Hepatic Fat Content and Mitochondrial Flux in Obese Youth Before and After Bariatric Surgery
Conditions: Hepatic Steatosis, Bariatric Surgery Candidate, Adolescent Obesity, Obesity
Sponsor: University of Colorado, Denver

 
Study Title: 
 
Hepatic fat content and mitochondrial flux in 
obese youth before and after bariatric surgery 
(MANGO Study) 
 
Protocol Version Date: 2/14/2022 
 
NCT03587727 

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w 
COMIRB Protocol 
 
PROTOCOL #: 
18-0479  
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: 
Hepatic fat content and mitochondrial flux in obese youth before and after bariatric 
surgery 
 
Version Date: 2/14/2022  
 
Principal Investigator:  Melanie Cree Green, MD, PhD  
 
 
 
 
  
 
I. Hypotheses and Specific Aims:   
 
NAFLD has become a major public health problem in the United States because of its high 
prevalence, association with serious cardiometabolic complications and progression to cirrhosis 
and liver failure. NALFD is characterized by increased intrahepatic triglyceride content (>5.5% 
of the liver volume or more than 5% of hepatocytes contain triglyceride), and progression to 
inflammation and fibrosis.1-3 NAFLD is associated with increased risk of developing type 2 
diabetes (T2D), hypertension, and coronary heart disease.2,4 Obesity is an important risk factor 
for NAFLD; NAFLD occurs in ~70% of adults with obesity and in ~35% of children with obesity.5-
7  However, data from several studies suggest the histological 
manifestations and progression to fibrosis are different in adults 
and children with NAFLD7.  Differences between the ages suggest 
distinct pathology as i) Youth with NAFLD may progress to liver 
failure fairly rapidly, by early adolescence, as compared to a more 
indolent course in adults and ii) biopsy criteria are distinct with 
periportal steatosis and fibrosis in youth and sinusoidal ballooning 
and fibrosis in adults.7 Alterations in mitochondria function, 
secondary to an influx of substrates from the diet and dysfunction 
muscle and adipose tissue are thought to contribute to NAFLD6,8. 
We hypothesize that alterations in mitochondrial energy and 
substrate metabolism are involved in the pathogenesis and 
pathophysiology of NAFLD and these factors differ in children and 
adults. A better understanding of intrahepatic regulation of energy 
and intermediary metabolism in children and adults with NAFLD is needed to provide a 
framework for future studies designed to prevent and treat NAFLD across the lifespan.    
Nonalcoholic fatty liver disease (NAFLD), characterized by an increase in intrahepatic 
triglyceride (IHTG) content (i.e. steatosis) with or without inflammation and fibrosis (i.e. 
steatohepatitis), is a common complication of obesity. It is estimated that 70% of adults and 
35% of children with simple obesity have NAFLD, with rates of 50% in polycystic ovarian 
disease and even higher in diabetes7,9. In particular, youth with severe obesity who are 
candidates for bariatric surgery have even high rates of NAFLD, in excess of 50%. Further it is 
not clear if bariatric surgery decreases the risk of progression of NAFLD, as the influx of free 
fatty acids during rapid weight loss may actually lead to worsening of NAFLD, if the liver cannot 
metabolize these increased substrates. 
      Data from a series of studies conducted in animal models and in people suggest hepatic 
mitochondrial metabolism is directly involved in the pathogenesis and pathophysiology of 
NAFLD. Animal and human studies have demonstrated that both the anaplerotic and 
cataplerotic cycles of hepatic mitochondria are upregulated in NAFLD.10,11 Excess carbon from 

