Maximizing the Dietary Pattern of Older Adults: the Effects of Protein Intake on Protein Kinetics
Phase: NA
Conditions: Sarcopenia
Sponsor: University of Arkansas

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 1 of 23 
 
University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:  
Maximizing the dietary pattern of older adults: the 
effects of protein intake on protein kinetics. 
 
Principal Investigator: 
Arny A. Ferrando, PhD, Professor 
Center for Translational Research in Aging & Longevity 
University of Arkansas for Medical Sciences 
4301 West Markham Street, Slot 806 
Little Rock, AR 72205 
Phone: 501-526-5711 
Email: aferrando@uams.edu 
 
Sub-Investigators:  
Gohar Azhar, M.D. 
 
 
 
 
David D. Church, Ph.D. 
 
 
 
 
Katie R. Hirsch, Ph.D. 
University of Arkansas for Medical Sciences 
4301 West Markham Street 
Little Rock, AR 72205 
 
Clinical Research Staff: Taylor Gregory, PA-C 
 
 
 
 
 
Scott Schutzler, R.N. 
 
 
 
 
 
 
Study location: 
 
UAMS Reynolds Institute on Aging 
 
 
 
 
629 Jack Stephens Drive 
 
 
 
 
Little Rock, AR 72205 
 
Funding Support:  
Foundation for Meat and Poultry Research and Education 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 2 of 23 
 
Table of Contents 
List of Abbreviations ................................................................................................................. 4 
1.0 Protocol Summary ................................................................................................................. 5 
2.0 Background ............................................................................................................................ 6 
2.1 Rationale and Significance ........................................................................................... 6 
3.0 Hypothesis .............................................................................................................................. 9 
4.0 Study Population .................................................................................................................... 9 
4.1 Inclusion Criteria ............................................................................................................. 9 
4.2 Exclusion Criteria ............................................................................................................ 9 
4.3 Subject Recruitment ..................................................................................................... 10 
4.4 Subject Compensation................................................................................................. 10 
4.5 Run-In Meals ................................................................................................................... 11 
5.0 Study Visits ........................................................................................................................... 11 
6.0 Blinding .................................................................................................................................. 13 
7.0 Randomization ..................................................................................................................... 13 
8.0 Biological Samples .............................................................................................................. 13 
8.1 Blood................................................................................................................................. 14 
8.2 Urine .................................................................................................................................. 14 
8.3 Saliva ................................................................................................................................ 14 
8.4 Muscle .............................................................................................................................. 15 
9.0 Sample and Data Processing ............................................................................................ 15 
9.1 Blood................................................................................................................................. 15 
9.2 Urine .................................................................................................................................. 15 
9.3 Saliva ................................................................................................................................ 16 
9.4 Muscle .............................................................................................................................. 16 
9.5 Dual Energy-Xray Absorptiometry ............................................................................ 16 
9.6 Multi-frequency Bioelectrical Impedance Analysis .............................................. 16 
10.0 Statistical Analysis – Data Use ....................................................................................... 17 
10.1 Statistical Analysis ..................................................................................................... 17 
10.2 Dissemination of Data ................................................................................................ 17 
10.3 Data Handling and Recordkeeping......................................................................... 17 
10.4 Data Access .................................................................................................................. 18 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 3 of 23 
 
10.5 Power Calculations ..................................................................................................... 18 
11.0 Risks and Benefits ............................................................................................................ 18 
11.1 Stable Isotope Ingestion ........................................................................................... 19 
11.2 Blood Sampling ........................................................................................................... 19 
11.3 Percutaneous Vastus Lateralis Muscle Biopsy .................................................. 19 
11.3 Study Meals .................................................................................................................. 19 
11.4 DXA Scan....................................................................................................................... 19 
11.5 Confidentiality .............................................................................................................. 19 
11.6 Data Safety Monitoring Plan..................................................................................... 20 
12.0 Ethical Considerations ...................................................................................................... 20 
13.0 References ......................................................................................................................... 21 
 
