Mitochondrial Function in Circulating Cells and Muscle Tissue
Conditions: Healthy, Obesity, Insulin Sensitivity, Women
Sponsor: University of Arkansas

Official Title: Mitochondrial function in circulating cells and muscle tissue 
NCT02700698 
Document Date: 03/21/2019 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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Study Title:  
Mitochondrial function in circulating cells and muscle tissue  
 
 
Principal Investigator:  
Elisabet Borsheim, Ph.D. 
Office: 
501-364-3053 
Cell: 
409-392-4203 (cell) 
 
 
Co-Investigators:  
Eugenia Carvalho, Ph.D. 
Richard Frye, M.D. 
Gohar Azhar, M.D. 
Sean Adams, Ph.D. 
 
 
Study Physician: 
Gohar Azhar, M.D. 
 
 
Study Location:  
Arkansas Children's Nutrition Center (ACNC), 
Arkansas Children’s Hospital Research Institute,  
15 Children’s Way 
Little Rock, AR 72202  
and 
University of Arkansas for Medical Sciences 
Reynolds Institute on Aging, Little Rock, AR 72205  
 
 
Funding: 
Arkansas Children's Nutrition Center (ACNC) 
United States Department of Agriculture (USDA) 
 
 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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Background 
Data from human and animal models strongly suggests that reduced tissue mitochondrial function can 
be an early marker for detection of dysfunctional metabolism [1].  It is unclear if tissue mitochondrial 
dysfunction can be detected using less invasive methodology.  In this project, we plan to study 
mitochondrial bioenergetics in circulating cells and paired muscle biopsies in two metabolic disparate 
groups of women: healthy lean vs. overweight/obese and/or insulin resistant women of similar age. 
Mitochondria in circulating blood cells sense oxidative stress during capillary passage and react by 
producing reactive oxygen species (ROS) [2].  Bioenergetic profiling of readily available circulating 
cells has recently been proposed to reflect systemic mitochondrial health/fitness, physical function and 
inflammation [3, 4].  Thus, the mitochondria function in circulating cells may potentially inform on the 
overall metabolic health in an individual.  The “bioenergetic health index” (BHI) in peripheral blood 
mononuclear cells (PBMC) and/or platelets has therefore been described as a potential new biomarker 
for assessing patient health [3, 5, 6]. 
Studies to date have, however, not compared results of mitochondrial function in circulating cells 
and in muscle tissue in the same persons.  Instead, a relation has been shown indirectly by an association 
between the bioenergetics of PBMC and physical function and inflammation in overweight/obese older 
adults [4].  Thus, there is a need for establishing this relation by comparing bioenergetics in circulating 
cells and in muscle tissue from the same individual, and in individuals with contrasting metabolism. 
By studying healthy lean and overweight/obese and/or insulin-resistance women of the same age, we 
expect to discover differences in muscle mitochondrial function in the two groups, with compromised 
function in the overweight/obese and/or insulin resistant group.  Obesity is associated with metabolic 
impairments in peripheral tissues and dysfunctional adipose tissue unable to buffer the flux of dietary 
lipids, leading to ectopic lipid deposition and lipotoxicity in other organs.  Lipid accumulation in skeletal 
muscle is associated with insulin resistance and overall cellular dysfunction, with mitochondrial 
dysfunction as one of the underlying mechanisms [7].  We hypothesize that PMBC mitochondrial 
function in the overweight/obese and/or insulin resistant group is compromised compared to the healthy 
lean group.  Finally, we hypothesize that there will be a relation between PBMC and muscle 
mitochondria function across the two groups.  

