Nutritional Biomarkers of Sarcopenia
Phase: NA
Conditions: Sarcopenia
Sponsor: University of Arkansas

Study Protocol: Nutritional Biomarkers of Sarcopenia 
NCT Number: NCT05117112 
Document Date: May 31, 2022

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
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Study Pro 
University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:   
 
Nutritional Biomarkers of Sarcopenia 
 
Principal Investigator: 
David D. Church, Ph.D. 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St., Slot #806 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
Office Telephone:  
 
 
 
 
Email: Dchurch@uams.edu 
 
Sub-Investigators:  
Donald L. Bodenner, M.D., Ph.D. 
 
 
 
 
Arny A. Ferrando, Ph.D. 
 
 
 
 
Robert R. Wolfe, 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): 
UAMS & TRI. 
 
 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
Page 3 
 
Table of Contents 
1.0 
Background and Rationale ....................................................................................................... 4 
1.1 
Introduction: ................................................................................................................................ 4 
1.2 Previous research on the topic .................................................................................................... 5 
2.0 Hypotheses ....................................................................................................................................... 7 
3.0 Study Design and Procedures ..................................................................................................... 7 
3.1 Study Visits ..................................................................................................................................... 8 
3.2 Subject Compensation ................................................................................................................ 10 
3.3 Recruitment .................................................................................................................................. 11 
3.4 Study Registration ....................................................................................................................... 11 
3.5 Sample Storage ........................................................................................................................... 11 
3.6 Retention of Samples for Future Use ....................................................................................... 11 
4.0 Study Population ........................................................................................................................... 12 
4.1 Inclusion Criteria .......................................................................................................................... 12 
4.2 Exclusion Criteria ......................................................................................................................... 12 
5.0 Risks and Benefits ........................................................................................................................ 13 
5.1 Blood sampling: ........................................................................................................................... 13 
5.2 DEXA, In Body scans:................................................................................................................. 13 
5.3 Physical and Functional Testing: .............................................................................................. 13 
5.4 Study supplement: ....................................................................................................................... 13 
5.4.1 Conflict of Interest ................................................................................................................ 13 
5.5 Stable Isotope Ingestion: ............................................................................................................ 14 
5.6 Confidentiality: .............................................................................................................................. 14 
5.7 Breath Sampling .......................................................................................................................... 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 
7.3 Sample Processing and Analyses ............................................................................................ 15 
7.4 Grouping ....................................................................................................................................... 15 
8.0 Ethical Considerations ................................................................................................................ 16 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
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9.0 Dissemination of Data .................................................................................................................. 16 
10.0 References .................................................................................................................................... 17 
 
Table of Events 
 
Procedure 
Visit 1 
Visit 2 
informed consent 
x 
 
medical history 
x 
 
list of medications 
x 
 
measure height 
x 
 
measure weight 
x 
 
DEXA scan1 
x 
x 
In Body scan1 
x 
x 
Dominant handgrip strength 
x 
 
5-rep chair stand 
x 
 
10-meter gait speed 
x 
 
SARC-F questionnaire 
x 
 
periodic blood sampling 
 
x 
frequent breath sampling 
 
x 
ingest study product 
 
x 
 
1If subject meets the requirements to perform the DEXA scan and In Body scan on visit 1, 
these tests will be performed at that time. If they do not, these tests will be performed at Visit 2. 
 
 
1.0 Background and Rationale 
 
1.1 Introduction:  
Sarcopenia increases the risk of a number of deleterious health conditions and represents a 
major financial cost to our healthcare system. Among older adults who are hospitalized, those 
with sarcopenia on admission are 5-fold more likely to incur higher hospital costs than those 
without (1). The operational definition of sarcopenia is defined as meeting the criteria for all of 
the following: low muscle strength, low muscle quantity or quality, and low physical 
performance. The diagnosis of sarcopenia requires techniques that are both expensive and 
operator-dependent. Simple measurements, such as BMI, do not necessarily identify 
sarcopenia. Importantly, current techniques can only identify sarcopenia after a 
physical/functional impairment has occurred. Skeletal muscle amino acid kinetics predict 
muscle health and functionality (2). Altered amino acid kinetics lead to decrements in muscle 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
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mass, quality, and performance. Our laboratory, as well as others, have documented that the 
muscle response to circulating essential amino acids (EAA) determines muscle amino acid 
kinetics (2). Thus, we propose to characterize skeletal muscle amino acid kinetics to an EAA 
challenge, i.e., an oral amino acid tolerance test (OATT), in order to determine the state of 
muscle health. Analogous to the oral glucose tolerance test (OGTT) used to characterize 
alterations in glucose metabolism, the proposed OATT represents a potential low-cost solution 
to classifying patients’ skeletal muscle health. The extrapolation of this work is the 
development of a simple analytical tool that would provide clinicians the ability to discern 
alterations in muscle amino acid kinetics prior to a loss of function. 
 
