Cocoa to Improve Walking Performance in Peripheral Artery Disease
Phase: NA
Conditions: Peripheral Artery Disease
Sponsor: Northwestern University

 
Page 1 of 41 
 
PROTOCOL TITLE: 
 
Cocoa to Improve Walking Performance in Peripheral Artery Disease: The 
COCOA-PAD Study 
 
PRINCIPAL INVESTIGATOR: 
Mary McDermott, MD 
Department of General Internal Medicine 
312-503-6419 
mdm608@northwestern.edu 
 
NCT NUMBER: 
NCT02876887 
 
 
VERSION NUMBER: 
Version #15. 
 
VERSION DATE: 
5.24.18 
 

 
Page 2 of 41 
 
Table of Contents 
 
1.0 Objectives……………………………………………………………… 3 
2.0 Background…………………………………………………………….. 4 
3.0 Inclusion and Exclusion Criteria……………………………………….. 8 
4.0 Study-Wide Number of Subjects………………………………………. 10 
5.0 Study-Wide Recruitment Methods…………………………………….. 10 
6.0 Multi-Site Research……………………………………………………. 10 
7.0 Study Timelines………………………………………………………... 10 
8.0 Study Endpoints………………………………………………………... 10 
9.0 Procedures Involved……………………………………………………. 11 
10.0 Data and Specimen Banking………………………………………….. 17 
11.0 Data and Specimen Management……………………………………... 18 
12.0 Provisions to Monitor the Data to Ensure the Safety of Subjects…….. 20 
13.0 Withdrawal of Subjects……………………………………………….. 21 
14.0 Risks to Subjects……………………………………………………… 21 
15.0 Potential Benefits to Subjects………………………………………… 23 
16.0 Vulnerable Populations………………………………………………. 23 
17.0 Community-Based Participatory Research…………………………… 23 
18.0 Sharing of Results with Subjects……………………………………... 23 
19.0 Setting………………………………………………………………… 24 
20.0 Resources Available…………………………………………………... 24 
21.0 Prior Approvals……………………………………………………….. 25 
22.0 Recruitment Methods…………………………………………………. 25 
23.0 Local Number of Subjects……………………………………………. 27 
24.0 Confidentiality………………………………………………………... 27 
25.0 Provisions to Protect the Privacy Interests of Subjects……………….. 27 
26.0 Compensation for Research-Related Injury…………………………... 27 
27.0 Economic Burden to Subjects………………………………………… 27 
28.0 Consent Process………………………………………………………. 28 
29.0 Process to Document Consent in Writing…………………………….. 28 
30.0 Drugs or Devices………………………………………………………28 
31.0 References…………………………………………………………….. 29 

 
Page 3 of 41 
 
1.0 Objectives 
1.1 Purpose, Objectives, Specific Aims 
Our prior work demonstrates that older people with lower extremity 
peripheral artery disease (PAD) have greater functional impairment and 
faster functional decline compared to those without PAD (1-4). PAD-related 
functional limitations are associated with increased healthcare costs and loss 
of independence (5-10). Yet few medical therapies have been shown to 
improve functioning or prevent mobility loss in older people with PAD. 
 
People with PAD have impaired endothelial function, impaired calf muscle 
perfusion, reduced skeletal muscle mitochondrial activity, and reduced calf 
muscle mass and strength (11-20).  Preliminary pre-clinical and human 
evidence show that cocoa and its major flavanol component, epicatechin, 
have therapeutic properties that reverse the pathophysiologic impairments 
that are present in PAD.  Specifically, cocoa and epicatechin improve 
endothelial function, increase skeletal muscle mitochondrial function, 
increase skeletal muscle angiogenesis, reduce skeletal muscle levels of 
myostatin, and increase skeletal muscle levels of follistatin (21-31).  
Myostatin is a growth differentiation factor that promotes muscle wasting.  
Follistatin blocks myostatin’s action, thereby promoting muscle growth.  By 
reducing myostatin and increasing follistatin, epicatechin-rich cocoa has 
potential to increase muscle mass and strength (22,25).  A small study in 
humans with type 2 diabetes and heart failure showed that epicatechin-rich 
cocoa increased skeletal muscle mitochondrial biogenesis, increased 
mitochondrial cristae abundance, and improved grip strength (25).  A small 
randomized cross-over trial in PAD patients showed that dark chocolate 
(>85% cocoa), but not milk chocolate (<35% cocoa), acutely increased 
treadmill walking time, improved endothelial function, and reduced 
oxidative stress two hours after chocolate ingestion (31).  However, no 
randomized trials have studied whether chronic daily epicatechin-rich cocoa 
improves walking ability in patients with PAD.   
 
The COCOA-PAD Study is a pilot double-blind, randomized controlled trial 
to test our hypothesis that epicatechin-rich cocoa improves endothelial 
function, increases calf skeletal muscle mitochondrial function and 
biogenesis, and favorably alters skeletal muscle levels of myostatin, 
follistatin, and mitochondrial transcription factor-alpha (TFAM), thereby 
improving lower extremity functioning in older people with PAD.  If our 
proposed pilot study supports our hypotheses, results will be used to design 
a large, definitive randomized clinical trial of epicatechin-rich cocoa in older 
people with PAD.   
 
Forty-four participants with PAD age 60 and older will be randomized to 
epicatechin-rich cocoa vs. an epicatechin-free and cocoa-free placebo 
(matched in appearance and taste) daily for six months.  Our primary 

 
Page 4 of 41 
 
outcome is the six-minute walk, a well-validated measure of walking 
endurance in PAD (32-34).  Secondary outcomes are pain-free and maximal 
treadmill walking performance, brachial artery flow-mediated dilation 
(FMD), accelerometry-measured physical activity, calf muscle biopsy 
measures, and MRI-measured calf muscle perfusion. To distinguish between 
the acute vs. chronic effects of cocoa, the six-minute walk and brachial 
artery FMD measures will be obtained while off of study beverages for 2.5 
hours (acute) and 24 hours (chronic), respectively. .  The treadmill stress test 
will be measured while off of study beverages for 48 hours. Remaining 
outcomes will be measured while still taking study beverages whenever 
possible.  
 
Primary Aim.  Among 44 participants with PAD age 60 and older, we will 
determine whether epicatechin-rich cocoa daily for six months improves six-
minute walk performance compared to placebo. 
 
Secondary Aims.  1. Among 44 participants with PAD age 60 and older, we 
will determine whether epicatechin-rich cocoa daily for six months 
improves maximal and pain-free treadmill walking time, increases brachial 
artery FMD, and increases accelerometer-measured physical activity 
compared to placebo.  2. Among 44 participants with PAD age 60 and older, 
we will determine whether epicatechin-rich cocoa daily for six months 
favorably improves the following measures obtained with calf skeletal 
muscle biopsy: activity of Cytochrome C Oxidase (COX) and citrate 
synthase, calf muscle levels of peroxisome proliferative activated receptor-γ 
co-activator 1α (PGC-1α), capillary density, myostatin, follistatin, and 
mitochondrial transcription factor-alpha (TFAM), compared to placebo.  3.  
Among 44 participants with PAD age 60 and older, we will determine 
whether epicatechin-rich cocoa daily for six months increases calf skeletal 
muscle perfusion, measured by magnetic resonance imaging (MRI), 
compared to placebo.  4. Among participants with PAD age 60 and older, 
we will determine whether epicatechin-rich cocoa daily for six months 
increases calf skeletal muscle biopsy measures of calf skeletal muscle 
regeneration and reduces oxidative stress, compared to placebo.  Specific 
measures of calf skeletal muscle regeneration that we will study consist of 
satellite cell number and activation state and centrally nucleated and 
embryonic myosin heavy chain-expressing fibers.  Specific measures of calf 
skeletal muscle oxidative stress will be nitrotyrosine and 4-hydroxynonenal 
(4-HNE).   
 
Exploratory Aims.  Among participants with PAD age 60 and older 
randomized to epicatechin-rich cocoa, we will determine whether the degree 
of improvement in six-minute walk performance and treadmill walking 
performance, respectively, is correlated with the degree of improvement in 
a) calf skeletal muscle perfusion; b) calf skeletal muscle measures of 

 
Page 5 of 41 
 
regeneration; and c) calf skeletal muscle measures of oxidative stress. 
 
 
 
1.2 Hypotheses. 
We hypothesize that: 
1. PAD participants randomized to epicatechin-rich cocoa will achieve 
greater increases in six-minute walk performance at six-month follow-up, 
compared to those randomized to placebo. 
2. PAD participants randomized to epicatechin-rich cocoa will have greater 
increases in treadmill walking time, brachial artery FMD, and physical 
activity at six-month follow-up, compared to placebo. 
3. Epicatechin-rich cocoa will improve each calf skeletal muscle measure 
listed above, compared to placebo.  
4. PAD participants randomized to epicatechin-rich cocoa will achieve 
greater increases in MRI-measured calf muscle perfusion at 6-month follow-
up, compared to those randomized to placebo. 
5. PAD participants randomized to epicatechin-rich cocoa will have more 
favorable changes in each calf muscle biopsy measure, compared to those 
randomized to placebo. 
 
2.0 
Background 
2.1 Relevant prior experience and gaps in current knowledge 
The COCOA-PAD Study builds on our prior work. We have successfully 
completed large randomized trials of PAD patients. Our prior work 
demonstrates that people with PAD have greater functional impairment and 
more rapid functional decline than those without PAD (1-5).  The functional 
impairment in PAD is associated with loss of independence, increased 
mortality, and poor quality of life (6-10,35).  
 
Few medical therapies are available to improve functional impairment in 
PAD.  Only two medications, pentoxifylline and cilostazol, are FDA-
approved for treating PAD-associated walking impairment.  Of these, 
pentoxifylline is usually ineffective and benefits from cilostazol are modest 
(36-39).  New therapies are urgently needed to improve walking 
performance in patients with PAD.   
 
2.2 Relevant Preliminary Data. 
 
A. Calf skeletal muscle fibers and mitochondria are damaged in PAD and 
contribute to functional decline. Endothelial function is impaired in PAD. 
First, our work shows that people with PAD have smaller calf muscle area, 
increased calf muscle fat content, and poorer leg strength compared to 
people without PAD (19,20,40,41).  Greater severity of these calf muscle 
impairments are associated with greater functional impairment and 
increased mobility loss in PAD (41-43).  Second, electron microscopy 

 
Page 6 of 41 
 
demonstrates pathologic changes in myofibrils, mitochondria, and 
sarcolemma of calf myofibers in patients with PAD (16,44,45).  More 
severe oxidative stress, such as that associated with walking-related 
ischemia reperfusion of calf muscle, is associated with more severe 
myofiber damage (44).  Third, PAD patients have reduced calf muscle 
mitochondrial energy production, measured by reduced adenosine 
triphosphate (ATP) production, compared to people without PAD (44-50). 
Lower ATP production is associated with greater functional impairment in 
PAD (18,44).  Fourth, people with PAD have impaired endothelial function 
compared to people without PAD. Greater endothelial function impairment 
in PAD is associated with lower physical activity and increased 
cardiovascular events (11,12,51).  Epicatechin-rich cocoa targets all of these 
pathophysiologic changes that are present in PAD.   
 
B. A5.  Pre-clinical evidence suggests that cocoa-derived epicatechin 
improves skeletal muscle characteristics and vascular function.  Epicatechin 
is a flavanol that exists in high concentrations in cocoa.  In pre-clinical 
studies, epicatechin has therapeutic effects on skeletal muscle.  First, 
Hutteman and colleagues studied 21 rats randomized to one of three groups:  
Group 1:  epicatechin for 30 days; Group 2: epicatechin for 30 days 
followed by 15 days with no therapy; Group 3:  Placebo.  At 30 day follow-
up, Group 1 had increased skeletal muscle mitochondrial biogenesis and 
mitochondrial function, measured by increases in PGC-1α, TFAM, 
mitochondrial cristae quantity, and capillary density (21).  PGC-1α is a key 
promoter of mitochondrial biogenesis and mitochondrial function.  TFAM 
increases mitochondrial transcription.  Greater mitochondrial cristae 
provides greater surface area for ATP production.  These favorable changes 
were sustained at 45 day follow-up in Group 2, 15 days after therapy was 
completed.  Second, Gutierrez-Salmean and colleagues administered 1 
mg/kg daily of cocoa-derived epicatechin to ten young (6 months old) and 
ten old (26 months old) mice (22).  At two-week follow-up, skeletal muscle 
levels of myostatin were reduced in the young and older mice by 15% and 
21%, respectively, while skeletal muscle levels of follistatin increased by 
56% in the older mice (22).  Myostatin is a protein and growth 
differentiation factor that promotes muscle wasting.  Follistatin blocks 
myostatin’s action, increasing muscle growth.   Thus, cocoa-derived 
epicatechin achieved favorable changes in myostatin and follistatin levels 
that increase muscle mass and strength.  Third, Nogueira and colleagues 
randomized 25 mice into one of four groups for 15 days:  Group 1:  Water 
alone; Group 2:  Water plus exercise; Group 3: cocoa-derived epicatechin 
alone; Group 4:  cocoa-derived epicatechin plus exercise (23).  The groups 
receiving epicatechin had greater increases in skeletal muscle capillary 
density, TFAM quantity, mitochondrial cristae abundance, treadmill running 
ability, and mitochondrial activity, compared to mice receiving water alone 
(23).  Groups receiving epicatechin also had less muscle fatigue.  In 
summary, preclinical evidence shows that epicatechin, the major flavanol 

 
Page 7 of 41 
 
component of cocoa, increases mitochondrial biogenesis, mitochondrial 
function, and capillary density in skeletal muscle.  Epicatechin improves 
muscle strength and running capacity in animals. 
 
