Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes
Phase: NA
Conditions: Healthy
Sponsor: University of Arkansas

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 1 of 13 
 
 University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:  
Whole Body Protein Metabolism of a Whey/EAA 
Supplement Using Continuous Oral Stable Isotopes 
 
Principal Investigator: 
Arny A. Ferrando, Ph.D., Professor  
                                           Donald W. Reynolds Institute of Aging 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St., Slot 806 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
 
Office phone: 501-526-5711 
 
 
 
 
Email: aferrando@uams.edu 
 
Sub-Investigators:  
Gohar Azhar, M.D. 
 
 
 
 
 
David D. Church, Ph.D. 
 
 
 
 
 
Katie R. Hirsch, Ph.D. 
 
 
 
 
 
Post-Doctoral Fellows 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St. 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
 
 
Study location: 
 
UAMS Reynolds Institute on Aging 
 
 
 
 
4301 West Markham St. 
 
 
 
 
Little Rock, AR 72205 
 
Funding Support:  
UAMS Geriatrics 
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 2 of 13 
 
Table of Contents 
List of Abbreviations ................................................................................................................. 3 
1.0 Protocol Summary ................................................................................................................. 4 
2.0 Background ............................................................................................................................ 4 
3.0 Specific Aims .......................................................................................................................... 5 
4.0 Study Population .................................................................................................................... 5 
4.1 Inclusion Criteria ............................................................................................................. 6 
4.2 Exclusion Criteria ............................................................................................................ 6 
4.3 Subject Recruitment and Consent .............................................................................. 6 
4.4 Subject Compensation................................................................................................... 6 
4.5 Study Visits ....................................................................................................................... 6 
4.6 Blinding .............................................................................................................................. 8 
4.7 Randomization ................................................................................................................. 8 
4.8 Study Products ................................................................................................................ 8 
5.0 Data and Specimen Collection and Management ............................................................ 8 
5.1 Blood Sample collection ............................................................................................... 8 
5.2 Sample Storage ................................................................................................................ 9 
5.3 Sample Analysis .............................................................................................................. 9 
5.4 Data Analysis & Statistics ............................................................................................. 9 
5.5 Dissemination of Data .................................................................................................... 9 
5.6 Data Handling and Recordkeeping ........................................................................... 10 
5.7 Data Access .................................................................................................................... 10 
6.0 Risks and Benefits ............................................................................................................... 10 
6.1 COVID-19 Protections .................................................................................................. 10 
6.2 Blood sampling/IV insertion ....................................................................................... 11 
6.3 Confidentiality ................................................................................................................ 11 
6.4 Study Products .............................................................................................................. 11 
6.5 Adverse Events .............................................................................................................. 12 
6.6 DXA Scan ......................................................................................................................... 12 
7.0 Ethical Considerations ........................................................................................................ 12 
8.0 References ........................................................................................................................... 13 
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 3 of 13 
 
List of Abbreviations 
 
CRF 
Case Report Form 
CTRAL 
Center for Translational Research in Aging and Longevity 
DXA 
Dual-energy X-ray Absorptiometry 
EAA 
Essential Amino Acid 
EDTA 
Ethylenediaminetetraacetic acid (a blood preservative) 
FDA 
Food and Drug Administration 
GC-MS 
Gas Chromatography-Mass Spectrometry 
GRAS 
Generally Regarded as Safe 
RIOA 
Reynolds Institute on Aging (UAMS) 
IRB 
Institutional Review Board 
LC-MS 
Liquid chromatography-mass spectrometry 
LOA 
Limits of agreement 
NB 
Net balance 
PB 
Protein breakdown 
PI 
Principal Investigator 
PS 
Protein synthesis 
SP 
study product or placebo 
TE 
Total error 
SEE 
Standard error of the estimate 
UAMS 
University of Arkansas for Medical Sciences 
IV 
Intravenous catheter 
 
 
 
 
Study Schema 
 
Study staff will recruit subjects via word of mouth to friends, family and colleagues. 
 
Informed consent process takes place during Visit 1, followed by DXA scan. 
 
Randomized subjects undergo two separate 7-hour study visits. 
 
Visits 2 and 3 entail serial blood sampling from an IV catheter, ingestion of stable 
isotopes of amino acids every 10 minutes, and ingestion of SP (study product or 
placebo). 
 
Blood samples analyzed at completion of study visits. 
 