 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 4 of 23 
 
List of Abbreviations 
 
3MH 
3-methyl-d3-L-histidine stable isotope 
AMDR 
acceptable macronutrient distribution range 
ANOVA 
analysis of variance 
AUC 
area under the curve 
BMI 
Body mass index 
CRF 
Case Report Form 
D2O 
Deuterated water 
DXA 
Dual-energy X-ray Absorptiometry 
EAA 
Essential Amino Acid 
GC-MS 
Gas chromatograph-mass spectrometry 
IRB 
Institutional Review Board 
MPS 
Muscle protein synthesis 
NB 
Net balance (of protein metabolism) 
NHANES 
National Health and Nutrition Examination Survey 
OPI 
Optimal Protein Intake 
PS 
Whole-body protein synthesis 
PB 
Whole-body protein breakdown 
RDA 
Recommended Daily Allowance 
RIOA 
Reynolds Institute on Aging 
UAMS 
University of Arkansas for Medical Sciences 
 
 
Study Schema 
 
Recruiting will be performed via word of mouth, TRI subject database (ARresearch.org), 
and the UAMS social media platforms. 
 
Visit 1: informed consent process takes place at the RIOA. Enrolled subjects undergo 
body composition test and DXA scan (or at Visit 2) using PI’s equipment. 
Randomization (1:1:1) occurs. 
 
Visit 2: subjects undergo body composition test and DXA scan if not done at Visit 1. 
Run-in meals are dispensed. 
 
Visit 3: Subjects report fasted to the RIOA to begin a 13-hour visit that entails stable 
isotope ingestion, blood/urine/saliva sampling and study meal ingestion. 
 
Visit 4: Subjects report fasted to the RIOA the morning after visit 3 to undergo blood, 
saliva, urine and muscle sampling approximately 24 hours after isotope ingestion. 
 
 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 5 of 23 
 
Study Calendar 
1Or at Visit 2. 
 
1.0 Protocol Summary 
 
This will be a randomized study to demonstrate how animal-based protein-rich food 
sources can be used by older adults to increase protein intake within pre-existing 
dietary patterns. In this regard, we propose to augment the current dietary pattern of 
older Americans by readily available quality protein sources. To this end, we will 
investigate the effects of recommended and common protein intakes on the 
maintenance of whole-body protein balance and potential for muscle protein anabolism. 
Plans to accomplish project goal(s): We will study 3 groups of 10 older subjects during 
daily protein intakes of 0.8 (recommended dietary allowance [RDA]), 1.1 (National 
Health and Nutrition Examination Survey [NHANES]), and 1.5 (optimal [OPI]) g/kg/d. 
Due to common eating patterns in older adults (1), these protein intakes will be 
structured within 2 meals per day. A 2-day dietary lead-in (matching the subject’s 
intervention paradigm) will proceed each dietary intervention. Whole-body protein 
balance will be determined during metabolic study days by oral ingestion of 2 grams of 
the stable isotope 15N-alanine, and a 24-hr integrated value of muscle protein synthesis 
will be determined by ingestion of deuterated water. Oral ingestion of 10mg of 3-Methyl-
d3-L-histidine and analysis of urine samples will permit measurement of muscle protein 
breakdown during this period. Integrated post-meal blood EAA will also be determined, 
as we have demonstrated a relationship between peripheral EAA response and protein 
anabolism. 
Study duration per subject will be about 2 weeks. 
 
Procedure 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Informed consent 
X 
 
 
 
DXA scan 
X1 
X 
 
 
Body composition testing 
X1 
X 
 
 
Randomization 
X 
 
 
 