C. Preliminary evidence from humans without PAD suggests that 
epicatechin-rich cocoa improves mitochondrial function, increases muscle 
mass, and improves strength.  Taub and colleagues administered chocolate 
squares and cocoa beverages daily for three months to five patients with 
type 2 diabetes and heart failure (age 41 to 71 years) (25).  Total daily 
epicatechin content in the cocoa products was 100 mgs.  In thigh muscle 
biopsies, nitric oxide, PGC-1α, TFAM, and mitochondrial cristae volume all 
increased following cocoa treatment.  In a separate uncontrolled trial, 
Gutierrez-Salmean administered cocoa-derived epicatechin to 6 healthy 
young people (mean age 28.5 years) and 6 older healthy people (mean age 
62) (22).  Plasma levels of myostatin decreased, follistatin increased, and 
grip strength improved following epicatechin treatment.  These preliminary 
studies in humans support our hypotheses that cocoa and epicatachin 
improve muscle mitochondrial function and biogenesis, muscle mass, and 
strength.  However, these studies were limited by small sample sizes and 
lack of control groups.  Further evidence is needed.      
 
D. Dark chocolate acutely improves endothelial function and treadmill 
walking in PAD.  Loffredo and colleagues randomized 20 PAD patients 
(mean age 69 years) to 40 grams of dark chocolate (>85% cocoa) vs. 40 
grams of milk chocolate (<35% cocoa) in a single-blind cross-over study 
(31). Two hours after ingesting dark chocolate, increases were observed in 
treadmill walking time (124.8 + 60.8 to 142.2+62.0 seconds, P<0.001), 
brachial artery flow-mediated dilation (FMD) (2.3%+2.2% to 6.3%+2.7%, 
P<0.001), and nitric oxide (11.0 umol/L+5.8 to 17.3 umol/L+5.8, P=0.001).  
Milk chocolate ingestion did not affect any of these outcomes.  These data 
demonstrate an acutely beneficial effect of dark chocolate in PAD.  
However, the effect of chronic daily administered dark chocolate on 
clinically important outcomes in PAD is unknown. 
 
In summary, pre-clinical evidence and small human studies show that cocoa 
and the cocoa-derived flavanol, epicatechin, target key pathophysiologic 
abnormalities in people with PAD  that are linked to functional impairment 
in PAD (21-28,31).   
 
2.3 Scientific background and significance of research based on existing 
 
literature. 
 
A. PAD is common and is associated with increased functional impairment 
and faster rates of functional decline compared to people without PAD.  
PAD affects 10-15% of community dwelling men and women age 60 and 

 
Page 8 of 41 
 
older (52-54) and will be increasingly prevalent as the U.S. population 
survives longer with chronic disease.   
 
B.  Cocoa is the dried and fermented seed from theobroma cacao, from 
which chocolate is made (see photo to left).  Epicatechins are flavanols, a 
major ingredient of cocoa, that have therapeutic effects on skeletal muscle 
and the vasculature (21-30).    In pre-clinical models, cocoa and/or its major 
flavanol, epicatechin, promote skeletal muscle mitochondrial biogenesis, 
increase capillary growth, improve endothelial function, and reduce 
ischemia/perfusion injury (21-24).  Cocoa and/or its major flavanol, 
epicatechin, also increase muscle mass and strength by favorably altering 
skeletal muscle levels of follistatin and myostatin (21,23,25).  
Pathophysiologic impairments in PAD that can be targeted by favorable 
effects of epicatechin-enriched cocoa are described below. 
 
Innovation of the research. 
 
A. Cocoa therapy has never been studied as a chronic, daily therapy for 
PAD.  Patients with PAD have reduced calf muscle mitochondrial energy 
production, reduced calf muscle mass and strength, and impaired endothelial 
function (13-20,40,41).  Cocoa and epicatechin improve mitochondrial 
energy production, increase mitochondrial biogenesis, increase skeletal 
muscle mass and strength, and improve endothelial function (11-20).  Thus, 
chronic, daily epicatechin-rich cocoa has the potential for benefit in PAD. 
 
B.  The COCOA-PAD Study will measure the effects of chronic daily 
epicatechin-rich cocoa on changes in calf muscle biopsy measures and 
endothelial function in PAD.  Calf muscle biopsies and brachial artery FMD 
at baseline and follow-up will delineate biologic pathways associated with 
improved walking performance in PAD.  Our calf skeletal muscle measures 
of TFAM, follistatin, and myostatin have never been studied before in 
people with PAD. 
 
C.  The study of therapeutic properties of a food-derived product is 
innovative.  This study has potential for far-reaching implications, since 
cocoa is widely accessible and acceptable to people with PAD. If the 
hypotheses of our proposed pilot trial are correct, a large definitive trial will 
be warranted.  
 
Justification for proposed dose. 
 
Our intervention is a dark cocoa beverage, naturally rich in epicatechin, 
manufactured by The Hershey Company.  Previous human study suggests 
that approximately 70 to 100 mgs of epicatechin per day is optimal for 
improving skeletal muscle mitochondrial biogenesis, mitochondrial 
function, and strength (22,25).  For example, in the study by Taub et al 

 
Page 9 of 41 
 
described in section A16 (25), the cocoa intervention contained 100 mgs of 
epicatechin.  We will prescribe three servings per day of the epicatechin-rich 
cocoa beverage, providing 75 mg daily of epicatechin.  The Hershey’s 
Company also produces a cocoa-free placebo drink that is packaged 
identically and appears and tastes identically to the cocoa beverage and has 
nearly identical nutritional content, including calories. Each beverage (cocoa 
and placebo) is prepared by dissolving the contents of a packet into water.  
Participants will be asked to drink three beverages per day.  
 
3.0 
Inclusion and Exclusion Criteria   
3.1 
Screening for eligibility  
  
Initial eligibility criteria will be assessed by telephone.  Potential 
participants who remain eligible after the telephone screening will be 
scheduled for a baseline visit, where they will undergo additional testing to 
determine their eligibility for randomization.  
 
3.2 Inclusion and Exclusion Criteria. 
Inclusion Criteria.  All participants will be age 60 and older. All 
participants will have PAD. PAD will be defined as follows:  
1. An ABI < 0.90 at baseline is an inclusion criterion for PAD. ABI 
< 0.90 is a well-accepted standard for defining PAD (55-58).  
2. Potential participants with an ABI > 0.90 who have vascular lab 
evidence of PAD or angiographic evidence of PAD will be eligible.  
Exclusion Criteria.   
1. Above- or below-knee amputation. 
2. Critical limb ischemia. 
3. Wheelchair-bound or requiring a cane or walker to ambulate.  
4. Walking is limited by a symptom other than PAD. 
5. Baseline six-minute walk value of < 500 feet or > 1,600 feet. 
6. Lower extremity revascularization, major orthopedic surgery, 
cardiovascular event, or coronary revascularization in the previous 
three months. 
7. Planned revascularization or major surgery during the next six months.  
8. Major medical illness including renal disease requiring dialysis, 
lung disease requiring oxygen, Parkinson’s disease, a life-threatening 

 
Page 10 of 41 
 
illness with life expectancy less than six months, or cancer requiring 
treatment in the previous two years. [NOTE: potential participants 
may still qualify if they have had treatment for an early stage cancer 
in the past two years and the prognosis is excellent. Participants who 
require oxygen only at night may still qualify.]  
9. Mini-Mental Status Examination (MMSE) score < 23 or dementia. 
10. Unwilling to attend three visits in one week for final outcome measures. 
11. Allergy to chocolate. 
12. Unwilling or unable to consume products manufactured on the same 
equipment that processes peanuts, tree nuts, egg, wheat, soy, and milk. 
13. Use of cocoa-containing dietary supplements. 
14. Unwilling to give up major dietary sources of epicatechin during the 
study.  
15. Symptoms of heart failure or angina that limit walking activity 
more than ischemic leg symptoms,, increase in angina, or angina at 
rest (i.e. unstable angina).  
16. Participation in or completion of a clinical trial in the previous 
three months. [NOTE: after completing a stem cell or gene therapy 
intervention, participants will become eligible after the final study 
follow-up visit of the stem cell or gene therapy study so long as at 
least six months have passed since the final intervention 
administration. After completing a supplement or drug therapy (other 
than stem cell or gene therapy), participants will be eligible after the 
final study follow-up visit as long as at least three months have 
passed since the final intervention of the trial.]  
17. Non-English speaking, a visual impairment that limits walking 
ability.  
18. In addition to the above criteria, investigator discretion will be 
used to determine if the trial is unsafe or not a good fit for the 
potential participant.    
 
Additional exclusion criteria for the muscle biopsy portion of the 
study: 
Participants who are taking anti-platelet therapy due to any history of 
TIA, stroke, or coronary stent or due to an acute coronary event in 

 
Page 11 of 41 
 
the past year will not be eligible to discontinue their anti-platelet 
therapy for muscle biopsy.  However, they may still be eligible for 
the muscle biopsy if Dr. Sufit and the patient agree to carry out the 
muscle biopsy while the participant remains on anti-platelet therapy. 
3.3 Special Populations.  
Vulnerable populations (fetuses, pregnant women, children, 
prisoners, and institutionalized persons) and adults unable to consent 
will not be included in this study.  
4.0 
Study-Wide Number of Subjects 
NA 
 
5.0 
Study-Wide Recruitment Methods 
NA 
 
6.0 
Multi-Site Research 
NA 
 
7.0 
Study Timelines 
Each participant’s part in this study will last approximately six 
months. We plan to enroll 44 eligible participants over 10 months. 
The estimated date to complete primary analyses is three years from 
the study start date. 
 
8.0 
Study Endpoints 
The primary study endpoint is the six-month change in the six-
minute walk data. 
Secondary study endpoints include a) six-month change in maximal 
and pain-free treadmill walking time; b)six-month change in brachial 
artery FMD; c) six-month change in accelerometer-measured 
physical activity; and d) six month change in the following calf 
skeletal muscle measures: activity of Cytochrome C Oxidase (COX) 
and citrate synthase, calf muscle levels of peroxisome proliferative 
activated receptor-γ co-activator 1α (PGC-1α), capillary density, 
myostatin, follistatin, and mitochondrial transcription factor-alpha 
(TFAM) 
Data will be collected at baseline and at the 6-month follow-up visit. 
9.0 
Procedures Involved 
9.1 Study design. 
Telephone screening. 

 
Page 12 of 41 
 
Initial eligibility criteria will be assessed by telephone via a telephone call 
script. The study will be described to the potential participant and the 
participant will be asked a series of eligibility questions to determine 
whether they qualify for a baseline study visit. If they qualify for a baseline 
visit, we will collect their name, address, telephone number(s), e-mail 
address, date of birth, gender, race, and ethnicity. 
 
In addition, some participants may be asked questions about their interest in 
supervised treadmill exercise programs, barriers to their participation in 
supervised exercise programs, and their ability to participate in supervised 
treadmill exercise programs.  These additional questions will be collected as 
pilot data for a subsequent study and may be administered at any time point 
during the COCOA-PAD trial. 
 
