/7 days a week. 
During the drug intervention period, participants will also be asked about their activity lev-
els, exertion levels, activity enjoyment, and barriers to activity as part of the weekly calls.

 
2.2.2 Study Timetable 
Study Phase 
Pre-Drug Assessments 
Midway 
Assess-
ment 
Post-Drug Assessments 
 
Con-
sent 
Visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 
Approximate 
Time  
between  
Study Visits 
 
Same 
day to 
+30 
days 
Visit 1 
+1 day 
to  
±14da
ys 
Same 
day to 
+7 days 
Previ-
ous 
Visit 
+1 day 
to±14d
ays 
6 weeks  
±5 days  
6 
weeks 
±5 
days 
Visit 6 
days 
±14 
days 
Same 
day to 
+7 
days  
Previous 
Visit to   
±14days 
Approximate  
Duration 
 of  
each visit 
1-2 
hours 
3-5 
hours 
3-4 
hours 
1-2 
hours 
3-4 
hours 
1-2 
hours 
3-4 
hours 
3-4 
hours 
1-2 
hours 
3-4 
hours 
Fasting  
prior to arrival 
 
 
 
 
X 
X 
 
 
 
X 
Consent 
X 
 
 
 
 
 
 
 
 
 
Medical  
History 
X 
 
 
 
 
 
 
 
 
 
Physical exam 
X 
X 
X 
X+* 
X 
X 
X 
X 
X+* 
X 
Vital Signs 
X 
X 
X 
X 
 
 
 
 
 
 
Vital Signs  
with 
Methemoglobin 
 
 
 
 
X 
X 
X 
X 
X 
X 
Max CPET* 
 
X 
 
 
 
 
X 
 
 
 
Sub-Max CPET*  
 
X 
 
 
 
 
X 
 
 
400-Meter Corri-
dor Walk*# 
 
X 
 
 
 
X 
X 
 
 
 
Steady-state 
walk*# 
 
 
X 
 
 
X 
 
X 
 
 
SPPB 
 
 
X 
 
 
X 
 
X 
 
 
Handgrip 
 
 
X 
 
 
X 
 
X 
 
 
Home Actigraph 
X 
* 
 
 
 
 
X 
 
 
 
Questionnaires 
X 
X 
 
 
 
 
X 
 
 
 

 
21 
3 Day  
Food Record 
 (3DFR)++ 
 
 
X 
 
 
 
 
X 
 
 
MRS 
 
 
 
X 
 
 
 
 
X 
 
Randomization 
 
 
 
 
X 
 
 
 
 
 
Muscle Biopsies 
1 & 2 
 
 
 
 
X 
 
 
 
 
X 
Blood Draw Ap-
proximate total 
quantity 
20 ml+ 
 
 
 
 
83  ml 
81 ml 
20  
mL 
 
 
81 ml 
CBC 
X+ 
 
 
 
 
X 
X 
 
 
 
Complete Meta-
bolic Panel 
(CMP) 
X+ 
 
 
 
 
 
X 
 
 
 
25-hydroxy vita-
min d (Vitamin 
D) 
X+ 
 
 
 
 
 
X 
 
 
 
HbA1c 
X+ 
 
 
 
 
 
X 
 
 
 
TSH 
X+ 
 
 
 
 
 
 
 
 
 
Nitrite/Nitrate 
 
 
 
 
X 
X 
 
 
 
X 
Inflammatory In-
dices 
 
 
 
 
X 
X 
 
 
 
X 
Additional Re-
search Bloods 
 
 
 
 
X 
X 
 
 
 
X 
*Assessment with NIRS 
#Assessment with ActiGraph 
+ Blood draw may be collected after signing consent and before randomization, blood 
draw may not include CMP and/or CBC/d if collected within the last 4 weeks 
++ 3 Day Food Record distributed at Visit 1 and Visit 6 and collected from subject at Visit 
2 and Visit 7 
+* If deemed necessary 
 
 
 
 

 
22 
 
 
 
2.3 STUDY DRUG 
 
Dr. Gladwin has developed nitrite capsules for oral dosing prepared using 14-nitrogen formula-
tion in collaboration with the University of Iowa’s drug development program and FDA approved 
under IND 115,926. Greater than 99.9% of naturally occurring nitrite exists as 14N nitrite. 
 
2.3.1. Study Drug Preparation and Dispensing  
The institutional Investigational Drug Service (IDS) pharmacy at the University of Pittsburgh will 
select study drug/placebo by the randomization sequence number. The independent research 
pharmacist at IDS will package all drugs/placebos to be similar in physical appearance. The 
study drugs are oral formulation of 20 mg sodium nitrite and matching placebo. Sodium nitrite of 
dose strengths and matching placebo will be supplied as capsules for oral administration. All 
capsule formulations will be identical in appearance (size, shape, color) and smell. The packag-
ing and labeling will be designed to maintain blinding to the Investigator’s team and to partici-
pants. 
 
2.3.2. Drug Administration 
IDS will be provided an updated order for dispensing drug/placebo at baseline and PRN. Partici-
pant will be instructed to bring remaining supply to follow up visit for interim monitoring counts.   
 
2.3.3. Dose Selection 
In the 2001 National Toxicology Program (NTP) Report summarizing 2-year rodent drinking wa-
ter studies, there was no evidence of carcinogenic activity of in male or female F344/N rats ex-
posed to up to 130 mg/kg/day in males and 150 mg/kg/day in females, or in male B6C3F1 mice 
exposed to up to 220 mg/kg/day. There was equivocal evidence of carcinogenic activity of so-
dium nitrite in the highest dose of 165 mg/kg/day in female B6C3F1 mice based on the positive 
trend in the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach. 
Exposure to sodium nitrite in drinking water resulted in increased incidences of epithelial hyper-
plasia in the forestomach. However, no chromosomal damage (genetic toxicity) was observed in 
three studies conducted in rats and mice in vivo.55 Taken together; these findings suggest mini-
mal carcinogenic nitrite-mediated risk. 
 
Multiple studies now demonstrate the predominantly positive safety profile of nitrate therapy in 
humans (most typically administered as beetroot juice).56 At the University of Pittsburgh, several 
pilot studies show safety and efficacy of nitrite, the active metabolite of nitrate, and show strong 
physiological rationale for its use.  
 
Kara Hughan is a co-investigator on this protocol. She has been studying utility of oral nitrite 
supplements (administered as capsules) to improve insulin sensitivity over 12 weeks in adults 
aged 18-60 years with metabolic syndrome in an NHLBI K23 funded study. Hughan’s first sub-
ject (healthy) was started on nitrate 1,000 mg once daily (with NO3 supplying a more prolonged 
source of plasma nitrite across the day via enterosalivary circulation until next dosing) in combi-
nation with nitrite 20 mg once daily.  
 
During the first subject’s drug course, PK plasma nitrate and nitrite concentrations, MetHb and 
BP data were assessed every other week. Given that only minor increases in MetHb were de-

 
23 
tected (that remained well within the safety threshold) and that the plasma nitrite rise after in-
gestion then fell to baseline after ~3-6 hours with nitrite dosing, requests were made to the IRB 
to increase nitrite dosing to 20 mg two times daily (bid)  this does not pose undue risk to partici-
pants.  
 
2.3.4. Treatment Period 
Participants will undergo study drug treatment duration of up to 16 weeks of nitrite vs. placebo 
three times daily; study drug during the 12 week intervention period and over the 2-4 week post 
intervention period. The two to four additional weeks during post intervention period are in-
cluded to:  
• 
allow continuation of the study drug/placebo through the final outcome collection series 
of visits 
• 
Range of two to four post intervention weeks accounts for minimum length of time be-
tween Visits 6 and Visits 9 and the maximum length of time allowed between Visit 6 
through Visit 9.  
 This is considered appropriate to study benefits of oral nitrite therapy. 
 
2.3.5. Breaking the Blind  
All participants and study personnel (except the study statistician, the dedicated providers, our 
safety officer and the study adjudicator) involved in assessments will be blind to treatment as-
signment. 
 
2.3.6. Medication Compliance 
At Visit 4, participants will self-administer their first dose of study medication under the supervi-
sion of the physician investigator or study clinician in the outpatient facility following completion 
of all baseline clinic assessments. After the pK (and participant is stable after first dose), partici-
pants will be dispensed their study medication for home use in prefilled seven-day labelled pill 
boxes to help with compliance and to help reduce burden or confusion. On the weekly phone 
calls, the study coordinator will review the daily diary card (study drug and symptoms), inquire 
about missed doses and review symptom assessments. Participants will return empty pill boxes 
when they return for study visits. Medication adherence will be reviewed. Participants will return 
any remaining capsules at their Visit 9 for final accountability. In the event that the adherence 
rate is <80% at any time reported by the subject, participants will be re-educated on medication 
adherence. If medication compliance repeatedly falls outside of the acceptable range, the study 
investigators will discuss subject eligibility for continued participation in the study. Of note, ad-
herence during Dr. Forman’s pilot study was excellent with no subject approaching the 80% 
level of concern. 
 
