Bioenergetics and Metabolism in Pediatric Populations
Conditions: Obesity, Type 2 Diabetes Mellitus, Insulin Resistance
Sponsor: Arkansas Children's Hospital Research Institute

Study Title: 
Bioenergetics and Metabolism in Pediatric Populations 
 
Sponsor:  
ABI Discovery Acceleration Initiative and NIH Grants 
 
Principal Investigator:  
Eugenia Carvalho, Ph.D. 
UAMS Department of Geriatrics 
Arkansas Children’s Research Institute 
13 Children’s Way, Slot 317 
Little Rock, AR 72205 
Office phone (8 am-5 pm): (501) 364-3057 
Email: ECarvalho@uams.edu  
 
 
Co-Investigator:  
Shannon Rose, Ph.D.  
 
UAMS Department of Pediatrics 
 
Arkansas Children’s Research Institute 
 
  
 
Study locations: 
 
Arkansas Children’s  
 
 
1 Children’s Way 
 
 
Little Rock, AR 72202 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version #:  10 
Date:  23FEB2018 
Page 1 
 

Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
Background and Rationale 
This research aims to better understand bioenergetics and metabolism in childhood obesity and 
type 2 diabetes (T2D). Obesity is a chronic condition that can lead to several serious health 
conditions. The prevalence of obesity in U.S. children has increased dramatically over the past 
several decades, and as a result, T2D is being diagnosed in a growing number of young 
children1. Oxidative stress and mitochondrial dysfunction have been implicated in obesity and 
the development of insulin resistance in adults2-5. However, the exact molecular mechanisms 
and their contribution to T2D development are not well understood and have been understudied 
in children. A better understanding of the pathophysiology of obesity and the development of 
insulin resistance in children is imperative for the development of effective prevention and 
treatment strategies, to prevent the progression into more serious health concerns and to 
improve the lives of children. 
 
An imbalance between cellular antioxidant defenses and endogenous reactive oxygen species 
(ROS) production results in oxidative stress. Numerous markers of oxidative stress are altered 
in obese children and adolescents2, 6-10. Glutathione (GSH) is the major intracellular 
antioxidant/redox buffer against macromolecular oxidative damage, while cysteine, the rate 
limiting amino acid for GSH synthesis, and the oxidized form, cystine, serves as the major redox 
couple in the plasma for circulating immune cells. Limited evidence indicates reduced GSH 
antioxidant/redox capacity in obese adults11-14. Mitochondrial function in skeletal muscle is 
essential for insulin-stimulated glucose uptake and fatty acid metabolism.  In adults, studies of 
muscle mitochondrial function have shown decreased activities in key enzymes15-21. In addition, 
reduced skeletal muscle mitochondria size and numbers have been reported in obese and to a 
greater extent in T2D adults, and the expression of nuclear encoded ETC genes in skeletal 
muscle was also decreased22-24.   
 
While mitochondrial dysfunction is known to play a role in obesity and the development of T2D 
in adults, a major research gap remains in obese children irrespective of insulin resistance or 
T2D. Decreased resting energy expenditure (REE) has been shown to predispose to obesity 
and T2D in adults25, but relatively little is known about REE in pediatric populations. Reduced or 
inefficient fatty acid oxidation (FAO) is also associated with obesity and insulin resistance5, 26; 
however, this has not been studied in a pediatric population. Given advances in stable isotope 
technologies, it is now possible to perform non-invasive FAO studies in children.  
 
Circulating blood cells are an easily obtainable source to examine bioenergetics and would be 
a preferred non-invasive alternative to collecting tissue biopsies, especially in children. 
Bioenergetic profiling of circulating cells has been used as a measure of systemic mitochondrial 
fitness, physical function and inflammation27-29. While circulating blood cell bioenergetics has not 
been compared between lean and obese individuals, peripheral blood monocyte bioenergetics 
was compared in morbidly obese adults before and after bariatric surgery, with observed post-
surgery increase in basal and maximal mitochondrial respiration that was also correlated with 
increased skeletal muscle maximal respiration30. Another recent report indicated that the 
monocyte and platelet maximal respiratory capacity correlated with the bioenergetics in both 
skeletal and cardiac muscle31. These studies suggests that bioenergetic profiling of circulating 
cells largely reflects the bioenergetic capacity of other tissues.  
 
