Role of the Protein Matrix in the Anabolic Response to a Ground Beef Patty as Opposed to the Impossible (Vegi-) Burger
Phase: NA
Conditions: Protein Metabolism
Sponsor: University of Arkansas

Study Title: Role of protein matrix in the anabolic response to a ground beef patty as opposed 
to the Impossible (vegi-) Burger 
 
Version: 4.0 
 
Date: 05May22 
 
NCT#: 05197140 
 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
Page 2 
 
University of Arkansas for Medical Sciences (UAMS) Clinical Protocol 
 
Study Title:  
Role of the protein matrix in the anabolic response to a ground 
beef patty as opposed to the Impossible (vegi-) Burger 
 
Principal Investigator: 
Robert R. Wolfe, Ph.D. 
 
Co-Investigators:  
Arny A. Ferrando, Ph.D 
 
 
 
 
 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St., Slot #806 
 
 
 
 
Little Rock, AR 72205 
 
 
 
 
 
Sub-Investigators:  
Gohar Azhar, M.D. 
 
 
 
 
Katie R. Hirsch, Ph.D. 
 
 
 
 
University of Arkansas for Medical Sciences 
 
 
 
 
4301 West Markham St. 
 
 
 
 
Little Rock, AR 72205 
 
Study Clinical Staff: 
Scott Schutzler, R.N. 
 
Study location: 
 
Donald W. Reynolds Institute on Aging @ UAMS 
 
 
 
 
629 Jack Stephens Drive 
 
 
 
 
Little Rock, AR 72205 
 
Support (Funding): 
National Cattlemen’s Beef Association. 
 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
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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 ..................................................................................................... 8 
3.1 Study Visits ..................................................................................................................................... 8 
3.2 Subject Compensation ................................................................................................................ 10 
3.3 Recruitment .................................................................................................................................. 11 
3.4 Study Registration ....................................................................................................................... 12 
3.5 Sample Storage ........................................................................................................................... 12 
3.6 Randomization ............................................................................................................................. 13 
4.0 Study Population ........................................................................................................................... 13 
4.1 Inclusion Criteria .......................................................................................................................... 13 
4.2 Exclusion Criteria ......................................................................................................................... 13 
5.0 Risks and Benefits ........................................................................................................................ 14 
5.1 Blood sampling: ........................................................................................................................... 14 
5.2 DEXA, In Body, ultrasound scans: ............................................................................................ 14 
5.3 Study foods: .................................................................................................................................. 15 
5.4 Stable Isotope Infusion: .............................................................................................................. 15 
5.5 Confidentiality: .............................................................................................................................. 16 
5.6 Intravenous Catheter insertion: ................................................................................................. 16 
5.7 Muscle Biopsy: ............................................................................................................................. 16 
5.8 Covid-19 Related risks: .............................................................................................................. 16 
6.0 Data Handling and Recordkeeping ........................................................................................... 17 
7.0 Data Analysis.................................................................................................................................. 17 
7.1 Statistical Analysis plan .............................................................................................................. 17 
7.2 Sample Size Calculation & Power Analysis ............................................................................ 18 
7.3 Sample Processing and Analyses ............................................................................................ 18 
8.0 Ethical Considerations ................................................................................................................ 19 
9.0 Dissemination of Data .................................................................................................................. 20 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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10.0 References .................................................................................................................................... 20 
 