2.3.7. Medication Storage and Accountability 
The study investigators or the study coordinator will document the amount of study drug/placebo 
dispensed and/or administered to participants, the amount returned by participants, and the 
amount received from and returned to the UPMC-IDS. The study drug/placebo accountability 
records will be maintained throughout the course of the clinical trial. 
 
2.3.8 Concomitant Medications 
Potential participants will be asked during phone screening if prescribed medication on the ex-
clusion criteria list. Potential participants will be asked to provide a list of concomitant medica-
tions at screening Visit 1. All candidates using vasoactive medications, unable to withhold war-
farin (or use bridging therapy) prior to muscle biopsy, or unable to withhold thienopyridine medi-
cations for 5 days prior to muscle biopsy will be excluded. Participants taking daily aspirin, non-
steroidal anti-inflammatory medications, or direct oral anticoagulants will be asked to hold 3 

 
24 
days prior to biopsy. If unable to hold aspirin, PI/study clinician will hold pressure after biopsy to 
prevent further bleeding for an additional 3 minutes.  
 
2.3.9 Rescue Medications 
Not applicable 
 
2.3.10 Randomization and Blinding:  
We will use the high quality pseudo-random deviate generator in SAS® (SAS Institute, Inc., 
Cary, North Carolina) to randomize participants to NO or placebo in 1:1 ratio. Because of the 
moderately large sample size and the resulting opportunity for participant characteristic imbal-
ance between the arms and the threat it would pose to internal validity, we will employ an adap-
tive randomization scheme to ensure balance by design rather than chance with respect to 
some key characteristics. Specifically, stratified by study site, we will use a variation of the mini-
mization algorithm that can account for both continuous and categorical variables to ensure bal-
ance with respect to gender and walking speed with 80% biased coin probability; and simultane-
ously guard against heavy sample size imbalance.68 Such adaptive schemes have been shown 
to be valid alternatives in trials. Study personnel will call in all prescriptions to the independent 
research pharmacist at the institutional Investigational Drug Service (See Resources and Envi-
ronment), who will package all drugs/placebos to be similar in physical appearance. We have 
successfully employed the same process in other recently completed trials. Therefore, all partic-
ipants and study personnel (except the study statistician and the dedicated provider of clinical 
care related to study safety) involved in assessments will be blind to treatment assignment. 
 
2.4 STATISTICAL ANALYSIS 
2.4.1 Sample Size Justification 
The principle focus of the proof of mechanism trial is the impact of nitrite on mitochondrial ener-
getics. To detect a significant difference in mitochondrial state 3 respiration in permeabilized fi-
bers (the primary outcome for Aim I), the planned sample size is 35 per group (a total of 70 par-
ticipants). Respiration data from a previous multi-site RTC that was co-led by Dr. Coen, showed 
a baseline values of 270.30±99.80 pmol/min/mg (mean ± standard deviation) for State 3 respira-
tion and a pre- to post-intervention change of 68.5±97.61 pmol/min/mg58 We conservatively an-
ticipate a dropout rate of approximately 14%. Using published methods59-62 implemented in 
commercial software (PASS 2012®, Number Cruncher Statistical Systems, Kaysville, Utah), with 
70 patients and 60 anticipated completers, we will be able to detect statistical significance of a 
difference as small as 71.80 pmol/min/mg in pre- to post-nitrite change with 80% power in a 2-
tailed test at the α=0.05 level. The criterion for a minimally clinically important difference (MCID) 
has not been authoritatively established for the measure, but the above detectable difference 
corresponds to a moderate to large effect size (d=0.74) by Cohen’s criterion.63 Therefore, the 
planned sample size affords an appropriate level of statistical sensitivity to address the aims in a 
mechanistic study with proximal biological outcomes such as ours. 
For Aim 3, with the anticipated 30+30 completers, we will be able to detect statistical signifi-
cance of correlations as small as 0.49 and 0.35, respectively, between changes in mitochondrial 
energetics and changes in physical activity/function and fatigability with 80% power in two-tailed 
tests at α=0.05 conducted without and with stratification by treatment group.  As such, the 
planned sample size will afford sufficient statistical sensitivity to detect even moderate associa-
tions between mitochondria respiration and clinical endpoints. 
 
2.4.2 Data Analysis 
Overview: All statistical analyses will be performed and overseen by the study statistician Dr. 
Perera (see LOS) using SAS® version 9 (SAS Institute, Inc., Cary, North Carolina). Participant 

 
25 
flow will be summarized using a CONSORT diagram. Data will be summarized by intervention 
arm and time point as well as baseline to follow-up change using appropriate descriptive statis-
tics.  First, the baseline participant characteristics will be compared between the two arms.  Any 
significant differences will be noted and accounted for as additional covariates in the sensitivity 
analyses. Second, main analyses to address the aims will be performed as outlined below using 
multiple imputation for missing data. The primary analysis will be performed to test the primary 
hypotheses H1a about the primary outcome of mitochondrial respiration (high resolution respi-
rometry). Secondary analyses will be performed for remaining hypotheses, secondary outcomes 
and other exploratory analyses. Third, we describe below our primary approach to missing data 
and a set of sensitivity analyses by including additional covariates and ignoring missing data.  
Baseline Comparison: Due to the adaptive randomization scheme, it is unlikely that baseline 
participant characteristics will be significantly different between the arms. If we do find any, they 
will be included as additional covariates in the sensitivity analyses. We will not alter the primary 
analytic strategy to preserve its a priori nature and predictability. We will use independent sam-
ples t- or Wilcoxon rank sum tests, as appropriate based on distributional properties, to compare 
continuous baseline characteristics between the intervention arms. For categorical baseline par-
ticipant characteristics, we will use chi-square and Fisher’s exact tests, as appropriate. Statisti-
cal significance of the intervention term will be interpreted as indicating the need to include them 
as covariates in sensitivity analyses.  
Aim 1 Analysis: Analysis of covariance (ANCOVA) model using the SAS® GLM procedure with 
baseline to follow-up change in mitochondrial respiration (high resolution respirometry) as the 
dependent variable, intervention arm (nitrite/placebo) as the effect of main interest, and the 
baseline value of mitochondrial bioenergetics as a covariate. The magnitude and statistical sig-
nificance at α=0.05 of the nitrite vs placebo contrast will serve as the formal test of the primary 
hypothesis H1a.    
We will employ the same analytic strategy described above for the primary outcome mitochon-
drial respiration for each of our remaining secondary continuous outcomes of mitochondrial bio-
energetics, skeletal muscle perfusion and clinical measures collected only pre- and post-inter-
vention. The magnitude and statistical significance of the nitrite vs placebo contrasts will serve 
as formal tests of the hypotheses H1b-c and H2a-b.  
Aim 2: For physical function and activity measures additionally assessed midway, and hypothe-
sis H3a, we will instead employ a linear mixed model to accommodate multiple post-randomiza-
tion assessments and the resulting stochastic non-independence of observations. Specifically, 
we will fit a series of linear mixed models with change from baseline in each measure of physi-
cal function/activity as the dependent variable; intervention arm (nitrite/placebo), follow-up time 
(mid-course assessments and12 weeks) and their interaction as fixed effects; baseline value of 
the dependent variable as a fixed-effect covariate; and a participant random effect to account for 
multiple observations from each participant. We will appropriately construct means contrasts to 
make comparisons between intervention arms at each of the follow-up time-points, and interpret 
their statistical significance as supporting hypothesis H3a.     
Aim 3 (hypothesis H3b) Analysis: We will examine whether changes in mitochondrial energetics, 
muscle perfusion and physical function/activity/fatigability tend to move at least partially in 
unison. We will compute Pearson and Spearman rank correlations between pre- to post-treat-
ment changes in the above measures over the treatment period. We will consider correlations 
both with and without stratification by treatment group. We will also consider adjusting for base-
line values of the measures considered and obtain partial correlation coefficients to isolate the 
associations in changes above and beyond any cross-sectional associations at baseline and 
comparing the correlations between groups using the Fisher’s z-transformation. Finally, we will 
explore multivariable linear regression models with change in measures of physical function/ac-

 
26 
tivity as the dependent variable; treatment group, and baseline and changes in measures of mi-
tochondrial energetics as independent variables, taking care not to exceed the number of inde-
pendent variables that could be realistically afforded by the effective sample size.  
Sensitivity Analyses: We will perform a series of sensitivity analyses to ensure the robustness of 
our results against various assumptions. One such analysis will involve inclusion of gender and 
baseline participant characteristics significantly different between groups as additional covari-
ates in the ANCOVA and linear mixed models above. Another will involve sensitivity of results to 
missing data handling techniques. In addition, if analyses of residuals from the models show vi-
olations of statistical assumptions, we will consider fitting models after Box-Cox transforming the 
continuous variables. 
 