Version #:  10 
Date:  23FEB2018 
Page 2 
 

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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
obese children with T2D or insulin resistance.  
 
Study Design and Procedures  
This study consists of: 
1. An observational cross sectional bioenergetic and metabolic analysis of several groups 
of children including lean, obese, and T2D.   
2. A longitudinal bioenergetic and metabolic analysis of obese children that will be followed 
for 12 months, repeated measures will be taken at 12 months post-baseline.  
3. A prospective bioenergetic and metabolic analysis of obese insulin resistant children (Aim 
3) and newly diagnosed children with T2D or insulin resistance who will be prescribed 
metformin as part of their clinical care (Aim 4).   
 
A prospective subject and his/her parent will come for the study visit that will last up to 4 hours. 
A subject (and/or his/her parent) will be asked questions regarding the subject’s medical history 
that will be reviewed by study staff to identify patients who fulfill inclusion/exclusion criteria. In 
addition, study staff may review medical records from ACH or other healthcare practitioners 
(such as physicians or pharmacists) outside ACH in order to confirm the inclusion/exclusion 
criteria. Information from medical records may be collected from the time of consent up until the 
time that data analysis for this study is complete and results have been published. Study staff 
will ask parents about demographic information including biologic gender, date of birth, 
race/ethnicity, and parental occupations as well as grade in school. 
 
Prior to the study visit, study staff may contact the parent and ask for medications that the 
participant is currently taking and may ask of any new health diagnoses. In the event that the 
participant is taking medications or has a new health diagnosis that may cause him/her to be 
ineligible to participate in the study visit, the study visit may be rescheduled or canceled. Study 
staff may contact a parent and send the IRB approved consent form and information regarding 
the scheduled visit to the parent(s) prior to the scheduled date. If the consent form has been 
revised since the last informed consent discussion, the new IRB approved consent form may be 
sent to parents. If applicable, the parent will be informed of the blood draw and advised that if 
the parent wishes their child to complete the blood draw at the visit, the child will need to be 
fasting including no food or liquid after 12 am, except for water and medications the day of the 
visit. 
 
At the initial study visit eligible participants will be met by study staff at the designated study 
location. Participants will arrive after an overnight fast of no food or liquid after 12 am, except for 
water and medications the day of the visit. The study visit may include collection of 
anthropometric measurements, tanner stage assessment, physical activity questionnaire, 
depression scale, resting energy expenditure, body composition by bioelectrical impedance, 
saliva, buccal swab, blood, stool, and urine collection. Attempts to repeat anthropometric 
measurements, body composition by bioelectrical impedance, and resting energy expenditure 
will occur if a reading is not given the first time or if the child is unable to comply during the first 
measurement. The parent and subject will not have to stay at the research location for the entire 
Version #:  10 
Date:  23FEB2018 
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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
visit in order for the samples to be obtained. Instructions will be given to the parents for collection 
of samples if the parent and child decide not to remain at the research location for the entire 
visit. 
 
If a procedure(s) (see below) is not successfully completed, the parent and, if warranted, the 
participant may be asked to return to the study site for an additional visit to re-attempt the 
procedure. The decision to ask the parent/participant, to return for another attempt will be made 
by the principal investigator and/or co-investigator. When a decision is made to re-attempt the 
procedure, study staff will contact the parent and explain what procedure they would like to re-
attempt and if the parent is willing, schedule the visit. If a blood draw or fat oxidation estimation 
will be re-attempted, the parent will be re-advised that if the parent wishes their child to complete 
the procedure, the child will need to be fasting including no food or liquid after 12 am, except for 
water and medications the day of the visit. The study visit will be conducted in the same manner 
as the initial visit, with only the desired procedure(s) being re-attempted. 
 