Table of Events 
 
Procedure 
Visit 1 
Visit 2 
Visit 3 
informed consent 
x 
 
 
medical history questionnaire 
x 
 
 
list of medications 
x 
 
 
measure height 
x 
 
 
measure weight 
x 
 
 
Blood/urine to LabCorp 
x 
 
 
DEXA scan1 
x 
x 
 
In Body scan1 
x 
x 
 
dispense run-in meals 
 
x 
 
stable isotope infusion 
 
 
x 
periodic blood sampling 
 
 
x 
muscle biopsy x 3 
 
 
x 
ingest study food 
 
 
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:  
According to the USDA Economic Research Service Food Availability Data, beef consumption 
has been on a general decline for the past 40 years, with current per capita consumption 
approximately 35% lower in 2020 than in 1980. Some of this decline over the past few years 
can be attributed to politically motivated claims that carbon emissions from cattle represent a 
major threat to the global environment. However, the major reason for declining beef 
consumption is the widely promulgated claim that beef is unhealthy. The hamburger in 
particular has become a poster child for the notion that beef consumption is responsible for the 
increasing occurrence of obesity in the United States. The trend of decreasing beef 
consumption due to perceived health concerns, particularly obesity and associated health 
problems, has led major fast-food chains such as Burger King and McDonalds, to offer 
vegetarian burgers as healthy alternatives to the traditional beef burger. However, examination 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Page 5 
 
of the components of the macronutrient matrix of a typical vegetarian burger gives reason to 
question the health benefits as compared to a traditional beef patty-hamburger. 
 
1.2 Previous research on the topic: 
One of the principal nutritional benefits of dietary protein in a food source is the stimulation of 
muscle protein turnover and the net gain of body protein (the anabolic response) (1). 
Table 1 shows key aspects of the macronutrient matrix of the Impossible Burger and an 80/20 
percent beef patty, with particular emphasis on the protein/essential amino acid (EAA) content. 
The Impossible Burger was chosen as a representative vegetarian burger because soy, which 
is the highest quality plant-based protein, is the principal protein source (2). Four-ounce 
servings of a beef patty and of an Impossible Burger each provide 19 g of protein. However, a 
closer inspection of the macronutrient matrix of the two food sources reveals that the 4 oz beef 
patty contains more than twice the amount of essential amino acids (EAAs) as compared to a 
4 oz Impossible Burger. We recently completed an NCBA-supported study in which the 
anabolic responses to seven different “ounce equivalents” of protein food sources, as 
described by the USDA My Plate, were quantified using stable isotope tracers. Rather than 
being metabolically equivalent, as implied by their designation by My Plate as “ounce 
equivalent” protein food sources, we found that there was a linear relation between the net 
gain in body protein and the EAA content of the protein food source (Figure 1A) (3). Moreover, 
the slope of the relation between EAA content and increase in net protein balance was 
approximately 1, meaning that any difference in EAA content directly corresponded to an 
equivalent difference in net body protein gain (Figure 1B). 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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These results support the notion that the EAA content of a protein food source is principally 
responsible for the magnitude of the anabolic response. This fundamental concept is the basis 
for the scoring of protein quality by the Digestible Indispensable Amino Acid Score (DIAAS) 
(4). With relevance to the comparison of a beef patty and an Impossible Burger, we found that 
consumption of 2 ounces of beef resulted in an anabolic response that was approximately 4 g 
net gain of protein greater than the response to consumption of 2 ounces tofu (Figure 1) (3). 
The difference in anabolic responses corresponds to the difference in the EAA content of the 
two protein food sources (Figure 1A). Muscle protein fractional synthesis rate was also greater 
in those consuming beef as opposed to tofu. Further, compilation of our in-house data set 
across various populations and food sources indicates that the EAA content of a protein food 
source explains 80.4% of the variance in the total amount of body protein gained (5). This 
large dataset has allowed us to develop a predictive equation that indicates the total amount of 
body protein gained will be 7.6 g from 4.2 g of EAA (the Impossible Burger) and 16.5 g from 
8.9 g of EAA (beef patty). Stated differently, the greater EAA content of a 4 oz beef patty is 
predicted to elicit more than double the anabolic response to the same amount of an 
Impossible Burger. 
Plant-based burgers such as the Impossible Burger are being marketed as a one-for-one 
replacement for beef-based burgers, but the disregard for protein quality is misleading and 
creates a false perception of equivalent nutritional benefit of the two protein food sources. In 
 