Pick up run-in meals 
 
X 
 
 
24-hour stable isotope 
procedure 
 
 
Start 
Finish 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 6 of 23 
 
2.0 Background 
 
2.1 Rationale and Significance 
Sarcopenia is the age-related loss of skeletal muscle mass and function. Sarcopenia is 
a complex multifactorial syndrome associated with increased risk of morbidity, mortality, 
and healthcare cost. Despite being associated with advanced age groups, the loss of 
skeletal muscle mass and function begin in the third and fourth decades of life, 
respectively (2). Thus, treatments that prevent age-related losses of skeletal muscle 
mass and function are essential for all aging populations. 
Protein turnover is an important regulatory process of muscle growth and function and is 
influenced by daily protein intake in order to supply precursor amino acids to activate 
protein synthesis and attenuate protein breakdown. The current RDA for protein for 
adults is 0.8 g/kg/d, which is the minimum protein intake necessary to avoid malnutrition 
(3). However, the acceptable macronutrient distribution range (AMDR) was developed 
to express dietary recommendation in the context of a complete diet. For protein the 
AMDR is 10 – 35% of total calories. Assuming a 2000 Kcal healthy US-style eating 
pattern, the AMDR provides 1.05 – 3.67 g/kg/d when reference body weights of 57 and 
70 kg are assumed for women and men, respectively (4). In this regard, even the lower 
end of the AMDR intake is higher than the RDA. Moreover, many nutritional 
organizations (3,5) have recommended intakes above the RDA (1.2 - 2 g/kg/d) for older 
adults. Despite this level of support, an increase in the RDA for the general population, 
or for older adults, has not materialized. 
Increasing the RDA would represent a physiologically accurate acknowledgment of an 
increased protein requirement with aging. The adequacy of protein intake is made 
further difficult as older adults tend to consume lower food/caloric intakes as they age. 
This age associated decrease in food/caloric intake has been attributed to reduced 
appetites, changes in chemo-sensory abilities, and deteriorations in dentition, manual 
dexterity, and gastro-intestinal function (6). Unfortunately, studies suggest that the 
consumption of animal-based protein-rich foods is impacted to a greater degree (6). 
This is of concern as animal-based protein-rich foods not only provide a better protein to 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 7 of 23 
 
calorie ratio, requiring less total food intake to meet protein requirements, but also 
provide unique nutritional profiles that cannot be obtained through supplementation or 
substitution (7). Furthermore, we have demonstrated (Figure 1) that complete protein 
ingestion leads to a greater increase in peripheral EAA, and further, that this increase is 
directly related to the increase in muscle protein synthesis and whole-body net protein 
balance. It is apparent that the use of animal-based protein-rich food sources are 
imperative for older adults to meet their elevated protein requirements. Thus, it is crucial 
to identify nutritional strategies that require minimal changes in eating behaviors of older 
adults yet improve the potential for protein homeostasis. 
Older adults have decreased motor abilities, which impacts their ability to cook and 
prepare their own food. Animal-based protein-rich food sources often require more time 
and effort to prepare than plant sources. This aspect contributes to inadequate protein 
intakes as plant sources have a lower protein to calorie ratio. Thus, an important 
strategy to increase daily protein intake is to improve the convenience and minimize the 
effort required to prepare animal-based protein-rich food sources. This can be readily 
accomplished by using minimally and further processed meat and poultry products 
within older adults’ diet. The reduced effort and time required to incorporate these 
products into a pre-existing dietary plan would improve the potential of successful 
implementation. 
One common approach to increase dietary protein in older adults has been to define an 
“optimal” within-day protein intake distribution. Based upon skeletal muscle responses 
to various doses of protein intake, it was concluded that an even distribution of protein 
intake, as opposed to an uneven distribution, was preferable (8). The primary issue here 
is that saturable protein dose limits are not well-defined, and arguably non-existent. 
Research consistently supports the concept that muscle protein synthesis is maxed out 
with around 10-15 grams of EAA when supplied in liquid format following resistance 
exercise (9–13). However, we have observed (Figure 1) that greater protein intake is 
required when provided in whole-food format as opposed to liquid drinks. 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 8 of 23 
 
 
Figure 1: A greater digestibility requirement (whole-food protein sources) results in a 
lower stimulation of muscle protein synthesis. These data also demonstrate that greater 
peripheral EAA are required to increase muscle protein synthesis. 
 
Basing protein distribution optimization upon saturable protein dose studies reflects the 
“muscle-full” effect theory, which recently was shown to be an artifact of the rapid and 
transient rise in circulating amino acids rather than a true physiological effect (14). 
Furthermore, protein recommendations are based upon whole-body protein 
requirements, not muscle requirements. This is an important consideration as we (15) 
and others (16) have demonstrated that whole-body protein balance does not plateau 
with protein intake; i.e., greater improvements in whole-body net protein balance are 
observed with increasing doses of protein. 
The above highlight an incorrect underlying assumption of evenly distributing protein 
intake throughout the day. More pertinent to the issue is that studies directly testing 
protein distribution do not provide strong evidence for an even protein distribution being 
superior to an uneven protein distribution. Despite some promising data (17) in younger 
adults (36.9 ± 3.1 years of age), our recent studies (18,19) in older adults (50 – 75 years 
of age) demonstrated no superior effect of an even protein distribution on whole-body 
protein balance or muscle protein synthesis over a 24-hour period. More importantly, 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 9 of 23 
 