The collection of stool, saliva, and buccal swab samples is optional, and these samples may not 
be collected if the subject or parent does not want the sample(s) to be used for future research.  
 
Study Procedures to be conducted at each visit:  
 
1. 
Intake of Food and Liquid: A parent will be asked by the study staff about the food and 
drink that the subject eats and drinks the day of and day following the visit (up until 
the last urine sample is collected for fat oxidation estimation). This is important 
because the diet will influence the fat oxidation estimation.  
 
2. 
Anthropometrics: Anthropometric measurements including height, weight, waist 
circumference, blood pressure, and pulse will be obtained using standardized 
techniques. BMI-for-age will be calculated and plotted on CDC growth charts to 
confirm lean/obese stratification. 
 
3. 
Tanner (Puberty) Stage Assessment: Parents will be asked to complete a form with 
drawings depicting pubertal development. This form will be used to determine pubertal 
status. A physician may examine the subject for Tanner Stage (if the parent agrees) 
during the visit in order to confirm the parental report and for purposes of analyzing 
study results. The questionnaire given to parents to complete is subjective and a 
parent 
could 
potentially 
incorrectly 
assess 
Tanner 
Stage. 
While 
the 
inclusion/exclusion criteria prevents the inclusion of those with puberty development, 
if a parent incorrectly assesses his/her child’s Tanner Stage, then this may alter the 
results or may cause the exclusion of many potential participants based on the parents 
response, especially in obese participants. Because the results from the samples are 
reliant on the participants being pre-pubertal with no signs of maturation, conclusions 
may be incorrectly drawn from the study data if a child has pubertal development. At 
the beginning stages of puberty, insulin levels rise dramatically showing insulin 
resistance. Thus, if the study determines the child to have insulin resistance but insulin 
resistance is actually due to development of puberty instead of being MUO then this 
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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
could alter the results and conclusions made from the study. Because of this, it is 
important to the study and the results to have the correct Tanner Stage level for each 
participant. 
 
4. 
Physical Activity Questionnaire (Elementary School): A parent or participant will 
complete a form indicating the level of physical activity the participant had during the 
previous week in order to provide general estimates of physical activity levels for each 
subject during a 7-day period prior to the study. In addition, the parent or participant 
will be asked about the participant’s physical activity from the previous months leading 
up to the study visit. 
 
5. 
Depression Scale: With assistance from the parent, a child will complete a form 
(Center for Epidemiological Studies Depression Scale for Children (CES-DC)) used 
as a depression scale. This scale will be used to account for depression as a 
confounding factor that could affect oxidative and bioenergetics parameters.  
 
6. 
Saliva and Cheek Cells: We will ask each participant if he/she is willing to provide a 
sample of saliva and have his/her buccal mucosa swabbed. Cheek cells will be 
collected by study staff  using a soft swab to gently swipe the inside of the child’s 
cheek bilaterally for approximately one minute each, per cheek. The swabs and saliva 
will be labeled with the study ID number of the participant and the study acronym. 
These samples will remain on ice or in a refrigerator until transported to the PI’s 
laboratory. After the sample has been processed, the sample will be stored at -80°C 
in the PI’s laboratory. This sample will be used for future research studies on pediatric 
nutrition and may be retained until it is used up. This is optional and is not a 
requirement for completion of the study visit. 
 