1.2 Previous research on the topic 
The rate of loss of muscle with aging in any given individual is variable and may be influenced 
by genetic factors, level of activity, amount of protein in the diet, total body weight, and 
occurrence of serious diseases that directly affect muscle mass and function. Nonetheless, the 
estimate from cross-sectional data that between 0.5-1.0 % of muscle mass is lost per year 
after the age of 70 years is reasonable (3). The physiological basis for loss of muscle over time 
results from an imbalance between the rates of muscle protein synthesis and breakdown. 
Consumption of dietary protein results in the absorption of amino acids and the activation of 
protein synthetic processes throughout the body, particularly muscle protein synthesis (4). 
There is a direct relationship between the amount of dietary protein intake and the net gain in 
body protein in young healthy individuals (5). Anabolic resistance refers to the circumstance in 
which dietary protein has a diminished stimulatory effect on the net production of new muscle 
protein. Critical illness (6) or the response to a major burn injury (7) provide clear-cut examples 
of anabolic resistance. In anabolic resistance associated with these pathological conditions, 
the normal linear relationship between dietary protein intake and net protein synthesis is 
capped, meaning that beyond a certain level of protein intake there is no further gain in net 
protein balance. For example, beyond a level of protein intake of 1.1 g protein/kg/day there is 
no net gain in body protein in critically ill patients (6). Once the maximal rate of protein 
synthesis is reached, there is no further gain in net protein synthesis, as the rate of protein 
breakdown is not affected by dietary protein consumption in severely catabolic patients. The 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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consequence of exceeding the amount of dietary protein intake that maximizes the net gain in 
body protein is the oxidation of the “extra” amino acids. As a result of the oxidation of a large 
percentage of dietary amino acids, urea and ammonia production increase significantly (7). 
Since this clinical population often has multi-organ failure, including impaired kidney function, 
an increase in urea and ammonia production can have adverse physiological consequences.   
Aging-related anabolic resistance is different than anabolic resistance associated with critical 
illness. Differences in the basic metabolic responses of protein metabolism in the two 
circumstances can explain the basis for the different nature of anabolic resistance. In critical 
illness, the loss of muscle protein is driven by a high rate of protein breakdown, which may be 
as much as 3 fold above normal (8). The high rate of protein breakdown floods the 
intramuscular pool with free amino acid precursors for protein synthesis, driving up the rate of 
protein synthesis. As a result, although protein synthesis is significantly less than the 
accelerated rate of breakdown in critical illness, protein synthesis is already driven close to the 
maximum rate without any intake of protein or amino acids (8). Since precursor supply is 
already high because of accelerated protein breakdown, provision of dietary protein or amino 
acids in critically ill patients has only a small effect on precursor availability and thus muscle 
protein synthesis. In healthy elderly individuals, basal rates of muscle protein synthesis and 
breakdown are not measurably different from younger individuals. As a result, protein 
synthesis can be increased by consumption of even small amounts of amino acids (9). In 
contrast, older adults are less responsive to the anabolic stimulus of low doses of amino acid 
or protein intake compared to younger individuals (10). Limited responsiveness of muscle 
protein synthesis at lower levels of intake but a similar maximal protein synthetic response 
suggests that the slope of the line relating the increase in muscle protein synthesis relative to 
increasing amounts of dietary protein consumption is less in elderly, but ultimately reaches the 
same maximum value as in younger individuals. However, behavior change, in this case 
increasing dietary protein to maximize muscle protein synthesis to blunt age-related losses of 
skeletal muscle, are difficult in elderly adults (11). Therefore, identification of individuals at risk 
for sarcopenia sooner in life are required. Considering changes in muscle protein kinetics 
precede functional outcome changes (e.g. muscle strength), assessment of postprandial 
amino acid metabolism represents a likely solution. The relationship between protein intake 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
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and muscle protein synthesis has been demonstrated to be driven by the change in plasma 
essential amino acid concentrations (2). Stated another way, the changes in plasma essential 
amino acid concentrations following the ingestion of dietary protein drive the increase in 
muscle protein synthesis. The analogous parallel to amino acid and muscle is the use of the 
oral glucose tolerance test used to classify individuals with prediabetes and type-2 diabetes. 
Therefore, demonstrating that the essential amino acid response to an oral amino acid 
challenge can differentiate between elderly and young populations is a worthwhile first step in 
developing a new clinical diagnostic test to identify sarcopenia. 
 