2.4.3 Safety Analysis 
We will describe and descriptively compare proportions of those with clinical endpoints/adverse 
events such as hospitalizations, urgent clinic/ER visits, and death between the treatment 
groups, as using chi-square and Fisher’s exact tests for dichotomous outcomes with such a 
small number of participants is unlikely to reliably demonstrate statistical significance. 
 
2.4.4 Handling Missing Data 
The best approach for handling missing data is prevention. Despite our best efforts, missing 
data will occur. To minimize missing data, we will use a multi-pronged approach:  
 
1. Adherence to protocol measurement completion by participants. We will compensate partici-
pants for participation in trial by assessment.   
 
2. Completion of protocol measurements by staff. We employ an experienced group of research 
staff, familiar with the measurement protocols described. We utilize a double check system dur-
ing participant visits to assure all data are collected. We have available back-up systems for 
most of the outcome assessments, should we experience equipment failure. Staff is cross-
trained to allow for planned or inadvertent absences.  
 
3. We will clearly document those with missing data and reasons in the CONSORT diagram and 
compare those with missing data to complete data with respect to available data.  Statistical 
guidelines for handling missing data recommend methods such as multiple imputation, which 
consider the uncertainty involved in imputing missing data.  Multiple imputation is arguably the 
best available objective method to analytically account for missing data under the ignorable or 
missing-at-random (MAR) assumption.  Specifically, we will generate M=5 imputed values for 
each missing value, analyze the 5 datasets as though complete, and finally combine the results 
appropriately so that they reflect the uncertainty involved in imputation. SAS® MI and MIANA-
LYZE procedures will be used. Other approaches to missing data, including the naïve ap-
proaches of ignoring the missing values and last-value-carried-forward (LVCF) will also be con-
sidered in sensitivity analyses and robustness of the results to using these approaches will be 
examined. 
 
2.4.5 Data Management 
The PI will oversee all aspects of data management. With the consultation of the Data Center 
(DC), the PI and coordinator will develop an operations manual to standardize all procedures and 
staff training in areas such as patient recruitment, measurement, assessment, and data entry, 
management, and security.  
 

 
27 
Data management will follow the policy and procedures of the DC. The data entry system will be 
created using .NET 2.0, Microsoft Access, or SQL. We will create a tracking database that will be 
linked to the data entry database that will monitor enrollment, and track follow-up rates and the 
data entry process, providing up to date status reports. The database will include routine data edit 
checks for consistency both within and between forms. DC will work closely with investigative 
team and other study personnel to ensure that protocols are being followed, data integrity and 
confidentiality are maintained, and that the data contains a minimum amount of missing data. All 
study files residing in designated network folders will be backed-up daily and archived weekly. 
The weekly data are stored in a safety deposit off site (> 1 mile off campus). The weekly archived 
files are maintained for 1 year until the data are erased. All screened study participants will be 
assigned unique study identifiers that will appear on all data collection instruments, tapes, docu-
ments, and files used in the statistical analysis and manuscript preparation. Only authorized team 
members will have access to personal information needed for tracking and informed consent. 
Other data quality assurance measures will include detailed documentation of computer opera-
tions and data editing procedures and regular meetings with project staff to review any changes 
in procedure. The DC also has specific data quality measures that will be implemented.  These 
include data verification, built in data validation mechanisms such as logic and out of range data 
checks, and repeated evaluation of the data collection and entry process.   
 
3. HUMAN SUBJECT 
3.1 PARTICIPANT POPULATION 
Potential participants who meet the inclusion criteria, and have none of the exclusion criteria, will 
be enrolled without restriction as dictated by the study protocols. We will make efforts to enroll 
participants in this research in a distribution that mirrors the study population of the Pittsburgh 
area.  
3.2 INCLUSION CRITERIA 
• 
Age ≥70 years 
• 
Sedentary (<1 hour/week of volitional exercise activity) 
• 
Clinically stable (euvolemic; baseline HR <100 bpm) and without hospitalization or inva-
sive cardiac procedure for 6 weeks 
3.3 EXCLUSION CRITERIA  
 
• 
Blood pressure <110 or >160/95 mmHg 
• 
Participant is at greater risk of hypotensive adverse events caused by sodium nitrate fol-
lowing review of past medical history and physical exam. 
• 
Participants with GDPD deficiency in whom sodium nitrate might increase the risk of he-
molytic crisis.  
• 
Orthopedic or other chronic condition which limits physical activity or exercise testing as-
sessments 
• 
If valve replacement has been performed, patient may not be enrolled for 12 months after 
this procedure 
• 
Severe peripheral or pulmonary artery disease  
• 
Anemia: Hgb <11.0 (♂),10.0 (♀) gm/dl  
• 
Participants with diabetes whose HgbA1c >10.0%  

 
28 
• 
Chronic alcohol (>14 drinks ETOH a week) or drug (any cocaine, methamphetamine, and 
cannabis ≥4 x week) dependency  
• 
Allergy to lidocaine; Allergy to Red Dye 
• 
Chronic use of oral corticosteroids or other medications that affect muscle function  
• 
Current use of organic nitrates or phosphodiesterase type 5 (PDE5) inhibitors  
• 
Unable to hold warfarin, direct-acting oral anticoagulants (DOACs), non-steroidal anti-in-
flammatory medications (NSAIDs) or aspirin for 3 days prior to muscle biopsy, or to hold 
thienopyridine medications for 5 days prior to muscle biopsy. Participants unable or un-
willing to hold will follow the modified ASA hold plan 
• 
Any bleeding disorder that would contraindicate biopsy such as history of clinically signif-
icant bleeding diathesis (e.g., Hemophilia A or B, Von Willebrand's Disease or congenital 
Factor VII deficiency)  
• 
Unstable psychiatric diagnosis that would affect adherence and ability to complete the 
protocol  
• 
Dementia or inability to give informed consent or follow study protocol 
• 
End-stage disease 
• 
Other chronic unstable disease such as active neoplasm, end stage chronic kidney, liver 
or other organ disease  
Relative Exclusions  
• 
Participants with a non-3T MRI compatible pacemaker, implantable cardiac defibrillator, 
stent or other identified metal in their body will be excluded from the Magnetic Resonance 
Spectroscopy study.  
• 
Subject who use PDE5s for erectile dysfunction and are willing to withhold use 24 hours 
prior and during entire dosing period may be enrolled.  
4.   IRB APPROVAL AND FDA AMENDMENTS 
The Investigator will obtain, from the University of Pittsburgh Institutional Review Board 
(IRB), prospective approval of the clinical protocol and corresponding informed consent 
form(s); modifications to the clinical protocol and corresponding informed consent forms, 
and advertisements (i.e., directed at potential research participants) for study recruitment.    
The only circumstance in which a deviation from the current IRB-approved clinical proto-
col/consent form(s) may be initiated in the absence of prospective IRB approval is to elimi-
nate an apparent immediate hazard to the research subject(s).  In such circumstances, the 
Investigator will promptly notify the University of Pittsburgh IRB of the deviation. The Investi-
gator should also notify the sponsor of this event.   
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 CFR 
Parts 50 and 21 CFR 56, and in conformance with applicable International Conference on 
Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
In the event that the University of Pittsburgh IRB requires, as a condition of approval, sub-
stantial changes to a clinical protocol submitted under an FDA-accepted IND application, or 
in the event of the Investigator’s decision to modify the previously accepted clinical protocol: 
• 
For Phase 2 and 3 clinical studies:  The Sponsor will submit (i.e., in advance of imple-
menting the change) a Protocol Amendment to the IND describing any change to a 
Phase 2 or Phase 3 protocol that significantly affects the safety of participants, the scope 

 
29 
of the investigation, or the scientific quality of the study.  Examples of Phase 2 and 3 
clinical protocol changes requiring the submission of a Protocol Amendment include: 
i. 
Any increase in drug dosage or duration of exposure of individual participants to 
the investigational drug beyond that described in the current protocol, or any sig-
nificant increase in the number of participants under study. 
ii. Any significant change in the design of the protocol (such as the addition or dele-
tion of a control group). 
iii. The addition of a new test or procedure that is intended to improve monitoring 
for, or reduce the risk of, a side effect or adverse event; or the dropping of a test 
intended to monitor the safety of the investigational drug. 
 