7. 
Blood Collection: A pediatric nurse or trained phlebotomist will collect blood from the 
participant under fasted conditions. Depending on the child’s weight, up to the 
maximum allowed per the guidelines provided by Arkansas Children’s for the 
maximum allowable single blood draw volumes will be collected. The volumes are 
derived using a conservative 3.0 ml/kg estimate as well as a 5% of total body volume 
estimate. Numbing cream or a pain relieving device (Buzzy) may be used for the blood 
draw. Blood samples may be used for: 
a. Isolation of PBMC and platelets for bioenergetics analysis using the Seahorse 
Extracellular Flux (XF)96 Analyzer, protocols for such have been established by 
Rose and Frye 34, 35. Platelets and PBMC bioenergetics may be analyzed 
immediately, or cells may be cryopreserved and analyzed at a later time. Leftover 
cells may be cryopreserved for repeated bioenergetic profiling. 
b. Analyses of insulin, glucose, lipid levels, and other analytes including, but not 
limited to, redox metabolites, hormones and inflammatory markers. 
c. Leftover blood samples collected during this study may be stored in the PI’s 
laboratory and used for future research studies on pediatric nutrition.  
 
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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
8. 
Urine collection: Urine collection may occur at the study location or the sample may 
be collected at home and brought back to study staff. When possible, attempts will be 
made to collect the first-morning urine. Urine collection will be attempted in a sterile 
specimen cup or in a hat and then transferred to a specimen cup. Samples collected 
during the study visit will be placed on ice, in an ice bath, or in a refrigerator until they 
can be delivered to the research site laboratory. Samples collected by 
subjects/parents after the study visit may be stored in a tightly closed specimen cup 
in a refrigerator until they are able to return to the research site, to occur within 6 
weeks of collection. Samples delivered to the research site will be stored in a 
refrigerator until they can be transferred to the research laboratory and stored at -
80°C. This sample will be used for this study and may be used for future research 
studies involving pediatric nutrition.  
 
9. 
Stool collection: Stool collection may occur at one of the study locations or the sample 
may be collected at home and brought back to study staff. Attempts will be made to 
collect the entire sample using a hat and then transferring it to a sterile specimen 
cup(s). Samples collected during the study visit will be placed on ice, in an ice bath, 
or in a freezer until they can be delivered to the research site laboratory. Samples 
collected by subjects/parents after the study visit may be stored in a tightly closed 
specimen cup in a freezer until they are able to return to the research site, to occur 
within 6 weeks of collection. Samples delivered to the research site will be stored in a 
freezer until they can be transferred to the research laboratory and stored at -80°C. 
This sample will be used in future research studies on pediatric nutrition. This sample 
is optional and is not required for the completion of the study visit. 
 
10. 
Fat oxidation estimation: Fat Oxidation may be measured by administering deuterated 
palmitate. Breakfast, lunch and snacks may be provided during this visit to the 
participant to ensure all participants are consistent. After obtaining a baseline urine 
sample, the stable isotopes will be administered. Follow up urine samples will be 
obtained up to 3 times within 48 hours following the administration of the stable 
isotopes. Recommended collection times for the 3 samples will be approximately 2 (± 
30 minutes), 6 hours (± 1 hour) post dosing, and the morning following the study visit, 
depending on the compliance of the child and parent. The urine samples will be 
obtained to measure the labeling in total body water that may be used to estimate 
dietary fat oxidation. The stable tracer (2H31-palmitic acid) may be administered orally 
at a dose of 15 mg/kg in a drink and/or mixed with food (i.e. yogurt, etc.). The parents 
will be asked to collect some of these samples at home. They will be given thorough 
instructions about how to collect, label, record the time and store these samples. A 
specimen collection bowl (hat) may be used for collection of urine. The use of these 
stable isotopes in children to measure fat oxidation has been previously approved 
(IRB# 202870, 202954, 203770). If a baseline urine sample is not collected, then the 
stable isotopes will not be administered and the study visit will not be completed.   
 
11. 
Body composition may be assessed by bioelectrical impedance analysis using the 
Tanita Body Composition Analyzer. A device (InBody 570 Body Composition 
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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
Analyzer) similar to the Tanita, is routinely used in the clinic. The Tanita requires 
participants to stand still with bare feet on the weighing platform while a small amount 
of electrical current is used to measure impedance.   
 