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 4 of 13 
 
Study Calendar 
 
Procedure 
Visit 1 
Visit 2 
Visit 3 
Informed consent 
x 
 
 
Fasted prior to visit 
 
x 
x 
Blood sampling 
 
x 
x 
IV 
 
x 
x 
Ingest stable isotopes 
 
x 
x 
Ingest SP 
 
x 
x 
 
 
1.0 Protocol Summary 
 
This will be a double-blinded, randomized, crossover pilot study to evaluate the validity 
of a continuous oral sip-feeding of stable isotope tracer method for the evaluation of 
whole-body protein metabolism. This method will be evaluated using two different 
amounts of an essential amino acid (EAA) + whey protein supplement on up to ten 
younger, healthy subjects. Stable isotope preparations will be continuously consumed 
by subjects throughout visits 2 and 3 to enable whole body protein metabolism to be 
measured. A DXA scan will be performed to quantify subject’s lean mass. 
 
2.0 Background 
 
Stable isotope tracers are routinely used to study protein metabolism in free-living 
individuals in the fields of nutrition and exercise (1, 2). Whole-body rates of protein 
synthesis (PS), breakdown (PB), and net balance (NB) in response to nutrition and 
exercise interventions are commonly evaluated using a primed-constant infusion of 
essential amino acid (EAA) tracers, typically 2H5-phenylalanine and 2H4-tyrosine (1). 
This method has many advantages. The primed-constant infusion allows for 
determination of basal protein kinetics, in addition to the acute physiological response to 
an intervention, which allows for each subject to serve as their own control. Although an 
effective method, administration of the primed-constant infusion requires involvement 
from multiple trained individuals, including research pharmacists and nurses who 
compound and administer the EAA tracer. This limits the ability to apply these methods 
in field-type settings outside of a hospital or lab. The cost associated with purchase and 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 5 of 13 
 
compounding of the tracer, in addition to involvement from multiple specialists, makes 
these types of studies very expensive and difficult to conduct.  
 
An alternative to primed-constant infusion is to orally ingest amino acid tracers in sip 
fashion to achieve a similar plateau enrichment. Oral bolus ingestion of amino acid 
tracer has been used to quantify 24-hour whole-body protein metabolism in a variety of 
healthy and clinical populations for over 50 years (3). This method is one of the most 
conducive field-methods for determining whole-body protein metabolism in free-living 
subjects and has minimal participant burden. However, full integration of the tracer from 
a single bolus ingestion into the entire protein pool takes at least 10 hours. This limits 
the ability to quantify acute changes in protein kinetics of acute exercise or nutritional 
interventions. However, ingestion of a priming bolus, followed by continuous ingestion of 
EAA tracer may achieve steady-state equilibrium similar to constant infusion of the 
isotope. Thus, oral ingestion may enable the determination of acute changes in protein 
kinetics in the same respect as constant infusion.  If shown to be valid, this method 
would significantly reduce the rigor and expense of pharmacy-prepared infusions, and 
more importantly, allow for quantification of acute physiological protein responses within 
the field setting. 
 
3.0 Specific Aims 
 
SA1: To evaluate the validity of a continuous oral sip-feeding method of stable isotope 
tracer for the evaluation of whole-body protein metabolism in response to two different 
doses of an EAA/whey protein supplement (Low=6.3g and High=12.6g). We will 
compare kinetic responses with oral isotope ingestion to those previously determined by 
constant infusion with the same SP (4).  
 
4.0 Study Population 
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 6 of 13 
 
Up to 10 subjects of any gender or ethnicity will be enrolled with a target of n=10, 50% 
female for study completion. Subjects’ duration of participation is approximately 2 
weeks. 
 
4.1 Inclusion Criteria 
1. Aged 21-40 years. 
2. Body weight of ≤ 80kg (176 pounds). 
 
4.2 Exclusion Criteria 
1. History Chronic kidney disease. 
2. History of dairy allergy. 
3. History of lactose intolerance. 
4. History of whey allergy. 
5. History of gastric reduction/bypass surgery. 
6. Pregnant females. 
 
 
4.3 Subject Recruitment and Consent 
Study staff will recruit subjects via word of mouth to friends, family and colleagues. 
Those who are interested will schedule Visit 1 with study staff, where the informed 
consent discussion and determination of eligibility will take place. Those who are eligible 
will have Visit 2 scheduled. 
 
4.4 Subject Compensation 
Subjects will be compensated $15 for Visit 1 and $100 each for Visits 2 and 3. They will 
be mailed a check through UAMS SAP for their amount based upon visits attended. 
 
4.5 Study Visits 
 
Visit 1: This visit will take place at the UAMS RIOA.   
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 7 of 13 
 
At this visit, informed consent discussion is held. Study staff will use appropriate PPE 
with surgical mask and gloves. Subjects will also be required to wear a mask and if they 
do not have one, a mask will be provided to them by the study staff. If a subject enrolls, 
their eligibility will be determined. Eligible subjects will be randomized and be scheduled 
for Visit 2. A whole body DXA scan will be performed by study staff using the PI’s 
machine for quantification of lean mass. 
 