our work indicated that total protein intake throughout the day has a direct influence on 
anabolism (18–21). The take home message from these studies is that total protein 
intake, not intake patterns, dictates protein homeostasis. This is an important message, 
as NHANES data indicates that only 17.3% of adults eat more than two meals a day 
containing ≥30 grams of protein per meal (1).  Thus, the issue becomes one of 
increasing protein intake in older adults within their pre-existing dietary patterns. 
Incorporating minimally and further processed meat and poultry products into current 
dietary patterns provides an easily accessible strategy for older adults to meet higher 
protein requirements required to maintain protein mass. Therefore, demonstrating the 
efficacy of these products to meet body protein demands within a preexisting dietary 
pattern of two meals a day is the focus of this project. 
 
3.0 Hypothesis 
 
We hypothesize a stepwise increase, RDA<NHANES<OPI, in muscle protein synthesis 
and whole-body protein balance with increasing doses of daily protein intake. 
 
4.0 Study Population 
 
Up to 50 subjects of any gender or ethnicity will be enrolled with a target of n=30 for 
study completion. Subjects’ duration of participation is approximately 2 weeks. 
 
4.1 Inclusion Criteria 
1. Men and post-menopausal women. 
2. Ages 50-70 years. 
3. BMI ≤ 35 kg/m2. 
4. Capable of providing informed consent. 
5. COVID-19 negative and/or asymptomatic. 
 
4.2 Exclusion Criteria 
1. Subject who does not/will not eat animal protein sources. 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 10 of 23 
 
2. Body mass index >35. 
3. Complete blood count lab results that indicate anemia or abnormal white blood 
cell counts. 
4. History of chemotherapy or radiation therapy for cancer in the 6 months prior to 
enrollment. 
5. Use of insulin to control blood sugar level. 
6. Currently receiving androgen (e.g., testosterone) or anabolic (e.g., GH, IGF-I) 
therapy. 
7. Currently using prescription blood thinning medications. 
8. Unable or unwilling to suspend aspirin use for 7 days prior to Visit 3. 
9. Unwilling to avoid using protein or amino-acid supplements during participation. 
10. Subjects who are unwilling to fast overnight. 
 
 
4.3 Subject Recruitment 
Recruiting will be performed via word of mouth and the UAMS social media platforms. 
Study staff will also utilize the ARresearch.org database through the UAMS TRI. 
Interested subjects will then be scheduled for Visit 1 where the informed consent 
process will take place. 
 
4.4 Subject Compensation 
Subjects will be compensated for participation as shown in the table below. They will be 
mailed a check to the address they provide through UAMS SAP program. Parking 
passes will be provided to all subjects for all visits at no cost to subjects. 
 
Visit 1 
$20 
Visit 2 
$20 
Visit 3 
$360 
Visit 4 
$120 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 11 of 23 
 
4.5 Run-In Meals 
Subjects will be provided with 3-days’ worth of ready-to-eat meals designed and 
prepared by the study dietician. Subjects will be instructed to eat only the food that is 
labelled for each particular meal over the run-in days. They may add non-caloric 
condiments and seasonings and have their usual beverages. Caloric requirements are 
determined by the Harris-Benedict formula based on subject’s weight and sex and are 
matched to the subject’s intervention group (e.g. 0.8 or 1 or 1.5g protein/kg per day). 
Each meal’s containers are placed into a labelled plastic zippered storage bag, with the 
meals for one day placed into a larger labelled bag. Subjects are asked to store them in 
a refrigerator. 
 
5.0 Study Visits 
 
Subjects will be required to wear a mask. If they do not have one, a mask will be 
provided to them by the study staff.  
 
Visit 1: This visit will take place at the UAMS RIOA. At this visit, informed consent 
discussion will be held. This visit is expected to take less than one hour. If a subject 
consents, their height and weight will be measured to determine their BMI. Questions 
will be asked about their medical history and medication use. Eligible, enrolled subjects 
will be shown a menu of the foods they will be asked to eat to elicit any food allergies or 
intolerances. If the subject is fasted for 8 hours, has not had caffeine in 12 hours, and 
has not strenuously exercised in 24 hours, they will undergo body composition testing 
and DXA scan. If these criteria cannot be met during this visit, subjects will perform 
these two tests at Visit 2. A blood sample will be drawn for Complete Blood Count 
(Labcorp). Randomization will occur. Future study visits are scheduled. 
 
