Effect of an Essential Amino Acid/Protein Composition on Protein Metabolism
Phase: NA
Conditions: Sarcopenia
Sponsor: University of Arkansas

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 1 
 
University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:  Effect of an essential amino acid/protein composition on protein 
metabolism. 
 
NCT03502941 
 
Principal Investigator: 
Arny A. Ferrando, Ph.D. 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St., Slot #806 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
Office Telephone: 501-526-5711 
 
 
 
 
Email: aferrando@uams.edu 
 
Sub-Investigators:  
Gohar Azhar, M.D. 
 
 
 
 
Sanghee Park, 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): 
Adesso, LLC. 
 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 2 
 
 
Table of Contents 
1.0 Background and Rationale ........................................................................................................... 4 
1.1 Product to be tested ...................................................................................................................... 4 
1.2 Product composition ...................................................................................................................... 4 
1.3 Previous research on the topic .................................................................................................... 5 
2.0 Hypothesis ........................................................................................................................................ 7 
3.0 Study Design and Procedures ..................................................................................................... 7 
3.1 Study Visits ..................................................................................................................................... 8 
3.2 Second study (visit 3, for EAA/protein Arm) ............................................................................ 10 
3.3 Subject Compensation ................................................................................................................ 10 
3.4 Randomization for EAA/protein Arm ........................................................................................ 11 
3.5 Blinding .......................................................................................................................................... 11 
3.6 Sample Storage ........................................................................................................................... 11 
4.0 Study Population ........................................................................................................................... 11 
4.1 Inclusion Criteria .......................................................................................................................... 12 
4.2 Exclusion Criteria ......................................................................................................................... 12 
5.0 Risks and Benefits ........................................................................................................................ 12 
5.1 Blood sampling: ........................................................................................................................... 12 
5.2 DEXA scan: .................................................................................................................................. 13 
5.3 Muscle samples: .......................................................................................................................... 13 
5.4 Study supplements: ..................................................................................................................... 13 
5.5 Confidentiality: .............................................................................................................................. 13 
5.6 Stable Isotope Infusion: .............................................................................................................. 14 
6.0 Data Handling and Recordkeeping ........................................................................................... 14 
7.0 Data Analysis.................................................................................................................................. 15 
7.1 Statistical Analysis plan .............................................................................................................. 15 
7.2 Sample Size Calculation & Power Analysis ............................................................................ 15 
8.0 Ethical Considerations ................................................................................................................ 15 
9.0 Dissemination of Data .................................................................................................................. 16 
10.0 References .................................................................................................................................... 17 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 3 
 
11.0 Appendices ................................................................................................................................... 20 
11.1 CTRAL Muscle Biopsy SOP .................................................................................................... 20 
11.2 Stable Isotope Infusion SOP ................................................................................................... 23 
 
 
 
Table of Events 
 
Procedure 
Visit 1 
Visit 2 
Visit 3* 
informed consent 
x 
 
 
medical history 
x 
 
 
list of medications 
x 
 
 
measure height 
x 
 
 
measure weight 
x 
 
 
Blood for CBC 
x 
 
 
Urine pregnancy test 
x1 
 
 
Physical exam 
x2 
 
 
vital signs 
x2 
x 
x 
DEXA scan 
x3 
x3 
 
stable isotope study 
 
x 
x 
periodic blood sampling 
 
x 
x 
muscle biopsies 
 
x 
x 
ingest study product 
 
x 
x 
follow-up call 
 
x 
x 
 
*Only for subjects who have enrolled into the crossover arm of the study. 
1 For women of child bearing potential, sent to LabCorp. 
2 Can be done at Visit 1 or Visit 2. 
3 DEXA scan will be performed on female subjects of childbearing potential after it is confirmed 
they are not pregnant. 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 4 
 
1.0 Background and Rationale 
 
1.1 Product to be tested 
The product to be tested is designed to stimulate the net synthesis of body protein, including 
muscle protein. The composition is protected by intellectual property and is based on a 
specially formulated mixture of essential amino acids (EAAs) and whey protein.  The overriding 
metabolic goal of the composition is to maximally stimulate the rate of net muscle protein 
synthesis. The stimulation of protein synthesis is the metabolic basis for increases in muscular 
strength and function, and is crucial for increased biogenesis of mitochondria. 
 
