Effects of 12 Weeks of Nutritional Therapy Interventions in Heart Failure
Phase: NA
Conditions: Heart Failure NYHA Class II
Sponsor: University of Arkansas

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 1 
 
University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:  Effects of 12 Weeks of Nutritional Therapy Interventions in Heart Failure 
 
Principal Investigator: 
Gohar Azhar, M.D. 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St., Slot #749 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
Office Telephone: 501-526-5935 
 
 
 
 
Email: azhargohar@uams.edu 
 
Sub-Investigators:  
Jeanne Wei, M.D. 
 
 
 
 
Arny A. Ferrando, Ph.D. 
 
 
 
 
Bryce Marquis, Ph.D. 
 
 
 
 
Sanghee Park, Ph.D. 
 
 
 
 
Nookaew, Intawat, Ph.D. 
 
 
 
 
Eugenia Carvalho, Ph.D. 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St. 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
 
Study location: 
 
Donald W. Reynolds Institute on Aging @ UAMS 
 
 
 
 
629 Jack Stephens Drive 
 
 
 
 
Little Rock, AR 72205 
 
Support (Funding): 
NIH STTR grant to Essential Blends, LLC.; subcontract to UAMS 
 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 2 
 
Table of Contents 
Table of Events ..................................................................................................................................... 4 
1.0 Background and Rationale ........................................................................................................... 5 
2.0 Hypotheses ....................................................................................................................................... 7 
3.0 Study Design and Procedures ..................................................................................................... 7 
3.1 Study Visits ..................................................................................................................................... 7 
3.2 Subject Compliance ...................................................................................................................... 9 
3.3 Subject Compensation ................................................................................................................ 10 
3.4 Randomization ............................................................................................................................. 10 
3.5 Blinding .......................................................................................................................................... 10 
3.6 Study Products ............................................................................................................................. 11 
3.7 Sample Storage ........................................................................................................................... 11 
4.0 Study Population ........................................................................................................................... 12 
4.1 Inclusion Criteria .......................................................................................................................... 12 
4.2 Exclusion Criteria ......................................................................................................................... 12 
5.0 Risks and Benefits ........................................................................................................................ 13 
5.1 Blood, urine and fecal sampling: ............................................................................................... 13 
5.2 DEXA scan: .................................................................................................................................. 13 
5.3 Exercise testing (strength and functional): .............................................................................. 14 
5.4 Study supplements: ..................................................................................................................... 14 
5.5 Confidentiality: .............................................................................................................................. 14 
5.6 Oral isotope ingestion: ................................................................................................................ 14 
6.0 Data Handling and Recordkeeping ........................................................................................... 14 
7.0 Data Analysis.................................................................................................................................. 15 
7.1 Statistical Analysis plan .............................................................................................................. 15 
7.2 Significant Interaction Analysis Plan ........................................................................................ 15 
7.3 Sample Size Calculation & Power Analysis ............................................................................ 16 
7.4 EAA mixture or placebo Effect .................................................................................................. 16 
7.5 Time Effect .................................................................................................................................... 17 
7.6 Urine analyses ............................................................................................................................. 17 
7.7 Fecal analyses ............................................................................................................................. 17 
7.8 Blood analyses ............................................................................................................................. 18 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 3 
 
8.0 Ethical Considerations ................................................................................................................ 18 
9.0 Dissemination of Data .................................................................................................................. 19 
10.0 References .................................................................................................................................... 19 
11.0 Appendices ................................................................................................................................... 21 
11.1 Study Product Information ....................................................................................................... 21 
 
 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 4 
 
Table of Events 
Event 
V.1 
V.2 
V.3 
V.4 
V.5 
V.6 
V.7 
V.8 
V.9 
V.10 
V.11 
V.12 
V.13 
V.14 
Informed 
Consent 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
SLUMS 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Medical 
History 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Meds used 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ht & Wt. 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Temp & BP 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood 
sample1 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ingest D3-cr. 
 
x 
 
 
 
 
 
 
 
 
 
 
x 
 
Phys. exam2 
(x) 
(x) 
 
 
 
 
 
 
 
 
 
 
 
 
Collect urine 
for 3 days 
 
x3 
 
 
 
 
 
 
 
 
 
 
x3 
 
Fasting 
before visit 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
Blood 
sample4 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
Urine sample 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
Stool 
sample5 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
Strength & 
functional 
tests 
 
x 
 
 
 
 
 
x 
 
 
 
 
 
x 
Practice 
strength test 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
SF-36 
questionnaire 
 
x 
 
 
 
 
 
x 
 
 
 
 
 
x 
DEXA scan 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
Issue diet 
records 
 
x 
 
 
 