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surplus calories (glucose or free fatty acids (FFA)) are directed to the TCA cycle in the 
mitochondria, but the TCA cycle cannot serve as a carbon sink, and thus cataplerosis is also 
increased, providing substrates for fatty acid esterification (FAE) and de novo lipogenesis.12,13 In 
addition, increased TCA cycle activity increases the production of reactive oxygen species  
(ROS), which cause inflammation and subsequent fibrosis1 (Fig. 1). Pediatric and adult NAFLD 
are not the same; hepatic steatosis is located primarily in the periportal areas of the liver in 
youth but in perisinusodal areas in adults, and youth typically have more rapid progression to 
fibrosis. The mechanism(s) responsible for these differences is not known, but could be related 
to differences in mitochondrial dysfunction.  Together, these studies underscore the need for 
sophisticated metabolic studies in both children and adults to better understand the factors 
involved in the pathogenesis and pathophysiology of NAFLD.  
      Methods used to assess hepatic mitochondrial energy metabolism include: i) obtaining liver 
tissue and assessing ex vivo mitochondrial ATP production and ii) 31phosphorus magnetic 
resonance spectroscopy (31P-MRS) to measure phosphate concentrations.14  The existing 31P-
MRS procedure is limited because it assesses non-dynamic concentrations of 31P metabolites 
or the response to a specific dietary challenge, but does not provide a direct measure of global 
hepatic mitochondrial energy metabolism.14-16  In addition, this procedure requires excessive 
participant time in the magnet which is burdensome, particularly for children, and expensive. 
Therefore, there is an unmet need for a non-invasive research method that can assess global 
dynamic hepatic mitochondrial energy metabolism.  
    New methods that magnetically remove the γ-phosphate of ATP via inversion transferase 
sequences allow for dynamic measurement of phosphate kinetics.17 This methodology has been 
successfully used to assess ATP production in vivo in skeletal muscle tissue, but has not yet 
been applied to the liver.17 This cross-institution project will allow the development of a non-
invasive in vivo methods to study hepatic energy metabolism. We will combine the novel 31P-
MRS method with concomitant ex vivo analysis of mitochondrial functional capacity by using the 
Oroboros analysis of liver tissue obtained during bariatric surgery to provide a unique and 
comprehensive analysis of hepatic energy and substrate metabolism in human participants 
across the lifespan. Metabolic status will also be categorized at that time. A follow-up study in 
individuals a year later will allow for evaluation of the long-term effects of bariatric surgery on 
NAFLD risk and progression and or regression in this youth population. 
 
In the wake of our national obesity epidemic, bariatric surgery has become a critical 
option for many youth with severe obesity for management of obesity and related complications 
such as risk for type 2 diabetes and NAFLD. Bariatric surgery is very effective in inducing weight 
loss, although the effects on NAFLD are unclear, and may vary by time post-operatively. 
Following bariatric surgery, there is adipose tissue breakdown in the negative energy state, with 
very large influxes of free fatty acids to the liver. These FFA can be oxidized in the mitochondria 
for ATP generation, or stored in the liver18. Especially in individuals who have underlying 
mitochondrial defects in the liver, as is known to occur in NAFLD19, this can lead to excess 
deposition of FFA in the liver, and potential worsening on NAFLD state prior to resolution. One 
limited study in 20 youth with repeat biopsies showed 100 percent improvement in glucose and 
NAFLD markers and 90% improvement in fibrosis, although fill assessment of diabetes risk was 
not performed20. NAFLD status is the best predictor for development of type 2 diabetes in youth 
and thus it is important to assess the 2 components of diabetes risk- insulin resistance and 
insulin secretion. Predictors for improvement of NAFLD beyond weight loss are not clear, 
especially in youth. A better understanding of the post-prandial nutrient handling following 
bariatric surgery is needed to further optimize post-surgery therapies to improve NAFLD. 
 
HYPOTHESIS:  
1) NAFLD status will be improved by 1-year post-bariatric surgery and this may relate to 
post-prandial glucose and FFA concentrations and hepatic mitochondrial metabolism. 

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2) 31P-MRS, blood and liver isotopomer analysis and metabolomics can be used as 
surrogate measures to assess hepatic function in NAFLD 
 
Specific Aims: 
Aim 1: Determine the effects of bariatric surgery on NAFLD status, substrates available for the 
development of NAFLD (glucose and FFA) and surrogate markers of NAFLD status, 1-year 
post-surgery 
Hypothesis: NAFLD status will be better 1-year post-surgery. 
Rationale: Data from adults indicates that NAFLD status can worsen with bariatric surgery. This 
is thought to relate to the excessive free fatty acid delivery to the liver, however, less is known in 
youth with severe obesity, and clinical measures such as ALT, which are not very accurate have 
been used for follow-up testing in youth. 
Methods: A study identical to the one performed pre-surgery will be performed, with the 
exception of the liver biopsy. Alterations in glucose and fat metabolism will be assessed with a 
mixed meal tolerance test (MMT). 
31P-MRS will be used to assess hepatic oxidative phosphorylation, MRI for hepatic fat fraction 
and MR elastography for liver stiffness. Serum for metabolomics for preidentified markers will be 
performed.  
 