1.2 Product composition 
The product composition is protected by intellectual property. It contains a combination of 
ingredients designed to stimulate muscle protein synthesis and to ultimately induce long-term 
improvements in muscle function and size. 
Muscle Protein Synthesis: EAAs are the key to rapid recovery of muscle. EAAs are the “active” 
components of dietary protein, meaning that they are the amino acids contained in dietary 
protein that actually stimulate the production of new muscle protein. In fact, EAAs are the only 
dietary macronutrients that are required for survival. They are “essential” because the body 
cannot produce them. There are 9 dietary EAAs. There are 11 more dietary non-essential 
amino acids that are not required in the diet because they can be produced in the body. The 
composition is based on a blend of 8 of the 9 EAAs. In addition to being the “active” 
components of dietary protein, EAAs also have the advantage over intact protein because free 
EAAs are absorbed from the intestine more quickly and more completely than amino acids 
contained in protein, which require digestion before absorption. Finally, the relative amounts of 
each EAA can be tailored for any specific metabolic function. In the case of the composition to 
be tested, the EAA formulation is designed to maximally stimulate muscle protein synthesis in 
healthy individuals under the age of 50 y. 
Intact Protein: Intact proteins are popular dietary supplements to stimulate muscle protein 
synthesis. While EAAs have a distinct advantage over intact protein in terms of the amount of 
muscle protein produced following exercise, the response to intact protein is sustained over a 
longer time than free EAAs because of the slower absorption. The composition combines the 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 5 
 
benefits of free EAAs as well as intact protein. The unique combination of EAAs and intact 
protein cause an immediate increase in muscle protein synthesis (due to the EAAs), and the 
response is sustained by the slower entry into the body of the EAAs contained in the intact 
protein. 
 
1.3 Previous research on the topic 
The primary goal of the composition is to promote muscle protein synthesis and the balance 
between synthesis and breakdown. Muscle protein synthesis is most effectively stimulated by 
EAAs. The response of protein synthesis following EAAs is more than twice as large as a 
comparable dosage of whey protein (1). Oral ingestion of 2 x 6 g doses of EAAs, taken one 
hour apart after exercise, induces a greater response of muscle protein turnover than any 
other nutritional strategy (2). The magnitude of response to EAAs after exercise is dose 
dependent. The anabolic response to 6 g of EAAs was twice that of the response to 3 g (2,3). 
Addition of non-essential amino acids to EAAs provides no extra benefit in terms of muscle 
protein synthesis (4,5). 
We assert that the optimal profile of EAAs for young, healthy subjects differs from that for 
elderly individuals. In contrast to the case for older individuals, there is no requirement for a 
disproportionate amount of leucine above and beyond to fulfil its requirement as a precursor 
from protein synthesis. Whereas a disproportionately large dose of dietary leucine relative to 
the other EAAs amplifies the stimulation of muscle protein synthesis in metabolically-
challenged circumstances such as aging (6), a large dose of leucine is not necessary in 
younger individuals. A large dose of leucine activates mTOR and other factors that are 
involved at the molecular level in regulating the rate of muscle protein synthesis (7). While this 
action of leucine is helpful when the activity of mTOR and associated factors are blunted, such 
as occurs in aging. However, mTOR activity is not rate-limiting in young, healthy individuals. 
Therefore, a high proportion of leucine in the composition would be redundant in terms of 
activation of mTOR. By reducing the proportion of leucine in the composition, the profile of the 
EAAs in the composition more closely reflects the profile of EAAs in muscle protein. This is 
advantageous since the limiting step in muscle protein synthesis in healthy young individuals is 
the availability of EAA precursors in proportion to the profile of EAAs in muscle protein (8). 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 6 
 
The profile of EAAs of the composition is specifically designed for maximal stimulation of 
muscle protein synthesis following exercise. 
The use of free EAAs is advantageous in terms of being able to determine the exact profile of 
the amino acids. Also, unlike intact protein, EAAs are rapidly and extensively absorbed (9). 
The rapid peak response in plasma EAAs is a key reason for their effectiveness (10). On the 
other hand, the total duration of the response is limited, because just as the concentrations of 
EAAs in the blood rise rapidly, they fall rapidly as well (2). For this reason, the composition 
contains protein in addition to the EAAs to prolong the anabolic response in the recovery 
period. 
EAAs are the “active” components of dietary protein that are primarily responsible for the 
stimulation of protein synthesis. Non-essential amino acids (NEAAs) are not required in the 
short term. This is because the NEAAs are normally produced in the body at fast enough rates 
to avoid deficiencies. On the other hand, studies performed in livestock suggest that maximal 
long-term animal growth and development is achieved with a balance of about 20-30%% 
NEAAs and 70- 80%EAAs (11). The addition of a small amount of intact protein to a mixture of 
EAAs is the most efficient way to incorporate the appropriate amount of NEAAs into a dietary 
supplement designed to stimulate muscle protein synthesis over the long-term. A number of 
studies have determined that whey protein is the most effective intact dietary protein in terms 
of stimulation of muscle protein synthesis after exercise (12,13). Whey protein is approximately 
50% EAAs and 50% NEAAs. By combining a small amount of whey protein with EAAs, the 
necessary intake of NEAAs is provided in a format (I,e,. intact protein) that, while not as 
effective as the EAA mixture, nonetheless has its own stimulatory effect on muscle protein 
synthesis. The combination of free EAAs and intact protein give an immediate response (from 
the free EAAs) which is prolonged over time by the slower digestion of the intact protein. 
The idea of combining a specific formulation of EAAs with an intact protein to obtain both an 
immediate as well as a more sustained response of protein synthesis is novel. Previous 
studies have added leucine to whey protein, but the inventor’s laboratory found this approach 
to not be effective (14). If only leucine is added to intact protein, an imbalance in the availability 
of the other EAAs is created that limits precursor availability for protein synthesis. The same 
limitation is true in the case of providing only branched chain amino acids. A unique aspect of 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 7 
 