12. 
Resting energy expenditure by indirect calorimetry: A metabolic cart (Microlife 
Medgem Analyzer may be used to make gas exchange measurements (i.e. volume of 
oxygen consumed and volume of carbon dioxide produced by the participant).  
Measurements will be performed in a quiet, temperature-monitored room.  Children 
will be required to lie still without falling asleep for the duration of the measurement.  
The data obtained will be used to assess whole-body substrate oxidation rates. 
 
 
Participants in the obese insulin-resistant and newly prescribed metformin groups may be asked 
to participate in additional visits. The additional study visits will include the same procedures as 
the initial visit. For the newly prescribed metformin group, participants/parents will be asked to 
verify their compliance with metformin by bringing their medication to the visit to show study staff 
and asking the parent/participant to report their compliance. In addition, if a procedure(s) is not 
successfully completed, the parent and, if warranted, the participant may be asked to return to 
the study site for an additional visit to re-attempt the procedure. The decision to re-attempt the 
procedure(s) will be made by the principal investigator and/or co-investigator and the same steps 
as described for the re-attempt of a procedure from the initial visit followed. 
 
• Participants in the obese-insulin resistant group may be asked to complete one 
additional study visits to occur at 12 months (± 2 weeks) after the initial study visit. 
This is to follow changes in bioenergetic and metabolic parameters over time to 
assess validity and will help establish not only the degree of change in the studied 
outcomes, but also their predictive value for T2D development, in addition to 
distinguish between metabolically healthy obese (MHO) and metabolically unhealthy 
obese (MUO) phenotypes. We may request that all obese insulin resistant participants 
complete one additional study visit; however, this additional study visit is optional as 
we do not want to discourage potential enrollment due to the possibility of another visit 
or lengthy visit. We anticipate roughly 43% of the 70 obese insulin resistant 
participants will decline a 2nd study visit with a goal of 40 subjects completing a second 
visit at 12 months (± 2 weeks).  
• Participants in the newly prescribed metformin group who are prescribed metformin 
as part of their clinical care will be asked to complete a second study visit to occur six 
months (± 2 weeks) after beginning metformin therapy prescribed as part of their 
clinical care. We may request that all 20 participants in this group complete the 
additional study visit; however, the additional study visit is optional as we do not want 
to discourage potential enrollment due to the possibility of multiple visits or lengthy 
visits. We anticipate 50% of the 20 participants in this group will decline a 2nd study 
visit with a goal of 10 subjects completing a 2nd visit at 6 months (± 2 weeks). 
 
A participant/parent pair will be compensated $100.00 in gift cards for each study visit completed. 
Completion of the study visit may include participation in parental study forms, anthropometric 
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Title:  Bioenergetics and Metabolism in Pediatric Populations 
PI:  Eugenia Carvalho, Ph.D. 
 
measurements, fat oxidation estimate measured by deuterated palmitate, blood sample, urine 
samples, body composition assessment, and resting energy expenditure by indirect expenditure.     
Because the parent and participant do not have to stay at the study location for the entire visit, 
completion of the study visit would occur once the samples are obtained by the research 
personnel. Parents/participants who are healthy with a normal weight and also healthy obese 
children, will be given a one-time $100.00 in gift cards for participation in the research study visit. 
Parents/participants with T2D or insulin resistance who are prescribed metformin therapy at their 
normally scheduled doctor visit or part of the obese with insulin resistance group will be asked 
to complete two study visits and will be paid up to $200.00 in gift cards ($100.00 in gift cards at 
the first study visit and $100.00 in gift cards at the second visit). For each study visit, a $50 gift 
card will be provided to the study participants/parents after completion of the procedures to be 
completed at the research site (e.g., anthropometric measurements, Tanner Stage Assessment, 
physical activity questionnaire, depression scale, and blood collection) and another $50 gift card 
will be given to the study participants/parents upon return of the study samples and intake of 
food and liquid form from the visit. Those participants/parents who are asked to return to re-
attempt procedure(s) not successfully completed at a study visit (initial or second) will be given 
a $25 gift card after re-attempting to complete the procedure(s). Participants/parents may be 
given up to $20 in ACRI merchandise. Breakfast, lunch and snacks may be provided during 
study visits. 
 