5. RECRUITMENT AND INFORMED CONSENT PROCEDURES 
5.1 RECRUITMENT METHODS:  
A variety of mechanisms will be used to recruit participants for this protocol 
a. Potential candidates will be identified through screening of inpatients at the UPMC Presby-
terian, UPMC Montefiore, UPMC Mercy, and UPMC Shadyside hospitals. Potential candi-
dates will be identified through screening of inpatients’ medical records for subjects 70 years 
and older with past medical history relative to inclusion and exclusion criteria. Recruiter will 
request bedside nurse to ask patient if it is okay to approach and explain study. Those inter-
ested in participating further will be provided the study brochure and study contact infor-
mation or the option to complete the initial screening in person.  
b. UPMC patients can be referred by their physician and/or cardiologist from the UPMC Cardi-
ology Center, General Cardiology clinic, Benedum Geriatric Center, the Comprehensive Pul-
monary Hypertension (PH) Program or other UPMC or community facility. For UPMC Pres-
byterian/Montefiore-based clinics, we will obtain physician and HRPO approval for screening 
of subject medical records for identification of eligible candidates. Dr. Forman currently al-
ready has and will confirm continued approval from UPMC physician groups to screen their 
patients. He will engage with additional cardiologists and geriatricians for permission to 
screen from those clinics. Once identified as preliminarily eligible, the study coordinator/staff 
will reaffirm with the subject's physician on the clinic visit day that recruitment of the subject 
is medically appropriate. Staff will also request direction from the attending physician as to 
the timing of speaking to the subject, i.e., before or after the subject has seen the physician 
for their visit. This will be done on a case-by-case basis. Study staff will speak with candi-
dates at clinic visits or by telephone call to assess interest and review medical history to as-
sess eligibility.  
Participants referred from facilities outside the UPMC Epic medical record system will be 
provided a medical record release for their cardiologist/physician to release needed docu-
ments to confirm eligibility. 
Participants can also be identified from the inpatient Cardiology service. The study coordina-
tor will follow the same procedure as for outpatients by screening the inpatient service for 
participants 70 years of age and older. Such participants will need to be followed for 6 
weeks of disease stability before enrollment, but recruitment and ascertainment of interest 
can be initiated, with consideration of medical circumstances in each case. As noted, per-
mission to speak with participant will be obtained from the Cardiology attending. 
c. Research Registries 

 
30 
Two research registries are available to the PI. The HRPO submission will include the re-
quest to use the Clinical Translational Science Institute’s PittPlusMe Research Registry, 
which includes a database of over 90,000 individuals who have indicated their interest in 
participating in research studies. The PittPlusMe initiative includes the use of engaging par-
ticipants via social media. 
Second, the Claude D. Pepper Center has a Research Registry of over 3000 community 
dwelling participants that are >60 years of age and is also available once HRPO and Pepper 
Registry approves.  
d.  Advertisements 
An approved study flier and recruitment brochure will be placed in key places and/or be dis-
tributed to physician offices, related clinics, or on other occasions/venues that present as 
an opportunity to recruit (e.g., a PI speaking engagement or a community outreach event to 
reach minority participants). Potential participants can self-refer by contacting the study 
staff via a telephone number/email address that is provided on these advertisements. Study 
staff will utilize the approved phone screening script when responding to interested candi-
dates. With subject permission, they will be screened on the phone to make a preliminary 
assessment of eligibility. We will obtain permission to access their medical records in the 
UPMC database or request records from their provider as needed to further document eligi-
bility.  
Advertisement, such as on radio, television, or print copy in newspapers, or bus signs may 
also be utilized depending on recruitment rates.  
e. Once participants have been determined to be eligible from medical record review and at-
tending approval, recruitment and enrollment procedures will then follow, including: 
i. Confirmation of eligibility by PI 
ii. Updating participants’ cardiologist and confirming management of holding anti-coagulants 
medication pre-muscle biopsy per protocol, if needed.  
iii. Setting dates for patient visits with subject and staff will also occur prior to screening visit 
to maintain timeliness of visits per protocol 
Scheduling outpatient screening visits (Consent and Visit 1) where study risks and potential 
benefits, and rights as a research subject will be described in detail, informed consent will be 
obtained and where final eligibility will be confirmed 
5.2 INFORMED CONSENT PROCEDURES  
Participants must provide informed consent. The information about this study will be given to the 
subject in language understandable to them. Either the physician investigator or a non-physician 
member of the research team will present the study. They will verbally present a general outline 
of the research plan, including inclusion and exclusion criteria, to the prospective participant. 
The consent form, outlining the design of the study, will include the risks and benefits of partici-
pating, and will be reviewed and the physician investigator and/or non-physician member of the 
research team will answer any questions. Prospective participants may take as much time as 
required to make an informed decision. Written informed consent will be obtained from each 
participant and the physician investigator prior to performing any research study procedures. 
 
6.  POTENTIAL RISKS AND BENEFITS 
6.1 POTENTIAL RISKS 

 
31 
6.1.1 Risk of Experimental Drug Intervention 
Nitrite: Numerous studies have evaluated acute, subacute and chronic drinking water exposures 
of nitrate and nitrite in laboratory animals and drinking water and dietary exposures in humans. 
Recent studies are available using high doses of nitrite by oral route in the form of beet root 
juice. Recent studies have evaluated acute exposures of oral preparations of nitrite and nitrate 
on PK and BP and are characterized below. More extensive human data is available on paren-
teral sodium nitrite as it is currently available and approved by the FDA for use in the emer-
gency treatment of cyanide poisoning. It is also notable that neutrapharmaceutical preparations 
are currently being sold with levels of nitrite (12.7 mg per tablet) and nitrate (3.9 mg per tablet). 
Sodium nitrite has been used commercially as a food preservative, an anti-corrosive agent, a 
coloring agent, and an anti-anginal agent, with additional uses in laxatives, burn ointments, and 
liniments. Amyl nitrite has been inhaled or ingested as a euphoric stimulant. Nitrite has also 
been found as a contaminant in well water. Literature searches generated case reports of nau-
sea, vomiting, abdominal pain, dizziness, headache, flushing, cyanosis, tachypnea, dyspnea, 
hypotension and death attributed to excess nitrite (high-dose) exposure from these sources as a 
consequence of methemoglobinemia due to oxidation of heme-iron in oxyhemoglobin. Normal 
background methemoglobin production is 1-3%. If levels of methemoglobin rise above approxi-
mately 30% of total hemoglobin, a subject may appear cyanotic and experience dyspnea, due to 
the reduced oxygen carrying capacity of hemoglobin (methemoglobin cannot bind oxygen). Lev-
els above 50% can cause seizures, hypotension, coma and death. Sodium nitrite administration 
for cyanide poisoning at the labeled dosage of 300 mg causes methemoglobinemia, a desirable 
effect, as methemoglobin binds to cyanide, thus protecting cellular mitochondria. A standard 
dose of nitrite used for cyanide poisoning is 300 mg up to 600 mg. Note that methemoglobin lev-
els have never risen higher than 3% at the currently used therapeutic doses (<75 mg) in 80 vol-
unteers in phase I studies at the NIH. 
The Sponsor of this study protocol, Dr. Gladwin, has previously held an IND for sodium nitrite 
(IND # 70,411) for cardiovascular applications and currently has an approved IND for the use of 
sodium nitrite for lung transplant recipients (IND # 111,643). The cardiovascular IND involved 
the administration of sodium nitrite to 69 normal volunteers in 4 phase I-II clinical trials without 
observed adverse effects. He has also treated 11 participants with sickle cell disease on this 
IND without observed adverse effects. The lower doses of nitrite used in these investigational 
treatment regimens – 60-120 mg daily or 20-40% of the dose (300 mg) used in the emergency 
treatment of cyanide poisoning–do not produce methemoglobin levels greater than 3% and 
have not been associated with clinically significant hypotension. There have been no adverse 
events noted in the 80 treated normal human volunteers and patients with sickle cell disease.67 
In another study by Gladwin et al., 18 healthy adults received an infusion of sodium nitrite total-
ing 75 mg (15 minutes each x 2 infusions). This was associated with a 7 mm Hg decrease in 
mean arterial pressure, a peak methemoglobin of less than 3% and no other significant effects. 
Note this single dose is 1.9 times the larger single dose (tid) we plan to use in this trial.  
In Dr. Forman’s current pilot study, 20 mg tid were used, which were lower or similar to the 
doses of nitrite cited and used safely in any of the noted studies. 
To summarize, we anticipate the following symptoms by organ system and likely frequency of 
risk: 
Gastrointestinal 
• 
Common:  none 
• 
Frequent:  none 
• 
Infrequent:  dry mouth 
• 
Rare:  nausea, abdominal pain and vomiting 

 
32 
Hematologic 
• 
Common:  none 
• 
Frequent:  none 
• 
Rare:  methemoglobinemia 
Cardiovascular 
• 
Common:  none 
• 
Frequent:  hypotension in older participants on higher nitrite doses 
• 
Rare:  flushing, tachycardia, hypotension in healthy controls 
Neurologic 
• 
Common:  none 
• 
Frequent: none 
• 
Rare:  headache, dizziness, seizure, coma 
Respiratory 
• 
Common:  none 
• 
Frequent:  none 
• 
Rare:  tachypnea, dyspnea, cyanosis 
Renal 
• 
Nocturia: infrequent 
 