2.0 Hypotheses 
 
We hypothesize that plasma amino acid response will be altered in elderly sarcopenic adults 
as compared to young and elderly healthy subjects. 
We also hypothesize that the pharmacokinetics of plasma amino acids can be used to 
distinguish sarcopenic elderly from young and elderly healthy. 
 
3.0 Study Design and Procedures 
 
We will enroll up to 35 subjects with a goal to study up to 30 healthy male and females: a 
group of 10 whose age is between 18 and 30 years, a group of 10 whose age is 70-89 years 
without the presence of sarcopenia, and a group of 10 whose age is 70-89 years with 
sarcopenia. We will use a 5-hour (one hour prior and 4 hours post-consumption of an amino 
acid supplement) period to completely characterize the plasma amino acid response to 10 
grams of free-form essential amino acids of which 10% of the leucine content has been 
enriched with L-[1-13C] leucine. The principal end-point will be plasma amino acid response 
over the 4 hours following the consumption of the study product. 
Secondary endpoints include body composition and total body water measurements. Small 
shifts in fluid from the extracellular to intracellular space likely correspond with EAA 
appearance and disappearance in plasma. BIA is a non-invasive, rapid assessment that is 
sensitive to acute shifts in fluid that are associated with disease and malnutrition states, such 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
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as sarcopenia. As an exploratory analysis, we will measure acute shifts in total body, 
intracellular, and extracellular fluid using multifrequency BIA throughout visit 2. This will serve 
as preliminary data on a potential non-invasive method to evaluate muscle quality, anabolic 
resistance, and a proxy indicator of sarcopenia. 
 
3.1 Study Visits 
Visit 1: subjects will come to the UAMS TRI 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. Females of child bearing capacity will be asked 
to provide a urine sample for pregnancy testing prior to the DEXA scan. Exceptions will be 
made for women who have recently (past week) or currently are menstruating, have an 
implanted uterine device, or women who are celibate. Urine samples will be sent to LabCorp. 
Subjects will be asked to perform the following tests and measurements at visit 1: 
Standing height without shoes (in cm), 
Body weight without shoes (in kg), 
Handgrip strength testing of their dominant hand (3 reps, in kg), 
10-meter usual gait speed test, 
5-rep timed chair stand test, 
SARC-F (paper) questionnaire (older subjects only). 
 
If subject meets the requirements (fasted for at least 8 hours, caffeine-free for at least 12 
hours, and did not perform strenuous exercise during the past 24 hours) to perform the DEXA 
scan and In Body scan on visit 1, these tests will be performed at that time. If they do not, 
these tests will be performed at Visit 2. 
 