the composition to be investigated is that a balanced mixture of all the EAAs is added to the 
whey protein source. With this approach, the rapid response of muscle protein synthesis to the 
free EAAs is sustained by the more gradual absorption of the EAAs from the whey protein. The 
unique combination of whey protein and a specially formulated EAA mixture that coincides with 
the proportionate demand for precursors for stimulated muscle protein synthesis provides a 
rapid and sustained response. 
The stimulation of both the number and function of the mitochondria is central to improved 
functional capacity of muscle. The EAA leucine in the composition acts as a nutraceutical to 
stimulate mitochondria biogenesis (15), and the free amino acids and protein stimulate the 
synthesis of new mitochondrial proteins (16). Therefore, the combination of free EAAs 
increases the ability of the muscle mitochondria to produce energy. 
 
2.0 Hypothesis 
 
We propose that consumption of the EAA/protein composition will stimulate the net gain (net 
protein synthesis) of body protein in a dose-dependent manner. We further propose that the 
magnitude of increase in net protein synthesis will be greater than induced by the consumption 
of the same amount of whey protein isolate, which is currently the most popular supplement on 
the market targeting the stimulation of muscle protein synthesis. 
 
3.0 Study Design and Procedures 
 
We will study a total of up to 20 healthy male and females between 18 and 50 years of age. 
We will use a randomized, two-period, stable isotope (Cambridge Isotope Labs, Tewksbury, 
Mass.) infusion study: 4.5-hour basal fasted period and 4-hour post-meal period (total 8.5-h 
time period). The principal end-point will be the total anabolic response (whole body protein 
synthesis minus breakdown) and the secondary end point will be muscle protein fractional 
synthetic rate (FSR) over the 4 hours following the consumption of either the test composition 
or whey protein. The plasma amino acid response will also be a secondary end point. 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 8 
 
We will utilize two distinct arms within this protocol. Arm 1 will consist of a group of subjects 
who will perform a randomized, single blind cross-over (two stable isotope studies) between 
the two doses of the EAA/protein study supplement, with a ≥ oneweek washout period 
between stable isotope studies. Arm 2 will consist of a group of subjects (with similar gender 
composition) who will undergo one stable isotope study during which they will ingest the whey 
protein isolate study supplement. Subjects will be enrolled into the double stable isotope study 
arm,  utilizing the specific consent form. After Arm 1 enrollment is completed, unique future 
subjects will be enrolled into Arm 2 – the single stable isotope arm, using the specific consent 
form. 
 
3.1 Study Visits 
Visit 1: subjects will come to the UAMS IOA 3rd floor for informed consent discussion. Once 
consent is obtained, subsequent study procedures will be performed. A medical history 
including allergies and list of current medications will be obtained (printed from Epic if they are 
a UAMS patient). Subject height and weight will be measured. A blood sample will be drawn 
for complete blood count [CBC]. Females of child bearing capacity will be asked to provide a 
urine sample for pregnancy testing. Blood and urine samples will be sent to LabCorp for the 
screening tests. A physical exam and vital signs will be performed at this visit or at visit 2. A 
DEXA scan for whole-body analysis will be performed in the PI’s lab at UAMS (or at visit 2). 
Subjects can get a copy of their CBC and DEXA scan upon request. Any abnormal findings will 
be discussed with them by the study nurse and/or physician. Based upon the results of the 
screening blood sample, visit 2 will be scheduled. 
 