6.1.2 Risk of Study Procedures 
Risks of exercise testing assessments: (stress test on cycle, 6-minute walk, steady-state on 
treadmill, strength and balance test):  
These tests require people to apply effort. Although careful medical supervision will help to 
lessen the difficulty of doing these tests, some people might still find them unpleasant. The exer-
cise test may cause muscle soreness or fatigue (common). Some people get anxious while 
breathing through a mouthpiece or mask(common) or experience a fall (rare). Another risk is 
redness, skin chafing or irritation from the EKG electrodes used during exercise testing. If an 
abnormal rise in blood pressure or changes in the electrical pattern of the participants heart is 
noted, or if the participant develops chest pain, the exercise will be stopped immediately. 
Rarely, exercise may cause muscle sprains, muscle strains, or broken bones. Other risks in-
clude abnormal blood pressure (infrequent), fainting, dizziness (infrequent), disorders of heart 
rhythm (infrequent), and in very rare instances, heart attack, stroke, or even death (rare). In 
adults without a known history of heart disease, the risk of heart attack or death from maximal or 
sub-maximal exercise bouts is rare. The relative risk of exercise testing for older adults has not 
been clearly defined. However, a survey of more than 2,000 clinical exercise testing laborato-
ries, in which more than 600,000 tests were performed, showed a death rate of approximately 
0.5 per 10,000.  The PI/ study clinicians, and study staff are trained to minimize risks and to 
treat appropriately if any instability should develop.   
Cardiopulmonary and Functional Assessments:  

 
33 
The cardiopulmonary exercise test (CPET) entails symptom limited (maximal) exercise provoca-
tion. The exercise stimulus is associated with a 1 in 10,000 chance of significant untoward out-
come (e.g., myocardial infarction, arrhythmia), including the possibility of death. However, all en-
rolled will have a physical exam immediately before the test to best insure they are stable. All 
physicians and study exercise testing personnel are ACLS trained and a code cart is in the im-
mediate vicinity. In addition, patients’ cardiologists will be notified before patients are enrolled, 
and will be asked to approve only those patients they deem to be stable. 
During the other physical activity assessments (e.g., 400-meter corridor walk, 5 minute steady-
state treadmill walk, SPPB, and handgrip) there are also inherent exercise-associated risks, yet 
since CPET will be completed first, that initial assessment provides some certainty that partici-
pants are stable for the walking based assessments thereafter.  
Skeletal Muscle biopsy 
• Muscle biopsies are associated with a chance of bruising (about 1 in 100). Participants 
are instructed in what signs or symptoms at the biopsy site warrant contacting the study 
coordinator post biopsy. 
• Vasovagal reaction due to anxiety, 
• Bleeding and infection are rare side effects.  Participants will be provided with signs and 
symptoms, educated on when to contact the study team and when to seek emergency 
medical treatment.  
• Muscle biopsies are also associated with a small chance of infection (less than one in 
1000) and pain.  
Infection: Careful steps to keep the area free of germs will minimize infection risks. At the 
biopsy visit, participants will be given instructions for care and signs or symptoms of in-
fection for which they should contact study staff.  
Pain: lidocaine numbing medicine is used to reduce any possibility of pain, but sometimes 
the lidocaine itself produces a brief burning feeling as it enters through the skin. Additional 
numbing cream may be used in participants who report lower pain thresholds or inade-
quate response to numbing in the past. The PI/study clinician will evaluate the need and 
safety for this in select cases as needed. 
• Holding of anti-coagulant and anti-thrombin medications. With the permission of the pre-
scribing physician (if deemed necessary), blood-thinning medicines such as aspirin (81 
mg - 3 days) as well as warfarin (3 days) or others will be held before the muscle biopsy, 
and restarted post muscle biopsy. However, if the primary cardiologist prefers, bridging 
therapy using a different blood thinning medication (enoxaparin) will be used while the as-
pirin and warfarin are on hold. Holding the anti-coagulant regimen which is in place for 
prevention of cardiovascular events theoretically increases the risk of cardiovascular 
events, albeit a very small amount. 
• Additional unusual risks include allergic reactions to the elastic bandage wrap, leg numb-
ness that would indicate the elastic bandage had been applied too tightly; and skin red-
ness, irritation, and chafing from the Steri-strip™. Participants allergic to the Steri-strip™ 
will require a stitch. 
Risks of withholding anti-diabetic medication. 
Participants may be asked to withhold their anti-diabetic medication before or on the day of 
the exercise tests, biopsies. It will depend on the type of medication (and for the exercise 
test, current glycemic control), anticipated time of test and next meal. This may result in a 
transient increase in blood glucose which will self-resolve and does not carry long term risk.  

 
34 
Genetic testing 
As a broad concept, results of genetic testing on muscle and blood are thought to influence 
future employment or insurability for participants or their blood relatives if new diagnoses are 
identified. The results of these genetic tests are de-identified, subject to privacy laws and 
are not available to future employers or insurers. Further, because the results of the genetic 
tests performed in this study will only be linked to a disease condition that is already known, 
it is unlikely that the results of these genetic tests will have any significant impact on partici-
pants’ current health profile. The usefulness of these results in providing treatment for medi-
cal conditions has not been determined because they are research tests. 
Risk of lidocaine 
 
This FDA-approved numbing medicine, lidocaine, is injected into the site before the biopsy pro-
cedure. Participants with prior difficulty with this local anesthetic will be excluded from the study. 
A possible side-effect is an anaphylactic reaction. Participants will be questioned before enroll-
ment about prior experiences with the local anesthetic lidocaine. While the risks of having a reac-
tion to lidocaine used for this purpose are rare, it is always possible to have an unexpected serious 
reaction to any medication. 
 
Magnetic resonance spectroscopy  
• 
The MRRC has extensive experience with MRS studies in diverse populations and is 
well-trained to reassure participants. During these MRS sessions, even with previously 
scanned participants, measures are taken to insure subject comfort. If any participant 
exhibits any significant clinical findings on any of the exams (e.g., tumor on MRI), they 
will be referred to appropriate clinicians. In the case of an emergency during participation 
in the study, the staff will initiate the appropriate emergency procedures as per standard 
operating procedure at UPMC. All staff at UPMC involved in MRI/MRS scanning are 
trained yearly in safety and emergency protocols.  
• 
MRS is not associated with any known adverse effect except for people with metal or 
magnetic implants (such as metallic clips in the brain or cardiac pacemakers).  
 
Metal objects within the body can heat to potentially dangerous temperatures or possibly 
move in the patient’s body.  
 
Some types of tattoos (home-made) can also heat and cause discomfort. Any partici-
pants with the possibility of these risk factors will not be tested in the MRI/MRS studies. 
 
Metal objects can also become projectile when placed near the magnetic field. This has 
been reported, but it is a very rare occurrence. Protection from magnetic objects can be 
safeguarded by the usual safety techniques that are practiced in MRI/MRS sessions, in-
cluding having participants and researchers take all metal objects off of the subject be-
fore entering the environment. 
• 
Thus, any candidate who meets inclusion/exclusion criteria with the exception of a metal 
or possibility of metal in their body will be invited to participate, but will forego the MRI 
testing. 
Double screening of participants, by the study coordinator and again on the day of test-
ing will be completed for thorough assurance that the subject is safe for MRI. 
• 
Another potential risk is claustrophobia caused by being in the enclosed space of the 
MRI/MRS scanners. Participants who may become anxious or uncomfortable during any 
part of the procedure will be immediately taken out of the scanner and not tested. Partici-
pants are instructed to talk to the experimenter if they feel uncomfortable for any reason 

 
35 
during the testing procedures. If participants feel uncomfortable, they are immediately 
taken out of the scanner. 
• 
Noise levels in the magnet can be uncomfortable for participants and they will be wear-
ing earplugs through all MRI/MRS procedures. There is also microphone in the bore of 
the magnet. As above, participants who feel uncomfortable are taken out of the scanner. 
Near Infrared Spectroscopy (NIRS) 
• 
Participants may feel uncomfortable during the placement of the NIRS device onto their 
legs although it is not painful. There are some risks for minor skin irritation, redness, and 
chafing associated with the use of the doubled-sided tape to attach the NIRS sensors to the 
thighs. Though no other adverse effects from non-ionizing LED light source have been re-
ported, it is possible that effects not yet reported may occur. 
Actigraphy 
• 
Participants will be instructed on wearing an ActiGraph GT3X+, non-invasive activity monitor 
for one week at the beginning and end of the study and during the cycle CPET and all walk-
ing testing performed. There may be the inconvenience of having to wear a device around 
the wrist, and a small chance of causing a rash or irritation of the skin but should be no more 
than that of watch wristband. It has a small flashing light that may slightly bother some peo-
ple.  
Questionnaires  
• 
Patients will answer questionnaires as part of the study assessments which require re-
sponses about daily activities, diet, quality of life, medication, health history, cognitive 
ability and sleep which in some cases may be a source of emotional distress. Efforts will 
be made to keep the environment and support by staff to be reassuring and pleasant.  
Peripheral Blood sample 
• 
Blood samples for nitrite and nitrate monitoring as well as serological assessments for 
the study entail needle sticks and/or IV placement. There is a small chance of infection 
and pain. Careful steps to keep the area free of germs will minimize infection risks. 
• Common risks of blood sampling by venipuncture or intravenous line placement include 
temporary pain, bruising which may last for several days, redness, and swelling. Infrequent 
risks include feeling lightheaded or faint when blood is drawn. This is usually due to nerv-
ousness (not due to the amount of blood), and it is not usually serious. Rare risks include 
infection and bleeding. 
• A risk of drawing blood is anemia, from a lower hemoglobin level. A common symptom of 
anemia is fatigue (feeling tired or weak). The amount of blood to be drawn over the course 
of this investigation is an approximate maximum of 224 ml over approximately 16 weeks. 
This is not likely to cause anemia, especially with blood draws extended over 6-8 weeks. 
Also note that we are screening and excluding for anemia at Visit 1. 
 