Baseline testing. 
Participants will be asked to come to Northwestern Memorial Hospital at 
baseline for the purposes of providing informed consent.  In addition, at the 
baseline study visits, we will obtain the ankle brachial index (ABI) 
measurement, to ensure they are eligible for the study.  We will administer 
study questionnaires to obtain information about medical history, including 
presence of comorbidities, medication use, smoking status, and other health 
characteristics. We will perform baseline physical functioning tests 
including the six-minute walk test, chair stands, balance testing, and the 
treadmill walking performance test.  We will take a blood sample, measure 
participants’ height and weight, and measure the blood pressure in their arm. 
We will perform a test of brachial artery flow-mediated dilation (FMD). 
Physical activity levels will be measured using an accelerometer. In 
addition, participants will be asked to undergo an MRI and a calf muscle 
biopsy at baseline. However, potential participants not eligible for MRI 
testing will still be allowed to participate.  Participation in the calf muscle 
biopsy is optional.  Baseline study visits will be performed over multiple 
days. Participants who have a muscle biopsy will be telephoned at 
approximately seven days after the muscle biopsy.  Participants who report 
any complaints about their biopsy site (such as significant pain or redness) 
will be scheduled for an evaluation of their biopsy site by a study physician.  
 
Details of baseline visit procedures are described in section 9.2. 
 
Randomization.  Participants will be randomized to either epicatechin-rich 
cocoa intervention or placebo using a SAS computer program. We will use 
block randomization with block sizes randomly selected from 4 and 6 to 
ensure balance. Randomization will be stratified according to baseline six-
minute walk performance. 
 
Monthly assessments to monitor adherence.  Participants will attend 
monthly study visits to receive their monthly supply of beverages. 

 
Page 13 of 41 
 
Participants will be asked to bring any remaining beverage packets from the 
previous month. Packets will be counted and the number of packets 
remaining from the previous month will be recorded. Participants will be 
asked to keep a log of their study beverage consumption and will be asked 
to turn in their log at each monthly visit. Participants will be asked to bring 
their current medications to these monthly meetings and changes in 
medication from the previous month will be recorded. In addition, weight 
and blood pressure will be measured at each monthly visit. If participants 
are having difficulty adhering to the beverages, staff may call participants in 
between monthly visits to check in on adherence and to remind them to fill 
out their monthly adherence log. 
 
Monitoring diet and weight gain.  Participants will receive a handout 
about monitoring and controlling their weight during the study.  Participants 
in the intervention and control groups will be asked to weigh themselves 
approximately daily and record their weight on a data collection form.  
Participants who do not have access to a scale will be provided with a scale. 
They will be asked to bring this weight chart to each monthly visit.  
Participants observed to gain five percent or more of their body weight 
compared to baseline during the study will be evaluated by the study team 
for possible causes of weight gain.  If the weight gain is determined due to 
increased caloric intake (rather than fluid weight gain), they will receive 
dietary counseling in effort to avoid additional weight gain and to advise 
them on dietary methods to help with weight control. Study beverages will 
be reduced or discontinued if a participant gains 20 or more pounds during 
the study. 
 
Six-month follow-up testing. After six months of participation in the study, 
participants will be asked to return for follow-up testing. All measures and 
tests that were conducted at baseline will be repeated.  
 
The six-minute walk and brachial artery FMD will be measured while off of 
study beverages for 2.5 hours and 24 hours, respectively.  . The treadmill 
stress test will be measured while off of the study beverages for 48 
hours.The calf muscle biopsy will be performed  after other outcome 
measures are complete and while on study beverages whenever possible.  If 
extenuating circumstances prevent outcome measurement in these defined 
time frames, the measurement will be obtained as close as possible to the 
desired time point/length of time after the final cocoa dose. 
 
Some or all study measures may be repeated at baseline or follow-up for 
data quality (e.g. if a treadmill test must be stopped due to extremely high 
blood pressure before the patient completed the test is one potential example 
of why a measure may need to be repeated).  
 

 
Page 14 of 41 
 
In some cases, participants may be asked to take study beverages for longer 
than six months. For example, if a participant has an illness that prevents 
them from returning at six months for follow-up testing or if they are out of 
town during their six-month follow-up window.  In these instances, 
participants will be asked to continue taking their study beverages for more 
than six months. Participants may be asked to take up an additional two 
months of study beverages. 
9.2 Research procedures. 
Ankle Brachial Index (ABI).  After the participant rests supine for five 
minutes, the right brachial, dorsalis pedis (DP), posterior tibial (PT) and left 
DP, PT, and brachial artery pressures are measured using a hand-held 
Doppler probe. Pressures are measured twice. The ABI is calculated for 
each leg by dividing the average of the DP and PT pressures by the average 
brachial pressure (59).  If a participant qualifies for a heel-rise ABI based on 
their initial ABI, they will be asked to perform 50 heel-rises at a rate of one 
per second followed by additional measurements of the right and left 
brachial, DP, and PT pressures. 
 
Questionnaire Administration.  Participants will be administered IRB-
approved study questionnaires by a trained and certified study coordinator.   
 
Six-minute walk.  Participants will be asked to walk back and forth along a 
100-foot hallway for six minutes.  They will be instructed that the purpose 
of the six-minute walk test is to measure the distance they can walk in six-
minutes.  A script will be read to describe the study procedure.  Participants 
will be asked whether they feel the test is safe to try and whether they have 
any questions. Participants who cover a distance of less than 500 feet or 
more than 1600 feet during the baseline six-minute walk will be excluded 
from the study.  In a pilot study to determine whether the six-minute walk 
distance is longer if a participant uses a 60 foot course instead of a 100 foot 
course, a subset of participants may be asked to return to the medical center 
on a separate day in order to perform the six-minute walk test using a 60 
foot course.  Note that this additional testing will not be performed within a 
month before the six-minute walk test performed as the primary outcome 
measure.  Only the six-minute walk test performed on the 100 foot course 
will be used for the study’s primary outcome measure. 
  
 
Treadmill testing.  The Gardner graded treadmill exercise test is the 
standard, accepted treadmill protocol for measuring change in maximal 
treadmill walking time in response to interventions among PAD participants 
(34,60,61-63).  In the Gardner exercise protocol, speed is maintained at 2.0 
miles per hour (mph) and treadmill grade increases by 2.0% every two 
minutes. If patients cannot walk at 2.0 mph, treadmill speed is started at 0.50 
mph and increased by 0.50 mph every 2 minutes until the participant reaches 
2.0 mph, after which the treadmill grade is increased every two minutes 
(34,60,61-63).  The treadmill stress test results will be read by the study 

 
Page 15 of 41 
 
cardiologist. If the results of the stress test are abnormal, participants will be 
referred back to their own physician for follow-up.  These potential 
participants will be evaluated individually to determine whether it is safe 
and appropriate for them to continue in the study.  It is noted that a regular 
exercise stress test does not have a high specificity for coronary artery 
disease.  Therefore, some participants will be allowed to continue in the 
COCOA-PAD Study.  This will be determined on a case-by-case basis.  For 
example, if the electrocardiographic tracing is considered a ‘false positive’ 
based on a more specific stress test result or based on an angiogram result 
ordered separately by the participant’s physician, then the participant will be 
allowed to continue in the study.  If the participant has stable angina or no 
angina symptoms and his/her physician considers a six-minute walk test 
safe, then the participant will be allowed to continue in the study, so long as 
he/she meets other eligibility criteria.  These are some examples of when it 
may be appropriate for a participant with an abnormal baseline exercise 
stress test to continue in the COCOA-PAD Study.   
 
Brachial Artery FMD.  Brachial artery imaging will be performed by a 
Registered Diagnostic Cardiac Sonographer using established methods 
(34,51,64).  With the participant supine, a blood pressure cuff over the upper 
arm is inflated for five minutes at a supra-systolic pressure according to 
protocol. The brachial artery is imaged (B-mode and Doppler) 5 to 9 cm 
above the antecubital fossa using a linear array vascular ultrasound 
transducer (Siemens Medical Solutions). FMD is calculated as the percent 
change in brachial artery diameter 60 seconds after cuff release.  Changes in 
FMD will be read in Dr. James Stein’s laboratory by a reader blinded to 
participant group assignment. 
 
Accelerometry.  Physical activity will be measured objectively and 
continuously in all participants over 7 days at baseline and follow-up, using 
the well-validated ActiGraph accelerometer (65-67).  Participants wear the 
ActiGraph on their beltline for seven days, removing it only for bathing or 
sleeping.  If the monitor needs to be worn following the biopsy, 
measurements will not begin until at least seven days have passed after the 
biopsy. By this time, any discomfort from the muscle biopsy that may limit 
activity has resolved.   
 
Blood collection and long-term storage. At baseline and six-month follow-
up study visits, participants will have approximately 45 mls of blood drawn 
for processing and long-term storage at -70 degrees Celsius. Approximately 
10% of participants selected by chance will have an additional set of blood 
drawn for quality assurance, for a total of 90 mL at each visit. Blood 
obtained will be collected in serum, plasma EDTA, and plasma citrate 
tubes.  Stored blood will await later analyses for biomarkers and other 
emerging blood markers related to peripheral artery disease that may change 
in response to the intervention.  Genetic testing may also be performed on 

 
Page 16 of 41 
 
stored DNA if the participant agrees to this optional study element on the 
consent document. Results of the genetic testing on the sample will be 
stored with other data collected. Samples will be labeled with the 
participant’s study identification number and will not be stored with other 
health or identifying information. Information associated with the sample 
will be stored a secure database on password protected computers that are 
secured by Northwestern University firewalls. Access is limited to study 
staff. If the samples are shared with other researchers not part of the current 
study, the PI will grant permission to the other researchers to analyze the 
samples after receiving IRB approval. Samples will be identified with a 
study identification number and the other researchers will not have access to 
PHI. Results of testing on the blood samples will not be shared with the 
study participants. 
 
Calf muscle biopsies.  Biopsies will be performed by Robert Sufit, MD, a 
board-certified neurologist with > 30 years of experience performing muscle 
biopsies.  He completed all biopsies for our pilot study.  The muscle biopsy 
will be obtained from the medial head of the gastrocnemius muscle in the 
leg with lowest ABI, at the point that is 67% of the distance between the 
medial malleolus and the medial aspect of the proximal tibia.  This site 
represents greatest calf muscle diameter in >95% of individuals (68).  
Anesthesia is achieved with subcutaneous lidocaine.  Subcutaneous and 
adipose tissue are dissected.  Approximately 300 mgs of muscle tissue is 
removed. Approximately 50 mgs is mounted and snap frozen in liquid 
nitrogen-cooled isopentane for immunohistochemistry and the remainder is 
frozen directly in liquid nitrogen and stored at -70 degrees Celsius for 
protein isolation.  At 6-month follow-up, we will repeat the biopsy, adjacent 
to the baseline incision site.  In the open biopsy, muscle tissue is directly 
visualized, providing substantial advantages over blind needle biopsy.     
 
Muscle biopsy specimens frozen at -70 degrees C will be shipped to Dr. 
Leeuwenburgh’s laboratory at the University of Florida and Dr. Peterson’s 
laboratory at University of Kentucky for analyses.  Enzyme activities will be 
measured in whole muscle homogenates using established methods (69).  
COX activity will be determined spectrophotometrically at 30°C as the 
maximal rate of oxidation of fully reduced Cytochrome C, measured by the 
change in absorbance at 550 nm. (69). Citrate Synthase activity will be 
measured using a kit (Sigma-Aldrich; Catalogue # CS0720), per the 
manufacturer’s instructions.  Protein expression of PGC-1α, TFAM, 
myostatin, and follistatin in whole tissue homogenate will be measured with 
Standard Western Blot (70-71).  Capillary density will also be measured 
using standard methods (72).  Satellite cell number will be quantified by 
counting Pax7+DAPI+ nuclei and activation state by counting 
MyoD+Pax7+ DAPI+ nuclei in cryosections and expressed per muscle fiber 
(51).  Regenerating fibers will be identified using an antibody against 
embryonic myosin heavy chain and by central nucleation to measure 

 
Page 17 of 41 
 
changes in response to epicatechin-rich cocoa administration.  
Immunohistochemistry will be performed using well standardized, validated 
methods with excellent test re-test reliability (78,79).  Nitro-tyrosine and 4-
hydroxynonanal will be measured using Standard Western Blot techniques 
as described previously (80,81). 
 
Other measures related to skeletal muscle quality and function may also be 
performed at Northwestern or at other institutions as designated by the 
Principal Investigator. 
 
MRI.  We will use arterial spin labeling with cardiovascular magnetic 
resonance imaging, methods developed by co-investigator Dr. Christopher 
Kramer and successfully implemented at Northwestern, to measure changes 
in calf skeletal muscle perfusionat 3 Tesla between PAD participants 
receiving epicatechin-rich cocoa vs. placebo. A thigh cuff is inflated in the 
leg with lowest ABI up to 250 mm Hg.   After five minutes, the blood 
pressure cuff is rapidly deflated.  Seven control-tagged image pairs are 
acquired over 60 seconds using PASL pulse sequence with single-shot echo-
planar imaging readouts (field of view 200x200 mm, matrix 64x64, 
repetition time 4000 ms, echo time 32 ms, slice thickness 10 mm).  
Perfusion is measured and quantified on a Siemens Healthcare workstation 
by selectively drawing regions of interest of the hyperemic areas on the 
perfusion maps.   
 