Figure 1. Relation between EAA serving and net gain in body protein of a variety of protein food sources. 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
Page 7 
 
addition to the greater EAA content per gram protein, the profile of EAAs in beef protein more 
closely reflects the dietary requirements for individual EAAs, as reflected by a DIAAS 
approximately 40% greater than soy protein. In addition, the overall macronutrient matrix of a 
protein food source can potentially influence the anabolic response by affecting the temporal 
EAA response. Both the peak concentration of EAAs after consumption as well as the time to 
reach the peak concentration affect the anabolic response (5). The Impossible Burger contains 
various binders and starches that could potentially interfere with the absorption of the EAA 
from the protein matrix, with a resultant slowing of the rate of absorption and blunting of the 
maximal concentrations of EAAs in the blood and the speed at which the peak concentrations 
are reached. These factors likely have a corresponding negative impact on the magnitude of 
the anabolic response. Since no such issues impede the digestibility of beef protein, it is 
reasonable to propose that the plasma EAA levels will reach higher peak EAA concentrations, 
and achieve the peak concentrations more rapidly, than ingestion of the same amount of EAAs 
in the matrix of the Impossible Burger. As a result of both the advantageous profile of EAAs in 
beef protein and the more rapid absorption of EAAs, we propose that a beef patty will not only 
provide a greater anabolic response than an iso-nitrogenous amount of Impossible Burger, but 
that also consumption of 4 oz of beef patty will also elicit a greater anabolic response than 
consumption of two Impossible Burgers. 
The caloric value of the Impossible Burger and a beef patty is also relevant when considering 
the relative nutritional value and nutrient density of the two protein food sources. The caloric 
value of the Impossible Burger is almost twice that of a beef patty when normalized for the 
amount of EAAs (Table 1). We therefore anticipate that differences in anabolic responses 
between a beef patty and Impossible Burger will be amplified when normalized by the caloric 
values of the two protein food sources. 
 
2.0 Hypotheses 
 
1. Consumption of a 4 oz beef patty will result in a greater anabolic response than 
consumption of a 4 oz Impossible Burger, including greater increases in plasma EAA 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Page 8 
 
concentrations, a greater balance between whole body protein synthesis and breakdown, and 
a greater increase in muscle protein fractional synthetic rate (FSR). 
2. Consumption of a 4 oz beef patty will result in a greater anabolic response than 
consumption of two 4 oz Impossible Burgers, including greater increases in plasma EAA 
concentrations, greater balance between whole body protein synthesis and breakdown, and 
greater increase in muscle protein FSR. 
 
3.0 Study Design and Procedures 
 
We will enroll up to 28 subjects with a goal to study up to 24 healthy male and females. We will 
use a 10-hour period to measure the protein metabolism in response to the study food (one 
beef patty, one vegi-burger, or two vegi-burgers).  
 
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 questionnaire 
including allergies and list of current medications will be completed. Females of child bearing 
capacity will be asked to provide a urine sample for pregnancy testing. About 4mL of venous 
blood will be drawn to send for complete blood count for study criteria. Blood and 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), 
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. 
Run-in meals will be discussed with subjects to elicit food allergies or intolerances. 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Visit 2: Subjects return to the UAMS TRI. If they will undergo the DEXA and InBody scans, the 
must have 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, DEXA, ultrasound scans will be performed if these were not done 
at visit 1. Two days’ of run-in meals will be dispensed with instructions on their preparation. 
Meals will be consumed for the two days immediately preceding Visit 3. 
 
Visit 3: Subjects return to the UAMS TRI having fasted overnight. Study staff will ask about 
adverse events since their last visit. Vital signs (heartrate, blood pressure and oral 
temperature) will be measured. 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. A second IV catheter will be placed into a vein on of the subject’s 
other arm to use for stable isotope infusion. 
After an initial blood sample is obtained, the priming dose of isotopes are given IV push, the 
constant infusions are begun, and a timer is started. Blood and muscle samples are obtained 
according to the schedule below. 
The study food is served after the 4:00 blood/muscle sample are obtained. Subjects are asked 
to consume the study food within 10 minutes. 
At the conclusion of 10 hours elapsed time, the IV catheters are removed and the sites 
dressed with a bandage. Vital signs (heartrate, blood pressure and oral temperature) will be 
measured. 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) 
120 
Blood sample (~4mL into an EDTA tube), 
ingest study supplement, muscle sample 
from vastus lateralis 
150 
Blood sample (~4mL into an EDTA tube) 
180 
Blood sample (~4mL into an EDTA tube) 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Page 10 
 