Visit 2 (approximately 1 week after visit 1): Subjects return to the UAMS TRI having fasted for 
at least 8 hours, avoided caffeine for at least 12 hours, and avoided strenuous exercise during 
the past 24 hours. They will be asked about any adverse events since last visit. The In Body 
and DEXA scans will be performed if these were not done at visit 1. Prior to the IV insertion, an 
In Body scan will be performed if not already performed on this visit. 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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The study nurse will insert an IV catheter into a vein of one of the subjects’ arms to use for 
periodic blood sampling, warming the arm by means of a heating pad or a heated plastic box. 
After an initial blood sample is obtained, a timer is started and blood samples and In Body 
scans will be obtained according to the schedule below. The study product (dissolved in ~10 
ounces of water along with the stable isotope L-[1-13C] leucine) is served directly after the third 
blood sample. Subjects are asked to consume the drink within 1 minute. 
Using a mask that covers their mouth and nose, subjects exhaled breath will be collected to 
measure the appearance of the labelled carbon in the leucine isotope in expired CO2. The 
mask will be worn for approximately 2 minutes prior to every blood sample time point after the 
initial sample. A 1-2 minute period of breath analysis will occur at the designated blood sample 
collection times. The mask can remain off between collection periods. 
At the conclusion of 5 hours elapsed time, the IV catheter will be removed and the site dressed 
with a bandage. A snack and beverage will be offered to the subject. Subject will then be free 
to leave. 
Elapsed time (min., approximate) 
Procedure 
-0 
Blood sample (~4mL into an EDTA tube) 
30 
In Body scan 
60 
Blood sample (~4mL into an EDTA tube), , 
breath analysis, In Body scan 
75 
Blood sample (~4mL into an EDTA tube),  
ingest study supplement, breath analysis, In 
Body scan 
90 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
105 
Blood sample (~4mL into an EDTA tube) 
breath analysis 
120 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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3.2 Subject Compensation 
Subjects will accrue compensation according to the below table. They will be handed a 
Walmart ® gift card of $25 at the end of Visit 1. They will be handed a UAMS check for their 
Visit 2 compensation immediately after their participation in visit 2 ceases (whether completed 
or not). If they were to fully attend every visit, their total amount would be $125. If they stop 
participating prior to the completion of study visit 2, they will receive prorated pay of $20 per 
hour. 
135 
Blood sample (~4mL into an EDTA tube) 
breath analysis 
150 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
165 
Blood sample (~4mL into an EDTA tube) 
breath analysis 
180 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
200 
Blood sample (~4mL into an EDTA tube) 
breath analysis 
210 
In Body scan 
220 
Blood sample (~4mL into an EDTA tube) 
breath analysis 
240 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
270 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
300 
Blood sample (~4mL into an EDTA tube) 
breath analysis, In Body scan 
Visit 
Amount 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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3.3 Recruitment 
Potential subjects will be recruited from the Little Rock and central Arkansas areas by use of 
flyer, social media, ARResearch.org registry, and word of mouth. Individuals interested in the 
study will be scheduled for Visit 1. 
 
3.4 Study Registration 
This study will be registered on clinicaltrials.gov website as required. 
 
3.5 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 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. All blood samples shall 
be identified using a unique study acronym. None of a subject’s personal identifiers shall be 
present on any biological sample. 
 
3.6 Retention of Samples for Future Use 
With explicit permission via the consent form, plasma samples will be kept for future approved 
research. 
• Samples will only be released to investigators within or outside of UAMS who provide 
proof of appropriate IRB review and/or approval. 
• Samples will be kept in the PI’s freezers located on the 7th floor of the UAMS Reynolds 
Institute on Aging. This floor is controlled-access via approved UAMS badges. 
• All samples are labelled with an alpha-numeric code. The key to this code is kept on a 
secure UAMS server to which access is limited to study staff. 
• No personal identifiers are on the samples. 
1 
$25 (gift card in hand) 
2 
$100 (check) 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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• Samples will be kept for up to approximately 10 years, after which they will be placed 
into biohazard trash receptacles. 
• Samples will be retained for further analyses related to metabolic biomarkers (e.g., 
metabolomics, proteomics, inflammation markers, etc.). 
• Subjects can rescind their permission to keep their samples by contacting any of the 
study staff listed in the consent form.  
 
4.0 Study Population 
 
Up to 35 subjects will be enrolled in order to meet the goal of 10 completers in each of the 
three groups. Attempts will be made to balance sexes within groups. 
 
4.1 Inclusion Criteria 
• Ages 18 – 30 and 70-89 yrs. 
 
4.2 Exclusion Criteria 
• History of diabetes 
• History of malignancy or chemo/radiation therapy in the 6 months prior to enrollment 
• History of gastrointestinal bypass/reduction surgery (Lapband, gastric sleeve, etc.) 
• Pregnant females 
• Unwilling to wear the breath-collection mask 
• Subjects who cannot refrain from using protein or amino acid supplements for 7 days prior 
to Visit 2 
• Concomitant use of oral or injectable corticosteroids 
• Concomitant use of testosterone, IGF-1, or similar anabolic agent 
• 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 
 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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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 measure plasma amino acid concentrations and possibly stable isotope enrichment, 
glucose, and insulin. The total amount of blood taken will be approximately 60 mL. Subjects 
should have no noticeable effects from this volume. 
 