For pilot data collection, we will obtain MR images that allow us to quantify 
skeletal muscle and fat in calf muscle of participants. 
 
Medications.  Participants and their physicians will be asked not to change 
their medications during the study if possible.  Participants will be asked to 
bring all of their medications to baseline and follow-up visits and to monthly 
adherence visits.  We will systematically record names and doses of 
medications and inquire about medication changes at each of these visits.  If 
we find differences in the use of specific medications between study groups 
(such as statins or cilostazol), we will adjust for these differences.   
 
Other measures.  Body mass index (BMI) will be assessed at baseline and 
follow-up by objectively measuring height and weight. Weight will also be 
measured at monthly adherence visits.  Patient report will be used to 
document comorbidities.  A four-meter walk test will be administered at 
usual and fastest pace at the baseline and follow-up visits.  Participants will 
be asked to perform the usual paced four-meter walk at usual pace and the 
“fast paced” four meter walk at their fastest pace.  Each of these short walks 
will be performed twice.  Participants will be asked to complete a series of 
standing balance exercises and chair stands. Participants will have the blood 
pressure in their arm checked.  
 

 
Page 18 of 41 
 
Dietary counseling and weight monitoring.  Because of the potential for 
weight gain from study interventions, all participants will be weighed and 
receive dietary counseling at baseline and each monthly follow-up visit.  
Dietary counseling will consist of isocalorically removing approximately 
180 Kcal from other dietary sources to accommodate the beverage.  Total 
calories per day in the cocoa and placebo are comparable to a can of sugar 
sweetened soda or juice drink.  Participants who gain or lose five or more 
percent of their baseline body weight during the study will receive 
additional dietary counseling as appropriate. Participants who gain 20 or 
more pounds will have their study beverages reduced or discontinued. 
Participants will also be asked to refrain from eating foods high in 
epicatechin during the study. 
 
Pilot Data Collection. Some participants may be asked to complete a short 
questionnaire to allow investigators to better understand whether and how 
people with peripheral artery disease prefer to carry out walking exercise, 
and to collect data on whether weight loss is an important goal for people 
with peripheral artery disease.   
 
9.3 
Overview of protection against risks.   
 
Prior to beginning data collection, all study coordinators undergo training 
and are certified by Dr. McDermott using a detailed checklist for each data 
collection element. Research coordinators are certified in each element of 
the study visit. Dr. McDermott or a designee re-certifies coordinators 
approximately every six months to ensure continued adherence to protocol.  
Those not adhering to all aspects of the protocol undergo additional training 
followed by re-certification.  
 
All research staff members have completed human subjects training required 
by Northwestern’s institutional review board (IRB).  This training includes 
education about the importance of maintaining confidentiality of personal 
health information.  The study principal investigator or a co-investigator is 
available to answer questions that arise during the informed consent process 
as needed.   
 
Participants are asked to sign a study consent form prior to data collection.  
The research coordinator reviews study procedures, including risks and 
benefits associated with study participation.  The research coordinator 
answers participants’ questions.  Dr. McDermott and other study 
investigators are available to answer participants’ questions.  Dr. 
McDermott’s pager, direct telephone line, and home telephone number are 
provided to participants.   
              
Minimizing risks related to the epicatechin-rich cocoa beverage and 
placebo.  Exclusion criteria include a chocolate allergy.  Participants will be 

 
Page 19 of 41 
 
asked to return to the medical center once monthly.  At these visits, blood 
pressure and weight will be measured.  At each of these monthly follow-up 
visits, all participants will receive dietary counseling, under the direction of 
co-investigator Dr. Linda Van Horn.  Dietary counseling will consist of 
helping participants to identify removal of approximately 180 Kcal from 
other dietary sources to accommodate the calories associated with both the 
epicatechin-rich cocoa beverage and placebo (i.e. isocaloric).  Safety will 
also be monitored by our DSMB.  However, as described above, the 
epicatechin-rich cocoa is a food product derived from the theobroma cacao 
plant.  Thus we anticipate that adverse events will be very infrequent. 
 
 
 
Minimizing risks related to muscle biopsy.  The muscle biopsy procedure 
will be performed by Dr. Robert Sufit who has more than 30 years of 
experience performing these muscle biopsies, primarily as part of his 
clinical practice as a Board-Certified Neurologist.  As in our pilot study, 
completed in preparation for this proposal, the muscle biopsy procedures 
will be performed under sterile conditions using sterile technique.  Local 
anesthesia will be obtained using subcutaneous lidocaine.  All participants 
will be provided with written and verbal instructions about wound care and 
will be advised to contact Dr. McDermott immediately if any signs of 
wound infection occur. In addition, participants with a muscle biopsy will be 
telephoned approximately seven days after the muscle biopsy and will be 
asked about their muscle biopsy site.  Participants who report significant 
discomfort or redness at the site of the muscle biopsy site during this 
telephone call will return for a site visit.   
 
Many PAD participants take anti-platelet therapy to prevent cardiac and 
cerebrovascular events.  If potential participants are taking anti-platelet 
therapy, they will be asked to hold their anti-platelet therapy during the 
seven days leading up to the muscle biopsy procedure. Participants who are 
asked to hold their anti-platelet therapy during the week leading up to the 
muscle biopsy procedure may experience a heart attack or stroke related to 
the temporary discontinuation of the anti-platelet therapy.  Permission from 
the participant’s physician will be required before participants are asked to 
discontinue anti-platelet therapy. Participants who are taking warfarin or 
other anti-coagulant therapy will not be eligible for muscle biopsy.   
 
Minimizing risk related to baseline and follow-up testing.  All study 
coordinators undergo baseline training and are certified by Dr. McDermott 
before beginning data collection (See Appendix A for an example 
certification checklist).  Training and certification involves ensuring that 
coordinators are trained in methods to help minimize falls.  Dr. McDermott 
re-certifies coordinators every six months to ensure continued adherence to 
study protocol.  Those who are not adhering to protocol undergo additional 
training followed by re-certification.     

 
Page 20 of 41 
 
 
Minimizing risk related to loss of confidentiality.  The following methods 
will be employed to maintain confidentiality of participants.  First, study 
recruitment letters will be mailed, using IRB-approved methods, only after 
receiving permission from the participant’s physician.  The personal 
physician of each study participant will have the option of not allowing 
investigators to contact the potential participant.  Lists of potentially eligible 
participants will be obtained by individuals who normally have access to 
these lists as part of their daily work requirements.  Recruitment letters for 
potential participants identified from hospital and outpatient lists are 
prepared by research staff members whose job is to assist study investigators 
with recruitment.  These research staff members have completed training in 
the ethical conduct of human subject research, including maintaining 
participant confidentiality.  Recruitment letters to potential participants 
identified from medical center lists are mailed in sealed envelopes and 
addressed to the potential participant.  All potential participants who receive 
mailed information about the study after the approval from their physician 
will have the opportunity to call a voice-mail system to ask NOT to be 
further contacted about this study.  Secondly, only study investigators and 
trained research staff will have access to the study database.  Third, 
participants will be assigned a unique study identifier.  Individual names 
will ultimately be removed from the study database and only the unique 
study identifier will be used to distinguish participants in the database.  
Fourth, collected data will be maintained in locked computer files and file 
cabinets to which only study investigators have access.  Collected data will 
be used only for research purposes.  Any published data will not contain any 
individual identifiers. 
 
Minimizing risks related to MRI. Participants will be carefully questioned 
to ensure it is safe for them to undergo MRI testing.   
 
 
10.0 Data and Specimen Banking 
10.1 Storage of specimens. 
Muscle specimens and blood specimens for long-term storage will be stored 
in a freezer belonging to Dr. McDermott's research program at Northwestern 
University, in the freezer farm in the basement of Olson Pavilion. 
Specimens will be stored for up to 70 years, after which they will be 
destroyed. 
10.2 Data to be stored or associated with each specimen. 
Specimens will be coded; meaning that a key will exist that can link the 
codes back to the direct subject identifiers. Each participant will be assigned 
a unique study ID number that can be traced back to the study participant.  
The muscle specimens and the blood samples that are stored will be labeled 
with this unique identifier and the date and time of the blood collection.  

 
Page 21 of 41 
 
10.3 Procedures to release data or specimens. 
Only Dr. McDermott has control over release of study data or specimens.  
Any investigators seeking to analyze blood or muscle specimens must 
contact Dr. McDermott for permission.  Each request, if it occurs, will be 
considered on a case-by-case basis.  Dr. McDermott will obtain IRB 
approval prior to releasing any blood or muscle specimens for analysis, 
other than those tests specifically named in this application. 
 
11.0 Data and Specimen Management 
11.1 Data management. 
Data is recorded using preprogrammed instruments and an electronic case 
report form using secure, HIPAA-compliant REDCap database software on 
servers maintained by Northwestern’s Clinical and Translational Sciences 
Institute. We have substantial experience with REDCap.  
 
11.2 Power Estimates/Calculations.   
 
The COCOA-PAD Study is a pilot study intended to collect preliminary 
data to estimate an effect size and to assess the feasibility of our proposed 
methods.  Therefore, we calculated the sample size that allows us to identify 
an effect size estimate. Our power calculations anticipate a < 10% drop-out 
rate at 6-month follow-up, based on data from our completed GOALS and 
SILC randomized trials in PAD participants (34,60). For our primary aim, 
we will compare changes in six-minute walk performance at 6-month 
follow-up between the epicatechin-rich cocoa group and the placebo group. 
Comparisons will be made for measures obtained both while off of the study 
beverages for 2.5 hours and while off of the study beverages for 24 hours, 
respectively,  in order to assess both acute and chronic effects of cocoa on 
six-minute walk.  A total of 40 participants completing the trial will provide 
a two-sided 95% confidence interval (CI) with a margin of error of 0.64 
standard deviation (SD) for the effect size in change of six-minute walk 
performance between the cocoa (n=20) and the placebo groups (n=20). 
Using the estimated SD from our SILC trial (34), the margin of error of 0.64 
SD represents approximately 33 meters. Prior studies have defined clinically 
meaningful changes in the six-minute walk as 20 meters (small meaningful 
difference) and 50 meters (large meaningful difference) (73,74).   Similarly, 
40 participants completing the trial provides a two-sided 95% CI with a 
margin of error of 0.64 SD for the effect size in 6-month change of each 
secondary outcome between the cocoa and placebo groups.    
   
Statistical Analyses.  Data will be analyzed according to each participant’s 
originally assigned group, irrespective of whether the participant adheres to 
his/her assigned group (i.e. intention to treat principle).  For our Primary 
Aim, we will use a t-distribution to obtain the 95% CI for the difference in 
changes in six-minute walk performance at 6-month follow-up between the 

 
Page 22 of 41 
 
cocoa and placebo groups.  Comparisons at six months will be made to 
measure both the acute effects (2.5 hours after drinking the study beverages) 
and the chronic effects (24 hours after drinking the study beverages).  The 
characteristics of participants in the intervention and control group will be 
compared using t or chi-square tests.  If there is indication of major 
imbalance, we will perform analysis of covariance (ANCOVA) with change 
in six-minute walk distance as the response variable to evaluate differences 
between the two groups, adjusting for potentially imbalanced covariates.  If 
there is evidence that the normality assumption for the distribution of 
outcome measures is violated, we will either apply appropriate 
transformation to the original measures before conducting the 
aforementioned analyses or obtain a nonparametric estimate and 95% CI for 
the difference in median between the cocoa and placebo groups (74). These 
methods will also be used to analyze outcomes in our secondary specific 
aims. 
 
11.3 After recruitment was completed, and prior to reviewing results, 
investigators determined that a one-sided test with a significance level of 
0.10 will be used to in the statistical analyses. Correspondingly, the point 
estimator and one-sided 90% CIs will be obtained for the differences in all 
outcomes between the cocoa and the placebo groups. Since the trial is a 
pilot study collecting preliminary evidence on the potential benefit of 
epicatechin-rich cocoa on change in walking ability and other outcomes in 
PAD patients, one-sided tests are justified. For the same reason, the 
decision on whether a larger independent confirmatory study is warranted 
to prove the hypothesized treatment effect of cocoa will be made based on 
all relevant evidence in this pilot study collectively, which include but are 
not restricted to the statistical significance. Steps to secure data to maintain 
confidentiality during storage, use, and transmission. 
First, all research assistants must complete training in protection of subject 
privacy and prevention of disclosure of identifying information. 
 