Study Population 
 
Study investigators, research staff, or any qualified personnel will conduct recruitment of study 
participants using IRB approved advertisement. Advertisements may be distributed in the form 
of postcards or flyers via direct mail or to various locations, including pediatricians’ offices, health 
fairs, daycare centers, schools/universities, recreational centers, child retail stores, 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. On-hold phone messages, 
BoomText messages, screensavers, and radio/television advertisements may be used. In 
addition, research staff may contact parents who expressed an interest in our studies or who 
previously agreed to be contacted regarding future studies. The research staff will educate the 
parents and children about the study. We will also work with physicians at ACH to identify 
subjects that may qualify. They will provide study staff with contact information for the patient, 
and study staff will call and educate the parents and children about the study. The physicians 
will introduce the study to their patients; if the patient is interested the physician will obtain 
contact information so that the study staff can follow up and talk to the parents and children 
about the study. In this case, the only contact by study staff would be to introduce the study 
followed by informed consent in person. We will protect all personal information obtained during 
recruitment, enrollment, and testing processes, and maintain this in a locked drawer or cabinet. 
 
175 pre-pubertal children ages 5-9 years old or T2D/insulin resistant children ages 5-17 years 
old who have been newly prescribed metformin will be recruited and may enroll for this study 
with the goal that 130 subjects stratified across the following groups will complete the study: i) 
healthy lean (n=20); ii) healthy obese (n=20); iii) obese insulin resistant (n=70); obese with new 
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metformin prescription(n=20) (children with T2D or insulin resistant who have been newly 
prescribed metformin therapy as part of their clinical care). Every attempt will be made to 
ensure that groups are balanced with respect to sex and ethnicity.  
 
Inclusion Criteria:  
• Age 5-9 years and Tanner stage as reported by parent no greater than stage 1 OR Age 
5 years - 17 years 5 months, diagnosed with type 2 diabetes mellitus or insulin resistance 
and prescribed metformin (not yet taking) 
• Either healthy lean (BMI≥ 5th percentile and <85th percentile for age/sex) or obese (BMI ≥ 
95th percentile for age/sex) 
• For those with BMI≥ 95th percentile for age/sex, parental verbal confirmation will be 
obtained that the child had a history of BMI≥ 95th percentile for age/sex for at least six 
months prior to study enrollment 
 
Exclusion criteria: 
• Genetic or physical conditions impacting mobility over past year as determined by the PI  
• Having known chronic illnesses/disorders that may independently affect study outcome 
measures: type 1 diabetes mellitus, neurologic (e.g. epilepsy), developmental 
(developmental delay, autism spectrum disorder), endocrine (thyroid, Cushing’s), hepatic, 
autoimmune, cardiac and renal disorders.  Also, chronic lung disorders except well 
controlled asthma that does not require permanent use of inhaled/oral steroids 
• Taking any of the following medications that can affect study outcome: antipsychotics, 
thyroid hormone replacement therapy, inhaled/oral steroids, insulin, anabolic drugs 
(growth hormone replacement therapy and oxandrolone) and stimulants 
• Taking metformin prescribed as part of their clinical care at the time of enrollment (may 
begin metformin therapy prescribed as part of their clinical care while enrolled in the 
study) 
• BMI<5th percentile for age/sex (classified as underweight based on CDC growth charts) 
• Subjects determined ineligible by the PI. 
Risks and Benefits 
We do not foresee any adverse events associated with this protocol.  There is the potential risk 
of loss of confidentiality to study participants. Measures to protect the confidentiality of study 
participants will be implemented by applying the appropriate steps to secure the collected data 
as described in the Data Handling and Recordkeeping section below. The proposed stable 
isotopes to measure whole body fat oxidation is non-radioactive and presents no risk to humans. 
There is a small risk that participants will encounter bruising and/or infection after having blood 
taken, however, the use of well-established blood taking techniques, and sterile procedures, by 
trained phlebotomists or nurses, will ensure the risk is minimal. Also, numbing cream and/or a 
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pain relieving device (Buzzy) may be used to minimize the pain associated with the blood draw. 
There is the risk that researchers could develop ways in the future to link genetic information 
back to the subjects even though the samples do not include any personal identifiers, except for 
the assigned unique study code and the study acronym. Thus, we do not anticipate any adverse 
effects associated with the study.  However, any adverse effects or unanticipated problems will 
be reported to the study PI, the IRB, and the study sponsor in accordance with IRB Policy 10.2. 
There will be no direct benefits to the study participants; however, knowledge gained from the 
study could potentially benefit patients in the future. 
 