Aim 2: Utilize resting  31P-MRS method and stable istopomer analysis to measure hepatic 
mitochondrial metabolism 
Hypothesis: Markers of mitochondrial function can be assessed with 31 Phosphorus MR 
spectroscopy (31 P-MRS) and stable isotopomer analysis 
Rationale: A resting 31 P-MRS can give surrogate measures of mitochondrial function. 
Administration of a glycerol drink with stable isotope tracers and subsequent blood and liver 
analysis of serum glucose and glycerol labelling and liver TCA cycle intermediates can give 
direct results on hepatic metabolism. The MR methodology is non-invasive and the blood 
analysis is finally invasive, as compared to the current need for an invasive liver biopsy.  
Methods: The standard pulse/acquire hepatic 31 P-MRS protocol is already in use in a similar 
patient population (14-0542 and 16-2399) and well tolerated. The oral glycerol tracer has/is 
being used in a similar patient population (16-2399, 18-0803, 19-0636) with and identical blood 
sampling timeline in 18-0803 and 19-0636 . 
 
Aim 3: Determine hepatic mitochondrial activity in vivo using 31P-MRS, isotopomer and plasma 
metabolomic analysis and ex vivo using Oroboros in youth with severe obesity. NAFLD features 
will be further characterized using liver histology to assess steatosis and NASH and by using 
MR elastography to assess global hepatic fibrosis. Alterations in glucose and fat metabolism will 
be assessed with a mixed meal tolerance test (MMT). Comparisons between these surrogate 
markers and biopsy samples will help identify minimally invasive measures to predict NAFLD 
severity without the need for future biopsies. 
Hypothesis:  We hypothesize that alterations in mitochondrial energy and substrate metabolism 
are involved in the pathogenesis and pathophysiology of NAFLD. A better understanding of 
intrahepatic regulation of energy and intermediary metabolism in children with NAFLD is needed 
to provide a framework and identify minimally invasive biomarkers for future studies designed to 
prevent and treat NAFLD. 
Rationale: NAFLD is unfortunately an increasing common complication of pediatric obesity. 
Youth onset metabolic disease confers an early and more severe morbidity and earlier mortality 
than adult onset disease, emphasizing the urgency of treating this population21. 
Methods: We will compare multiple methods of non-invasive and minimally invasive measure of 
hepatic metabolism with the gold standard but invasive liver biopsy. 31P-MRS and isotopomer 
analysis will be used to assess hepatic oxidative phosphorylation, MRI for hepatic fat fraction 

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and MR elastography for liver stiffness. Serum for metabolomics analysis will be collected and 
metabolites which related to MR and biopsy measure identified (targeted analysis for fatty acids, 
acycarnitines and bile acids). Ex vivo measures of mitochondrial function will be measured in 
liver biopsy samples (already being collected during bariatric surgery for clinical NALFD staging) 
with oroboros technologies. NAFLD staging data from the pathology lab will also be collected. 
Alterations in glucose and fat metabolism will be assessed with a mixed meal tolerance test 
(MMT). 
 
III. Research Methods 
 
A.  Outcome Measure(s):   
Primary Outcome Measure(s): Post-prandial glucose, insulin, C-peptide, GLP-1 and glucagon 
dynamics as assessed with an MMTT. 
Secondary Outcome Measure(s):  Hepatic fat content per MR, and biopsy, Stiffness per MR 
elastography,and fibrosis per biopsy. . Exploratory Aims: ex vivo hepatic mitochondrial activity 
per biopsy, isotopomer analysis and Hepatic mitochondrial activity per 31P-MRS. Likely 
contributors to above measures: Lipid/glucose markers:(fasting C-peptide and lipid panel, 
HbA1c); hepatic markers:(c-reactive protein, glucagon, GLP-1 and leptin, adiponectin, AST, 
ALT, GGT) Body size and composition: (BMI, waist/hip ratio, Bodpod, hepatic visceral fat via 
MRI22-25), Whole body fat oxidation at rest as measured with a metabolic cart; Physical activity/ 
diet:(accelerometer, activity survey (3DPAR); Food frequency survey). Questionnaires for 
presence of obstructive sleep apnea. Questionnaires for perceived mental strengths and 
difficulty (note there is no assessment of suicidality on this tool).  
 