Visit 2: Subjects return to the UAMS IOA having fasted overnight from 10:00 P.M. They will be 
asked about any adverse events since last visit. If not performed at visit 1, the physical exam 
and/or DEXA scan will be performed at this visit. For subjects who enrolled into the double 
infusion arm, randomization will be performed as to the order of supplement dose ingestion. 
After vital signs are measured, the study nurse will insert an IV catheter into a vein on each of 
the subjects’ arms. One catheter is used to infuse the stable isotopes L-ring-D5 phenylalanine 
and 2H2-tyrosine. The other is to allow for periodic blood sampling, warming the arm by means 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 9 
 
of a heating pad or a heated plastic box. After an initial blood sample is obtained, the study 
nurse will infuse the priming doses of the two above isotopes as well as a priming dose of 2H4-
tyrosine. Constant infusion of isotopes commences immediately after the priming doses are 
completed. A timer will be started, and blood/muscle samples will be obtained according to the 
schedule below. The appropriate study supplement will be served directly after the second 
muscle biopsy procedure. Subjects will be asked to consume their supplement within 2 
minutes. 
At the conclusion of the 8.5-hour infusion study, the IV catheters will be removed and the sites 
dressed with a sterile bandage. Written and verbal instructions regarding the care of the 
muscle biopsy site will be provided. A snack and beverage will be offered to subjects. Vital 
signs will be measured, and subjects will then be free to leave. A follow-up phone call will be 
made within 72 hours after subjects have left the facility to assess for adverse events related to 
the stable isotope study visit. 
Elapsed time (min., approximate) 
Procedure 
-0 
Blood sample (~6mL) 
120 
Blood sample (~6mL) 
150 
Blood sample (~6mL), muscle sample 
(subject’s choice of left or right vastus 
lateralis ~150mg) 
180 
Blood sample (~6mL) 
210 
Blood sample (~6mL) 
240 
Blood sample (~6mL) 
270 
Blood sample (~6mL), muscle sample 
(~150mg), ingest study supplement 
290 
Blood sample (~6mL) 
310 
Blood sample (~6mL) 
330 
Blood sample (~6mL) 
360 
Blood sample (~6mL) 
390 
Blood sample (~6mL) 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 10 
 
420 
Blood sample (~6mL) 
450 
Blood sample (~6mL) 
480 
Blood sample (~6mL) 
510 
Blood sample (~6mL), muscle sample 
(~150mg) 
 
3.2 Second study (visit 3, for EAA/protein Arm) 
After approximate minimum 1-week washout period, subjects who enrolled into the 
EAA/protein arm will undergo a second stable isotope study as described above. The order of 
consumption of each beverage will be randomized. The opposite leg will be used for muscle 
protein synthesis measurements. No further DEXA will be performed. 
 
3.3 Subject Compensation 
Subjects will accrue compensation for every visit according to the below table. They will be 
mailed a check for their total compensation approximately 2-3 weeks after their participation 
ceases (whether completed or not). If they were to attend every visit, their total amount would 
be $325 for the whey arm and $625 for the EAA/protein arm. If they stop participating during a 
stable isotope study visit, they will receive prorated pay of $25 per hour. 
 
 
Visit 
Amount 
1 
$25 
2 
up to $300 
3 (if applicable) 
up to $300 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 11 
 
3.4 Randomization for EAA/protein Arm 
Study staff will strive to maintain gender balance between the two arms. A randomization list 
will be implemented to reveal which dosage of EAA/protein will be tested first in the case of the 
cross-over study (Arm 1). 
 
3.5 Blinding 
This will be blinded to the extent feasible, given that different beverages will look and taste 
different. Since the subject has no conscious control over their rate of muscle protein 
synthesis, total blinding is of no concern. Nonetheless, subjects will not be told which dose of 
the product they are consuming. The data analyst will need to know the amount and nature of 
the product being consumed to calculate the rate of protein breakdown. 
 
3.6 Sample Storage 
Blood and muscle samples will be kept frozen at -80 degrees Centigrade or colder once the 
initial processing has taken place. 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. With explicit permission 
via the ICF, all biological samples will be kept indefinitely for future IRB-approved uses. All 
blood and muscle samples shall be identified using a unique study acronym. None of a 
subject’s personal identifiers shall be present on any biological sample. 
 
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 study staff will place IRB-approved flyers around the Little Rock community. 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. 
. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 12 
 
4.1 Inclusion Criteria 
 Ages 18-50 yrs. 
 
4.2 Exclusion Criteria 
 History of diabetes 
 History of malignancy in the 6 months prior to enrollment 
 History of gastrointestinal bypass/reduction surgery (Lapband, gastric sleeve, etc.) 
 History of chronic inflammatory condition or disease (Lupus, HIV/AIDS, etc.) 
 Pregnant females 
 Subjects who report regular resistance training exercise > once per week 
 Hemoglobin < 9.5 g/dL at the screening visit 
 Platelets < 150,000 at the screening visit 
 Subjects who cannot safely stop using aspirin for 7 days prior to muscle biopsies 
 Concomitant use of corticosteroids (ingestion, injection or transdermal) 
 Any other disease or condition that would place the subject at increased risk of harm if they 
were to participate, at the discretion of the study physician 
 