Data Handling and Recordkeeping 
All study subject material will be assigned a unique identifying code or number.  The electronic 
key to the code will be kept on a secure sharepoint site designed specifically for the study. 
Sharepoint is beneficial in that UAMS Techsource controls access for the site so that only select 
individuals are able to view the site. Only the PI and research personnel will have access to the 
code and information that identifies the subject in this study. 
The PI and research study personnel will complete and maintain appropriate CITI training. 
Source documents and CRFs will be stored in a locked drawer. Documents will be archived 
according to UAMS/ACH/ACNC policies regarding destruction of research records.  
Data will be entered into a research database. Access to the study database(s) is password 
protected and will be limited to study personnel and regulatory authorities. 
At the time of collection, samples will be kept on ice or refrigerated (with the exception of stool 
which will be kept in a freezer) in the pediatric clinical research unit or in Dr. Rose’s laboratory 
(Rm 4015 of ACRI) until they can be transported to the research laboratory. Samples will be kept 
at -80°C once the initial processing and analyses have taken place. Samples will be stored in 
Room N2002 Freezer N2002FT6 or N2002FT18 of ACRI in Dr. Carvalho’s laboratory. These are 
monitored continuously for proper temperature and working condition. None of a subject’s 
personal identifiers will be present on any biological sample, except the unique subject ID and 
the study acronym. The samples may be used until they are used up for research on pediatric 
nutrition.  
 
At any time if the subject decides that he/she does not want to participate anymore, that subject’s 
data will be included as part of the planned analysis of study data. No more information will be 
collected after withdrawal, however all data and samples collected prior to withdrawal will still be 
used. 
 
Information pertaining to this study will be made publically available through the 
ClinicalTrials.gov website. 
 