B. Description of Population to be Enrolled:   
Study staff aims to enroll 16 obese boys and girls 13-20 years old. 
 
Ethnic Categories 
Gender 
 
Females 
Males 
Total 
Hispanic or Latino 
7 
6 
13 
Not Hispanic or Latino 
6 
6 
12 
Ethnic Categories: Total of All Participants 
13 
12 
25 
  
Racial Categories 
 
 
 
American Indian/Alaska Native 
 
 
 
Asian 
 
 
 
Native Hawaiian or Other Pacific Islander 
2 
2 
 
Black or African American 
2 
2 
 
White 
9 
8 
 
Racial Categories: Total of All Participants * 
13 
12 
25 
 
Inclusion Criteria: 
1. Obese youth (>95th percentile for age and sex) ages 13-20 years, 
scheduled for bariatric surgery 
2. BMI 35-60 kg/m2 
3. Largest body circumference <200cm (in order to fit into MRI machine) 
 
Exclusion Criteria: 
1. Use of medications known to affect insulin sensitivity or hepatic outcomes: 
oral glucocorticoids within 10 days, atypical antipsychotics, 

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immunosuppressant agents, HIV medications, PPAR-γ or PPAR-α, 
metformin. 
2. Infectious hepatitis 
3. Alcohol abuse, (defined by the America Association for the Study of Liver 
Diseases as >21 drinks/week for males >14 drinks/week for females, with a 
drink considered 14 grams of alcohol) 
4. Mitochondrial disease 
5. Currently pregnant or breastfeeding women. Development of pregnancy 
during the study period will necessitate withdrawal from the study. 
6. Severe illness requiring hospitalization within 60 days 
7. Diabetes, defined as Hemoglobin A1C > 6.4% 
8. Anemia, defined as Hemoglobin < 10 mg/dL 
9. Diagnosed major psychiatric or developmental disorder limiting informed 
consent 
10. Implanted metal devices that are not compatible with MRI 
 
C. Study Design and Research Methods   
The study consists of 3 outpatient visits. The Baseline Visit would be completed within two 
months prior to bariatric surgery, and the Post-surgery visit will be scheduled approximately 1-
year post-surgery. 
*The glycerol drink will be taken the day of the Baseline visit, and in the morning of operation, 
then 1 year later  
NOTE: Depending on scheduling and availability, the MRI scans can be completed on a 
separate visit within 10 days of the MMTT. 
 
SCREENING VISIT  
Pediatric CTRC Outpatient Unit:  Chart review of bariatric patients will be conducted for 
inclusion/exclusion criteria evaluation. Participants whom appear to meet eligibility criteria would 
then be approached about a screening visit.  During this visit, patients will review and complete 
consent documents, have demographics and medical history confirmed, assess allergies and 
further evaluation of inclusion/exclusion criteria, and have anthropometrics completed for 
eligibility assessment.  
Study Calendar 
Screening 
Visit 
Baseline Visit:  
MMTT and MRI 
Post-Surgery Visit:  
MMTT and MRI 
Consenting and Eligibility Assessment 
X 
 
 
History & Physical 
 
X 
X 
Urine Pregnancy Test (females only) 
 
X 
X 
Questionnaires- SEARCH Food 
frequency, Activity 3DPAR, Strengths and 
Difficulties and Sleep assessments 
 
X 
X 
Bodpod 
 
X 
X 
Mixed Meal Tolerance Test (MMTT) 
 
X 
X 
MRI of abdomen and liver  
 
X 
X 
31P-MRS of Liver  
 
X 
X 
Metabolic Cart 
 
X 
X 
Glycerol drink - optional 
 
X* 
X 
Total Time of visit (approximately) 
30 minutes 
6-8 Hours 
6-8 Hours 
Location of Visit 
CHCO CTRC 
Outpatient 
CHCO CTRC 
Outpatient, and Brain 
Imaging Center 
CHCO CTRC 
Outpatient, and Brain 
Imaging Center 

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BASELINE VISIT 2 (FIGURE 2: MMTT AND MRI) 
 
 
Overall Plan:  
MRI at UCD Research (Brain Imaging Center):  
1. The imaging will be of the liver. Participants will undergo a standard MRI of the 
mid-abdomen to assess the amount of subcutaneous fat, visceral fat and percent 
liver fat. MR elastography will be performed to assess liver stiffness. Finally, 31P 
spectroscopy to measure concentrations of phosphate molecules including PDE, 
PME and PCr. 
 