Risks of fasting 
8 hours before the fasting visits indicated in Study Flowchart section 2.2.1 
Fasting for blood work is common and carries little risk. Participants may feel tired, hungry or 
irritable until they are served the standardized breakfast upon arrival.  
Participants who are diabetic have relatively greater risks of hypoglycemia, but the PI/study 
clinician will guide these individuals, and possibly adjust their medications as needed to min-
imize these risks.  
 

 
36 
Overall subject burden  
• 
Subject burden is a possibility due to the number of visits and particularly during the 
comprehensive physical assessment battery that is completed as part of the protocol. 
However, as noted in the Methods, precautions have been integrated with the assess-
ments to minimize risk to the participants. Rest periods are provided after each active 
functional assessment, moreover, active functional assessments alternate with ones that 
are completed at rest (questionnaires). Furthermore, at the end of each assessment and 
before the next functional assessment, and the rate of perceived exertion (RPE) is reas-
sessed to evaluate fatigability and to make sure that the subject has returned to baseline 
of physiological and subjective parameters prior to the next test. The consent language 
includes that participants may withdraw at any time. The consent form includes language 
and PI and the study coordinator will be sure that participants understand the number, 
duration and activity for each visit, so as to fully inform potential participants on this is-
sue. 
 
6.2 ALTERNATIVE TREATMENTS 
The alternative treatments for the participants participating in this investigation are to continue 
their medical care under the direction of their primary physicians. 
 
6.3 POTENTIAL BENEFITS 
There may be no direct medical benefit to participants in the study. It is hoped that researchers 
will learn more about the effectiveness of nitrite capsule supplements to improve strength, gait 
speed and balance. Oral nitrite may also increase efficiency of work by reducing their oxygen 
uptake required for the same work intensity. We hope the information learned from this study 
will benefit other older patients in the future. 
 
7. RISKS MANAGEMENT PROCEDURES 
7.1 PROTECTION AGAINST RISKS   
General Risks of Study Protocol and Procedures 
All research interventions/activities will be conducted in private patient care areas. The collection 
of sensitive information about participants is limited to the amount necessary to achieve the aims 
of the research, so that no unneeded sensitive information is being collected. 
All demographic and clinical information about the subject will be stored in an electronic pass-
word-guarded study database under the supervision of the Investigator for this protocol.  
The Center for Research on Health Care Data Center (CRHC-DC) at the University of Pitts-
burgh  
 
Data will be maintained by the CRHC Data Center with MS SQL Server. SQL Server is de-
signed for large relational databases and is a stable platform in which to house large volumes of 
data.  All tables within SQL will be linked with a key identifier.  SQL enables the system pro-
grammers to quickly generate custom datasets based on specific analyses.  Additionally, SQL 
has a reporting mechanism that enables one of our system programmers to quickly generate 
reports as needed for the study. Data entered by clinical site personnel will be automatically 
added to the relational database stored on Data Center servers.   
 

 
37 
The EWI (Enterprise Web Infrastructure) is backed up with the Symantec NetBackup sys-
tem.  The backup images are processed through an IBM ProtectTier appliance and stored on 
IBM DS8870 storage arrays.  All EWI servers get a daily operating system backup in the form of 
a full or differential backup.  The backup schedule has daily differential and bi-weekly full back-
ups for all EWI servers.  The backup images have a retention period of 60 days.  They are repli-
cated to a secondary IBM DS8870 storage array located at Posvar Hall.   This protects the 
backups images in the event of a catastrophic event at the RIDC data center. 
 
EWI sites with a backend database server also receive a daily backup.  All EWI database serv-
ers have a daily full backup and daily log backups.  EWI database servers running MS SQL 
Server utilize a specialized NetBackup SQL backup agent.  Any EWI database server running 
MySQL have a daily backup in the form of daily local dump and then have the exported data-
base files backed up during the daily operating system backup.  These again are replicated to 
the Posvar site. The backup methods used are proven methods for recovery.  Partial file re-
stores and full site restores have been performed upon a request from a department. 
 
The Data Center is complaint with the FDA 21 CFR part 11 protocols. Access to data will be 
controlled through password and authentication policies. Only approved individuals will have ac-
cess to data. Password policies control the length and variability of the user password selection. 
Audit trails will be implemented to log date, time, individual, changed values, and rationale for all 
data changes. 
 
We have three high-end machines dedicated to statistical analyses.  These machines contain 
up to 8 Xeon processors and up to 32gb or RAM with stripped raided SCSI high end perfor-
mance hard drives. This provides a framework to manipulate and analyze very large datasets in 
an efficient and time effective manner.  
 
The CRHC-DC biostatistical group uses current versions of SAS, R, and Stata for all data ma-
nipulation, statistical analyses, and reporting with set guidelines for code documentation and re-
producibility.  In addition, the statisticians use M-Plus and StatXact for specific types of analyses 
or simulations.    
Specimens will be stripped of subject identifiers and stored according to a similar coding proto-
col as described above. These specimens will be stored safely in the custody of the Investigator 
responsible for the individual assays. The Investigators will limit future access to any remaining 
sample to only those investigators with prior IRB approval for their studies. 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety.  
The Investigator will retain the data for the entire period of this study and will retain the specified 
records and reports for up to two years after the marketing application is approved for the inves-
tigational drug; or, if a marketing application is not submitted or approved for the investigational 
drug, until two years after investigations under the IND have been discontinued and the FDA so 
notified. The Investigator may continue to use and disclose participants’ de-identified infor-
mation for the purpose of this study for a minimum of seven years after final reporting or publi-
cation of the study. If the subject and/or legal representative decide to withdraw or be withdrawn 
from study participation, they may request that the study data and samples be destroyed. Sub-
ject names or other directly identifiable information will not appear on any reports, publications, 
or other disclosures of clinical study outcomes. 
If applicable, any clinical study data that will be recorded directly on the CRF or electronic data-
base whereupon the CRF/electronic data is to be considered the Source Data. Source Data are 
the clinical findings and observations, laboratory and test data, and other information contained 

 
38 
in Source Documents.  Source Documents are the original records (and certified copies of origi-
nal records; including, but not limited to, hospital medical records, physician or office charts, 
physician or nursing notes, subject diaries or evaluation checklists, pharmacy dispensing rec-
ords, recorded data from automated instruments, x-rays, etc.  When applicable, information rec-
orded on the CRF shall match the Source Data recorded on the Source Documents.  Describe 
the procedures for accounting for any missed, unused, and/or spurious data. 
 
7.2 PROTECTION AGAINST POTENTIAL RISKS OF EXPERIMENTAL INTERVENTION 
• 
Involvement by trained staff/investigators with experience in the administration of the 
study drug: Dr. Forman, PI, and physician Co-I, Dr. Hughan are principally responsible 
for monitoring and protecting the safety of participants’ use of sodium NO2. Their com-
bined experience in the use of the study drug is adequate to protect against (the rela-
tively low) risks. 
• 
Continuous monitoring by the Data and Safety Monitoring Board. 
• 
Required Education in the Protection of Human Research Participants: The Principal In-
vestigator and all sub-investigators listed on the University of Pittsburgh HRPO approved 
protocol are required to complete the Collaborative Institutional Training Initiative (CITI) 
courses in research fundamentals, including Human Participants Research and Respon-
sible Conduct of Research. Staff will complete these courses as well, prior to any subject 
contact. Investigators and staff with subject contact will also complete the Good Clinical 
Practice module. Investigators with an identified Conflict of Interest (COI) will complete 
the COI module. These web-based tutorials are a requirement of the HRPO for investi-
gators prior to protocol submission 
 
8. ADVERSE EVENTS  
The proposed study will use the FDA definition of adverse events (AE) and serious adverse 
events (SAE). Any SAE, which is unexpected and related to study intervention, will be reported 
immediately to the IRB and FDA and will include all known details regarding the nature of the 
SAE. In the event that a participant either withdraws from the study or the investigators decide 
to discontinue a participant due to a SAE, the participant will be monitored by the PI and treating 
investigator until (a) a resolution is reached (e.g., the problem has resolved or stabilized with no 
further change expected), (b) the SAE is determined to be clearly unrelated to the study inter-
vention, or (c) the SAE results in death. Outcomes of SAEs not previously reported will be re-
ported to the sponsor, IRB and FDA via a follow-up report.  A summary of the SAEs that oc-
curred during the previous year will be included in the FDA annual progress report as well as in 
the annual IRB renewal. 
The severity of adverse changes in physical signs or symptoms will be classified as follows: 
• 
Grade 1 (Mild): asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated. 
• 
Grade 2 (Moderate): minimal, local or noninvasive intervention indicated; limiting age-
appropriate ADL (Activities of Daily Living). 
• 
Grade 3 (Severe): medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-
care/ADL. 
• 
Grade 4 (Life-threatening): consequences; urgent intervention indicated. 