Visit 2: This visit will take place at the UAMS IOA. Subjects will be instructed to fast 
(consuming only water) from 10:00 P.M. the evening prior to this visit. Study nurse will 
insert an IV into the subject’s preferred arm. At scheduled intervals, study staff will draw 
a ~2mL blood sample from the IV for research purposes, warming the arm by means of 
a heating pad or a heated plastic box. Blood will be transferred into a blood collection 
tube that contains EDTA, cooled on wet ice, centrifuged at ≥ 3000 RPM for ≥ 6 minutes, 
and have the resultant plasma transferred to a labelled cryo tube for freezing at ≤ -80 
degrees Centigrade. 
After the initial blood sample is drawn, subjects will begin ingesting their oral isotope 
preparations, beginning with a priming dose (per study-specific stable isotope 
calculations). A timer will start when the priming dose is ingested. At 10-minute 
intervals, subjects will ingest a smaller volume of stable isotope preparations that will 
continue throughout the duration of the study visit (approximately 7 hours). 
 Approximately three hours after study initiations, subjects will ingest their randomly 
chosen amount of SP. Blood sample collection will continue at specific intervals 
throughout the remainder of Visit 2. 
At the conclusion of the 7-hour study visit, the IV will be removed and a bandage placed 
over the site. Subjects will be provided with snacks and a beverage and be free to leave 
the site. Visit 3 is scheduled. 
 
Visit 3: Subjects will come back to the RIOA having fasted overnight as for Visit 2. All 
study procedures undertaken during Visit 2 will be repeated at Visit 3, with subjects 
ingesting the remaining SP amount. 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 8 of 13 
 
 
4.6 Blinding 
This will be a double-blinded study. Both SP amounts are dissolved into equal volumes 
of water so that subjects will not know which amount they are ingesting. Blood samples 
are coded so that lab staff and investigators will not know which amount of SP was 
consumed at each visit. Investigators will be unblinded after all samples have been 
processed and data analysis has taken place. 
 
4.7 Randomization 
SP grouping will be predetermined before the first enrollment takes place. Study staff 
will document enrollment information into a spreadsheet that contains the SP group 
designations. SP labels will contain these designations. In the event that subject 
dropouts or screening failures skew the balance of groups, the groups will be modified 
toward the end of the enrollment period so that balance will be regained at the 
conclusion of the study. 
 
4.8 Study Products 
SP arrives packaged in a small tub. It is stored at room temperature. Study staff will 
weigh the doses of SP: 6.3g and 12.6g. Prior to ingestion, SP is dissolved into 
approximately 120mL of cool water. This product has been approved and used in 
previously approved studies (IRB#217658). It is provided at no cost by Adesso, LLC 
(Delaware). 
 
5.0 Data and Specimen Collection and Management 
 
5.1 Blood Sample collection 
Study staff will collect approximately 2mL of whole venous blood at each occasion and 
place it into an EDTA tube. A total of 15 blood samples per study visit will be drawn for 
this study. Approximately 60 mL of blood will be drawn per subject if they complete both 
visits 2 and 3. 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 9 of 13 
 
 
5.2 Sample Storage 
Blood 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 PI’s 
laboratory, located in a restricted area inside the UAMS RIOA building. Said freezers 
are monitored continuously for proper temperature and working condition. Samples will 
be destroyed only after all data has been analyzed. All blood samples are coded using a 
unique identifier. None of a subject’s personal identifiers are present on any biological 
sample. 
 
5.3 Sample Analysis 
Plasma samples are analyzed for amino acid concentrations and stable isotope levels 
(enrichment). The analyses are performed in the CTRAL by study staff. 
 
5.4 Data Analysis & Statistics 
Separate independent sample t-tests will be used to evaluate the difference in average 
whole-body protein metabolism measured using stable isotope infusion versus oral sip-
feeding method for both the low- and high-dose supplement. To assess validity of the 
oral method compared to the infusion method, total error (TE), standard error of the 
estimate (SEE), and Pearson’s correlation coefficient, in addition to mean difference 
and 95% limits of agreement (LOA), will also be evaluated for PS, PB, and NB. Data 
using the infusion method was collected on a previous cohort of participants of similar 
demographics (n=8; 60% female; Age=21.4 ± 0.5 years; Weight=73.8 ± 4.8 kg; percent 
body fat=21.1 ± 2.2%) using the same supplement product and dosing (4). 
 
5.5 Dissemination of Data 
Results of this study may be used for presentations, posters, grant applications, or 
publications. The publications will not contain any identifiable information that could be 
linked to a subject. 
 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 10 of 13 
 
5.6 Data Handling and Recordkeeping 
Source documents and paper CRFs will be stored in a secure area of the PI’s 
laboratory. Access will be limited to study personnel. When eventually destroyed, paper 
documents will be shredded per UAMS disposal guidelines. At no time shall Protected 
Health Information be released to non-study personnel. Coded data (results from blood 
sample analyses) will be stored in an Excel file kept on the UAMS/RSCOA server 
(requires secure access). These data files do not contain PHI or identifiers; the code to 
subjects is also kept in an Excel file on the UAMS/RSCOA server. These files will be 
kept indefinitely. 
 