 
 
3.2 Subject Compensation 
Subjects will accrue compensation according to the below table. They will be handed a $20 gift 
card at the end of visit 1. They will accrue $20 for visit 2 and up to $300 for visit 3. A check will 
be mailed via UAMS SAP for their accrued compensation approximately 3-4 weeks after their 
participation ceases (whether completed or not). If they were to fully attend every visit, their 
total amount would be $340. If they stop participating prior to the completion of study visit 3, 
they will receive prorated pay of $25 per hour. 
210 
Blood sample (~4mL into an EDTA tube) 
240 
Blood sample (~4mL into an EDTA tube), 
muscle sample from vastus lateralis 
serve study food 
270 
Blood sample (~4mL into an EDTA tube) 
300 
Blood sample (~4mL into an EDTA tube) 
330 
Blood sample (~4mL into an EDTA tube) 
360 
Blood sample (~4mL into an EDTA tube) 
390 
Blood sample (~4mL into an EDTA tube) 
420 
Blood sample (~4mL into an EDTA tube) 
450 
Blood sample (~4mL into an EDTA tube) 
480 
Blood sample (~4mL into an EDTA tube) 
510 
Blood sample (~4mL into an EDTA tube) 
540 
Blood sample (~4mL into an EDTA tube) 
570 
Blood sample (~4mL into an EDTA tube) 
600 
Blood sample (~4mL into an EDTA tube),  
muscle sample from vastus lateralis 
Visit 
Amount 
1 
$20 
2 
$20 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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3.3 Recruitment 
Potential subjects will be recruited from the Little Rock and central Arkansas areas by use of 
UAMSHealth social media, word of mouth, the ARResearch.org registry, and contacting prior 
subjects who indicated they wanted to be contacted about future studies. UAMS students and 
employees will not be targeted for participation but will not be refused. Study staff will ask 
enrolled subjects to recruit others if they are comfortable doing so, and study staff will seek 
subjects within their social circles. Individuals interested in the study will be scheduled for Visit 
1. 
Facebook Script: 
The Center for Translational Research in Aging and Longevity at UAMS is recruiting healthy 
individuals ages 18-40 for a study about measuring how blood and muscle proteins respond to 
either a beef patty or vegi-burger(s).  
  
Participants will be compensated for their time. If interested, please contact us: 
Email sseale@uams.edu or call 501-526-5734 for more information. 
  
Twitter Script: 
Our research team is recruiting individuals ages 18-40 for a study about how blood and muscle 
proteins respond to a beef patty vs vegi-burger. 
Participants will be compensated! Contact us for more info: email sseale@uams.edu, or call 
501-526-5734 
 
During the initial conversation (in person or via phone), the following items will be discussed in 
non-technical language: 
• this study is completely voluntary. 
• the goal of the study: to see if beef or vege-burgers are more efficient at making 
proteins in our body. 
• the number of visits to UAMS study site and their duration: 2 visits that are less than an 
hour, and 1 visit that lasts a bit over 10 hours. 
• study procedures: must have 2 IV catheters placed and undergo 3 muscle biopsies from 
one incision on their outer thigh, eat our food for almost 3 full days, fast overnight twice, 
3 
$300 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
Page 12 
 