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
Version 9 
03/21/2019 
 
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The main goal of this project is to determine whether mitochondrial function in circulating cells is 
related to that measured in the muscle fibers of the same subjects, and if differences can be observed 
when comparing metabolic dissimilar groups. Considering the difficulty of obtaining muscle biopsies 
from various populations, especially pediatric populations, it would be a major step if a blood sample 
can inform about overall/tissue mitochondrial health. 
Study Design: 
Twenty (20) healthy lean, and 20 overweight or obese and/or insulin resistant women of similar age will 
be recruited, with the goal of 15 women/group completing the study. 
Inclusion criteria:  
• Women. 
• 25-35 years of age.  
• Either healthy lean (BMI<25), or overweight (25≤BMI<30) or obese (BMI≥30) and/or insulin 
resistant. 
Exclusion criteria:  
• Pre-existing medical conditions (e.g., chronic renal failure, bleeding disorders, heart disease, self-
reported hepatitis or HIV, thyroid disorders) that can affect study outcomes, as determined by the PI 
and/or designee. 
• Self-reported possibility of current pregnancy. 
• Regular smoking. 
• Alcohol or drug abuse.   
Enrollment/Recruitment: Various advertisements will be distributed by direct mail or to various 
locations, including but not limited to healthcare offices, weight loss clinics, health fairs, 
schools/universities, recreational centers, websites (ACH, ACRI, ACNC, UAMS, and others as 
applicable) and churches. Also, print or digital ads may appear in newspapers, magazines, social media 
and circulars. Recorded phone messages that play when the phone is answered and the person is on hold, 
BoomText messages, screensavers, and radio/television advertisements may be used. We will also work 
with physicians, who have patients that may qualify as potential volunteers. They will in that case 
administer a flyer to the potential subject and ask them to contact us if they are interested in participation. 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
Version 9 
03/21/2019 
 
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A standardized screening script will be used to determine whether potential participants qualify for the 
study.  Potential participants will be asked if their information can be saved for consideration in future 
studies and their response will be documented.  We will protect all personal information obtained during 
recruitment, enrollment, and testing processes, and maintain this in a closed office. 
Informed Consent Process: During the informed consent process, the PI, co-Is, or a research staff 
member will review all study procedures and the informed consent documents with the potential 
participant.  If eligible and interested in enrollment, informed consent from the participant will be 
obtained and documented.  The participant will be given ample time to review the consent form, ask 
questions and receive clarification prior to signing any documents. 
Study Procedures: The participants that qualify and sign consent for this study will come only once to 
the Reynolds Institute on Aging at UAMS or to the ACNC where the visit will take place, depending on 
room and physician’s availability. 
The visit will take 2-3 hours and the participant will arrive after an overnight fast.  At the visit, vitals 
anthropometrics will be recorded, body composition may be measured, a blood sample will be collected 
and a muscle biopsy sampled. 
• Vitals and Anthropometric measures: The subject’s blood pressure, pulse, temperature, body 
mass, height, and waist and hip circumferences will be obtained using standardized techniques.   
• Physical activity questionnaire: A questionnaire (attached) will be administered to record 
physical activity. This will enable us to control for levels of physical activity between 
participants. 
• Dual-Energy X-ray Absorptiometry (DXA):  Body composition may be measured using a DXA 
device (Hologic Inc, MA, USA).  This technology involves very small amounts of radiation.  The 
subject will be asked to lie down without moving during the entire length of scanning (about 5 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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min).  The scanning process will be aborted at the request of the participant or if any movement 
occurs.  This procedure may be repeated if the first one is invalid or is aborted.   
• Blood collection: Blood will be collected by a trained phlebotomist. In total, ~50 ml (~3.5 
tablespoons) will be drawn for isolation of PBMC and platelets, and for analyses of insulin, 
glucose, lipid levels, and other analytes such as metabolites or hormones.   
• Muscle biopsy: After the blood draw, a muscle tissue biopsy will be taken from m. vastus 
lateralis, about 4-6 inches above the knee by Gohar Azhar, M.D. Before any incisions are made, 
the skin will be cleansed with an antiseptic scrub solution and allowed to dry, and the skin and 
tissue below injected with lidocaine.  A small incision about the size of this dash “—–“ will then 
be made, through which a needle about the size of the letter “O” is gently pushed into the muscle.  
A piece of the muscle is then removed with the needle.  The skin will thereafter be closed with 
dermabond glue, and a light dressing applied. The biopsy site will be iced, a biopsy procedure 
note recorded, and the subject will be given thorough oral and written instructions about how to 
care for the biopsy site (please see attachments).  
The biopsy will immediately be placed in a BIOPS buffer and analyzed for mitochondrial 
function within a few hours after sampling.  We have established the protocol for determination 
of mitochondrial function in permeabilized muscle tissue using high resolution respirometry 
(Oxygraph O2k, Oroboros Instruments, Innsbruck, Austria) [8].  Similarly, the co-investigator 
Dr. Richard Frye, MD, at ACHRI has established protocols on determination of mitochondrial 
function in cells, using the Seahorse Extracellular Flux (XF) 96 Analyzer (Seahorse Bioscience, 
North Billerica, MA, USA). 
Sample Storage: Blood and muscle samples will be kept at -80°C once the initial processing and analyses 
have taken place.  Samples will be stored in appropriate freezers in the PI’s laboratory.  These are 
monitored continuously for proper temperature and working condition.  Samples will be destroyed only 
after all data has been analyzed and reported.  All blood and muscle samples will be using a unique study 
acronym. None of a subject’s personal identifiers will be present on any biological sample. 
Statistical and Power Analyses: Forty women between 25-35 years old (20 healthy lean and 20 insulin-
resistant and/or overweight or obese) will be recruited to participate in this study.  Assuming a moderate 
dropout rate of 25%, we anticipate the final sample size to be 30 (15 healthy lean and 15 insulin resistant 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
Version 9 
03/21/2019 
 