Abdominal/Hepatic Imaging and Elastography 
Imaging will be performed on a Siemens 3 Tesla MRI magnet. Hepatic fat fraction will be 
performed using modification of the Dixon method as in our previous studies9,26. Visceral 
adiposity will be measured using the gold standard of an MRI slice at L4-L59. Hepatic 
fibrosis will be measured with a standard clinical elastography sequence. 
 
 
31P MRS of the Liver  
MRS Data acquisition: MRS will be performed on a Siemens 3 Tesla MRI magnet with 
research capabilities. A custom 1H/31P abdominal coil will be used for imaging and MRS 
(Clinical MR Solutions, Brookfield, WI) as in our previous 31P work27. The coil has a 
concentric probe with an inner coil 16-18 cm in diameter (for 31P) and a 20 cm outer coil 
(for 1H scout imaging and shimming). Resting phosphate concentrations can be 
measured with a pulse/acquire sequence.  
 
MRS Data Analysis: For the 31P data, peak positions and areas of interest 
[phosphocreatine (PCr), inorganic free phosphate (Pi), β-ATP(3 peaks), α-ATP(2 peaks), 
γ-ATP(2 peaks), and PME] will be determined by time domain fitting with jMRUi 28,29, 
utilizing AMARES (A Method of Accurate, Robust and Efficient Spectral fitting), a 
nonlinear least-square-fitting algorithm using our previously built prior knowledge files 30. 
Figure 2: Baseline Visit 
 
 

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We have utilized this method for muscle 31P analysis for the previous 6 years, and have 
extensive experience with this analysis.  Percent PME relative to all other phosphate 
peaks will be calculated.  
 
2. MMTT at Pediatric CTRC Outpatient unit:  Participants will be asked not to have caffeine or 
exercise for 3 days prior to this visit. After an overnight fast, participants will ingest a UC13 
glycerol tracer, then 3 hours later a liquid meal consisting of (45g carbohydrates,14g fat, and 
14g protein). The metabolic response to the tracer will be measured for 3 hours fasting, and 
then the MMT will be measured for 4 hours. Body composition will be measured by BodPod.  
Accelerometer: One week prior to the MMTT visits, the participant will be provided two 
accelerometers (GT3X BT by Actigraph and ActiWatch by Philips Respironics) to be 
worn for seven days to measure level of habitual physical activity, which affects insulin 
sensitivity, and sleep patterns. Accelerometers are effective tools for the objective 
measurement of physical activity 31 because they have the ability to continuously record 
physical activity data and such data can be used to estimate METs of activity. They 
provide more detailed information than pedometers, which only measure walking steps, 
and help get around the recall bias of questionnaires. We are currently using the GT3X 
BT Actigraph in adolescents in our other diabetes studies; therefore, we are familiar with 
their use in this population and have the necessary computer software and interpretation 
skills. The Actiwatch is being used as a tool for objective measurement of sleep patterns. 
The Actiwatch is fitted with a LED monitor that detects multiple spectrums of light to 
better assess sleep patterns in this population. 
 
*One week prior the MMTT, participant will receive via mail the accelerometer, sleep watch, 
sleep diary, and food log.  
 
 *Note: The day of the surgery, participants will injest the UC13 glycerol (small volume 30 ml, 
clear) 2 hours prior to anesthesia time. This is within the standard pre-surgical patient 
recommendations for small volume of clear liquids up to 2 hours pre-anesthesia. Participants 
will have a liver biopsy during their bariatric surgery as part of their clinical standard of care. 
Participants will be asked to give their consent for this study to use a small sample of the liver 
tissue for research purposes. We will also collect approximately 7 teaspoons of blood during 
surgery to measure glucose, insulin glycerol, FFA, triglycerides and isotopomer analysis at the 
time of the liver biopsy.  
 
POST-SURGERY VISIT 3 (FIGURE 3: MMTT AND MRI):  
Same procedures as Baseline Visit will be repeated 1-year post-surgery (with exception of liver 
biopsy sample). 