 
39 
• 
Grade 5 (Death): event is a direct cause of death.  
 
8.1 REPORTABLE ADVERSE EVENTS 
For this study, a serious adverse event is any untoward clinical event that is thought by either 
the Principal Investigator, Dr. Forman or the sponsor, Dr Gladwin to be unexpected and at least 
possibly related to the study and results in any of the following: 
1. Death 
2. A life-threatening adverse event 
3. Inpatient hospitalization or prolongation of an existing hospitalization 
4. A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
5. A congenital anomaly or birth defect 
6. Important medical events that may not result in death, be life threatening, or require hos-
pitalization. 
7. Hospitalization may be considered serious when, based upon appropriate medical judg-
ment, they may jeopardize the patient, or subject, and may require medical, or surgical 
intervention to prevent one of the serious outcomes listed above. 
If clinically important and unexpected adverse experiences or clinically important study-related 
adverse experiences occur, they will be recorded on the adverse event case report form.    
 
8.2 ADVERSE EVENTS REPORTING TIMELINES 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug or 
procedures must be reported to the DSMB Chair and to the IRB within 24 hours of discovery of 
the incident with subsequent follow-up submission of a detailed written report. 
The FDA will be notified by telephone or facsimile transmission of a human adverse event that 
is fatal or life-threatening no later than 7 calendar days after receiving the respective human ad-
verse event information, followed by the subsequent submission of a written IND Safety Report. 
Serious and unexpected adverse events associated with the use of the study drug or proce-
dures will be reported within 48 hours to the DSMB Chair and the IRB with subsequent follow-up 
submission of a detailed written report in accordance with the respective policies and proce-
dures of the IRB. Written IND Safety Reports will be submitted to the FDA as soon as possible 
and, in no event, later than 15 calendar days following the investigator-sponsor’s receipt of the 
respective adverse event information. All AE and SAE will be reported to the DSMB quarterly.  
A summary report of the findings will be prepared and submitted to the regulatory agencies as 
required. 
 
9. DATA SAFETY MONITORING  
9.1 DATA SAFETY MONITORING BOARD 
The DSMB will also be expected to meet as needed, but not less than, every six months to pro-
vide an overall summary status report to the regulatory agencies. An emergency meeting of the 
DSMB may be called at any time by the Chair should participant safety questions or other unan-
ticipated problems arise.  

 
40 
In addition, the DSMB Report addressing the following information will be submitted to the IRB 
at the time of continuing review annually or more often as required: 
• 
A list of the research personnel who participated in the data and safety monitoring.  
• 
The frequency of monitoring that took place during the renewal intervals and/or the dates 
that data and safety monitoring was conducted.  
• 
A summary of cumulative data related to unanticipated problems (including adverse 
events) including a determination of causality and whether the risk to benefit assessment 
has changed.  
• 
If appropriate, a summary of pertinent scientific literature reports, therapeutic develop-
ments, or results of related studies that may have an impact on the safety of study par-
ticipants or the ethics of the research study.  
• 
A summary of the outcome of reviews conducted to ensure subject privacy and research 
data confidentiality.  
Final conclusions regarding changes to the anticipated benefit-to-risk assessment of the study 
participation and final recommendations related to continuing, changing, or terminating the 
study. 
 
9.2 DATA SAFETY MONITORING PLAN 
Monitoring of safety and data quality in the proposed study will be the responsibility of all per-
sonnel on the project, with primary responsibility and supervision by the Investigator.  The Insti-
tutional Review Board will approve the Statement of Informed Consent for the study and provide 
institutional oversight of data and safety issues. The study protocol will be approved prior to re-
cruiting or obtaining consent from any participants. Moreover, the study will be reviewed at a 
minimum of annual basis (or more frequently as deemed necessary) by the IRB committee. 
Each participant will sign the Informed Consent Form described above prior to participating in 
the study. To ensure participant safety, once participants are enrolled in the study, study staff 
will immediately report all adverse and serious adverse events to one of the Investigators. The 
Investigator will, per institutional requirements, report them to the IRB for their review. These 
events should also be communicated to the sponsor of the IND.  With regards to the monitoring 
of data quality and protected health information, all required personnel proposed for this project 
will have the required human participants and confidentiality training, which includes information 
about maintaining data integrity and security. Confidentiality will be guarded using established 
procedures such as storing data in locked cabinets within locked offices or locked data rooms, 
coding by study identification numbers rather than any personally identifying information to 
avoid revealing the identity of participants, and aggregating data across participants. The key 
linking names and study identification numbers will be kept separately from the data sets with 
limited access by study personnel. Only study personnel will have access to the data sets on 
protected servers. In order to maintain the highest standard of data entry quality, all data will be 
double entered, with discrepancies highlighted so that they can be reviewed by the project coor-
dinator. Oversight of all aspects of data management will occur with the Investigator. 
 
9.3 PARAMETERS TO BE MONITORED 
The following progress will be monitored throughout the course of the research to ensure the 
safety of participants as well as the integrity and confidentiality of their data. 
• 
An evaluation of the progress of the research study, including subject recruitment and 
retention, and an assessment of the timeliness and quality of the data. 

 
41 
• 
A review of collected data (including adverse events, unanticipated problems requiring 
reporting and those captured on the non-compliance log, and subject withdrawals) to de-
termine whether there is a change to the anticipated benefit-to-risk assessment of study 
participation and whether the study should continue as originally designed, should be 
changed, or should be terminated. 
• 
An assessment of external factors or relevant information (e.g. pertinent scientific litera-
ture reports or therapeutic development, results of related studies) that may have an im-
pact on the safety and study participants or the ethics of the research study. 
• 
A review of study procedures designed to protect the privacy of the research participants 
and the confidentiality of their research data. 
 
9.4 FREQUENCY OF MONITORING 
Dr. Forman will review subject safety data as it is generated. Dr. Forman and the research staff 
will meet with bi-weekly and/or monthly (as they get further into the study) intervals to re-evalu-
ate study goals, subject recruitment, data coding and retention, documentation and identification 
of adverse events, complaints and confidentiality of participants. There will be an evaluation of 
the progress of the research study, including assessments of data quality, timelines, participant 
recruitment, accrual, and retention. Dr. Forman will also review the outcome and adverse event 
data to determine whether there is any change to the anticipated benefit-to-risk ratio of study 
participation and whether the study should continue as originally designed or should it be re- 
evaluated and changed 
 
10. WITHDRAWAL OF PARTICIPANTS AND STOPPING CRITERIA 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug or 
procedures must be reported to the HRPO within 24 hours of discovery of the incident with sub-
sequent follow-up submission of a detailed written report. The FDA must be notified by tele-
phone or facsimile transmission of a human adverse event that is fatal or life-threatening no 
later than 7 calendar days after receiving the respective human adverse event information, fol-
lowed by the subsequent submission of a written IND Safety Report. 
Serious and unexpected adverse events associated with the use of the study drug or proce-
dures must be reported to the HRPO with subsequent follow-up submission of a detailed written 
report in accordance with the respective policies and procedures of the HRPO. Written IND 
Safety Reports will be submitted to the FDA as soon as possible and, in no event, later than 15 
calendar days following the investigator-sponsor’s receipt of the respective adverse event infor-
mation.  
All reports noted in this section will also be provided to the NIA Program Officer and the DSMB. 
Consent: During the consent process and when deemed appropriate by study staff throughout 
study, participants will also be informed that they are free to withdraw from the study at any time 
without any consequences. 
Intervention Phase:  
Outpatient: If participants report change in skin tone and dizziness or other signs of methemo-
globinemia for >24 hours, the need for an interim visit with the above described steps will be fol-
lowed. The visit can be conducted in the UPMC Montefiore GRC or TRC, or in the Pepper Cen-
ter-SMART Center. 

 
42 
At this visit, methemoglobin will be assessed. If greater than 5%, the PI will be unblinded to drug 
or placebo assignment. If participants are on nitrite, PI will evaluate data and make a determina-
tion regarding discharge on 20 mg tid. If discharged on 20 mg tid, participants will be contacted 
via phone for the next 2 days for symptom re-assessment. If symptoms remain resolved, partici-
pants will continue on 20 mg tid. If symptoms persist, participants will be requested to come in 
for repeat methemoglobin testing after 3 days.  If level persists above 5%, participation in the 
study for the subject will be discontinued. 
 