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and/or overweight or obese).  A two-sample t-test will be used to assess differences between means of 
the two groups for either the blood or the muscle measurements.  In addition, correlation and 
concordance analyses will be performed between the blood and the muscle results across the two groups 
to assess their correlation and agreement.  Outcome variables will be checked to verify that the 
distributional assumptions (normality) for the t-test are met. When substantial deviation from 
assumptions is encountered, and if a suitable data transformation is not found, groups will be compared 
using the nonparametric Wilcoxin/Mann-Whitney rank-sum test. 
Power analysis: Using the 2-sample t-test with equal variance, a sample size of 30 (with 15 subjects per 
group) achieves 80% power to detect a standardized difference of 1.06 units between the two group 
means versus the null hypothesis of equal means at a 5% α-level.  Alternatively, without any dropout, a 
sample size of 40 (with 20 subjects per group) achieves 80% power at 5% α- Although the effect sizes 
are estimated to be moderate to large, it will provide valuable information regarding whether a larger 
study is warranted, and will form a basis for statistical approaches to other planned follow-up studies. 
Data Safety Monitoring Plan 
All investigators, co-investigators and study staff will complete and maintain appropriate CITI training. 
All data and communications will be recorded in standardized case report forms to ensure a paper copy 
trail. Source documents and 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/ACH/ACNC policies 
regarding destruction of research records. At no time shall Protected Health Information be released to 
non-study personnel. 
Research data will be entered into a clinical database.  This database will be housed on a shared drive 
backed up nightly off-site Monday – Friday. Access to our server is password protected, as is access to 
the study database(s). Access will be limited to study personnel. 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
Version 9 
03/21/2019 
 