 
 
x 
 
 
 
 
x 
 
Collect diet 
records 
 
 
x 
 
 
 
 
 
x 
 
 
 
 
x 
Dispense 
study product 
and diary 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Return study 
product and 
diary 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
1 For criteria-related testing. 
2Can be done on visit 1 or 2. 
3 Between visits, at home. Calls will be made to remind subjects of the time for collection. 
4 For protocol endpoints. 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 5 
 
5 If subjects unable to provide at this visit, they can take home a collection kit to return at next visit. 
 
1.0 Background and Rationale 
 
Heart failure develops when cardiac muscle becomes weakened and consequently is 
compromised in its ability to contract, relax, or both. Impaired heart function leads to reduced 
exercise capacity, which in turn leads to progressive muscle weakness and a vicious cycle of 
sedentary behavior, weight gain, and subsequent development of metabolic abnormalities and 
sarcopenia. Approximately 6-10% of individuals over the age of 65 suffer from heart failure (1). 
The risk of death is 35% in the first year after diagnosis, and decreases to about 10% per year 
thereafter (2). There are two principal forms of heart failure. Most commonly, heart failure has 
referred to reduced force of ejection (systolic heart failure). Heart failure may also result from 
an impaired ability of the heart to relax (diastolic heart failure) (3). Systolic heart failure is often 
accompanied with diastolic dysfunction (1-2). However, more often the contractile capacity of 
the heart is not affected, or only slightly impaired, and thus (unlike the case with systolic heart 
failure) the ejection fraction is usually preserved. This form of heart failure is termed heart 
failure with preserved ejection fraction (HFPEF), and is the predominant form of heart failure 
world-wide (4,5). Not only are there different types of heart failure, there is a wide range of 
potential causes of heart failure, including the natural process of aging (6). Regardless of the 
specific underlying cause or type of heart failure, there are common pathophysiological 
responses. Most prominently, impaired exercise capacity, shortness of breath, fatigue and 
muscle strength are hallmarks of heart failure that lead to decreased physical function, and 
ultimately sedentary behavior and development of insulin resistance (2). Heart failure is also 
associated with fluid retention and even edema of the intestinal wall which resultant 
malabsorption of nutrients (7).  Poor health of the intestinal mucosa in heart failure might 
potentially also influence the gut microbiome. Some long-term consequences of reduced 
exercise tolerance and malabsorption in long-standing heart failure are loss of muscle mass 
and the development of cardiac cachexia (7). These responses not only contribute to morbidity 
and mortality and financial strain on the health care system, but to decreased health-related 
quality of life. 
Treatment of the underlying cause of heart failure would be optimal. Indeed, pharmacological 
treatment of heart failure predominantly targets the improvement of cardiac performance. 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 6 
 
Heart failure may be treated with varying degrees of success with a variety of drugs, including 
angiotensin-converting (ACE-inhibitors, angiotensin II, beta blockers, diuretics, digoxin, nitrates 
and others). However, treating systolic heart failure pharmacologically may be quite complex in 
the elderly. More than 50% of individuals over age 65 with heart failure have at least four co-
morbidities (8), and these may complicate therapy of heart failure. Adverse responses to 
pharmacological therapy are not uncommon, including the fact that both ACE inhibitors and 
beta blockers can adversely affect muscle function (9). Further, HFPEF is becoming more 
widely recognized, and to date, despite the expenditure of millions of dollars and years of time 
and effort, not one large-scale clinical study of a single drug therapy has demonstrated a 
substantial beneficial outcome. This disappointing reality may reflect the difficulty of treating a 
syndrome with diverse causes, interacting complex pathogenesis, and multiple co-morbidities 
with an entirely drug-oriented approach. Reduced exercise capacity is a prominent clinical 
feature of heart failure. This leads to progressive muscle weakness and a vicious cycle of 
sedentary behavior, weight gain, and subsequent development of metabolic abnormalities and 
sarcopenia. 
In this study we will perform a randomized clinical trial of a commercially produced nutritional 
supplement (EAA mixture Essential Blends, Fairbanks, AK) as compared to a placebo in order 
to determine effects on physical function and health-related quality of life. Subjects will ingest 
either the EAA mixture product or placebo every day for 12 consecutive weeks. Outcomes will 
be determined by comparing the results of physical and functional tests from weeks -1 to 6 and 
12. 
In a pilot study 18 overweight/obese subjects with heart failure (all exceeded 40% body fat) 
were studied. Nine subjects received 12 weeks of dietary supplementation with 20 g of whey 
protein consumed daily, while the other nine were controls. Consistent with the acute data 
showing a limited stimulation of muscle protein synthesis with dietary amino acids in the profile 
of whey protein in even healthy elderly (i.e., anabolic resistance, Figure 1), supplementation 
with whey protein failed to improve functional performance. The lack of a demonstrable effect 
of whey is consistent with the diminished responsiveness to the stimulation of muscle protein 
synthesis by whey protein in heart failure (10). In contrast, a 2016 UAMS pilot study found that 
the EAA mixture is better at overcoming anabolic resistance than Ensure Heart Health (see 
below). 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 7 
 