Second, all data collection forms are maintained in a secure office space. 
 
Third, our study databases are maintained in locked computer files or on 
secure hard-drives that are password protected; to which only authorized 
staff have access. Dr. McDermott or a study manager must provide 
permission for programmers and research assistants to access study 
databases. 
 
Fourth, a study identification number will be assigned to each participant. 
This identification number will be used to label blood specimens, for 
example. In addition, most pages of our data collection forms will have only 
the study identification number listed (and not the participant's name, for 
example). 

 
Page 23 of 41 
 
11.4 Quality Control.   
Health interviewers will be trained by a senior coordinator and certified by 
Dr. McDermott in each data collection element, using a detailed checklist.  
Health interviewers are rigorously evaluated for adherence to protocol, 
delineated in our manuals, prior to beginning data collection.  They are re-
certified approximately every six months by a project manager or Dr. 
McDermott.  When deficiencies are identified, interviewers undergo 
additional training and re-assessment. Ten percent of participants are 
randomly identified for quality control.  This subset has their ABI measured 
twice by independent examiners and may have a split sample of muscle 
tissue (i.e. two samples from the same participant) sent for analyses.  The 
second muscle sample is designated an arbitrary identification number to 
which the technician is blinded.  Thus, quality control is monitored 
continuously.  A 20% subset of collected MR images will be interpreted by 
two independent evaluators. For the skeletal muscle measures, a 10% subset 
of participants will have an additional quality control muscle specimen 
analyzed.  To achieve this, up to 40% of individuals may have up to 400 
mgs of tissue removed. 
Blinding for data collection.  The study design is double-blinded. 
Participants, study investigators, and the study coordinator collecting 
outcome data will be unaware of group assignment.  
Missing Data.  First, missing data may occur if participants are lost to 
follow-up.  However, in our prior randomized trials of PAD participants, 
drop-out rates were <10% at 6-month follow-up (34,60,75).  Thus, drop-out 
should be small.  Second, we recognize that not all participants may return 
for a second muscle biopsy at follow-up.  However, 95% of participants 
who completed a muscle biopsy in our pilot study indicated that they were 
willing to return for a second biopsy.  Thus, loss to follow-up should be 
small. Third, some participants may not adhere to their beverage. We will 
measure adherence and will perform a sensitivity analysis that includes only 
those who adhered to 80% or more of their assigned beverage.  This pilot 
study will determine likely adherence rates for a larger trial. 
 
12.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
A Data Safety Monitoring Board (DSMB) will monitor safety throughout 
the study.  Please see separate Data Safety Monitoring Board (DSMB) 
charter.  The DSMB charter will be a living document that can be modified 
during the study.   
The DSMB will meet at least every six months during the study.  The 
DSMB will review and approve the protocol prior to beginning data 
collection.  They will decide on stopping criteria for the study. The 
biostatisticians and data manager will work with the DSMB to perform 
interim analyses.  Adverse events will be monitored continuously 

 
Page 24 of 41 
 
throughout the study and will be reported to the DSMB in a timely manner 
according to pre-specified requirements.  Analyses for each DSMB meeting 
will be completed according to the requests of the DSMB. Investigators and 
DSMB members will be blinded to group assignment. Adverse event rates 
and interim study results will be reviewed and discussed by the DSMB at 
the DSMB meetings.  At least four categories of adverse events will be 
defined:  a) death; b) cardiovascular events (myocardial infarction, stroke, 
and coronary arrhythmias); c) hospitalizations; and d) injury or illness 
causing chronic disability.  We will report all serious adverse events to the 
DSMB in a timely fashion.  In addition to these serious adverse events, we 
will monitor participant weight and blood pressure during monthly visits 
throughout the study.  We will use a designated data collection form to 
record these events.  As noted above, however, we anticipate that that 
epicatechin-rich cocoa beverage will have few significant adverse events. 
13.0 Withdrawal of Subjects 
13.1 Anticipated circumstances under which subjects will be withdrawn 
from the research without their consent, including stopping 
participation for safety reasons.  We anticipate that subject 
withdrawal from the research without their consent will be 
infrequent.  However, a potential example is if a participant develops 
symptoms during the study and the Principal Investigator feels that 
the symptoms could make the study unsafe for the participant to 
continue. In this circumstance, the participant would be advised to 
follow-up with their physician.  If the participant refuses to follow-
up with their physician, it may be necessary for the participant to be 
withdrawn without their consent.  
13.2 Procedures when subjects withdraw from the research. Subjects may 
withdraw from the research at any time. If they decide to leave the research, 
they should contact the principal investigator, Dr. Mary McDermott. 
If they stop being in the research, already collected data may not be 
removed from the study database. They will be asked whether the 
investigator can collect data from their routine medical care. If the subject 
agrees, this data will be handled the same as research data. 
 
14.0 Risks to Subjects 
 
The placebo beverage has the same caloric intake and nutritional 
characteristics as the cocoa beverage except that the placebo drink contains 
no cocoa or flavanols.  In general, older patients with PAD have a high 
prevalence of comorbid diseases, particularly coronary artery disease, 
cerebrovascular disease, diabetes mellitus, and pulmonary disease.  Thus the 
patient population is likely to be of generally poorer health than that of older 
men and women without PAD in the general population.   

 
Page 25 of 41 
 
 
Risks associated with cocoa therapy.  Cocoa is the dried and fermented 
seed from theobroma cacao, from which chocolate is made. Epicatechins 
are flavanols and a major ingredient of cocoa that has therapeutic effects on 
skeletal muscle and the vasculature (21-30).  Our cocoa-beverage 
intervention is a food product, manufactured by the Hershey Company. 
Similar cocoa drinks are widely available for commercial use.  The flavanol 
epicatechin, when administered in therapeutic doses for research purposes, 
can cause lowering of blood pressure (76).  Thus, blood pressure levels will 
be measured monthly during the study.  The epicatechin-rich cocoa drink 
and the placebo will each deliver approximately 180 calories per day in the 
doses prescribed (three drinks per day).  Weight will be monitored monthly 
throughout study participation.  
 
Risks associated with the muscle biopsy.  The muscle biopsy is associated 
with several potential risks.  These include discomfort during the muscle 
biopsy procedure and immediately afterward, scarring from the muscle 
biopsy skin incision, bleeding, and infection. In addition, potential 
participants who are asked to hold their anti-platelet therapy during the week 
leading up to the muscle biopsy procedure may experience a cardiovascular 
event related to the temporary discontinuation of the anti-platelet therapy.  
First, to minimize risk related to muscle biopsy, all participants undergoing 
muscle biopsy will receive a written hand-out regarding signs to watch for 
that may indicate wound infection.  They will also be verbally instructed in 
this.  Each participant will be instructed to call Dr. McDermott immediately 
if any signs of infection occur. Participants will be telephoned 
approximately seven days after the muscle biopsy and asked for any 
symptoms that may suggest an infection or delayed healing.  Second, 
permission from the participant’s physician will be required before 
participants are asked to discontinue anti-platelet therapy.    
 
Six-minute walk test, four-meter walks, balance, and chair stands.  The 
physical functioning tests may be associated with muscle fatigue or 
soreness.  These symptoms typically resolve with rest.   
 
These tests may be associated with the risk of falling or coronary ischemia 
or dyspnea due to heart failure or lung disease.  Rarely, falling may result in 
a fracture.  However, the research assistant who will collect these data has 
been trained to prevent falling.  The risk of a fracture secondary to a fall 
during the testing is less than 1 in 5,000.  If a participant experiences chest 
pain, research assistants are trained to page Dr. McDermott immediately.  If 
the chest discomfort does not immediately resolve with rest, participants are 
escorted to Northwestern’s Emergency Department, which is located in the 
same building as the location of the tests.  Dr. McDermott facilitates follow-
up, by contacting participants’ physicians, for those who experience new 

 
Page 26 of 41 
 
chest discomfort during testing.  In our experience, the risk of chest 
discomfort is approximately 1 in 750.   
 
Risks associated with ABI measurement.  The ankle brachial index 
measurement consists of measuring systolic blood pressure in each 
extremity using a hand-held Doppler.  The ABI is non-invasive, safe and 
does not have any known lasting side effects.  During the ankle brachial 
index test, participants may experience discomfort from the inflated blood 
pressure cuff.  However, this discomfort resolves immediately when the cuff 
is released. 
 
Risks associated with questionnaire administration.  Participation 
includes a risk of loss of confidentiality regarding personal health 
information.  However, all research staff have undergone formal human 
subjects training.  They are trained to protect the privacy of research subject 
participants.   
 
Risks associated with drawing blood.  The potential risks of drawing 
blood include a bruise at the site of vein puncture, inflammation of the vein, 
and infection. Participants undergoing a blood draw may experience 
lightheadedness, dizziness, or fainting. 
 
Risks associated with MRI testing.  The MRI scanner makes loud banging 
noises while doing a measurement.  Participants will be provided with 
earplugs or earphones to protect against the noise.  MRI testing can be 
difficult for people with claustrophobia.  Potential participants who are 
claustrophobic will not be encouraged to participate in MRI testing. 
 
15.0 Potential Benefits to Subjects 
Participants who are randomized to the intervention could experience 
improved functional performance and prevent mobility loss, if our 
hypotheses are correct.   
If the COCOA-PAD Study supports our hypotheses, results will be used to 
design a large, definitive randomized controlled trial of epicatechin-rich 
cocoa to improve lower extremity functioning and prevent mobility loss in 
the large and growing number of older people who are disabled with PAD. 
16.0 Vulnerable Populations 
NA 
 
17.0 Community-Based Participatory Research 
NA 
 
18.0 Sharing of Results with Subjects 

 
Page 27 of 41 
 
Participants will receive results of their ankle brachial index (ABI) test and 
will be provided with a “result letter” at the end of their baseline visit.  ABI 
and stress test results will be mailed to the participant’s physician by 
request. They will not be provided with other study results routinely. 
However, participants will be notified of abnormal stress test results or 
abnormal blood pressure results. 
19.0 Setting 
 
 
 
Baseline and follow-up data collection will take place at Northwestern 
Memorial Hospital in the Galter Pavilion (675 N. St. Clair) on floors 11 or 
18, 680 N. Lake Shore Drive on the 14th floor, or at 750 N. Lake Shore 
Drive.   
 
20.0 Resources Available 
Our multidisciplinary team includes internationally recognized investigators 
in PAD (Drs. Criqui, Kibbe, McDermott), functional decline (Drs. Ferrucci, 
Guralnik, McDermott), skeletal muscle molecular and cellular biology (Drs. 
Ferrucci, Peterson, Leeuwenburgh, Sufit), cocoa/epicatechin (Drs. 
Villarreal, Van Horn), and MRI (Dr. Kramer).  Dr. James Stein, an 
internationally recognized expert in endothelial function, and Dr. Linda Van 
Horn, an internationally recognized diet and nutrition expert, have joined the 
investigative team.  Dr. Van Horn will assist with dietary counseling to 
prevent study-related weight gain.  Dr. Christopher Kramer is internationally 
recognized for his work developing MRI methods for measuring calf muscle 
perfusion in patients with PAD. We have successfully completed 
observational studies of functioning in PAD participants (R01-HL58099, 
R01-HL64739, and R01-HL71223L) (1-5,7,19,22,32) and randomized 
clinical trials of exercise or behavioral interventions in PAD participants:  
(R01-HL073351, R01-HL073912, and R01-HL088589) (34,60,75).  We 
have experience with all outcomes proposed.  We are an ideal investigative 
team to conduct the COCOA-PAD Study. 
 
We have substantial experience with calf muscle biopsies in PAD 
participants.  Since September 1, 2013, we have completed 54 muscle 
biopsies in a pilot study of people with and without PAD.  Biopsies were 
performed by co-investigator Dr. Sufit, who has more than 30 years of 
experience performing muscle biopsies.  Most of the muscle specimens 
remain frozen at -70 degrees Celsius, since resources are not available for 
analyzing all of them.  Available resources allowed us to analyze specimens 
from 17 participants (9 with PAD) in Dr. Leeuwenburgh’s laboratory.  Our 
results show that more severe leg ischemia is associated with poorer 
mitochondrial activity and that poorer mitochondrial activity is associated 
with poorer six-minute walk.  Citrate synthase activity, a measure of 
mitochondrial function and quantity, and COX activity each correlated with 
PAD severity, measured by the ankle brachial index (ABI) 
(correlations=0.492 and 0.769, respectively).  Among PAD participants, 

 
Page 28 of 41 
 
citrate synthase and COX activity were correlated with the six-minute walk 
(0.411 and 0.397, respectively).   
 