Visit 2: This visit will occur in a fasted state if the subject did not meet the body 
composition testing criteria at visit 1. Subjects will undergo body composition testing 
and DXA scan. Study meals eaten during the 2-day run-in phase will be dispensed. A 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 12 of 23 
 
10mg dose of 3MH will be provided with instructions to mix it with water, ingest it at 
1900, and record the time on the evening prior to study visit 3. Subjects are instructed to 
eat all of the provided food and add nothing but non-caloric seasonings and 
condiments. They may have their usual beverages but must not ingest any protein or 
amino acid supplements during this time. This visit will take place at the UAMS RIOA. 
 
Visit 3: This visit will take place at the UAMS RIOA on the morning after subject 
completes the 2-day run-in meals. At this visit, subjects will report having fasted for 10 
or more hours. This visit is expected to take about 13 hours. During this visit, subjects 
will be asked to provide blood/urine/saliva samples. They will ingest deuterated water 
(~3mL per kg body weight) at specific times (total dose divided into 10 aliquots and 
ingested every hour up to study hour 9:00) and 2g of a stable isotope of alanine (15N-
alanine) dissolved in water. Two (group-specific) study meals will be served, one in the 
morning and one in the afternoon/evening. See Figure 2 for a timeline. After the second 
saliva sample has been collected, subjects will go home and return to the study site the 
following morning having fasted overnight. 
 
Visit 4: This visit will take place at the UAMS RIOA on the morning after visit 3. Subjects 
will fast overnight between visits 3 and 4. Approximately 24-hours after isotope 
ingestion, the final biological samples will be collected and the subject offered a snack 
and beverage. They will then be dismissed.  
 
Figure 2: 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 13 of 23 
 
 
6.0 Blinding 
 
The study is a single-blind, randomized, controlled trial (RCT). While participants cannot 
be completely blinded as to the amount of protein in their meals, key investigators and 
outcomes assessors will be blinded to treatment assignment, making this a single-blind 
RCT from the investigators’ perspective. 
 
7.0 Randomization 
 
Subjects will be randomized equally to one of three groups after they are determined to 
be eligible using a generated list of group designations that corresponds to subject ID. 
Efforts will be made to balance the sexes within each group. The three groups utilized 
are:  
1) recommended dietary allowance for protein (RDA; 0.8g/kg/day); 
2) habitual protein intake consistent with population level norms (NHANES; 
1.1g/kg/day); 
3) optimal protein intake (OPI; 1.5g/kg/day). 
 
8.0 Biological Samples 
Samples will be handled exclusively by study staff. All samples will be identified using 
the subject’s unique study code; the key to which is kept on a secure UAMS server 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 14 of 23 
 
(\\rscoa\nmel\AAFerrando\262424 2Meal study 2021 folder). All specimens will be 
stored in appropriate freezers in the PI’s lab on the 7th floor of the UAMS Reynolds 
Institute on Aging (a secured access floor). All specimens will be discard into biohazard 
receptacles within 7 years after conclusion of the study. With explicit written permission 
(on the ICF), muscle samples will be retained indefinitely for use in the PI’s lab for future 
non-commercial uses that have been explicitly permitted by the UAMS IRB. The key to 
subject identity-study code will be deleted within 7 years of the conclusion of the study 
(e.g. closure of the study in Clara), regardless of whether any samples will be kept for 
future use. 
8.1 Blood 
Blood samples (~5mL each) will be collected via venipuncture at the specific times 
mentioned in Figure 2 below (+/- 10 minutes). The blood will be placed into an EDTA 
(purple top) vacutainer tube, gently agitated 6 times, and placed onto wet ice until it is 
processed in a refrigerated centrifuge. Plasma will be pipetted into a labelled cryo vial 
for each specific time point and frozen at < -60 degrees C in the PI’s lab. Two samples 
will be drawn (~10 mL) during Visits 3-4 (1 each). 
 