 
 
2.0 Hypotheses 
 
1. We propose that 12 weeks of regular consumption of EAA mixture will improve physical 
function in individuals with heart failure as compared to placebo. 
2. We propose that regular consumption of EAA mixture will improve health-related quality 
of life as compared to placebo. 
 
3.0 Study Design and Procedures 
 
This is a randomized, double blind, prospective study examining the effects of 12 weeks’ 
ingestion of either EAA mixture or placebo on physical function. We will also examine several 
blood parameters and fecal microbiome in both groups of subjects (see section 5.1). Up to 80 
subjects ages 60 to 89 will be enrolled to obtain 60 completers (30 per group). 
 
3.1 Study Visits 
Visit 1: subjects will come to the UAMS IOA 3rd floor for informed consent discussion. Once 
consent is obtained, study staff will administer and score the SLUMS test for dementia. If 
subjects score >20, subsequent study procedures will be performed. A medical history and list 
of current medications will be obtained (printed from Epic if they are a UAMS patient). Subject 
height, weight, body temperature and blood pressure will be measured. A blood sample will be 
drawn (approx. 15 mL) for criteria-related tests if recent results are not available from their 
medical record. A brief training session will take place where subjects will be introduced to the 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 8 
 
isokinetic dynamometer (Cybex) machine and how it is used at the testing visits. They will 
undergo a practice test on one leg. 
A physical exam will be performed at this visit or at visit 2. Based upon the results of the 
screening blood sample, visit 2 will be scheduled.  
 
Visit 2: Subjects must come to this visit having fasted overnight. They will be asked about any 
adverse events since last visit. A fasting blood sample (~30 mL) will be drawn to determine 
baseline values (tests listed below), and a snack will be provided and coffee offered. A urine 
sample will be collected for dipstick proteinuria. Subjects will be asked to provide a stool 
sample, but if unable at this visit, they can take home a sample collection kit to use. They will 
be instructed to collect the stool sample at home on the day of Visit 2 or at UAMS. Subjects will 
be provided with a drink containing 30mg of a stable isotope of creatine (D-3, Cambridge 
Isotopes Laboratory, Tewksbury, MA) dissolved in water. They will be asked to collect 3 
separate urine samples over the next 72 hours at approximately 24-hour intervals, place them 
into their freezer, and return them to UAMS at their next visit. 
If not performed at visit 1, the physical exam will be performed at this visit. Randomization will 
be performed as mentioned below. The following assessments will be performed: functional (6-
minute walk distance), SF-36 health questionnaire, a whole-body DEXA scan for lean mass 
measurement, strength (Cybex testing of maximal voluntary contraction of each leg; bilateral 
hand grip strength). Subjects will be provided with forms and training for recording their dietary 
intake over a 3-day period during the next week. Study staff will dispense approximately 10 
days’ worth of study product and a consumption diary for subjects to complete after ingesting 
each dose. Future visits will be scheduled. 
 
Visits 3-7: Subjects will come to the IOA weekly to return their urine samples, empty 
supplement containers and consumption diary. They will be asked about any adverse events 
since last visit. Study staff will dispense more study product and a consumption diary. 
 
Visit 8: Subjects will be asked about any adverse events since last visit. They will return their 
empty supplement containers and consumption diary. In no particular order, the following 
assessments will be performed: strength (Cybex testing of maximal voluntary contraction of 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 9 
 
each leg; bilateral hand grip strength), functional (6-minute walk distance), SF-36 health 
questionnaire. Study staff will dispense more study product, a consumption diary, and another 
3-day diet diary. 
 
Visits 9-12: Subjects will come to the IOA weekly to return their empty supplement containers 
and consumption diary. They will be asked about any adverse events since last visit. Study 
staff will dispense more study product and a consumption diary. 
 
Visit 13: Subjects will come to the IOA to return their empty supplement containers and 
consumption diary. They will be asked about any adverse events since last visit. Study staff 
will dispense more study product and a consumption diary. Subjects will be provided with a 
drink containing 30mg of a stable isotope of creatine (D-3, Cambridge Isotopes Laboratory, 
Tewksbury, MA). They will be asked to collect 3 separate urine samples over the next 48 hours 
at approximately 24-hour intervals and return them to UAMS at their next visit. Subjects will be 
issued a stool sample collection kit to use just prior to visit 14 and another 3-day diet diary.  
 