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Purpose for lab test to be drawn: 
MMTT Labs 
Purpose 
Glucose  
Determination of IR 
Insulin 
Determination IR and beta cell function 
FFA 
Measure of lipolysis 
Glycerol 
Measure of lipolysis 
Glucagon 
Gut hormone known to influence meal response glycemia 
GLP-1 
Gut hormone known to influence meal response glycemia 
GGT 
Marker of hepatic disease 
C-peptide 
Measure of beta cell function, insulin clearance 
CMP 
Markers of hepatic disease (Alk phos, ALT, AST, albumin) 
Lipid panel 
Markers of serum lipids, hepatic function 
HbA1c 
Long term marker of glycemia 
Stored blood 
Future markers of glucose and fat metabolism, CVD or hormones 
related to NAFLD 
Isotopomer analysis 
Hepatic mitochondrial function 
 
 
 
 
Figure 3:   Post-Surgery Visit 
 
 

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Purpose for questionnaires being done: 
Questionnaire  
Description 
Study Validity 
Adolescent Sleep 
Hygiene Scale 
Measurement of 
sleep 
patterns/habits 
Cronbach’s alpha ranges from .46-.74; total scale alpha = 
.80 
LeBourgeois et al. 2005. The Relationship Between 
Reported Sleep Quality and Sleep Hygiene in Italian and 
American Adolescents 
Center for 
Epidemiological 
Studies Depression 
(CES-D) 
Measuring for 
depression 
See Table 3 below, adapted from: 
Stockings et al. 2015. Symptom screening scales for 
detecting major depressive disorder in children and 
adolescents: A systematic review and meta-analysis of 
reliability validity and diagnostic utility 
Cleveland 
Adolescent 
Sleepiness 
Questionnaire 
Measurement of 
sleepiness during 
a typical week 
alpha = .89 
Spilsbury et al. 2007. The Cleveland Adolescent 
Sleepiness Questionnaire: A New Measure to Assess 
Excessive Daytime Sleepiness in Adolescents 
SDQ: Strengths and 
Difficulties 
Questionnaire 
Behavioral 
screening 
questionnaire 
The internal reliability of the various self-report scales was 
assessed using Cronbach’s alpha coefficient. This was 
0.82 for the total difficulties, 0.75 for emotional symptoms, 
0.72 for conduct problems, 0.69 for hyperactivity, 0.65 for 
prosocial behavior, and 0.61 for peer problems. 
Goodman et al. 2003. The Strengths and Difficulties 
Questionnaire: a pilot study on the validity of the self-report 
version 
Sleep Disturbances 
Scale for Children 
Gain 
understanding of 
sleep-wake 
rhythm and any 
problems in sleep 
behavior 
Internal consistency ranged from .71-.79l test-retest 
reliability r = .71 
Bruni et al. 1996. The Sleep Disturbance Scale for Children 
(SDSC)Construct ion and validation of an instrument to 
evaluate sleep disturbances in childhood and adolescence 
3DPAR: Activities 
Scale 
Measuring 
activity in the 3 
days previous as 
a typical activity 
score 
Interrater and test-retest reliability was 0.99 and 0.98, 
respectively (P < 0.01). The correlation between relative 
energy expenditure from the PDPAR (kcal.kg-1.l.d-1) and 
pedometer and Caltrac counts was 0.88 (P < 0.01) and 
0.77 (P < 0.01), respectively. The correlation between 
percentage heart rate range (HRmax-HR-rest) and mean 
energy expenditure from the PDPAR was 0.53 (P < 0.01). 
The correlation between 1-min heart rates > 50% HRR 
sustained for 20 min and the number of 30-min blocks with 
a relative energy expenditure of at least four metabolic 
equivalent tasks (MET) was 0.63 (P < 0.01). The PDPAR 
provides valid and reliable estimates of physical activity 
and also accurately identifies bouts of moderate to 
vigorous activity. 
Weston et al. 1997 Validation of an instrument for 
measurement of physical activity in youth 
 