Dr. Christiaan Leeuwenburgh is an internationally recognized expert in 
skeletal muscle biology and mitochondrial function.  Dr. Leeuwenburgh’s 
laboratory will measure COX and citrate synthase activity and levels of 
protein markers of mitochondrial biogenesis (PGC-1α and TFAM) and 
proteins associated with skeletal muscle mass (follistatin and myostatin) as 
well as measures of muscle oxidative stress in samples obtained in the 
COCOA-PAD Study.  Dr. Leeuwenburgh’s laboratory performed all of the 
measures from our muscle biopsy pilot study.  Dr. Charlotte Peterson’s 
laboratory at University of Kentucky will perform immunohistochemical 
analyses of muscle regeneration. Dr. Peterson is internationally recognized 
for her work focusing on elucidation of cellular and molecular mechanisms 
that control skeletal muscle structure and function. Dr. James Stein of the 
University of Wisconsin Medical Center, will measure brachial artery FMD 
from images collected at Northwestern.  Dr. McDermott and Dr. Stein have 
worked together on previous randomized trials of PAD participants, funded 
by the NIH (34,51,77).  Addition co-investigators include Drs. Michael H. 
Criqui (University of California at San Diego), Jack M. Guralnik 
(University of Marlyand), Luigi Ferrucci (National Institute on Aging), and 
Francisco Villarreal (University of California at San Diego). Drs. Criqui, 
Guralnik, and Ferrucci have worked with Dr. McDermott on PAD studies of 
functional impairment for over eleven years and bring expertise in 
functional assessment, PAD, and clinical trials to the study team.  Dr. 
Villarreal is a nationally-recognized expert in cocoa and epicatechin therapy 
who has conducted pre-clinical studies and a small preliminary human trial 
of cocoa therapy for patients with diabetes and heart failure (22,23,25-27).     
 
21.0 Prior Approvals 
         NA 
 
22.0 Recruitment Methods 
22.1     When, where, and how potential subjects will be recruited. 
PAD participants will be identified from among individuals with PAD who 
have participated previously in research conducted by Dr. McDermott 
and/or who have expressed an interest in participating in future studies 
conducted by Dr. McDermott. 
 
In addition, some PAD participants may be identified from among 
consecutive patients diagnosed with PAD in the non-invasive vascular 
laboratory at Northwestern Medical Group (NMG). Dr. Mark Eskandari is 
medical director of the non-invasive vascular laboratory at NMG and will 
assist with identifying potential participants from the non-invasive vascular 
laboratory. As director of the vascular laboratory at NMG, Dr. Eskandari 

 
Page 29 of 41 
 
formally reads many of the non-invasive vascular laboratory tests. He 
maintains all non-invasive vascular test results in his vascular laboratory. As 
director of the vascular laboratory, Dr. Eskandari could conceivably contact 
the patients whose test results are maintained in his laboratory. However, 
Dr. Eskandari prefers that the contact of potential participants in studies 
come from the physicians referring him for testing. Lists of patients who 
have undergone lower extremity arterial testing in the non-invasive vascular 
laboratory are generated monthly and e-mailed from NMG to Dr. 
McDermott using an encrypted file. A research assistant, working on behalf 
of Dr. Eskandari, will contact referring physicians of potential participants 
identified from the vascular laboratory via fax, phone, page, or electronic 
message (EPIC or e-mail), to ask for permission to contact their patient 
about the study. If a reply is not received within three weeks, up to five 
letters are mailed from Dr. McDermott about the research study. We have 
substantial experience with our recruitment methods for our previous or 
ongoing studies. 
 
We also propose to obtain lists of consecutive patients with a diagnosis of 
lower extremity peripheral arterial disease and individuals at high risk for 
peripheral artery disease from Northwestern’s Enterprise Data Warehouse 
(EDW). EDW lists will be obtained by an individual who is employed by 
the Division of General Internal Medicine who has received training and 
permission to obtain the lists from the EDW. 
 
Similar methods will be used as those described above, in which the 
patient’s physician will be contacted via fax, telephone, page, or electronic 
message (EPIC or email) to ask for permission to contact their patient about 
the study. If a reply is not received within three weeks, up to five letters are 
mailed from Dr. McDermott about the research study.   
 
In the recruitment letters, recipients are asked to call us if they are interested 
in participation or if they do not want to be contacted further. Potential 
participants who do not call us within three weeks of the first mailed 
recruitment letter may be telephoned by study staff and invited to 
participate. 
 
In addition, we will use newspaper, television, and radio advertising to 
identify potential participants for this study. We will also use brochures, 
flyers, or posters that we will post in relevant office practices and public 
areas. We will use advertising on public transportation or online 
advertisements. 
 
We will obtain a list of patients who are in an eligible age range for the 
study and live in the Chicago area from a mass mailing company. Using 
this, we will send postcards to those individuals on the list. The postcards 
will instruct people to call a study number if they are interested.  

 
Page 30 of 41 
 
 
Participants who have participated in previous studies and indicated interest 
in future studies will be contacted. Participants who we screen for ongoing 
studies who may have PAD but are ineligible for that study and interested 
participating in a study may be screened for this study. 
 
We may also use the PCOR-NET for recruitment.  PCOR-NET is a PCORI-
funded network of institutions in the Chicago area. The purpose of the 
PCOR-NET is to assist investigators with recruitment for clinical trials.  
PCOR-NET has its own IRB (University of Illinois at Chicago).  PCOR-
NET uses ICD-9 codes and the electronic health record to identify potential 
participants who have PAD.  These patients with PAD receive a recruitment 
letter that describes the COCOA-PAD trial and invites the PAD patients to 
participate. 
 
22.2     Payment 
Participants will receive $25 for completing six-month follow-up 
testing and $25 per MRI.  
 
If the participant undergoes the optional muscle biopsy portion of the 
study, they will receive $100 per muscle biopsy. Therefore, participants 
will receive up to $275 for taking part in this research study. 
Participants will be given assistance and/or reimbursement for expenses 
related to travel such as parking, bus/train fare, taxi fare, and mileage, if 
requested. A receipt will be required for taxi fare reimbursement. 
Participants will be provided up to $90 per visit for travel 
reimbursement. If they require the use of our taxi service, we will 
estimate the fare on www.taxifarefinder.com. A one-way fare estimate 
must be less than or equal to $45 (i.e. round trip of $90) in order for the 
study to provide taxi service. 
 
23.0 Local Number of Subjects 
We will identify and randomize 44 eligible participants. 
 
24.0 Confidentiality 
NA 
 
25.0 Provisions to Protect the Privacy Interests of Subjects 
All research staff undergo training (human subjects training) in the 
protection of participant confidentiality and privacy.  Research staff have 
access to medical records only for the purpose of conducting research that is 
approved by the IRB. 

 
Page 31 of 41 
 
Questionnaires and all research procedures will be conducted in an enclosed 
space by a trained and certified research assistant.  Dr. McDermott 
personally certifies research assistants in data collection to help ensure that 
participants are treated with the highest level of professionalism.   
26.0 Compensation for Research-Related Injury 
If the subject needs medical care because of taking part in this research 
study, they should contact the investigator and medical care will be made 
available. This care will be billed to the subject, their insurance, or other 
third party. Northwestern University has no program to pay for medical care 
for research-related injury. 
 
27.0 Economic Burden to Subjects 
NA 
 
28.0 Consent Process 
 
The “SOP: Informed Consent Process for Research (HRP-090)” will be 
followed. Participants will be consented by a research assistant who has 
been trained and certified by Dr. McDermott in obtaining informed consent. 
A research assistant will explain the study to potential participants by 
telephone prior to their first study visit. When a potential participant arrives 
to the medical center for study participation, the research assistant will 
explain the full details of the research study, including risks and benefits. 
The informed consent process will take place at the initial baseline study 
visit in a private area on Northwestern’s medical campus.  
Potential participants will be provided plenty of time to read the consent 
form. The research assistant will answer questions and Dr. McDermott or 
another study investigator at Northwestern is also available to answer any 
questions that participants may have about the research. If the participant 
would like more time to discuss the research study with their physician or 
family member before signing the consent document, they will be allowed 
to do so and the study visit will be rescheduled for a later date.  
Potential participants who do not speak English, subjects who are not yet 
adults, cognitively impaired adults, and adults unable to provide written 
consent will not be eligible for study participation. 
29.0 Process to Document Consent in Writing 
The “SOP: Written Documentation of Consent (HRP-091)” will be 
followed.  
 
30.0 Drugs or Devices 
 
 

 
Page 32 of 41 
 
The Hershey Company will send cocoa-epicatechin and placebo beverages 
in identically appearing packets to Dr. McDermott. Packaging of the cocoa 
and placebo packets are identical and distinguished only by Lot ID number 
(D or F). Investigators and study staff and study participants are blinded to 
which group is ‘D’ vs. ‘F’. REDCap will randomize to either group D or F 
and participants will be provided with a month’s supply of cocoa packets 
labeled with either D or F according to their randomization assignment. 
 
Participants randomized to the intervention will consume three epicatechin-
rich cocoa beverages per day (for instance, participants may consume two in 
the morning and one in the afternoon/early evening). Participants 
randomized to placebo will take three identical-appearing placebo beverages 
per day (for instance, participants may consume two in the morning and one 
in the afternoon/early evening).  
Study beverage packets will be stored in a locked closet located on the 10th 
floor of 750 N. Lake Shore Drive. Study packets are stored at room 
temperature, not exceeding 90 degrees F. An electronic thermometer is used 
within the office space that tracks and records temperature daily and will 
alarm if the temperature exceeds 90 degrees. Packets will be counted and 
dispensed in a large handled bag to participants. A monthly supply will 
include 90 packets (3 packets per day X 30 days). Two individuals on the 
study team will independently check to ensure that the study randomization 
group assignment (Group D or F) matches the labeling on the packets before 
dispensing to the participant. A log will be kept to record the dates and 
number of packets that are dispensed throughout the study period. 
 
The Food and Drug Administration concluded that an IND is not required. 
 
 
 

 
Page 33 of 41 
 
31.0 References 
 
1. McDermott MM, Greenland P, Liu K, Guralnik JM, Criqui MH, Dolan NC, Chan C, 
Celic L, Pearce WH, Schneider JR, Sharma L, Clark E, Gibson D, Martin GJ. Leg 
symptoms in peripheral arterial disease: associated clinical characteristics and functional 
impairment. JAMA 2001;286:1599-606. PMID: 11585483. 
 
2.  McDermott MM, Liu K, Greenland P, Guralnik JM, Criqui MH, Chan C, Pearce WH, 
Schneider JR, Ferrucci L, Celic L, Taylor LM, Vonesh E, Martin GJ, Clark E. Functional 
decline in peripheral arterial disease: associations with the ankle brachial index and leg 
symptoms. JAMA 2004;292:453-61. PMID: 15280343. 
 
3.  McDermott MM, Greenland P, Liu K, Guralnik JM, Celic L, Criqui MH, Chan C, 
Martin GJ, Schneider J, Pearce WH, Taylor LM, Clark E. The ankle brachial index is 
associated with leg function and physical activity: the Walking and Leg Circulation 
Study. Ann Intern Med 2002;136:873-83. PMID: 12069561. 
 
4.  McDermott MM, Guralnik JM, Tian L, Liu K, Ferrucci L, Liao Y, Sharma L, Criqui 
MH. Associations of borderline and low normal ankle-brachial index values with 
functional decline at 5-year follow-up: the WALCS (Walking and Leg Circulation Study). 
J Am Coll Cardiol 2009;53:1056-62. PMID: 19298919. 
 
5.  McDermott MM, Guralnik JM, Tian L, Ferrucci L, Liu K, Liao Y, Criqui MH. 
Baseline functional performance predicts the rate of mobility loss in persons with 
peripheral arterial disease. J Am Coll Cardiol 2007;50:974-82. PMID: 17765125. 
 
6.  Fried LP, Guralnik JM. Disability in older adults: evidence regarding significance, 
etiology, and risk. J Am Geriatr Soc 1997;45:92-100. PMID: 8994496. 
 