Visit 14: Subjects must come to this visit having fasted overnight. They will be asked about any 
adverse events since last visit. They will return their diet records, empty supplement 
containers, any full supplement containers, their stool sample, and their consumption diary. 
Study staff will administer and score the SLUMS test for dementia. A fasting blood sample 
(~30 mL) will be drawn. After the blood sample is drawn, subjects will be offered a meal and 
beverages. A urine sample will be collected for dipstick proteinuria.  
The following assessments will be performed: functional (6-minute walk distance), SF-36 
health questionnaire, a whole-body DEXA scan for lean mass measurement, strength (Cybex 
testing of maximal voluntary contraction of each leg; bilateral hand grip strength). At the end of 
this visit, subject will have no further study visits. 
 
3.2 Subject Compliance 
If a subject consistently reports failing to drink at least 80% of the beverages, or fails to show 
up for the weekly visit multiple times, they will be dropped from the study. At the weekly visits, 
study staff will weigh the returning supplement containers and write the mass on the container. 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 10 
 
This will allow for calculation of expected decrease in mass over time, which will provide data 
as to subject compliance. 
 
3.3 Subject Compensation 
Subjects will accrue compensation for every visit according to the below table. They will be 
provided with a check based upon their compensation as of visits 7 and 14. If they were to 
attend every visit, their total amount would be $300. 
 
 
3.4 Randomization 
We will use computer randomization to assign subjects to one of two possible treatment 
groups. The randomization procedure will be stratified by gender. A randomization procedure 
will be implemented using an Excel spreadsheet. As a participant is identified and enrolled on 
study, the study coordinator will ascertain gender, which will determine the next randomization. 
 
A: subjects will consume 9g of EAA mixture supplement twice daily for 12 consecutive 
weeks. 
B: subjects will consume 9g of placebo twice daily for 12 consecutive weeks. 
 
3.5 Blinding 
This is a double-blinded study. EAA mixture will be provided by Prinova Inc. (Carol Stream, IL). 
Placebo will be purchased from online or local sources (Pure Protein website, Wal-Mart, etc.). 
Product containers will be coded so that only the unblinded study staff will know product 
Visit 
Amount 
1 
$25 
2 
$50 
3-7 (each) 
$10 
8 
$50 
9-12 (each) 
$10 
13 
$35 
14 
$50 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 11 
 
identity. After all study visits have been performed and all data entered into a database, the 
blind will be broken. Although the study products differ in taste, subjects will not be told which 
products are offered in which flavors. 
 
3.6 Study Products 
The active study product is 9g twice a day of EAA mixture, a proprietary formula of Essential 
Blends, LLC (Fairbanks, AK). Product is a blend of amino acids, flavorings, caffeine and 
sweeteners (see Appendix) in powder form; packaged in 200g sealed containers. Subjects will 
dissolve 1 scoop of supplement into ~7 ounces of water and stir/shake until fully dissolved. 
They will be provided with shaker cups free of charge. Servings will be ingested between 
meals so that they ingest 2 servings per day. EAA mixture is currently available in only one 
flavor (lemon/lime). 
The placebo product is 9g twice a day of Pure Protein 100% Whey protein (Worldwide Sport 
Nutritional Supplements, Inc., Bayport, NY). Unblinded study staff will repackage this product 
into 200g sealed containers which contain the appropriate scoops. See appendix for product 
information. Subjects will dissolve 1 scoop of supplement into ~7 ounces of water and 
stir/shake until fully dissolved. Servings will be ingested between meals so that they ingest 2 
servings per day. 
Subjects will be asked to save empty containers and return them to the study site for 
accountability purposes. Storage of both products is in a dry area at room temperature. 
Containers will be labelled identically, with the only difference being the study product code (A 
or B); the key to which is known only to the unblinded study staff until blinding has been broken 
at the end of data analysis. 
 
3.7 Sample Storage 
Blood and urine samples will be kept frozen at -60 degrees Centigrade or colder once the 
initial processing has taken place. Stool samples will be kept refrigerated at 4 degrees C until 
processed. Samples shall be stored in appropriate freezers in the PI’s laboratory, located in a 
restricted area inside the UAMS IOA building. Said freezers are monitored continuously for 
proper temperature and working condition. Samples will be destroyed only after all data has 
been analyzed and reported to the sponsor. All blood samples shall be identified using a 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 12 
 
unique study acronym. None of a subject’s personal identifiers shall be present on any 
biological sample. Urine samples collected during visits 2 and 14 will be discarded immediately 
after performing the dipstick test for proteinuria. 
 