5.0 Risks and Benefits 
 
There are no direct benefits for the subjects. 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 sampling:  
Blood samples will be collected solely for the purpose of experimentation. The blood will be 
used to determine eligibility and to measure glucose, insulin, plasma amino acid 
concentrations and stable isotope enrichment. The total amount of blood taken will be 
approximately 100 mL for subjects in the whey protein isolate arm, and 190 mL for subjects in 
the EAA/protein arm. Subjects should have no noticeable effects from these volumes. 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 13 
 
5.2 DEXA scan: 
The DEXA scan exposes subjects to approximately ½ of the radiation of one chest x-ray. They 
will undergo 1 DEXA scan. 
 
5.3 Muscle samples: 
Briefly, the risks related to the biopsy procedure include pain, bleeding, bruising, small scar 
formation at the biopsy site, Infection, and vaso-vagal response. Muscle biopsies are 
performed by a physician utilizing local anesthesia (using 1% plain Iidocaine) for pain 
management and strict sterile procedures. Subjects must meet platelet criteria prior to biopsy. 
See appendices for SOP and related documents. 
 
5.4 Study supplements: 
The test composition will be provided by Adesso, LLC. It consists of a proprietary blend of 
eight essential amino acids and whey protein. It is produced by Prinova, Inc, Carol Stream, Il. 
This product is not currently commercially available. All components of the composition have 
been ruled as Generally Regarded as Safe (GRAS) by the FDA. They are natural components 
of the normal diet. Prinova, Inc, has all necessary certifications for making products for human 
consumption. Two doses of the product will be tested (approximately 6.3 and 12.6 g of active 
components).  The product comes as powder and will be dissolved in water (8-16 oz) for 
consumption.   
The whey protein isolate is available commercially. The dosage (12.6 g) will be dissolved in 
water (12 oz). There are no known risks of adverse effects of either the test composition or the 
whey protein in the dosages used in this study.  All components are Generally Regarded as 
Safe by the FDA, and Prinova Inc has all the necessary certifications for producing and 
packaging products for human consumption  
 
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 Record keeping section below. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 14 
 
 
5.6 Stable Isotope Infusion: 
Briefly, the risk related to the isotope infusion is pyrogenic response. All isotope infusions are 
compounded and prepared by a licensed pharmacist at UAMS. An SOP for infusion 
procedures will be utilized (see Appendix). All stable isotopes are tested for sterility and 
pyrogenicity. The stable isotopes will be filtered during infusion through a sterile 0.22 micron 
(Millipore) filter placed in the infusion line.  Stable isotopes are naturally occurring compounds 
and are not radioactive and are already present in the body in varying amounts. The infusion 
will be in a 'tracer' dose, i.e., a dose only detectable by GCMS.  Infusion of these molecules 
will increase the level of naturally occurring isotopes by 7-10%. Any adverse reactions during 
the isotope infusion that suggest infection (urticaria, flushing, nausea, vomiting, sweating, 
chills, altered heart rate, hypo/hypertension, and hyperthermia) will be promptly addressed by 
the study physician. Depending on the seriousness of the reaction, the infusion study will be 
terminated. 
 
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 
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. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 15 
 
Sponsor (Adesso, LLC) will have monitoring and audit access to source documents and all 
collected and/or analyzed data. Any data sent to them will remain coded with the unique 
subject alphanumeric identifier. No identifiable information will leave UAMS. 
 
7.0 Data Analysis 
 
7.1 Statistical Analysis plan 
The purpose of this study is to estimate and compare the effect of two different nutritional 
beverages on total anabolic response (TAR) and muscle protein synthesis (MPS). To this end, 
an analysis of covariance (ANCOVA) model will be employed to compare the protein sources 
with respect to mean response (either TAR or MPS) after adjusting for basal measures. If a 
significant protein group effect is detected, then pairwise comparisons will be performed to 
determine if the two sources or two doses differ. In this case, the Tukey-Kramer method will be 
used to adjust for multiple comparisons. A 5% will be used to determine statistical significance. 
 
7.2 Sample Size Calculation & Power Analysis 
A total of N=16 subjects will be required for this 
study, 8 in each arm. With this sample size, the 
ANCOVA model will have 80% power to detect 
effect sizes of f = 0.484 or larger (Table 1). This 
estimate assumes the basal covariate explains 
50% of the variation in the response and a 5% α-
level is used to determine statistical significance. 
(Note that the effect size, f, is simply the ratio of the variation between the three group means 
and the between-subject variation). In the event the effect of the covariate is mis-specified, the 
following table presents effect sizes that can be detected as the magnitude of variation 
explained by the covariate increases from 10% to 90%.   
 