5.7 Data Access 
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 seven 
years after data analysis is completed. 
 
6.0 Risks and Benefits 
 
There are no guaranteed benefits for the subjects. Anticipated risks associated with this 
protocol are described in detail below. All experimental procedures will be performed by 
appropriately trained and credentialed personnel. The PI and/or study physician will be 
responsible for oversight of study procedures and evaluation of adverse events. 
 
6.1 COVID-19 Protections 
Subjects will undergo the current screening procedures in place for UAMS patients at 
the time of their visits. Visitors will not be permitted unless UAMS policy changes permit 
them. Subjects will be required to wear a suitable facemask for the entirety of their study 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 11 of 13 
 
visits except when ingesting their isotopes/SP. Study staff will wear their mandated 
masks and use universal blood-borne precautions while handing blood samples. 
Study visit areas are disinfected after study visits as per protocol. 
 
6.2 Blood sampling/IV insertion 
Blood samples will be collected solely for the purpose of experimentation. The total 
amount of blood taken will be up to 60 mL. Subjects should have no noticeable effects of 
this volume of sampling. Brief discomfort due to the IV insertion is common. There is also 
a risk of possible bruising, redness, and swelling at the IV site. There is also a possibility 
of feeling lightheaded when the IV is inserted or, less commonly, fainting. Finally, there is 
the rare risk of infection due to the IV. The subject’s heated arm will be monitored for 
signs of skin injury. Subject’s arm will be inside a pillowcase when using the hot plastic 
box method, with its thermostat set no higher than 55 degrees Centigrade. Heating pad 
will be used on the highest setting available. 
 
6.3 Confidentiality 
A potential risk to study participants is the loss of confidentiality.  Measures to protect 
the confidentiality of study participants will be implemented as described in the Data 
Handling and Recordkeeping section above. 
 
6.4 Study Products 
The SP contains whey protein and may contain a small amount of lactose. Subjects with 
a known allergy or intolerance to whey protein, milk or lactose should not participate. In 
subjects with these allergies/intolerances, side or adverse effects of SP would include: 
abdominal pain, bloating, rash or hives, red and watery eyes, a runny nose, sneezing 
and coughing, diarrhea, nausea, and intestinal cramps. 
 
The amino acids are naturally occurring required nutrients with no known adverse 
effects, even at much higher doses than will be used in this protocol. They are classified 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 12 of 13 
 
as GRAS by the FDA. Study staff are responsible for accountability and dispensing of 
SP. 
 
6.5 Adverse Events 
• Study staff will ask subjects about the occurrence of adverse events during visits 
2, 3, and the follow-up calls after these visits. 
• Possible side effects of the SP listed above will be documented as an adverse 
event. Any allergic reaction to the SP will be treated as an adverse event. 
• The PI and study physician are responsible for reviewing and evaluating adverse 
events. 
• Adverse events will be recorded in the CRF and on an excel spreadsheet that is 
submitted to the IRB at required intervals. 
• Serious adverse events will be reported to the IRB within 24 hours of their 
discovery. 
 
6.6 DXA Scan 
A whole body DXA scan will be performed one time per subject. The radiation exposure 
for a single scan is approximately half that of a chest x-ray. 
 
7.0 Ethical Considerations 
 
This study will be conducted in accordance with all applicable government regulations 
and University of Arkansas for Medical Sciences (UAMS) 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 

Whole Body Protein Metabolism of a Whey/EAA Supplement Using Continuous Oral Stable Isotopes 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
260813 protocol v.6 21Jul21 
Page 13 of 13 
 
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. 
 
8.0 References 
 
1. 
Kim, I.Y., Suh, S.H., Lee, I.K., and Wolfe, R.R. 2016. Applications of stable, 
nonradioactive isotope tracers in in vivo human metabolic research. Exp Mol Med 
48:e203. 
2. 
Schoenheimer, R., Ratner, S., and Rittenberg, D. 1982. Studies in Protein 
Metabolism. Nutr Rev 40:23-26. 
3. 
Waterlow, J.C., Garlick, P.J., and Millward, D.J. 1978. Protein turnover in 
mammalian tissues and in the whole body. Amsterdam ; New York New York: 
North-Holland Pub. Co.; sole distributors for the U.S.A. and Canada, Elsevier 
North-Holland. viii, 804 p. pp. 
4. 
Park, S., Church, D.D., Azhar, G., Schutzler, S.E., Ferrando, A.A., and Wolfe, 
R.R. 2020. Anabolic response to essential amino acid plus whey protein 
composition is greater than whey protein alone in young healthy adults. J Int Soc 
Sports Nutr 17:9.