5.2 DEXA, In Body scans: 
The DEXA scan exposes subjects to approximately ½ of the radiation of one chest x-ray. 
Subjects will undergo 1 DEXA scan. The In Body scans do not pose any risks. 
 
5.3 Physical and Functional Testing: 
The primary risks are fatigue and muscle soreness.  
 
5.4 Study supplement: 
The test composition is a patented mixture of 10 grams of amino acids (total of 17.8 grams of 
commercial product: XS Muscle Multiplier) dissolved into approximately 10 ounces of water. It 
is a commercially available product manufactured by Prinova™ Solutions (Prinovausa.com). 
All components of the composition have been ruled as Generally Regarded as Safe (GRAS) 
by the FDA.  
 
5.4.1 Conflict of Interest 
UAMS and Dr. Ferrando are listed on the approved patent for this particular amino acid 
supplement. There is an approved COI plan on file for Dr. Ferrando. He will not have access to 
raw data, only a summary of the compiled data. 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
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Date: 
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5.5 Stable Isotope Ingestion: 
There are no known risks of ingestion of the stable isotope of leucine. 
 
5.6 Confidentiality: 
A potential risk is the 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. 
 
5.7 Breath Sampling 
The only risk of breath sampling is potential claustrophobia and subsequent anxiety. 
 
6.0 Data Handling and Recordkeeping 
 
Source documents, paper questionnaires and consent forms, and CRFs will be stored in a 
secure area of the PI’s laboratory. Access will be limited to study personnel. Documents 
containing identifiers will be destroyed by shredding approximately 7 years after data analysis 
is completed or publication of data; whichever is longest. 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. 
The code file will contain subject initials, sex, subject ID, enrollment data, and anthropometric 
data; it will not include any identifiers. 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. 
 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
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Date: 
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7.0 Data Analysis 
 
7.1 Statistical Analysis plan 
Means by which experimental data will be analyzed or interpreted: Changes in the plasma 
amino acids area under the curve (AUC), maximal amino acid concentration, change from 
basal to maximal amino acid concentrations, rate to maximal amino acid concentration, and 
the clearance rate of amino acids from the plasma will be assessed with an one-way ANOVA 
with appropriate post hoc testing. Statistical significance will be accepted at an alpha level of p 
≤ 0.05. 
 
7.2 Sample Size Calculation & Power Analysis 
Statistical power and sample size are based on previous studies investigating differences in 
plasma amino acids in younger and older adults following the ingestion of 10 grams of free 
form essential amino acids (12). For peak EAA concentrations previous studies have 
demonstrated an effect size (Cohen’s D) of 1.6 between health young and individuals with 
incipient sarcopenia. This effect size indicates we need 8 individuals in each group to detect a 
significant difference at an alpha level of 0.05 with 80% power.  
 
7.3 Sample Processing and Analyses 
Blood samples will be processed and analyzed as described by PI’s lab (2). Blood samples will 
be analyzed via LCMS to determine the concentration of specific amino acids relevant to the 
hypothesis. If performed, glucose and insulin concentrations will be performed using a COBAS 
analyzer. Stable isotope concentrations will be measured using either GC-MS or LCMS 
methods, if performed. Breath samples will be analyzed in real time via the collection 
apparatus. After all data has been analyzed and verified, samples will be discarded into a 
biohazard trash bag and disposed in accordance with UAMS biohazardous waste policy. 
 
7.4 Grouping 
Subjects in the older groups will be divided into either sarcopenic or non-sarcopenic based on 
the results of the handgrip assessment at visit 1 and amount of lean tissue derived from the 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
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body composition measurements. The thresholds (per the Sarcopenic Definitions and 
Outcomes Consortium) are as follows: 
Men whose value is < 35.5 kg are considered to have low muscle strength 
Women whose value is < 20 kg are considered to have low muscle strength. 
 