4.0 Study Population 
 
Subjects will be recruited using these methods: 1) past subjects that indicated they wanted to 
be contacted about future studies will be called by study staff to elicit their interest in this study, 
and 2) the PI will refer potential subjects from her UAMS clinic population to study staff who 
will then contact the referrals via phone. Once a potential subject has agreed to come to 
UAMS for an informed consent discussion, an appointment will be made for them to meet with 
study staff in the research area of the 3rd floor of the Reynolds Institute at UAMS. This study 
will enroll up to 80 subjects aged 60-89 years inclusive. 
4.1 Inclusion Criteria 
• Ages 60-89 yrs 
• BMI between 18 and 45 kg/m2 
• Any ethnicity 
• Presence of mild-to-moderate heart failure (NYHA II or III symptomatology) as evidenced 
by prescribed diuretics or reported shortness of breath upon exertion. 
 
4.2 Exclusion Criteria 
• Allergic to milk or soy products 
• Hemoglobin <10 g/dL 
• eGFR < 30 
• Inability to perform strength and/or functional assessments 
• Myocardial infarction in the past 6 months 
• Unstable angina 
• Moderate-severe heart valve disease 
• Infiltrative, restrictive or hypertrophic cardiomyopathy 
• Dementia –determined by a SLUMS score of <20 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 13 
 
• Currently has inflammatory bowel disease 
• Received chemotherapy or radiation therapy within the past 12 months 
• Currently undergoing tube feeding 
• Currently receiving palliative care for end-of-life circumstance 
• Unwilling to refrain from using non-study protein/amino acid supplements during their 
participation in this study 
• If deemed medically unstable by the study physician for any other reason. 
 
5.0 Risks and Benefits 
 
There are no direct benefits for the subjects apart from the possibility getting knowledge of 
their own results of the assessments and blood tests. Expected risks associated with this 
protocol are described in detail below. All experimental procedures will be performed by 
appropriately trained and credentialed personnel. 
 
5.1 Blood, urine and fecal sampling:  
The total amount of blood taken will be approximately 100 mL. Subjects will have no noticeable 
effects. The risks of collecting blood samples include pain, bruising and bleeding. 
Urine samples will be tested using commercial dipsticks for proteinuria during visits 2 and 14. 
Urine collected between visits 1-2 and 13-14 will be analyzed in the PI’s lab for the isotope-
labelled creatinine to determine skeletal muscle mass (14). There is no risk of collecting voided 
urine. Fecal samples will be collected at visits 2 and 14. There are no risks associated with 
collecting fecal samples. 
 
5.2 DEXA scan: 
The DEXA scan exposes subjects to approximately ½ of the radiation of one chest x-ray. They 
will undergo 2 DEXA scans in total. 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 14 
 
5.3 Exercise testing (strength and functional): 
Subjects may become sore or fatigued during or after the exercise tests. Study staff will remain 
close to subjects for safety, in the case of dizziness or potential falls. Tests will be paused or 
stopped if the participant is tired or short of breath. Tests will also be stopped if, for any 
reason, there is concern about the subject’s safety. During the 6-minute walk test, subjects will 
wear a portable oxygen saturation monitor to allow study staff to observe for desaturation. 
 
5.4 Study supplements: 
There are potential allergens in the placebo: milk and soy. All components of EAA mixture 
(Table 4) are classified as Generally Regarded as Safe (GRAS) by the FDA. They will be 
provided by PrinovaUSA, which has all necessary certifications for safe production of dietary 
supplements. 
 
5.5 Confidentiality: 
A potential risk to study participants is the potential for loss of confidentiality.  Measures to 
protect the confidentiality of study participants will be implemented as described in the Data 
Handling and Recordkeeping section below. 
 
5.6 Oral isotope ingestion: 
There are no known risks to ingesting 30mg of stable isotope-labelled creatine. 
 
6.0 Data Handling and Recordkeeping 
 
Source documents and CRFs will be stored in a secure area of the PI’s laboratory. Access will 
be limited to study personnel. Documents containing identifiers (except the signed ICF) will be 
destroyed by shredding approximately 7 years after data analysis is completed or publication 
of data; whichever is longest. The original, signed ICF will be kept indefinitely. At no time shall 
Protected Health Information be released to non-study personnel. 
The Principal Investigator will carefully monitor study procedures to protect the safety of 
research subjects, the quality of the data and the integrity of the study.  All study subject 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 15 
 
material will be assigned a unique identifying code or number.  The key to the code (the 
instrument associating the data with subject identity) will be kept on a password-protected 
UAMS server, located behind locked doors in a restricted access area of the UAMS campus.  
Only those individuals listed on the title page of this protocol and their research staff members 
will have access to the code and information that identifies the subject in this study. This file 
will be deleted approximately 7 years after data analysis is completed. 
 