8.2 Urine 
During Visit 3 through 4, all voided urine will be collected using standard plastic urine 
jugs. During visit 3, at each void 1mL of urine will be placed into a labelled cryo tube 
and the time of voiding will be documented on a table in the CRF. At the completion of 
Visit 4, all urine will be intermixed and aliquots placed into labelled cryo tubes and 
frozen at < -60 degrees C in the PI’s lab. 
 
8.3 Saliva 
During Visit 3-4, three saliva samples will be collected at specific times (Figure 2) using 
absorbent collection devices (e.g. Salivette). They will be centrifuged and the saliva 
transferred into labelled cryo tubes and frozen at < -60 degrees C in the PI’s lab. 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 15 of 23 
 
8.4 Muscle 
At Visit 4, qualified and credentialed study staff (M.D. or PA) will perform a vastus 
lateralis muscle biopsy of the subject’s outer thigh using 1% plain lidocaine for 
anesthesia. Approximately 100mg of muscle tissue will be obtained using a Bergstrom 
needle. The tissue will be rinsed with normal saline and flash frozen using liquid 
nitrogen. The sample will be transferred into a labelled cryo tube and frozen at < -60 
degrees C in the PI’s lab. 
 
9.0 Sample and Data Processing 
 
All samples will be processed and analyzed in the PI’s lab in the RIOA. 
 
9.1 Blood 
Plasma EAA concentrations will be determined by liquid chromatography-mass 
spectrometry using the internal standard method, as we have done previously (30). 
Briefly, analytes will be derivatized with 9-fluorenylmethoxycarbonyl and quantification 
of each peak will be determined using MultiQuant software (version 2.1: AB Sciex). 
 
9.2 Urine 
Following ingestion of [15N] alanine, 24-hr dietary nitrogen intake and excretion will be 
measured. Isotopically labeled nitrogen from the urine samples will be used to 
determine nitrogen flux according to Fern et al. (24). Whole-body protein synthesis (PS) 
and breakdown (PB) are calculated from 24-hr urinary urea enrichment according to 
Stein et al. (25) and used to determine net protein balance and flux. The ingestion of 
3MH will increase the naturally-occurring 3MH in the body by less than 5%. This 
molecule is not reutilized in the body, but only excreted via the urine. Measurement of it 
in urine will allow determination of muscle protein breakdown. 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 16 of 23 
 
9.3 Saliva 
Body water enrichments following D2O ingestion will be determined through isotope 
exchange with acetone using GC-MS (29). 
 
9.4 Muscle 
MPS will be determined by oral D2O ingestion to assess 24-hr muscle protein synthesis 
using the single biopsy approach (26). In the interest of brevity, the sample preparation 
methods are standard in the PI’s laboratory and have recently been outlined in detail 
(27). Muscle preparations will be analyzed for incorporation of deuterated alanine by 
GC-MS using partial integration (28). The daily rate of muscle protein synthesis will be 
determined according to Gasier et al. (27). 
 
9.5 Dual Energy-Xray Absorptiometry 
This test will be used to measure body composition (fat mass, lean mass, and percent 
body fat). Subjects will be asked to remove all metal, thick clothing, and heavy plastic 
which could interfere with the DXA scans. The subject’s ID, age, ethnicity, height and 
weight will be entered into the computer prior to the scanning. The subject will be asked 
to lie down on the DXA table in the supine position. The participant will be centered on 
the table within the scanning area. The subject's shoulders and hips will be centered, 
and the hands will be placed by the side of the legs in a pronated position. Subjects will 
be instructed to remain still for the duration of the full-body scan. A trained DXA 
technician will perform and analyze all scans. Subjects will be asked to fast for 8 hours, 
avoid caffeine and alcohol for 12 hours, and avoid strenuous exercise for 24 hours prior 
to this procedure. 
 
9.6 Multi-frequency Bioelectrical Impedance Analysis 
This test will measure total body and segmental water, to be combined with the DXA 
measures for a more accurate body composition estimates. To obtain measurements, 
subjects are asked to stand barefoot on a calibrated scale to obtain subject weight. 
Once subject height, sex, and age are entered into the device, subjects are instructed to 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
Page 17 of 23 
 
hold onto two handles and remain still while an imperceptible electrical current runs 
between electrodes in the handles and scale. Total assessment time is <2 minutes. 
Subjects will be asked to fast for 8 hours, avoid caffeine and alcohol for 12 hours, and 
avoid strenuous exercise for 24 hours prior to this procedure. 
 