8.0 Ethical Considerations 
 
This study will be conducted in accordance with all applicable government regulations 
and University of Arkansas for Medical Sciences research policies and procedures. This 
protocol and any amendments will be submitted and approved by the UAMS Institutional 
Review Board (IRB) to conduct the study. 
The formal consent of each subject, using the IRB-approved consent form, will be obtained 
before that subject is submitted to any study procedure. All subjects for this study will be 
provided a consent form describing this study and providing sufficient information in language 
suitable for subjects to make an informed decision about their participation in this study. The 
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 offered to the participant, 
and the informed consent process will be documented in each subject’s research record. 
 
9.0 Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The publications 
will not contain any identifiable information that could be linked to a participant. 
 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
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10.0 References 
1. Goates S, Du K, Arensberg MB, Gaillard T, Guralnik J, Pereira SL. Economic Impact of 
Hospitalizations in US Adults with Sarcopenia. J Frailty Aging. 2019;8(2):93-99. doi: 
10.14283/jfa.2019.10. PMID: 30997923. 
2. Church DD, Hirsch KR, Park S, Kim IY, Gwin JA, Pasiakos SM, Wolfe RR, Ferrando 
AA. Essential Amino Acids and Protein Synthesis: Insights into Maximizing the Muscle 
and Whole-Body Response to Feeding. Nutrients. 2020 Dec 2;12(12):3717. doi: 
10.3390/nu12123717. PMID: 33276485; PMCID: PMC7760188. 
3. Mitchell WK, Williams J, Atherton P, Larvin M, Lund J, Narici M. Sarcopenia, dynapenia, 
and the impact of advancing age on human skeletal muscle size and strength; a 
quantitative review. Front Physiol. 2012;3:260. 
4. Wolfe RR, Miller SL, Miller KB. Optimal protein intake in the elderly. Clin Nutr 27:675-
684, Oct 2008. PMID: 18819733. 
5. Kim IY, Deutz NEP, Wolfe RR. Update on maximal anabolic response to dietary protein. 
Clin Nutr [Review] 2018 Apr; 37(2):411-418. PMID 28807333. 
6. Shaw, J. H., M. Wildbore, & R. R. Wolfe. Whole body protein kinetics in severely septic 
patients. The response to glucose infusion and total parenteral nutrition. Ann. Surg. 
205:288-294, 1987.  
7. Patterson BW, Nguyen T, Pierre E, Herndon DN, Wolfe RR. Urea and protein 
metabolism in burned children: effect of dietary protein intake. Metabolism 46, 5:573-
578, 1997. 
8. Wolfe RR. The 2017 Sir David P Cuthbertson lecture. Amino acids and muscle protein 
metabolism in critical care. Clin Nutr [Review] 2018 Aug; 37(4). PMID: 29291898. 
9. Wilkinson DJ, Bukhari SSI, Phillips BE, Limb MC, Cegielski J, Brook MS, Rankin D, 
Mitchell WK, Kobayashi H, Williams JP, Lund J, Greenhaff PL, Smith K, Atherton PJ. 
Effects of leucine-enriched essential amino acid and whey protein bolus dosing upon 
skeletal muscle protein synthesis at rest and after exercise in older women. Clin Nutr. 
2018 Dec; 37(6 Pt A):2011-2021. doi: 10.1016/j.clnu.2017.09.008. Epub 2017 Sep 23. 
PMID:29031484 

Nutritional Biomarkers of Sarcopenia 
PI: David D. Church, Ph.D. 
Institution: UAMS 
Sponsor: UAMS/TRI 
 
Version #: 10 
 
IRB# 262590 
Date: 
31May22 
Page 18 
 
10. Katsanos CS, Kobayashi H, Sheffield-Moore M, Aarsland A, Wolfe RR. Aging is 
associated with diminished accretion of muscle proteins after the ingestion of a small 
bolus of essential amino acids. Am J Clin Nutr 82:00-00, 1065-1073, Nov 2005. 
11. Appleton KM. Barriers to and Facilitators of the Consumption of Animal-Based Protein-
Rich Foods in Older Adults. Nutrients. 2016;8:187. 
12. Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, Atherton P, Wackerhage H, 
Taylor PM, Rennie MJ. Anabolic signaling deficits underlie amino acid resistance of 
wasting, aging muscle. FASEB J. 2005 Mar;19(3):422-4. doi: 10.1096/fj.04-2640fje. 
Epub 2004 Dec 13. PMID: 15596483.