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Discomforts and Risks 
The risks associated with the study procedures are as follow: 
• 
DXA:  The DXA instrument uses radiation to measure body composition.  The radiation exposure 
is small (< 0.1 mrem) corresponding to approximately 2 days’ worth of the naturally occurring 
radiation a person is exposed to while living in the Little Rock Area. 
• Blood Sampling: The total amount of blood drawn for this study will be approximately 50 ml 
(~3.5 tablespoons), which is only 1/5 of what is generally given during blood donations.  Aseptic 
technique will be followed which will minimize the risks of infection.  However, the risks related 
to blood draw include pain and bruising. 
• Muscle biopsy: The muscle biopsy procedure is performed under local anesthesia (1% lidocaine 
with a small amount of bicarbonate) for normal pain management, and possible formation of a 
bruise from local bleeding.  Bleeding from the muscle biopsy site is highly unlikely since no 
major arteries or veins are located in the region.  A possible complication of this procedure is 
infection due to lack of sterility during the procedure.  General precautions that will be taken 
include strict sterility during muscle biopsies.  At the end of the study, the small incision at the 
biopsy sites will be dressed with an antibiotic ointment to minimize risk of infection.  Further, 
pressure bandage and ice will be applied. 
Safety Reporting 
Definitions 
A.  Related: For this policy, an event is “related” if it was caused by participation in the research activity 
or there is a reasonable possibility that the event may have been caused by the procedures involved 
in the research. 
B.  Risk: The probability of harm or injury (physical, psychological, social or economic) occurring as a 
result of participation in a research study. 
C.  Unanticipated: A problem is “unanticipated” when it was unforeseeable at the time it occurred. 
D.  Unanticipated Problem Involving Risks to Subjects or Others (UPIRTSO): Any problem, event or 
new information that is: 
1) Unanticipated or unexpected; 
2) Related to the research; and 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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3) Involves new or increased risks to subjects or others. 
E.  Unexpected: An event is “unexpected” when its specificity, nature, severity or incidences are not 
accurately captured in the approved consent form. Examples include a lower rate of response or a 
side effect that is more severe than initially expected. 
(Serious) Adverse Events handling 
Any clinical study event that is judged to be an (S)AE, either reported spontaneously by the subject or 
observed by the investigator or her/his staff, is recorded on the (S)AE form during the course of the 
study. The investigator must ensure that this information, including onset, duration and nature of event, 
severity, and action taken, is captured. 
The severity of any (S) AE is scored as follows: 
- 
Mild: 
transient or mild discomfort; no medical intervention/therapy required. 
- 
Moderate: 
mild to moderate limitation in activity; some assistance may be needed; 
and/or minimal medical intervention/therapy required. 
- 
Severe: 
marked limitation in activity; some assistance usually required; and/or 
significant medical intervention/therapy/hospitalisation required. 
We do not foresee any adverse events associated with this protocol. In addition, testing can be aborted 
at any time at the participant’s request. All procedures are minimally to moderate invasive. However, 
any adverse effects or unanticipated problems will be reported to the study PI, the IRB, and the study 
sponsor in accordance with UAMS IRB Policy 10.2 Information that Must Be Reported to the IRB and 
IRB Actions. 
Risks/Benefits 
Participants will not directly benefit from this study. Future population may benefit from this research. 
Expected risks associated with this protocol are described in detail above. All experimental procedures 
will be performed by appropriately trained and credentialed personnel.. Potential risks from participation 
in this research protocol include problems associated with blood drawing and muscle biopsies. 
 
Participant Compensation: The reimbursement will be $100 for completing the study.  This will be 
mailed to them after study completion with a completion letter.   
 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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Possible Problems and Pitfalls:  We expect minimal problems with this study.  Both the physician that 
will perform the biopsies (Dr. Azhar, M.D.), and Dr. Borsheim and her staff have experience in collecting 
blood samples and performing muscle biopsies. 
 
Future plans: In future studies we plan to use these techniques to determine if children born to overweight 
and obese mothers or people at high risk for diabetes such as first degree relatives of diabetic subjects 
will show impaired mitochondrial function in circulating cells relative to control subjects. We may also 
use the techniques to examine whether physical activity and/or nutritional interventions affect these 
measurements. The information from the current protocol will inform as to whether or not the use of 
blood cell energetics measurements can supplant the need for muscle biopsies. 
 
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.   
 