7.  McDermott MM, Tian L, Liu K, Guralnik JM, Ferrucci L, Tan J, Pearce WH, 
Schneider JR, Criqui MH. Prognostic value of functional performance for mortality in 
patients with peripheral artery disease. J Am Coll Cardiol 2008;51:1482-9. PMID: 
18402904. 
8. Guralnik JM, Simonsick EM, Ferrucci L, Glynn RJ, Berkman LF, Blazer DG, Scherr 
PA, Wallace RB. A short physical performance battery assessing lower extremity 
function: association with self-reported disability and prediction of mortality and nursing 
home admission. J Gerontol 1994;49:M85-94. PMID: 8126356. 
9.  Guralnik JM, Ferrucci L, Simonsick EM, Salive ME, Wallace RB. Lower-extremity 
function in persons over the age of 70 years as a predictor of subsequent disability. N 
Engl J Med 1995;332:556-61. PMID: 7838189. 
10.  Guralnik JM, Ferrucci L, Pieper CF, Leveille SG, Markides KS, Ostir GV, Studenski 
S, Berkman LF, Wallace RB. Lower extremity function and subsequent disability: 

 
Page 34 of 41 
 
consistency across studies, predictive models, and value of gait speed alone compared 
with the short physical performance battery. J Gerontol A Biol Sci Med Sci 
2000;55:M221-31. PMID: 10811152. 
 
11.  Heinen Y, Stegemann E, Sansone R, Benedens K, Wagstaff R, Balzer J, Rassaf T, 
Lauer T, Kelm M, Heiss C.  Local association between endothelial dysfunction and 
intimal hyperplasia:  relevance in peripheral artery disease.  J Am Heart Assoc. 
2015;4:e0014672. Doi:10.1161/JAHA.114.001472. PMCID:PMC4345872. 
 
12.  Gokce N, Keaney JF Jr., Hunter LM, Watkins MT, Nedeljkovic ZS, Menzoian JO, 
Vita JA.  Predictive value of noninvasively determined endothelial dysfunction for long-
term cardiovascular events in patients with peripheral vascular disease.  J Am Coll 
Cardiol. 2003;41:1769-1775. PMID: 12767663. 
 
13.  Pipinos II, Judge AR, Zhu Z, Selsby JT, Swanson SA, Johanning JM, Baxter BT, 
Lynch TG, Dodd SL. Mitochondrial defects and oxidative damage in patients with 
peripheral arterial disease. Free Radic Biol Med. 2006;41:262-9. PMID: 16814106. 
 
14.  Pipinos II, Sharov VG, Shepard AD, Anagnostopoulos PV, Katsamouris A, Todor A, 
Filis KA, Sabbah HN. Abnormal mitochondrial respiration in skeletal muscle in patients 
with peripheral arterial disease. J Vasc Surg. 2003;38:827-32. PMID: 14560237. 
 
15.  Anderson JD, Epstein FH, Meyer CH, Hagspiel KD, Wang H, Berr SS, Harthun NL, 
Weltman A, Dimaria JM, West AM, Kramer CM. Multifactorial determinants of 
functional capacity in peripheral arterial disease: Uncoupling of calf muscle perfusion 
and metabolism. J Am Coll Cardiol. 2009;54:628-35. PMCID: PMC2768062 
 
16. Pipinos II, Judge AR, Selsby JT, Zhu Z, Swanson SA, Nella AA, Dodd SL. The 
myopathy of peripheral arterial occlusive disease: Part 2. Oxidative stress, neuropathy, 
and shift in muscle fiber type. Vasc Endovascular Surg. 2008;42:101-12. PMID: 
18390972. 
 
17. Pipinos II, Swanson SA, Zhu Z, Nella AA, Weiss DJ, Gutti TL, McComb RD, Baxter 
BT, Lynch TG, Casale GP. Chronically ischemic mouse skeletal muscle exhibits 
myopathy in association with mitochondrial dysfunction and oxidative damage. Am J 
Physiol Regul Integr Comp Physiol. 2008;295:R290-6. PMCID: PMC2494805 
 
18.  Hou XY, Green S, Askew CD, Barker G, Green A, Walker PJ. Skeletal muscle 
mitochondrial ATP production rate and walking performance in peripheral arterial 
disease. Clin Physiol Funct Imaging. 2002;22:226-32. PMID: 12076351. 
 
19.  McDermott MM, Tian L, Ferrucci L, Liu K, Guralnik JM, Liao Y, Pearce WH, 
Criqui MH. Associations between lower extremity ischemia, upper and lower extremity 
strength, and functional impairment with peripheral arterial disease. J Am Geriatr Soc 
2008;56:724-9. PMID: 18284536. 

 
Page 35 of 41 
 
 
20.  McDermott MM, Criqui MH, Greenland P, Guralnik JM, Liu K, Pearce WH, Taylor 
L, Chan C, Celic L, Woolley C, O'Brien MP, Schneider JR. Leg strength in peripheral 
arterial disease: Associations with disease severity and lower-extremity performance. J 
Vasc Surg 2004;39:523–30. PMID: 14981443. 
 
21.  Hüttemann M, Lee I, Perkins GA, Britton SL, Koch LG, Malek MH. (-)-Epicatechin 
is associated with increased angiogenic and mitochondrial signaling in the hindlimb of 
rats selectively bred for innate low running capacity. Clin Sci (Lond) 2013;124:663-74. 
PMID: 23252598. 
 
22.  Gutierrez-Salmean G, Ciaraldi TP, Nogueira L, Barboza J, Taub PR, Hogan MC, 
Henry RR, Meaney E, Villarreal F, Ceballos G, Ramirez-Sanchez I. Effects of (-)-
epicatechin on molecular modulators of skeletal muscle growth and differentiation. J 
Nutr Biochem 2014;25:91-4. PMID: 24314870. 
 
23.  Nogueira L, Ramirez-Sanchez I, Perkins GA, Murphy A, Taub PR, Ceballos G, 
Villarreal FJ, Hogan MC, Malek MH. (-)-Epicatechin enhances fatigue resistance and 
oxidative capacity in mouse muscle. J Physiol 2011;589:4615-31. PMID: 21788351. 
 
24.  Watanabe N, Inagawa K, Shibata M, Osakabe N. Flavan-3-ol fraction from cocoa 
powder promotes mitochondrial biogenesis in skeletal muscle in mice. Lipids Health Dis 
2014;13:64. doi:10.1186/1476-511X-13-64. PMID: 24708519. 
 
25.  Taub PR, Ramirez-Sanchez I, Ciaraldi TP, Perkins G, Murphy AN, Naviaux R, 
Hogan M, Maisel AS, Henry RR, Ceballos G, Villarreal F. Alterations in skeletal muscle 
indicators of mitochondrial structure and biogenesis in patients with type 2 diabetes and 
heart failure: effects of epicatechin rich cocoa. Clin Transl Sci 2012;5:43-7. PMID: 
22376256. 
 
26.  Ramirez-Sanchez I, Taub PR, Ciaraldi TP, Nogueira L, Coe T, Perkins G, Hogan M, 
Maisel AS, Henry RR, Ceballos G, Villarreal F. (-)-Epicatechin rich cocoa mediated 
modulation of oxidative stress regulators in skeletal muscle of heart failure and type 2 
diabetes patients.  Int J Cardiol 2013;168:3982-90. PMID: 23870648 
 
27.  Taub PR, Ramirez-Sanchez I, Ciaraldi TP, Gonzalez-Basurto S, Coral-Vazquez R, 
Perkins G, Hogan M, Maisel AS, Henry RR, Ceballos G, Villareal F. Perturbations in 
skeletal muscle sarcomere structure in patients with heart failure and type 2 diabetes: 
restorative effects of (-)-epicatechin-rich cocoa. Clin Sci (Lond)  2013;125:383-9. PMID: 
23642227 
 
28.  Balzer J, Rassaf T, Heiss C, Kleinbongard, Lauer T, Merx M, Heussen N, Gross HB, 
Keen CL, Schroeter H, Kelm M.  Sustained benefits in vascular function through 
flavanol-containing cocoa in medicated diabetic patients:  A double-masked, randomized, 
controlled trial.  J Am Coll Cardiol. 2008;51:2141-2149.  PMID: 18510961. 
 

 
Page 36 of 41 
 
29.  Flammer AJ, Hermann F, Sudano I, Speiker L, Hermann M, Cooper KA, Serafini M, 
Lüscher TF, Ruschitzka F, Noll G, Corti R. Dark chocolate improves coronary 
vasomotion and reduces platelet reactivity. Circulation 2007;116:2376-82. PMID: 
17984375.  
 
30.  Lee I, Huttemann M, Kruger A, Bollig-Fischer A, Malek MH. (-)-Epicatechin 
combined with 8 weeks of treadmill exercise is associated with increased angiogenic and 
mitochondrial signaling in mice. Front Pharmacol 2015;6:43. PMID: 25821434.  
 
 
31. Loffredo L, Perri L, Catasca E, Pignatelli P, Brancorsini M, Nocella C, De Falco E, 
Bartimoccia S, Frati G, Carnevale R, Violi F. Dark chocolate acutely improves walking 
autonomy in patients with peripheral artery disease. J Am Heart Assoc 2014;3:e001072. 
doi:10.1161/JAHA.114.001072. PMID: 24990275 
 
32.  McDermott MM, Ades PA, Dyer A, Guralnik JM, Kibbe M, Criqui MH.  Corridor-
based functional performance measures correlate better with physical activity during 
daily life than treadmill measures in persons with peripheral arterial disease.  J Vasc Surg 
2008;48:1231-7. PMCID: PMC2885131  
 
33.  McDermott MM, Guralnik JM, Criqui MH, Liu K, Kibbe MR, Ferrucci L.  Six-
minute walk is a better outcome measure than treadmill walking tests in therapeutic trials 
of patients with peripheral artery disease.  Circulation 2014;130:61-8. PMID: 24982117. 
 
34.  McDermott MM, Ades P, Guralnik JM, Dyer A, Ferrucci L, Liu K, Nelson M, 
Lloyd-Jones D, Van Horn L, Garside D, Kibbe M, Domanchuk K, Stein JH, Liao Y, Tao 
H, Green D, Pearce WH, Schneider JR, McPherson D, Laing ST, McCarthy WJ, Shroff 
A, Criqui MH. Treadmill exercise and resistance training in patients with peripheral 
arterial disease with and without intermittent claudication: a randomized controlled trial. 
JAMA 2009;301:165-74. PMID: 19141764. 
 
35.  McDermott MM, Tian L, Liu K, Guralnik JM, Ferrucci L, Tan J, Pearce WH, 
Schneider JR, Criqui MH. Prognostic value of functional performance for mortality in 
patients with peripheral artery disease. J Am Coll Cardiol 2008;51:1482-9. PMID: 
18402904. 
 
36.  Girolami B, Bernardi E, Prins MH, Ten Cate JW, Hettiarachchi R, Prandoni P, 
Girolami A, Buller HR.  Treatment of intermittent claudication with physical training, 
smoking cessation, pentoxifylline, or nafronyl: a meta-analysis.  Arch Intern Med 
1999;159:337-45. PMID: 10030306. 
 
37.  Dawson DL, Cutler BS, Meissner MH, Strandness DE Jr.  Cilostazol has beneficial 
effects in treatment of intermittent claudication:  Results from a multi-center, 
randomized, prospective, double-blind trial.  Circulation 1998;98:678-86. PMID: 
9715861. 
 

 
Page 37 of 41 
 
38.  Money SR, Herd JA, Isaacsohn JL, Davidson M, Cutler B, Heckman J,  Forbes WP.  
Effect of cilostazol on walking distances in patients with intermittent claudication caused 
by peripheral vascular disease.  J Vasc Surg 1998;27:267-74. PMID: 9510281. 
 
39.  Beebe HG, Dawson DL, Cutler BS, Herd JA, Strandness DE Jr, Bortey EB, Forbes 
WP.  A new pharmacological treatment for intermittent claudication:  results of a 
randomized, multi-center trial.  Arch Intern Med 1999;159:2041-50. PMID: 10510990. 
 
40.  McDermott MM, Hoff F, Ferrucci L, Pearce WH, Guralnik JM, Tian L, Liu K, 
Schneider JR, Sharma L, Tan J, Criqui MH. Lower extremity ischemia, calf skeletal 
muscle characteristics, and functional impairment in peripheral arterial disease. J Am 
Geriatr Soc 2007;55:400–6.  PMID: 17341243. 
 
41.  McDermott MM, Ferrucci L, Guralnik J, Tian L, Liu K, Hoff F, Liao Y, Criqui MH. 
Pathophysiological changes in calf muscle predict mobility loss at 2-year follow-up in 
men and women with peripheral arterial disease. Circulation 2009;120:1048-55. PMID: 
19738138. 
 
42.  Anderson JD, Epstein FH, Meyer CH, Hagspiel KD, Wang H, Berr SS, Harthun NL, 
Weltman A, Dimaria JM, West AM, Kramer CM. Multifactorial determinants of 
functional capacity in peripheral arterial disease: uncoupling of calf muscle perfusion and 
metabolism. J Am Coll Cardiol 2009;54:628-35. PMID: 19660694. 
 