7.0 Data Analysis 
 
7.1 Statistical Analysis plan 
A repeated measures analysis of covariance (ANCOVA) model will be used to evaluate the 
effects of EAA mixture and placebo on each parameter. The model will contain EAA mixture 
versus placebo and time main effects as well as their interaction. Baseline measures will be 
included in the model as a covariate to control for each subject’s starting point. The model will 
also include factors to account for lean body mass and gender. A non-significant interaction 
would imply that the EAA mixture or placebo and time effects are not dependent upon one 
another and that main effects can be tested. The significance of the interaction and main 
effects will be determined using a 5% α-level. Covariates that may need to be accounted for 
such as age and lean body mass may be added to the model as a separate exploratory 
analysis. 
 
7.2 Significant Interaction Analysis Plan 
A significant interaction suggests that the EAA mixture or placebo effect is dependent upon the 
time effect. This implies the EAA mixture or placebo effect will have to be assessed separately 
within each time point, and vice versa. Since the two EAA mixture and placebo groups are 
independent, an analysis of covariance (ANCOVA) model will be used to assess the EAA 
mixture or placebo effect for each time point (midpoint & final). A repeated measures analysis 
of variance (ANOVA) model will be used to assess the time effect for EAA mixture or placebo, 
where the repeated measure is time. A Bonferroni-corrected α-level of 2.5% will be used to 
determine the statistical significance of these tests. 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 16 
 
7.3 Sample Size Calculation & Power Analysis 
A total of 60 completed subjects will be required for this study with 30 subjects per EAA 
mixture and placebo group. The following power calculations are based on the analysis of the 
results of the six minute walk, leg strength, grip strength and 10 meter gait speed from our pilot 
studies. Insulin sensitivity and plasma lipids will be secondary end points. These calculations 
assume the “worst case” scenario where a significant EAA mixture or placebo-by-time 
interaction is detected and the covariates are not significant. In this case, the EAA mixture or 
placebo effect would be assessed separately within each time point, and the time effect would 
be assessed for each group. 
 
7.4 EAA mixture or placebo Effect 
The following power calculations are based on a two-sample t-test which is being used as an 
approximation of the ANCOVA model. With 30 subjects in each EAA mixture and placebo 
group and an α-level of 2.5%, a two-sample t-test will have approximately 80% power to detect 
effect sizes of 0.82 standard deviation units or larger. Table 4 presents the hypothesized group 
means for each endpoint as well as the corresponding EAA mixture or placebo effect and 
power estimate. These estimates correspond to approximately 25% (6-minute walk) to 50% 
(gait speed) of the differences in the values between individuals with heart failure vs age-
matched healthy controls as determined in our Phase I protocol. Our previous outcome studies 
with EAAs indicate that these levels of improvement are expected (e.g., 11, 12, 13). 
Table 4. Hypothesized group means and within group standard deviation 
used to estimate the power of the nutritional therapy effect when N=60 (30 
per group) are enrolled. The calculations assume that an α-level of 2.5% 
will be used to determine statistical significance. 
Endpoint 
Hypothesized Mean 
Changes# 
σΔ$ 
Effect 
Size 
Power (%) 
Control 
Nutritional 
product 
6 Minute 
Walka 
0 
105.1 
89.3 
1.18 
98.67 
Leg Strengtha 
0 
11.0 
13.3 
0.83 
81.31 
Grip Strengtha 
0 
4.1 
4.6 
0.89 
87.19 
10m Gait 
Speeda 
0 
0.23 
0.28 
0.82 
80.80 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 17 
 
#Means of the baseline and post-treatment changes for each group. 
$Estimated within group standard deviation of the changes. 
aEstimates of σΔ were obtained from pilot studies performed at the UAMS 
Pepper Center. 
 
7.5 Time Effect 
The following power calculations are based on a paired t-test which is being used as an 
approximation of the repeated measures ANOVA model. With 60 subjects within each time 
point, a paired t-test using a 2.5% α-level will have 80% power to detect effect sizes of 0.41 
standard deviations or larger. Note that the variance of the difference (𝜎𝜎𝛥𝛥
2) in the measures can 
be calculated using the following formula: 𝜎𝜎𝛥𝛥
2 = 𝜎𝜎𝑀𝑀
2 + 𝜎𝜎𝐹𝐹
2 −2𝜌𝜌𝜎𝜎𝑀𝑀𝜎𝜎𝐹𝐹, where ρ is the correlation 
between the mid-point (8 weeks) and final (16 weeks) measures, and σM and σF represent the 
standard deviations for the mid-point and 
final assessments, respectively. 
Assuming the standard deviations for the 
mid-point and final measures are similar, 
the equation can be  
 simplified to 𝜎𝜎𝛥𝛥
2 =  2𝜎𝜎𝛥𝛥
2(1 −𝜌𝜌). Using the 
simplified formula and our estimates of 
the standard deviations from Table 4, 
Table 5 presents the detectable 
differences that correspond to an effect 
size of 0.41. 
 