have an x-ray for body composition, must get an infusion of stable isotopes (and define 
what they are). 
• risks of procedures: pain, bruising, bleeding, infection, possible fever/chills, discomfort 
from fasting, small amount of radiation from x-ray, amount of blood taken (approx.. 5 
tablespoons), skin numbness and scar formation from biopsy incision. 
• compensation for time and travel: up to $340 if all 3 visits are completed. 
ARResearch.org: 
Using the age criteria, study staff will setup the study in the registry. The registry gatekeeper 
will send the announcement to a predetermined number of registrants (as determined by study 
staff, e.g. 250 for the initial volley). The submitted language will be emailed to the filtered 
registrants by the registry’s software. Interested registrants (respondents) will click a button to 
either allow the study staff to contact them via their preferred means or decline to be 
contacted. If/when a respondent replies to study staff’s contact, a brief discussion of the study 
overview will take place. Respondents who are still interested will schedule visit 1 with study 
staff. Recruitment metrics will be updated on the registry’s website through completion of 
enrollment. 
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. 
With explicit permission via the consent form, muscle and plasma samples will be kept 
indefinitely for future approved uses. No samples will be released to other investigators. 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Page 13 
 
3.6 Randomization 
Subjects will be stratified by sex and then randomized equally to one of three groups (one beef 
patty, one Impossible Burger, or two Impossible Burgers). 
 
3.7 Muscle Biopsy Procedure 
At Visit 3, qualified and credentialed study staff (M.D.) will perform three separate vastus 
lateralis muscle biopsies of the subject’s preferred outer thigh using 1% plain lidocaine for 
anesthesia. One incision will be used for all 3 biopsies. Approximately 100mg of muscle tissue 
per biopsy 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. Processing and analyses will take place in 
batches according to the procedures mentioned in 7.3 below. 
the incision will be closed with a medical glue, then dressed using a transparent adhesive 
bandage followed by a 6” wide elastic wrap that will protect the site from incidental contact and 
assist with hemostasis. Subjects are provided verbal and written instructions for care of the 
biopsy site, along with the phone number of the study nurse should they have any questions or 
concerns once leaving the study site. 
 
4.0 Study Population 
 
Up to 28 subjects will be enrolled in order to meet the goal of 8 completers in each of the three 
groups. Attempts will be made to balance sexes within groups. 
 
4.1 Inclusion Criteria 
• Ages 18 – 40 yrs. 
• Body Mass Index of 20-32 inclusive. 
 
4.2 Exclusion Criteria 
• Unwilling to eat animal proteins 
• History of diabetes that requires medication for control of blood glucose 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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• 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 
• Hemoglobin less than 12 g/dL at screening 
• Platelets less than 150,000 at screening 
• Subjects who cannot refrain from using protein or amino acid supplements for 7 days prior 
to Visit 3 
• 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 
 
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, stable isotope enrichment, glucose, and 
insulin. The total amount of blood taken will be approximately 72 mL (~5 tablespoons). 
Subjects should have no noticeable effects from this volume. 
 
5.2 DEXA, In Body, ultrasound 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 and ultrasound scans do not pose any risks. 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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Page 15 
 
5.3 Study foods: 
The run-in meals are comprised of frozen, ready-to-eat portions that are commercially 
available at most grocery stores (e.g. Stouffers). The ‘test’ foods are: 
• one 4 ounce (cooked weight) patty of 80/20 beef (from Kroger e.g.) 
• one 4 ounce Impossible Burger (https://impossiblefoods.com/products/burger) 
• two 4 ounce Impossible Burgers 
The risk of eating any of these foods is that of a food allergy. Subjects will be shown the list of 
foods and asked if any of them are intolerable. 
 
5.4 Stable Isotope Infusion: 
The primary risks of infusion of the stable isotopes are fever, chills, infection (from 
contamination). A 0.2 micron in-line filter will be used during the infusion. The isotopes will be 
purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA) and sent to the 
Center for Translational Research in Aging and Longevity at Texas A&M University (College 
Station, TX) for contractual compounding. Finished products will be shipped to the UAMS 
Research pharmacist who will store and dispense the isotopes to study staff as needed. 
Isotopes will be stored in glass vials and frozen until use. Two solutions will be produced: a 
mixture of L-[ring-2H5] phenylalanine + U-13C9-15N tyrosine (for constant infusion IVPB), and 
a mixture of L-[ring-2H5] phenylalanine + U-13C9-15N tyrosine + L-[ring-2H4] tyrosine (for 
priming doses IVP). 
Isotope rates of infusion: 
• L-[ring-2H5] phenylalanine: priming dose: 3.6µmol/kg. Constant infusion: 3.6 
µmol/kg/hour. 
• U-13C9-15N-tyrosine: priming dose: 0.113µmol/kg. Constant infusion: 0.113µmol/kg/hour. 
• L-[ring-2H4] tyrosine: priming dose: 0.31µmol/kg. 
IVP vials contain: 
IVPB vials contain: 
L-[ring-2H5] phenylalanine 
L-[ring-2H5] phenylalanine 
U-13C9-15N-tyrosine 
U-13C9-15N-tyrosine 
L-[ring-2H4] tyrosine 
 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
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5.5 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. 
 