43.  Herman SD, Liu K, Tian L, Guralnik JM, Ferrucci L, Criqui MH, Liao Y, 
McDermott MM. Baseline lower extremity strength and subsequent decline in functional 
performance at 6-year follow-up in persons with lower extremity peripheral arterial 
disease. J Am Geriatr Soc 2009;57:2246-52. PMID: 19874404. 
 
44.  Weiss DJ, Casale GP, Koutakis P, Nella AA, Swanson SA, Zhu Z, Miserlis D, 
Johanning JM, Pipinos II.  Oxidative damage and myofiber degeneration in the 
gastrocnemius of patients with peripheral arterial disease.  J Transl Med 2013;11:230. 
doi: 10.1186/1479-5876-11-230. PMID: 24067235. 
 
 
45.  Pipinos II, Judge AR, Zhu Z, Selsby JT, Swanson SA, Johanning JM, Baxter BT, 
Lynch TG, Dodd SL. Mitochondrial defects and oxidative damage in patients with 
peripheral arterial disease. Free Radic Biol Med 2006;41:262-9. PMID: 16814106. 
 
46.  Pipinos II, Sharov VG, Shepard AD, Anagnostopoulos PV, Katsamouris A, Todor A, 
Filis KA, Sabbah HN. Abnormal mitochondrial respiration in skeletal muscle in patients 
with peripheral arterial disease. J Vasc Surg 2003;38:827-32. PMID: 14560237. 
 
47.  Marbini A, Gmignani F, Scoditti U, Rustichelli P, Bragaglia MM, Govoni E. 
Abnormal muscle mitochondria in ischemic claudication. Acta Neurol Belg 1986;86:304-
10. PMID: 3811830. 
 

 
Page 38 of 41 
 
48.  Mäkitie J, Teräväinen H. Histochemical changes in striated muscle in patients with 
intermittent claudication. Arch Pathol Lab Med 1977;101:658-63. PMID: 579309. 
 
49.  Makris KI, Nella AA, Zhu Z, Swanson SA, Casale GP, Gutti TL, Judge AR, Pipinos 
II. Mitochondriopathy of peripheral arterial disease. Vascular 2007;15:336-43. PMID: 
18053417. 
 
50.  Pipinos II, Shepard AD, Anagnostopoulos PV, Katsamouris A, Boska MD. 
Phosphorus 31 nuclear magnetic resonance spectroscopy suggests a mitochondrial defect 
in claudicating skeletal muscle. J Vasc Surg 2000;31:944-52. PMID: 10805885. 
 
51.  Payvandi L,  Dyer A, McPherson D, Ades P, Stein J, Liu K, Ferrucci L, Criqui 
MH, Guralnik JM, Lloyd-Jones D, Kibbe MR, Liang ST, Kane B, Pearce WH, Verta M, 
McCarthy WJ, Schneider JR, Shroff A, McDermott MM. Physical activity during daily 
life and brachial artery flow-mediated dilation in peripheral arterial disease. Vasc 
Med. 2009;14:193-201. PMCID: PMC2749502 
52.  McDermott MM, Fried L, Simonsick E, Ling S, Guralnik JM.  Asymptomatic 
peripheral arterial disease is independently associated with impaired lower extremity 
functioning: the women’s health and aging study.  Circulation 2000;101:1007-12. PMID: 
10704168. 
 
53.  Newman AB, Siscovick DS, Manolio TA, Polak J, Fried LP, Borhani NO, Wolfson 
SK. Ankle-arm index as a marker of atherosclerosis in the Cardiovascular Health Study. 
Cardiovascular Heart Study (CHS) Collaborative Research Group. Circulation 
1993;88:837-4. PMID: 8353913.  
 
54.  Vogt MT, Cauley JA, Newman AB, Kuller LH, Hulley SB. Decreased ankle/arm 
blood pressure index and mortality in elderly women. JAMA 1993;270:465-9. PMID: 
8320785. 
 
55.  Lijmer JG, Hunink MG, van den Dungen JJ, Loonstra J, Smit AJ.  ROC analysis of 
noninvasive tests for peripheral arterial disease.  Ultrasound Med Biol 1996;22:391-8. 
PMID: 8795165. 
 
56.  Bernstein EF, Fronek A.  Current status of non-invasive tests in the diagnosis of 
peripheral arterial disease.  Surg Clin North Am 1982;62:473-87. PMID: 7089825. 
 
57.  Fung YC.  Blood flow in arteries: pressure and velocity waves in large arteries and 
the effects of geometric nonuniformity. In: Biodynamics: circulation. New York, NY: 
Springer-Verlag; 1984:133-136 
 
58.  Yao JS. New techniques of objective arterial evaluation.  Arch Surg 1973;106:600-4. 
PMID: 4572350. 
 
59.  Aboyans V, Criqui MH, Abraham P, Allison MA, Creager MA, Diehm C, Fowkes 

 
Page 39 of 41 
 
FG, Hiatt WR, Jonsson B, Lacroix P, Marin B, McDermott MM, Norgren L, Pande RL, 
Preux PM, Stoffers HE, Treat-Jacobson D, American Heart Association Council on 
Peripheral Vascular Disease.  Measurement and interpretation of the ankle-brachial 
index:  A scientific statement from the American Heart Association.  Circulation 
2012;126:2890-2909. PMID: 23159553.  
 
60.  McDermott MM, Liu K, Guralnik JM, Criqui MH, Spring B, Tian L, Domanchuk K, 
Ferrucci L, Lloyd-Jones D, Kibbe M, Tao H, Zhao L, Liao Y, Rejeski WJ. Home-based 
walking exercise intervention in peripheral artery disease: a randomized clinical trial. 
JAMA 2013;310:57-65. PMID: 23821089. 
 
61.  Gardner AW, Skinner JS, Cantwell BW, Smith LK.  Progressive vs. single-stage 
treadmill tests for evaluation of claudication. Med Sci Sports Exerc 1991;23:402-8. 
PMID: 2056896. 
 
62.  Gardner AW, Skinner JS, Cantwell BW, Smith LK.  Effects of handrail support on 
claudication and hemodynamic responses to single-stage and progressive treadmill 
protocols in peripheral vascular occlusive disease.  Am J Cardiol 1991;68:99-105. PMID: 
2058556. 
 
63.  Hiatt WR, Regensteiner JG, Hargarten ME, Wolfel EE, Brass EP.  Benefit of 
exercise conditioning for patients with peripheral arterial disease.  Circulation 
1990;81:602-9. PMID: 2404633. 
 
64.  Yeboah J, Folsom AR, Burke GL, Johnson C, Polak JF, Post W, Lima JA, Crouse 
JR, Herrington DM. Predictive value of brachial flow-mediated dilation for incident 
cardiovascular events in a population-based study: The multi-ethnic study of 
atherosclerosis. Circulation. 2009;120:502-9. PMCID: PMC2740975. 
 
65. Rothney MP, Brychta RJ, Meade NN, Chen KY, Buchowski MS. Validation of the 
ActiGraph two-regression model for predicting energy expenditure. Med Sci Sports 
Exerc. 2010;42:1785-92. PMCID: PMC2919650. 
 
66. Cooper AR, Page A, Fox KR, Misson J. Physical activity patterns in normal, 
overweight and obese individuals using minute-by-minute accelerometry. Eur J Clin 
Nutr. 2000;54:887-94. PMID: 11114687. 
 
67.  Kelly LA, McMillan DG, Anderson A, Fippinger M, Fillerup G, Rider J. Validity of 
ActiGraphs uniaxial and triaxial accelerometers for assessment of physical activity in 
adults in laboratory conditions. BMC Med Phys. 2013;13:5. PMID: 24279826. 
 
68.  Mitsiopoulos N, Baumgartner RN, Heymsfield SB, Lyons W, Gallagher D, Ross R. 
Cadaver validation of skeletal muscle measurement by magnetic resonance imaging and 
computerized tomography. J Appl Physiol (1985). 1998;85:115-22. PMID: 9655763   
 

 
Page 40 of 41 
 
69.  Adhihetty PJ, Taivassalo T, Haller RG, Walkinshaw DR, Hood DA. The effect of 
training on the expression of mitochondrial biogenesis- and apoptosis-related proteins in 
skeletal muscle of patients with mtDNA defects. Am J Physiol Endocrinol Metab 2007 
September;293(3):E672-E680. PMID: 17551003. 
 
70.  Joseph AM, Adhietty PJ, Buford TW, Wohlgemuth SE, Lees HA, Nguyen LM, 
Aranda JM, Sandesara BD, Pahor M, Manini T, Marzetti E, Leeuwenburgh C.  The 
impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of 
sedentary high- and low-functioning elderly individuals.  Aging Cell 2012;11:801-809. 
PMID: 22681576. 
 
71.  Wohlgemuth SE1, Lees HA, Marzetti E, Manini TM, Aranda JM, Daniels MJ, Pahor 
M, Perri MG, Leeuwenburgh C, Anton SD. An exploratory analysis of the effects of a 
weight loss plus exercise program on cellular quality control mechanisms in older 
overweight women. Rejuvenation Res 2011;14:315-24. PMID: 21631380. 
 
72.  Duscha BD, Robbins JL, Jones WS, Kraus WE, Lye RJ, Sanders JM, Allen JD, 
Regensteiner JG, Hiatt WR, Annex BH. Angiogenesis in skeletal muscle precede 
improvements in peak oxygen uptake in peripheral artery disease patients. Arterioscler 
Thromb Vasc Biol 2011;31:2742-8. PMCID: PMC3578302 
 
73.  Perera S, Mody SH, Woodman RC, Studenski SA.  Meaningful change and 
responsiveness in common physical performance measures in older adults.  J Am Geriatr 
Soc 2006;54:743-749. PMID: 16696738. 
 
74.  Puhan MA, Mador MJ, Held U, Goldstein R, Guyatt GH, Schunemann HJ. 
Interpretation of treatment changes in 6-minute walk distance in patients with COPD. Eur 
Respir J 2008;32:637–643. PMID: 18550610. 
 
75.  McDermott MM, Reed G, Greenland P, Mazor KM, Pagoto S, Ockene JK, Graff R, 
Merriam PA, Leung K, Manheim L, Kibbe MR, Olendzki B, Pearce WH, Ockene IS. 
Activating peripheral arterial disease patients to reduce cholesterol: a randomized trial. 
Am J Med 2011;124:557-65. PMID: 21605733. 
 
76.  Decker C.  Calculating a nonparametric estimate and confidence interval using SAS 
software.  In SAS Global Forum; 2000; Indianapolis, Indiana. 
http://www.lexjansen.com/pharmasug/2000/Coders/cc01.pdf. Accessed July 7, 2015. 
77.  Corti R, Flammer AJ, Hollenberg NK, Lüscher TF. Cocoa and cardiovascular health. 
Circulation 2009;119:1433-41. PMID: 19289648. 
 
78. White S, McDermott MM, Sufit RL, Kosmac K, Bugg AW, Gonzalez-Freire M, 
Ferrucci L, Tian L, Gao Y, Kibbe MR, Criqui MH, Leeuwenburgh C, Peterson CA. 
Walking performance is positively correlated to calf muscle fiber size in peripheral artery 
disease subjects, but fibers show aberrant mitophagy: an observational study. Journal of 
Translational Medicine 2016;14:284. PMID: 27687713. 

 
Page 41 of 41 
 
79.  Fry CS, Lee JD, Jackson JR, Kirby TJ, Stasko SA, Liu H, Dupont-Versteegden EE, 
McCarthy JJ, Peterson CA. Regulation of the muscle fiber microenvironment by 
activated satellite cells during hypertrophy. FASEB J 2014;28:1654-1656. PMID: 
24376025. PMCID: PMC3963024.  
 
80. Joseph AM, Adhihetty PJ, Buford TW, Wohlgemuth SE, Lees HA, Nguyen LM, 
Aranda JM, Sandesara BD, Pahor M, Manini TM, Marzetti E, Leeuwenburgh C. The 
impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of 
sedentary high- and low-functioning elderly individuals. Aging Cell. 2012;11:801-9. 
PMID: 22681576. PMCID: PMC3444680. 
 
81. Adhihetty PJ, Taivassalo T, Haller RG, Walkinshaw DR, Hood DA. The effect of 
training on the expression of mitochondrial biogenesis- and apoptosis-related proteins in 
skeletal muscle of patients with mtDNA defects. Am J Physiol Endocrinol Metab. 
2007;293:E672-80. PMID: 17551003.