Data Analysis 
Statistical and Power Analyses: 175 pre-pubertal children ages 5-9 years old will be recruited 
for this study assuming that 130 subjects stratified across the following groups will complete the 
study: i) healthy lean (n=20); ii) healthy obese (n=20); iii) obese insulin resistant (n=70); obese 
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with new metformin prescription (n=20).  A one-factor analysis of variance (ANOVA) will be used 
to compare mean outcome of interest across the groups of children (healthy lean, healthy obese, 
obese insulin resistant and obese T2D). Pair-wise comparisons using Tukey-Kramer post-hoc 
tests will be used to identify groups whose means differ significantly while retaining the family-
wise error rate at 5%. Outcomes will be also checked to verify that the assumptions of the 
parametric tests are met. When substantial deviation from assumptions are encountered, and if 
suitable transformation are not found, groups will be compared using the nonparametric Kruskal-
Wallis rank test, followed by Bonferroni corrected Wilcoxon rank-sum tests to identify groups 
that differ. Generalized linear model (GLM) will also be used to incorporate important covariates 
and effect modifiers into the analysis. 
For Aim 3: A paired t-test will be used to compare the mean change in the various outcomes 
described in SA1 and SA2 from baseline to 12 months for pre-pubertal insulin resistant obese 
children. If the assumptions for the paired t-test are not met, the non-parametric Wilcoxon 
matched-pairs signed-ranks test will be used. Alternatively, generalized linear mixed models 
(GLMM) will be used for Aim 3. GLMM account for the dependence between measurements 
from the same individual taken over time and allow the inclusion of fixed and time varying 
covariates. 
For Aim 4: A paired t-test will be used to compare the effects of metformin on the various 
outcomes on obese T2D/insulin resistant children with a new metformin prescription. If the 
assumptions for the paired t-test are not met, the non-parametric Wilcoxon matched-pairs 
signed-ranks test will be used. Alternatively, generalized linear mixed models (GLMM) will be 
used for Aim 4. GLMM account for the dependence between measurements from the same 
individual taken over time and allow the inclusion of fixed and time varying covariates. 
Sample Size Justification: 
For Aims 1 and 2, statistical power was computed based on a one-factor ANOVA with a total of 
110 children (70 obese insulin resistant, 20 healthy obese, 20 healthy lean).  Assuming 80% 
power and α = 0.05, the minimum detectable Cohen's f effect size (ES(f)) is 0.33. This is a 
medium effect size and should provide valuable information regarding whether a large study is 
warranted.   
For Aim 3, statistical power was computed based on a paired t-test with a total of 40 children. 
Assuming 80% power and α = 0.05, the minimum detectable Cohen's d effect size (ES(d)) is 
0.45 for those who completed the study. These are medium effect sizes and should provide 
valuable information regarding whether a large study is warranted.  
For Aim 4, statistical power was computed based on a paired t-test with a total of 10 children. 
Assuming 80% power and α = 0.05, the minimum detectable Cohen's d effect size (ES(d)) is 
1.0. Although this effect size is large, it should provide valuable information regarding whether a 
large study is warranted. 
 
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 
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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 will be signed by the parent or legally authorized 
representative. Written assent will be obtained from participants age 7-17. A copy of the signed 
consent will be given to the participant, and the informed consent process will be documented 
in each subject’s research record. A signed copy of the consent form may be placed in patients’ 
medical record. If consent is given for samples to be used for future research studies and if 
samples are still being used when a participant turns the age of 18, subjects will not be re-
contacted to sign a consent form and consent will be waived. However, subjects can contact 
study staff after they turn 18 to ask that any remaining samples be removed. In addition, 
participants may be recontacted about participating in future research studies. 
 
Initially, samples will be stored at Arkansas Children’s Research Institute. The information 
collected at the study visit, saliva, blood, urine, stool samples may be stored indefinitely and may 
be used for future research studies on pediatric nutrition. Prior to the information collected at the 
study visit and samples being used for future research studies, the PI will assess the ethics and 
scientific merit of the proposed research with the samples, and proposed future research will be 
reviewed by the IRB as may be required. The samples and health information collected for the 
study visit may be shared with researchers at the University of Arkansas for Medical Sciences, 
Arkansas Children’s Hospital, or Arkansas Children’s Research Institute. The samples may be 
shared with an outside group. The samples will only have a study ID number and study acronym 
to maintain confidentiality.  If participants decide they no longer want us to use their samples for 
future research studies, they may ask the study staff that the sample be removed. If the sample 
has been shared or if publication of results has occurred, then we may not be able to remove 
the sample. In addition, research results from the depression scale may be shared with the 
child’s PCP or if the child is actively followed at ACH the depression scale results may be shared 
with the child’s ACH physician if found to be significant and more information may be provided. 
If the child does not have a PCP or is not actively followed at ACH, then a referral will be made 
to a PCP for follow-up. For example, a score of 15 or greater on the CES-DC suggests that the 
participant expresses symptoms of depression. In this scenario, the child’s PCP or ACH 
physician would be notified of the depression scale result. If the child did not have a PCP, then 
a referral would be made to a PCP near the child’s home and the research results from the 
depression scale would be shared. In addition, the parent would be given information of 
resources to seek help for the child.   
 
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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.   
 
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