 
Hypotension and related symptoms 
During pK: 
If this occurs while in the clinic, care will be administered, including fluids and monitoring, to en-
sure that the problem resolves, and patient is stable. The physician investigator will factor rele-
vant subject symptoms and hemodynamics and then decide whether un-blinding is indicated 
and if it is appropriate for the subject to proceed with study participation prior to discharge.  
As outpatient: 
If participants experience lightheadedness or dizziness after visit 4 while they are on the study 
drug/placebo, we request that they call the study coordinator who will inventory their symptoms, 
compare to baseline levels (measured at pK), and discuss need for an interim visit with study 
physician. If a visit is indicated, participants will be asked to repeat drug dosing of 20 mg with 
follow up methemoglobin and BP pressure monitoring. A study physician will factor relevant 
subject symptoms and hemodynamics and will confirm a management plan including the: 
-need for un-blinding, 
-need to discontinue and drop from the trial.  
If participants proceed, they will be reassessed via phone each day for the next 2 days thereaf-
ter to ensure that the patient is stable.  However, if there are continued symptoms, the physician 
investigator will reevaluate the patient’s suitability for continued participation based on symp-
toms and/or hemodynamics. 
If participants who were initiated report symptoms as described above, they will be asked to 
complete an interim visit, methemoglobin and BP pressure assessment; the physician may have 
to withdraw the subject since a dose lower than 20 mg tid is not an option. 
 
Discontinuation of the Clinical Trial 
Stopping Rule: 
Extensive published literature and considerable local experience at University of Pittsburgh indi-
cates nitrite is not inherently dangerous at the doses targeted in this trial. Certain side effects of 
nitrite are idiosyncratic in older frail patients and it is anticipated that there will be participants 
who do not tolerate the treatment who may need to be withdrawn from the study. Nonetheless, if 
a subject enrolled in the study experiences a fatal event that is directly attributable to the NO2, 
the overall study treatment will be discontinued. 
 
 
 

 
43 
 
 
11. COSTS AND PAYMENTS 
11.1 COSTS 
Study drug/placebo and all research testing will be supported by ongoing research grants. All 
medications, lab tests, and any procedures described will not be billed to the participants and/or 
their health insurance company. 
 
 
 
 
 
 
11.2 PAYMENTS 
 
Participant Reimbursement Consent Visit and V1 (Baseline) 
$50 
Participant Reimbursement V2
$50
Participant Reimbursement V3
$50
Participant Reimbursement V4  
$100 
Participant Reimbursement #1
$250
Participant Reimbursement V5
$50
Participant Reimbursement #2 
$50 
Participant Reimbursement V6
$50
Participant Reimbursement V7 
$50 
Participant Reimbursement V8 
$50 
Participant Reimbursement V9 
$100 
Participant Reimbursement #3 
$250 
TOTAL: 
$550 
 
Participants who require any interim visits (including the separate Consent visit if neces-
sary) will be provided parking and compensated up to an additional $40 total at final re-
imbursement. 
 
12.  QUALIFICATIONS AND SOURCE OF SUPPORT 
 

 
44 
12.1 QUALIFICATIONS OF THE INVESTIGATORS 
 
Sponsor: 
Mark Gladwin, MD, is a Professor of Medicine, University of Maryland School of Medicine. He 
is an internationally recognized authority in the field of sodium NITRITE including both the basic 
science and a broad range of clinical applications in cardiovascular disease. He is a current IND 
holder for the investigation of sodium NITRITE in lung transplant. 
 
Principal Investigator: 
PI:  Daniel Forman, MD, is a Professor (Pending) of Medicine at the University of Pittsburgh 
School of Medicine, Department of Medicine and Chair, Geriatric Cardiology Section, Divisions 
of Geriatrics and Cardiology. Dr. Forman has a well-established track record of translational 
work focused on the interplay between skeletal muscle and physical function in older HF pa-
tients. Moreover, he is an expert in functional assessments in older HF patients. He is responsi-
ble for all aspects of this investigation and will work directly with research staff to screen pa-
tients, coordinate data collection and quality, and he will personally complete all muscle biopsies 
and supervise all functional evaluations. He will play a primary role in data analysis and publica-
tions. 
Sub-Investigators: 
. 
  
Sruti Shiva, PhD is an Associate Professor in the Department of Pharmacology and Chemical 
Biology and VMI at the University of Pittsburgh. Her research lab focuses on the mechanisms 
by which reactive nitrogen species (particularly nitrite and nitric oxide) regulate mitochondrial 
function, the factors that influence this regulation and the implications of this regulation on pa-
thology. 
 
Nancy Glynn, PhD is an Assistant Professor, Epidemiology, who completed formative work that 
established and validated indices of performance and perceived indices of fatigability, i.e., per-
formance fatigability which refers to the mechanistic property of declining function during a con-
stant physical activity stimulus, and perceived fatigability as a perception of tiredness associated 
with a physical activity stimulus. 
 
Subashan Perera, PhD is a Professor, Division of Geriatric Medicine and Department of Bio-
statistics at the University of Pittsburgh and has over 20 years of experience in providing statisti-
cal support to health science research at major academic medical institutions. He is a Senior 
Statistician and Co-Leader of Data Management, Analysis and Informatics Core of the NIA-
funded Pittsburgh Claude D. Pepper Older Americans Independence Center. In addition, he is 
the principal source of statistical support to numerous clinical trials and intervention studies 
funded by NIH, PCORI, AHRQ, CMS and VA. Dr. Perera will play a key role in randomization, 
data analyses and publications. 
 
Kara Hughan, MD is an Assistant Professor in the University of Pittsburgh School of Medicine, 
Department of Pediatrics, Division of Pediatric Endocrinology and Diabetes. Dr. Hughan has de-
signed and led the Phase I and early Phase II human subject trials with our IND-approved oral 
NITRITE at the University of Pittsburgh. She has extensive experience with evaluation of pK/pD, 
safety and efficacy of oral NO2. As Co-Investigator, she will play a role in data collected on oral 
NITRITE drug metabolism (plasma nitrate/nitrite concentrations, RBC nitric oxide concentra-
tions), safety (BP, MetHb) and efficacy (platelet and muscle mitochondrial function) and perform 
related statistical analyses on the data gained on these outcomes. Dr. Hughan will also assist 
with related trial regulatory/DSMB reporting. Dr. Hughan will work with Dr. Forman and his team 

 
45 
on implementing the nitrite therapy in this study. Dr. Hughan will provide leadership in protocol 
refinement, manuscript writing and publications. 
 
Anne Newman, MD, MPH is the Chairperson of the Department of Epidemiology, Graduate 
School of Public Health, University of Pittsburgh and has been a seminal leader in understand-
ing the relationship between lean body mass, relative proportions and configuration of fat and 
related impact on strength and health. Dr. Newman will interpret related data from the study and 
participate in manuscript preparation. 
 
Theodore Huppert, PhD is an Associate Professor in the Department of Radiology and has ex-
tensive experience in the area of non-invasive optical imaging (NIRS) both within the area of the 
human brain as well as muscle. He is the director of the NIRS Brain Imaging Laboratory at Uni-
versity of Pittsburgh Dr. Huppert is an biomedical engineer and will work with Dr. Forman and 
his team on implementing the NIRS measures in this study. Dr. Huppert will provide leadership 
in protocol refinement, manuscript writing and publications, and will participate on other study 
committees as needed. Dr. Huppert will also work closely with Dr. Forman on the collection and 
novel examination of NIRS data in association to fatigability.   
 
Frank Sciurba, MD is an Associate Professor of Medicine at the University of Pittsburgh School 
of Medicine and the Medical Director of the Pulmonary Physiology Laboratory in the Division of 
Pulmonary, Allergy and Critical Care Medicine. Dr. Sciurba's current leadership positions in-
clude his role as a Principle Investigator of the Network Management Core of the new National 
Heart, Lung, and Blood Institute-sponsored Pulmonary Trials Consortium which manages the 
execution of pragmatic, “real world” studies in a variety of chronic pulmonary conditions;  and 
his role as academic chair of the COPD Biomarker Qualification Committee, a group of aca-
demic, foundation and industry partners that work with U.S. Food and Drug Administration lead-
ership to address the need for new biomarkers to facilitate development of drugs and devices 
for chronic pulmonary conditions 
 
Paul Coen, PhD is an Associate Investigator at the Translational Research Institute Advent 
Health, Orlando, FL. He is the site PI at AdventHealth for this trial.  Dr. Coen is an expert in mi-
tochondrial bioenergetics both in respect to ex vivo and in vivo analytic techniques.  He leads 
the Advent Hospital study team, and he will work closely with Dr. Forman in all aspect of analy-
sis, presentation, and publication for this trial.  
 
12.2 SOURCE OF SUPPORT 
National Institutes of Aging Grant R01AG058883 
 
 
 
 
 
 

 
46 
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