7.6 Urine analyses 
Urine samples will be analyzed for the presence of D3-creatinine (mass spectroscopy in PI’s 
lab) and unlabeled creatinine (Labcorp, Dallas, TX). Subjects will be sent home with 3 labelled 
urine collection containers to use at appropriate intervals after ingestion. 
  
7.7 Fecal analyses 
A subset of 20 subjects (10 from each group) will be asked to provide a fecal sample using a 
standard stool collection kit (DNA/RNA Shield™ Fecal Collection Tube from Zymo Research).  
Table 5. Detectable differences associated with 
an effect size of 0.41. 
Endpoint 
ρ 
σΔ 
Detectable 
Difference 
 
6 Minute 
Walk 
0.25 
109.4 
44.9 
0.50 
89.3 
36.6 
0.75 
63.1 
25.9 
 
Leg 
Strength 
0.25 
16.3 
6.7 
0.50 
13.3 
5.5 
0.75 
9.4 
3.9 
 
Grip 
Strength 
0.25 
5.6 
2.3 
0.50 
4.6 
1.9 
0.75 
3.3 
1.4 
 
Gait Speed 
0.25 
0.34 
0.14 
0.50 
0.28 
0.12 
0.75 
0.20 
0.08 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 18 
 
Any participants who prefer to take the collection kit home will be provided a shipping container 
and explicit instructions on sample collection, storage and shipping. The stool collection kits 
contain a nucleic acid stabilizer solution that allows room temperature storage and shipment of 
collected samples. Once acquired by the laboratory, samples will be kept refrigerated at 4 
degrees C until processed, within 20 days. Fecal analysis will analyze the microbiome diversity 
in the subjects. Bacterial species (eg. bacteroidaceae, pseudomononadaceae, 
ruminocococacease) will be identified by sequencing the bacterial DNA. These results will be 
reported back to the investigators for analysis of microbiome gut diversity between heart failure 
patients on control supplement versus EAA supplement. Results will be stored for studies. 
The microbial DNA will be enriched and purified at a lab on the UAMS campus using a 
modification of the standard Zymobiomics DNA purification kit (Zymo Research) to obtain high 
quality and high molecular weight DNA. 
 
7.8 Blood analyses 
In addition to the blood testing for eligibility, blood samples will be collected solely for the 
purpose of experimentation. The blood will be used to measure brain naturetic peptide, 
creatinine, complete blood count (CBC), glucose, insulin, plasma lipids, profile of cardiac aging 
proteins including serum response factor, hemoglobin A1c, plasma acylcarnitines, 
mitochondrial function, and possibly amino acid concentrations. 
 
8.0 Ethical Considerations 
 
This study will be conducted in accordance with all applicable government regulations 
and University of Arkansas for Medical Sciences research policies and procedures.  This 
protocol and any amendments will be submitted and approved by the UAMS Institutional 
Review Board (IRB) to conduct the study. 
The formal consent of each subject, using the IRB-approved consent form, will be obtained 
before that subject is submitted to any study procedure.  All subjects for this study will be 
provided a consent form describing this study and providing sufficient information in language 
suitable for subjects to make an informed decision about their participation in this study.  The 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 19 
 
person obtaining consent will thoroughly explain each element of the document and outline the 
risks and benefits, alternate treatment(s), and requirements of the study.  The consent process 
will take place in a quiet and private room, and subjects may take as much time as needed to 
make a decision about their participation.  Participation privacy will be maintained and 
questions regarding participation will be answered.  No coercion or undue influence will be 
used in the consent process.  This consent form must be signed by the subject and the 
individual obtaining the consent.  A copy of the signed consent will be given to the participant, 
and the informed consent process will be documented in each subject’s research record. 
 
9.0 Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The publications 
will not contain any identifiable information that could be linked to a participant. 
 