8.0 Ethical Considerations 
 
Table 1. Expected Effect Size by Covariate Variation 
Variation Explained by 
Covariate (%) 
Detectable Effect Size, f 
10 
0.649 
30 
0.573 
50 
0.484 
70 
0.375 
90 
0.216 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 16 
 
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 
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. Adesso LLC, will 
have 90 days to review potential publications before submission. The ultimate decisions on 
content of publications will reside with UAMS investigators. The publications will not contain 
any identifiable information that could be linked to a participant. 
 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
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10.0 References 
 
1. 
Paddon-Jones D, Sheffield-Moore M, Katsanos CS, Zhang XJ, Wolfe RR. Differential 
stimulation of muscle protein synthesis in elderly humans following isocaloric ingestion 
of amino acids or whey protein. Exp Gerontol 2006 Feb; 41(2): 215-9. PMID: 16310330. 
2. 
Borsheim E, Tipton KD, Wolf SE, Wolfe RR. Essential amino acids and muscle protein 
recovery from resistance exercise. Am J Physiol Endocrinol Metab 2002 Oct; 283(4): 
E648-57. PMCID: 12217881. 
3. 
Miller SL, Tipton KD, Chinkes DL, Wolf SE, Wolfe RR. Independent and combined 
effects of amino acids and glucose after resistance exercise. Med Sci Sports Exerc 
2003 Mar; 35(3): 449-55. PMID: 12618575. 
4. 
Volpi E, Kobayashi H, Sheffield-Moore M, Mittendorfer B, Wolfe RR. Essential amino 
acids are primarily responsible for the amino acid stimulation of muscle protein 
anabolism in healthy elderly adults. Am J Clin Nutr 2003 Aug; 78(2): 250-8. PMID: 
12885705. 
5. 
Tipton KD, Gurkin BE, Matin S, Wolfe RR. Nonessential amino acids are not necessary 
to stimulate net muscle protein synthesis in healthy volunteers. J Nutr Biochem 1999 
Feb; 10:89-95. PMID: 15539275. 
6. 
Katsanos CS, Kobayashi H, Sheffield-Moore M, Aarsland A, Wolfe RR. A high 
proportion of leucine is required for optimal stimulation of the rate of muscle protein 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 18 
 
synthesis by essential amino acids in the elderly. Am J Physiol Endocriol Metab 2006 
Aug; 291(2): E381-7. PMID: 16507602. 
7. 
Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS, Kimball SR. Leucine 
stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-
sensitive pathway. J Nutr 2000 Oct; 130(10): 2413-9. PMID: 11015466. 
8. 
Wolfe RR, Miller SL. Amino acid availability controls muscle protein metabolism. 
Diabetes Nutr Metab 1999 Oct; 12(5): 322-8. PMID: 10741346. 
9. 
Adibi SA, Gray SJ, Menden E. The kinetics of amino acid absorption and alteration of 
plasma composition of free amino acids after intestinal perfusion of amino acid 
mixtures. Am J Clin Nutr 1967 Jan; 20(1): 24-33. PMID: 6017006. 
10. 
West DW, Burd NA, Coffey VG, Baker SK, Burke LM, Hawley JA, Moore DR, 
Stellingwerff T, Phillips SM. Rapid aminoacidemia enhances myofibrillar protein 
synthesis and anabolic intramuscular signaling responses after resistance exercise. Am 
J Clin Nutr 2011 Sep; 94(3): 795-803. PMID: 21795443. 
11. 
Heger J. Essential to non-essential amino acid ratios. 2nd. ed. In: JP Felix D’Mello, ed. 
Amino Acids in Animal Nutrition. CABI Publications. 2003; pp103-124. 
12. 
Devries MC, Phillips SM. Supplemental protein in support of muscle mass and health: 
advantage whey. J Food Sci 2015 Mar; 80 Suppl 1: A8-A15. PMID: 25757896. 
13. 
Tipton KD, Elliott TA, Cree MG, Aarsland AA, Sanford AP, Wolfe RR. Stimulation of net 
muscle protein synthesis by whey protein ingestion before and after exercise. Am J 
Physiol Endocrinol Metab 2007 Jan; 292(1): E71-6. PMID: 16896166. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 19 
 