 
LITERATURE  
 
1. 
Durhuus, J.A., C. Desler, and L.J. Rasmussen, Mitochondria in health and disease - 3rd annual 
conference of society for mitochondrial research and medicine - 19-20 December 2013 - 
Bengaluru, India. Mitochondrion, 2015. 20: p. 7-12. 
2. 
Ijsselmuiden, A.J., et al., Circulating white blood cells and platelets amplify oxidative stress in 
heart failure. Nat Clin Pract Cardiovasc Med, 2008. 5(12): p. 811-20. 
3. 
Chacko, B.K., et al., The Bioenergetic Health Index: a new concept in mitochondrial 
translational research. Clin Sci (Lond), 2014. 127(6): p. 367-73. 
4. 
Tyrrell, D.J., et al., Blood-cell bioenergetics are associated with physical function and 
inflammation in overweight/obese older adults. Exp Gerontol, 2015. 70: p. 84-91. 
5. 
Chacko, B.K., et al., The Bioenergetic Health Index is a sensitive measure of oxidative stress in 
human monocytes. Redox Biol, 2015. 8: p. 43-50. 
6. 
Kramer, P.A., et al., Decreased Bioenergetic Health Index in monocytes isolated from the 
pericardial fluid and blood of post-operative cardiac surgery patients. Biosci Rep, 2015. 35(4). 
7. 
Tumova, J., M. Andel, and J. Trnka, Excess of free fatty acids as a cause of metabolic dysfunction 
in skeletal muscle. Physiol Res, 2015. 
8. 
Porter, C., et al., Mitochondrial respiratory capacity and coupling control decline with age in 
human skeletal muscle. Am J Physiol Endocrinol Metab, 2015. 309(3): p. E224-32. 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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Muscle Biopsy Procedure Note 
Research Muscle Biopsy Procedure 
 
Study Name: 
 Mitochondrial function in circulating cells and muscle tissue 
 
Study PI:  Elisabet Borsheim, Ph.D. 
 
Subject Name/ID:   
 
 
 
 
 
Date:   
             
 
 
 
Location: Vastus Lateralis   
 
 
Biopsy #1 Side:    
        Right                Left 
 
 
 
 
 
 
 
 
Drug Allergies:  
 
 
 
 
 
 
 
 
 
 
 
Anticoagulant Medication:  
 
Heparin   ___ No  ___ Yes If yes, date/time last taken: __________ 
Coumadin  
_No  ___ Yes If yes, date/time last taken: __________ 
Plavix  
___ No  ___ Yes If yes, date/time last taken: __________ 
Aspirin 
___ No  ___ Yes If yes, date/time last taken: __________ 
 
Procedure: Muscle Biopsy #1 
After sterile preparation and adequate local anesthesia with           ml 1% lidocaine, the 
muscle biopsy procedure was performed with a 5 mm Bergstrom needle with/without 
complications. Pressure was applied to the biopsy site continuously until all bleeding ceased. 
The incision was bandaged with a sterile compression dressing.  
 
 
 
 
 
 
 
 
_____________________ 
Staff signature 
 
 
The subject was provided verbal and written instructions regarding wound care, as well as 
contact numbers in case of questions or problems. 
 
Duties performed, name:  
Biopsy:   
 
 
 
 
 
 
 
 
 
 Assistance:   
 
 
 
 
 
 
 
 
 
 
Comments:   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
___________________________________ 
 
 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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Center for Translational Research in Aging 
Biopsy Care Instructions 
Activity is good for your muscle after the biopsy; this prevents it from getting stiff. Your 
muscle may feel sore for a day or two, as if you have performed unaccustomed exercise; 
however, this is normal and will pass.   
A pressure dressing has been applied over the biopsy site.  Keep this on until you go to bed 
tonight. A special glue (Dermabond) was used to keep the incision closed, and a dressing 
applied over this. Keep this dressing on for 5-7 days following the biopsy. You can get the 
biopsy site/dressing wet in order to shower; otherwise, try to keep it dry to prevent the 
dressing from falling off prematurely. Please do not take any pain medication that may affect 
bleeding (e.g., aspirin). If necessary, clean the site with the alcohol wipes provided, and if 
the dressing falls off early, replace it with the ones provided. 
If you have any questions or problems with severe soreness, bleeding, or if the biopsy site 
becomes red or warm to the touch, please call Scott Schutzler, R.N. at (501) 388-1025 or 
Dr. Elisabet Borsheim at (409) 392-4203 (cell). 

IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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IRB# 205139: Mitochondrial function in circulating cells and muscle tissue 
Principal Investigator: Elisabet Borsheim, Ph.D.  
Arkansas Children’s Nutrition Center and United States Department of Agriculture 
 
 
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03/21/2019 
 
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