10.0 References 
1. 
McMurray FF, Pfeffer R. Heart failure. Lancet 2005; 365:1877-89. 
2. 
Chronic Heart Failure: National clinical guidelines for diagnosis and management in 
primary and secondary care: partial update [Internet]. National Clinical Guideline Centre 
(UK). London: Royal College of Physicians (UK) 2010 Aug; PMID:22741186 
3. 
Ferrari R, Bohm M, Cleland JG, Paulus WJ, Pieske B, Rapezzi C, Tavazzi L. Heart 
failure with preserved ejection fraction: uncertainties and dilemmas. Eur J Heart Fail 
2015; 17:665-71. 
4. 
Owan TE and Redfield MM. Epidemiology of Diastolic Heart Failure. Progress in 
Cardiovascular Disease 2005; 47:320-332. 
5. 
Bhatia RS, Tu JV, Lee DS, Austin PC, Fang J, Haouzi A, Gong Y, Liu PP. Outcome of 
heart failure with preserved ejection fraction in a population-based study. N Engl J Med 
2006 Jul; 355(3):260-9. 
6. 
Azhar G, Wei JY. The demographics of gaining and its impact on the cardiovascular 
health. Curr Cardiovasc Risk Report 2015; 4:1-6. 
7. 
Azhar G, Wei J. Cardiac cachexia. Currr Opin in Clin Nutr and Met Care 2005; 9:18-23. 
8. 
Braunstein JB, Anderson GF, Gerstenblith G, Weller W, Niefeld M, Herbert R, Wu AW. 
Noncardiac comorbidity increases preventable hospitalizations and mortality among 
Medicare beneficiaries with chronic heart failure. J Am Coll Cardiol 2003 Oct 1; 
42(7):1226-33. PMID:14522486 
9. 
Bacurau AV, Cunha TF, Souza RW, Voltarelli VA, Gabriel-Costa D, Brum PC. Aerobic 
exercise and pharmacological therapies for skeletal myopathy in heart failure: 
Similarities and differences. Oxid Med Cell Longev 2016; 437-467. PMID:26904163. 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 20 
 
10. 
Katsanos CS, Kobayashi H, Sheffield-Moore M, Aarsland A, Wolfe RR. Aging is 
associated with diminished accretion of muscle proteins after the ingestion of a small 
bolus of essential amino acids. Am J Clin Nutr 2005 Nov; 82(5):1065-1073. 
11. 
Ferrando AA, Paddon-Jones D, Hays NP, Kortebein P, Ronsen O, Williams RH, 
McComb A, Symons TB, Wolfe RR, Evans W. EAA supplementation to increase 
nitrogen intake improves muscle function during bed rest in the elderly. Clin Nutr 2010 
Feb; 29:18-23. 
12. 
Borsheim E, Bui QU, Tissier S, Kobayashi H, Ferrando AA, Wolfe RR. Effect of amino 
acid supplementation on muscle mass, strength and physical function in elderly. Clin 
Nutr 2008 Apr; 27(2):189-195. PMCID:PMC2430042 
13. 
Ferrando AA, Bamman MM, Schutzler SE, Spencer HJ, Evans RP, Wolfe RR, 
Increased nitrogen intake following hip arthroplasty expedites muscle strength recovery. 
J Aging: Research and Clinical Practice 2013; 2(4):369. 
14. 
Richard V. Clark, Ann C. Walker, Robin L. O'Connor-Semmes, Michael S. Leonard, 
Ram R. Miller, Stephen A. Stimpson, Scott M. Turner, Eric Ravussin, William T. Cefalu, 
Marc K. Hellerstein, and William J. Evans. Total body skeletal muscle mass: estimation 
by creatine (methyl-d ) dilution in humans. J Appl Physiol (1985). 2014 Jun 15; 116(12): 
1605–1613. Published online 2014 Apr 24. doi: 10.1152/japplphysiol.00045.2014 
 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 21 
 
 
11.0 Appendices 
 
11.1 Study Product Information 
 
EAA mixture 
 
 
His 
3.5%     
Ile 
7.1% 
Leu 
14.3% 
Lys 
6.0% 
Met 
3.3% 
Phe 
8.8% 
Thr 
5.3% 
Trp 
1.4% 
Val 
9.25% 
Glutamine 
1.2% 
Carnitine 
2.9% 
Nicotinamide 
1.5% 
Vitamin C                           
Caffeine 
Sucralose and other flavorings 
and preservatives                        
1.5% 
0.6% 
28.3% 
 
 
 
 
 
 
 
 

Effects of 12-Weeks of Nutritional Therapy Interventions in Heart Failure 
PI: Gohar Azhar, M.D. 
Institution: UAMS 
Sponsor: NIH subcontract 
Version #: 6 
 
IRB# 206313 
Date: 
27Jun18 
Page 22 
 
Placebo: Pure Protein – Vanilla Creme flavor