14. 
Tipton KD, Elliott TA, Ferrando AA, Aarsland AA, Wolfe RR. Stimulation of muscle 
anabolism by resistance exercise and ingestion of leucine plus protein. Appl Physiol 
Nutr Metab 2009 Apr; 34(2):151-61. PMID: 19370045 
15. 
Baum J. Leucine stimulates mitochondrialbiogenesis. In Preparation. 
16. 
Bohe J, Low JF, Wolfe RR, Rennie MJ. Latency and duration of stimulation of human 
muscle protein synthesis during continuous infusion of amino acids. J Physiol 2001 Apr; 
532 (Pt 2): 575-9. PMCID: 11306673. 
 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
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11.0 Appendices 
 
11.1 CTRAL Muscle Biopsy SOP 
 
Policy:  The following information is written to serve as a resource and guideline for the 
percutaneous vastus lateralis muscle biopsy procedure. This SOP will ensure that the 
procedure is performed in a consistent manner that protects participant safety. This procedure 
can be performed concurrently with a fat biopsy, provided that the protocol calls for samples at 
the same time-point. In this case, the fat bx shall be performed first so that the muscle bx site 
does not bleed during the performance of the fat bx. 
 
Purpose: The muscle biopsy procedure is conducted to obtain a sample (~100-150mg) of 
muscle tissue for metabolic (and possibly structural) analyses. 
 
Responsibility and Accountability:  
Principal Investigator:  Will oversee, direct, and be responsible for assuring adherence to the 
entire procedure.  
Study Physician: Responsible for overall medical supervision and performance of the 
procedure including handling of subject complaints or adverse events related to muscle 
biopsies.  
Study/staff nurse:  Will assist study physician with their performance of muscle biopsy 
procedure. Will educate subject about post-biopsy care per the written instructions. 
 
Materials:   
Equipment:  sterile 5mm (or 6mm) Bergstrom biopsy cannula. 
Supplies: 1 
betadine 3-swab pack, commercial preps, or chlorhexidine   
 
 
 
solution 
2 
drape sheets 
1 
fenestrated drape sheet 
1 
laceration tray 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
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Date: 
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1 
lidocaine 1% w/o epinephrine (from Rx) 
2pr 
sterile gloves 
2 
packs non-adhesive dressing sponges 
1 
Opti-lock IV extension tubing 
1 
sterile IV extension tubing ~24” long 
1 
#11 scalpel 
1 
Dermabond glue 
2 
clear Tegaderm-type dressing 
1 
50 or 60 mL syringe for suction 
1 
5mL syringe for rinsing muscle 
1 
18g needle for rinsing muscle 
1 
5mL sterile saline for rinsing muscle 
2 
6” ace bandage  
Pre-Procedure: 
1. Verify subject has already completed the consent and screening process. 
2. Nurse verifies that subject has not taken any blood-thinning medications recently.  
a. Prohibited medication classes: antiplatelet agents, anticoagulant agents. If 
subject uses these medications as prescribed by their physician, they will not 
be allowed to undergo a muscle biopsy. 
b. Aspirin or aspirin-containing compounds: subjects should wait 7 days after the 
last dose of aspirin was ingested before they undergo a muscle biopsy. 
3. Staff will set up a sterile field containing the above supplies and bx needle. 
4. Explain the procedure to subject in terms that they understand, including expected 
sensations and side effects. 
 
Procedure: 
1. The biopsy site (lateral thigh, 1/2 way between hip and knee) will be prepped with 
one of the prep solutions to ensure disinfection. 
2. Physician performing the bx and nurse will properly don sterile gloves. 
3. The prepped area will be draped using the supplied sterile materials. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
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4. Sufficient 1% lidocaine solution will be used to anesthetize the skin, subcutaneous 
tissue and fascia at the biopsy site using supplied syringe and needles. Limit 
lidocaine volume to less than 20mL per biopsy. Wait approximately 6-10 minutes 
after the initial injection before testing area for sensation. 
5. Using aseptic technique and sterile scalpel, a small incision (approximately ¼ inch) 
will be made in the skin after adequate anesthesia has been confirmed. 
6. The Bergstrom needle will be inserted through the incision site approximately ¾ to 1” 
past the fascia, and a muscle biopsy will be taken with the aid of suction. 
7. After the bx needle has been removed, a person wearing sterile gloves will apply 
firm compression for at least 7 minutes to the biopsy site. 
8. Muscle sample will be handed to study staff for appropriate processing. 
9. After hemostasis has been achieved, the biopsy site will be cleaned, and if this is the 
only or final biopsy to be performed, a medical adhesive will be used to close the 
incision. A pressure dressing will then be applied, and the subject will be advised to 
keep the pressure dressing on for approximately two hours. 
10. The subject will be provided written wound care instructions, and these will be 
reviewed verbally as well. 
Post Procedure and Discharge: 
1. Provide subject with written wound care instructions and ensure they understand the 
instructions. 
2. Call subject within 72 hours after the most recent bx to follow up for any problems or 
concerns. 
Emergency Procedures and Adverse Event Reporting: 
If bleeding persists, apply additional pressure to the site where muscle was biopsied. If 
necessary, apply ice pack to area to assist with hemostasis. Refer to study protocol for 
reporting procedures. 
Documentation: 
The study staff will complete the Muscle Biopsy Procedure note for each bx performed. Notes 
will be filed in the CRF. 
 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
Page 23 
 
11.2 Stable Isotope Infusion SOP 
 
Policy:  The following information is written to serve as a resource and guideline for the stable 
isotope infusion procedure. This SOP will ensure that the procedure is performed in a 
consistent manner that protects participant safety. 
 