5.6 Intravenous Catheter insertion: 
The risks of inserting an IV catheter are pain, bruising, bleeding, and infection. 
 
5.7 Muscle Biopsy: 
The risks of the muscle biopsy procedure include pain, bleeding, bruising, infection, residual 
numbness from the incision, scar formation, soreness lasting >48 hours, and hematoma. 
 
5.8 Covid-19 Related risks: 
Subjects will be COVID-19 negative and/or asymptomatic. 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 visits except when ingesting their meals. 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. 
 
5.9 Safety Monitoring Plan 
Study staff will monitor for adverse events and protocol deviations though the end of the study. 
The PI and study physician (if necessary) will be notified of adverse events shortly after their 
discovery. If a subject has an adverse event while on site, study staff will help get the care the 
subject needs. This may include first aid, emergency care, and/or follow-up care. 
Adverse events and protocol deviations will be recorded on an Excel spreadsheet that is filed 
with the IRB at specific intervals. Any “unexpected problems involving risks to subjects or 
others” (UPIRTSO) will be reported to the IRB within 24 hours of discovery. 
 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
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Date: 
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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. 
 
7.0 Data Analysis 
 
7.1 Statistical Analysis plan 
The post-prandial change in net protein balance (i.e., protein synthesis minus protein 
breakdown) relative to the baseline in each group will be the primary outcome. Responses of 
the rates of protein synthesis and breakdown will be secondary outcomes. Additional 
secondary endpoints will be muscle protein synthesis, plasma EAA and insulin area under the 
curve above baseline values, and protein kinetics normalized by caloric and EAA intake. 
Correlation analysis of the relation between protein kinetics and the EAA content of the protein 
food source will be additional secondary endpoints. 
One-way ANCOVA will be used to compare differences in protein kinetics (FSR, NB, PS, and 
PB) and protein kinetics normalized by caloric and EAA intake. Plasma EAAs and insulin area 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
Page 18 
 
under the curve above fasting valueswill be assess by a one-way ANOVA. All significant main 
effects of group will be followed with Bonferroni-adjusted pairwise comparisons. Partial 
Pearson’s correlation coefficients controlling for protein source (beef patty or Impossible 
Burger) will be used for the correlation analysis. Statistical significance will be accepted at p < 
0.05. 
 
7.2 Sample Size Calculation & Power Analysis 
A group size of 8 per group was a priori determined based upon an ANCOVA model to 
compare the protein sources and amounts with respect to mean response of net protein 
synthesis after adjusting for baseline measures. With this sample size, the ANCOVA model 
has 80% power to detect effect sizes of f = 0.484 or larger. This estimate assumes that the 
baseline covariate explains 50% of the variation in the response. A 5% α-level will be used to 
determine statistical significance. 
 