10.0 Statistical Analysis – Data Use 
 
10.1 Statistical Analysis 
Means by which experimental data will be analyzed or interpreted: Changes in whole-
body protein synthesis, breakdown, flux, net balance, muscle protein synthesis, and 
plasma EAA area under the curve (AUC) between groups will be assessed with a one-
way analysis of variance (ANOVA). In the event of a main effect of group, appropriate 
post-hoc comparisons will be carried out. Statistical significance will be accepted at an 
alpha level of p ≤ 0.05. 
 
10.2 Dissemination of Data 
The results of this project will demonstrate that daily protein intake from minimally and 
further processed food sources will serve as a pragmatic and efficient means for 
improving body protein status. 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 subject. 
 
10.3 Data Handling and Recordkeeping 
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. Paper source 
documents and Case Report Forms (CRFs) will be stored in a secure area of the PI’s 
laboratory. Access will be limited to study personnel. Documents will be archived 
according to UAMS policies regarding destruction of research records. Data (without 
identifiers) will be entered into an electronic spreadsheet or database (e.g. REDCap) to 
facilitate analyses. These files are maintained on secure password-protected UAMS 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
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Institution: UAMS 
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servers. Approximately 7 years after study completion, paper and electronic records will 
be destroyed per UAMS disposal guidelines. At no time shall Protected Health 
Information be released to non-study personnel. 
 
10.4 Data Access 
All subjects 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.  Only study 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. 
 
10.5 Power Calculations 
We propose a group size of 10 per group, based upon an ANOVA model to compare 
the nutrition interventions. With this sample size, the ANOVA model has 80% power to 
detect effect sizes of f = 0.484 or larger. This estimate assumes that the basal covariate 
explains 50% of the variation in the response and a 5% α-level is used to determine 
statistical significance. 
 
11.0 Risks and Benefits 
 
There will be no direct benefits to the study participants; however, knowledge gained 
from the study could potentially benefit patients in the future.  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 study 
physician will be responsible for oversight of study procedures and evaluation of 
adverse events. There are no known risks of body composition testing (InBody770, 
BioSpace, Seoul, South Korea), urine or saliva collection. 
 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
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11.1 Stable Isotope Ingestion 
The risks of drinking 70% deuterated water are temporary dizziness, nausea and 
possibly vomiting. Each dose is approximately 20-40 mL (1 ½ - 2 tablespoons). There 
are no known risks of ingestion of the stable isotopes of alanine or methyl histidine. 
 
11.2 Blood Sampling 
Up to 10 mL of venous blood will be drawn during visits 3 and 4 in total. This is less than 
a tablespoon. The risks of drawing blood include pain, bruising, bleeding, and small 
chance of infection from the needle sticks. One ~5mL sample will be drawn during visit 1 
to determine eligibility. 
 
11.3 Percutaneous Vastus Lateralis Muscle Biopsy 
The risks of this procedure include pain, bruising, bleeding, scar formation, and infection. 
This procedure will be done using aseptic technique by licensed study staff (MD or PA) 
after injection of local anesthesia (1% plain lidocaine). Approximately 100mg of tissue will 
be removed from the vastus lateralis muscle (subject’s choice of left or right leg). 
 
11.3 Study Meals 
The menu for study meals will be presented to subjects during Visit 1 to elicit whether 
any food allergies or intolerances are present. 
 
11.4 DXA Scan  
Subjects will undergo a DXA scan for whole-body composition. The radiation exposure 
for one DXA scan is approximately equal to ½ of the radiation from a chest x-ray. 
 
11.5 Confidentiality 
A potential risk to study subjects is the loss of confidentiality.  Measures to protect the 
confidentiality of study subjects will be implemented as described in the Data Handling 
and Recordkeeping section above. 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
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11.6 Data Safety Monitoring Plan 
• Study staff will ask subjects about the occurrence of adverse events during visits. 
• 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. 
 
 
12.0 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 
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 

Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
262424 protocol v.9 07Dec21 
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the informed consent process will be documented in each subject’s research record. 
 
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Maximizing the dietary pattern of older adults: the effects of protein intake on protein kinetics. 
PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: UAMS 
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Institution: UAMS 
Sponsor: UAMS 
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