Purpose: The stable isotope infusion procedure is conducted to measure in-vivo substrate 
kinetics (amino acid, glucose, fatty acids, etc) (1). 
 
Responsibility and Accountability:  
Principal Investigator:  Will oversee, direct, and be responsible for assuring adherence to the 
entire procedure.  
Study Physician: Responsible for overall medical supervision of the procedure. Will sign 
order(s) for related procedures/supplies, including infusions. 
Study/staff nurse:  Will provide immediate supervision throughout the procedure. Responsible 
for administering correct compound in correct dose to correct subject, as ordered (2). 
Pharmacist:  Will prepare infusions per physician’s order.  
 
Definitions:   
 
Stable isotope – a non-radioactive variant (heavier by electron mass) of a naturally-occurring 
substance, e.g. glucose, amino acids, etc. 
 
Materials:   
1. Equipment:  Calibration records for infusion pumps used will be maintained 
according to institutional policy.  
2. All infusions will be prepared by a licensed pharmacist.  
 
Pre-Procedure: 
1. Verify subject has already completed the consent and screening process. 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
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2. Nurse documents subject’s vital signs, verifies that subject has fasted as required by 
protocol, and has met any other protocol-specific requirements and notes this in the 
record.  
3. Nurse inserts blood sampling catheter and draws baseline sample and records these 
events in the record.  
4. Nurse inserts infusion line catheter. 
5. All syringes containing isotopes will be labeled by the institutional pharmacy for 
subject name/ID, isotope, dose, date and initials. 
6. A licensed medical person must perform the setup of infusions. 
7. The study physician will be notified of the date and time for each procedure and will 
be available by pager or cell phone throughout the procedure.  
8. A nurse will be responsible for maintenance of all catheters and be present during 
the procedure. 
 
Procedure: 
1. The subject will be advised to rest in a recumbent and/or supine position for the 
procedure. 
2. Blood and muscle samples will be obtained according to the study-specific CRF. 
Blood samples will be obtained by licensed medical personnel or specifically trained 
and credentialed technical staff. Muscle biopsies will be obtained according to SOP 
by UAMS physicians. 
3. Subjects will be monitored for adverse reactions to the infusion and blood and 
muscle sampling by study personnel and nurse. Any adverse reactions will be 
reported to the study physician immediately. If a reaction is suspected to be 
attributed to the infusion, it will be stopped and a sample preserved for later quality 
testing. 
4. Subjects will be asked to comment on their general well-being during the procedure. 
  
Post Procedure and Discharge: 

Effect of an essential amino acid/protein composition on protein metabolism. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: Adesso, LLC 
 
Version #: 4 
 
IRB# 217658 
Date: 
19Mar18 
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1. Immediately following the completion of the procedure, each subject will be offered a 
snack and will be encouraged to consume it. 
 
Emergency Procedures and Adverse Event Reporting: 
 
All stable isotope infusion procedures will be performed as described in this SOP to minimize 
the risk of an unanticipated event.  These procedures will be performed in the CRC at UAMS 
Central hospital. 
 
Emergencies will be handled according to institutional guidelines.  Adverse events will be 
reported by the PI to all relevant regulatory bodies (IRB, sponsor) pertaining to the specific 
protocol.  Incident reports will be filed according to individual institutional guidelines.  
 
 
Documentation:  The study nurse will sign the study flowsheet, which will be reviewed by the 
PI at the completion of the study. Documentation for outpatients will be according to 
institutional policy. 
 
Sources/References:  All drug products for any study shall be prepared in accordance with 
Arkansas Law (Regulation 07-02-0001; Drug Products and Descriptions; Standards for 
Compounding and Dispensing Sterile Products). 
 
1. 
Wolfe RR and Chinkes DL. Isotope Tracers in Metabolic Research: Principles and 
Practice of Kinetic Analysis. Wiley, New York, New York, 2004, 274 pages. 
 
2. 
Deglin JH and Vallerand AH. Davis’s Drug Guide for Nurses. 9th ed. F.A.Davis, 
Philadelphia, PA, 2005, xx-xxii.