7.3 Sample Processing and Analyses 
Calculation of protein kinetics: The calculation of whole-body protein kinetics (protein 
synthesis, protein breakdown, and net protein balance) is based on the determination of the 
rate of appearance (Ra) of phenylalanine (Phe) and of tyrosine (Tyr) into plasma and the 
fractional Ra of endogenous tyrosine resulting from phenylalanine hydroxylation. A two-pool 
model has been previously described and discussed in detail (6). Briefly, an isotopic steady 
state will be established in the baseline/fasted period, and protein kinetics are calculated 
accordingly (6). For the 6 hours after ingestion of the patty, the area under the curve (AUC) of 
plasma enrichments of phenylalanine and tyrosine tracers will be calculated. Ra of Phe reflects 
protein breakdown in the fasted state; the total appearance of Phe over the 6 post-prandial 
state reflects both protein breakdown and the appearance of protein from the ingested meal. 
The appearance of exogenous Phe in the peripheral circulation must be subtracted from total 
appearance of Phe to determine the rate of endogenous protein breakdown. The total 
appearance of exogenous Phe in peripheral blood is estimated from the amount of Phe in the 
dietary protein and the amount of protein consumed, the published value for the true ileal 
digestibility of Phe in the test protein, and the measured fraction of absorbed Phe hydroxylated 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
IRB# 273554 
Date: 
05May22 
Page 19 
 
to tyrosine (6). We have recently discussed in detail the validity of assumptions underlying this 
model of protein kinetics (6). Most importantly, this model does not introduce any systematic 
errors that might affect the comparison of the anabolic responses to the burgers (6). The rate 
of muscle protein synthesis will be calculated by dividing the increase in tracer enrichment in 
muscle protein over time by the precursor enrichment, taken to be the intracellular free 
phenylalanine enrichment. 
Analytic methods: Plasma samples will be processed as previously described for determination 
of enrichment by gas chromatography-mass spectrometry (7). Plasma amino acid 
concentrations will be determined by liquid chromatography-mass spectrometry using the 
internal standard method as described previously (3). Plasma insulin concentrations will be 
measured by commercially available human insulin ELISA kit (Alpco Diagnostics, Salem, MA). 
After all data has been analyzed and verified, samples for which permission was not granted 
for indefinite retention will be discarded into a biohazard trash bag and disposed in accordance 
with UAMS biohazardous waste policy. 
 
 
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 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
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Date: 
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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. 
 
10.0 References 
1. Wolfe RR. The underappreciated role of muscle in health and disease. Am J Clin Nutr 
[Review] 84(3):475-482, Sep 2006. 
2. Moughan PJ, Wolfe RR. Determination of Dietary Amino Acid Digestibility in Humans. 
J Nutr 149: 2101–2109, 2019. doi: 10.1093/jn/nxz211. 
3. Park S, Church DD, Schutzler SE, Azhar G, Kim I-Y, Ferrando AA, Wolfe RR. 
Metabolic Evaluation of the Dietary Guidelines’ Ounce Equivalents of Protein Food 
Sources in Young Adults: A Randomized Controlled Trial. J Nutr 151: 1190–1196, 2021. 
doi: 10.1093/jn/nxaa401. 
4. Wolfe RR, Rutherfurd SM, Kim IY, Moughan PJ. Protein quality as determined by the 
Digestible Indispensable Amino Acid Score: evaluation of factors underlying the calculation. 
Nutr Rev [Review] 2016 Sep; 74(9):584-99. PMID:27452871 
5. 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. 

Role of the protein matrix in the anabolic response to a ground beef patty as opposed to the Impossible (vegi-) Burger 
PI: Robert R. Wolfe, Ph.D., Co-PI: Arny A. Ferrando, Ph.D. 
Institution: UAMS 
Sponsor: NCBA 
 
Version #: 4 
 
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Date: 
05May22 
Page 21 
 
6. Wolfe RR, Kim I-Y, Church DD, Moughan PJ, Park S, Ferrando AA. Whole-body 
protein kinetic models to quantify the anabolic response to dietary protein consumption. 
Clinical Nutrition Open Science 36: 78–90, 2021. doi: 10.1016/j.nutos.2021.02.006. 
7. Wolfe RR and Chinkes DL. Isotope Tracers in Metabolic Research: Principles and Practice of 
Kinetic Analysis. Wiley, New York, New York, 2004, 474 pages. 
8. Symons TB, Sheffield-Moore M, Wolfe RR, Paddon-Jones D. A moderate serving of high-
quality protein maximally stimulates skeletal muscle protein synthesis in young and elderly 
subjects. J Am Diet Assoc 109(9):152-1586, Sep 2009.