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Food Frequency 
Questionnaire 
Measuring typical 
food intake over 
previous seven 
days. 
The mean correlations, adjusted for measurement error, of 
food groups and nutrients between the FFQ and true usual 
intake were 0·41 and 0·38, respectively, with 57 % of food 
groups and 70 % of nutrients exhibiting correlations >0·35. 
Correlations were high for low-fat dairy (0·80), sugar-
sweetened beverages (0·54), cholesterol (0·59) and 
saturated fat (0·51), while correlations were poor for high-
fibre bread and cereal (0·16) and folate (0·11). Reliability of 
FFQ intake based on two FFQ administrations was also 
reasonable, with 54 % of Pearson correlation coefficients 
≥0·5. Reliability was high for low-fat dairy (0·7), vegetables 
(0·6), carbohydrates, fibre, folate and vitamin C (all 0·5), 
but less than desirable for low-fat poultry and high-fibre 
bread, cereal, rice and pasta (0·2-0·3).  
Liese et al. 2015 Relative validity and reliability of an FFQ 
in youth with type 1 diabetes 
First described in 2006:  
Mayer-Davis et al. 2006. Search FFQ Dietary Intake among 
Youth with Diabetes: The SEARCH for Diabetes in Youth 
Study 
Optional 7 Day Food 
Log 
Optional record 
of 7-day diet to 
aid in food recall. 
Added per 
participant 
request to help fill 
out food 
frequency 
questionnaire. 
N/A 
Actigraphy Daily 
Sleep Diary  
 
Recording 
bedtime/wake 
time during 
actigraphy. 
Needed to 
corroborate 
watch collected 
data 
N/A 
   
PARTICIPANT RECRUITMENT/CONSENT/PAYMENT 
1. Participant Recruitment Plan 
Participants will be recruited from the bariatric surgery clinic at Children's Hospital Colorado. 
The PI and Co-I’s have a treatment relationship with the bariatric surgery patients from clinic or 
participants can call from study advertisements. Protected health information will only be 
accessible by study investigators. The initial patient contact will be made by personnel who 
have a treatment relationship with the participant if a chart review by treating physician reveals 
that they may qualify for the study.  
 
2. Informed Consent Plan 
Appropriately qualified and informed personnel who have completed the COMIRB and HIPPAA 
course requirements will fully explain the study protocol and consent form to the participant and 
guardian verbally in the language they understand.  The explanation will be conducted in a quiet 

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environment with adequate time given for the participant and guardian to review the study 
procedure before the commencement of the study.  Asking the participant to explain the study in 
their own words will assess the participant’s understanding.  If non-English speaking 
participants are enrolled in the study, the investigators will adhere to Section 10C of the 
COMIRB Instructions for Clinical Investigators regarding the consent of these participants. The 
consent form will also be translated into Spanish. The qualified personnel mentioned above will 
then obtain written consent from the guardian and assent from the participant, co-signed on the 
consent form, or in participants who are 18 years old, direct consent. The participant and 
guardian will be provided a copy of the consent form for better understanding and record 
purposes.  
Special Consent/Assent Plan 
Consent will be obtained from all participants in the study.  Following explanation, all 
participants under 18 years old will co-sign the consent form in addition to the parent/legal 
guardian signing the consent form.  All participants age 18 years old will sign the standard 
consent form. 
Participant Compensation, Incentives and Rewards  
Participants will be compensated with Target gift cards. They will receive a total of $100 for 
completing Baseline Visit: MRI/BodPod ($50) and MMTT/questionnaires ($50). Post-surgery 
visit is also a total of $100: MRI/BodPod ($50) and MMTT/questionnaires ($50). 
Total paid to participants for completing the full study is $200, but there may be additional 
payments listed below (max payment to one participant is $600). 
Participants may be paid an additional $50, if they have to come for an extra visit (ie MRI). For 
participants who live 50-99 miles from Children's Hospital Colorado, some travel expenses 
(mileage) may be eligible for reimbursement up to a maximum of $50. If they live 100 or more 
miles from Children's Hospital Colorado, some travel expenses (hotel stay, mileage, or a plane 
ticket) may be eligible for reimbursement up to a maximum of $300. 
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
 
Please note: with the emergence of COVID-19 we must follow hospital policies for screening 
hospital patients and visitors. This may include testing for COVID-19. This may involve a nasal 
swab, and the risks of this are discomfort and potentially bleeding from nose. To limit participant 
time in our clinics, portions of study visits may be done virtually (phone or Telehealth). 
 
1. IV Risks and Blood Sampling 
Description: One peripheral IV will be placed to draw blood during the MMTT. Blood will be 
drawn for labs relating to glucose and fat metabolism. 
Risk: Risk of pain, bruising at site of blood draw. There is temporary discomfort when the needle 
goes in and 10% of the time there is a small amount of bleeding under the skin that may 
produce a bruise. Rarely, there is a risk of a blood clot forming or infection.  We will use a low 
dose of a medication called heparin to try to prevent blood clotting. 
Justification/Minimization: Our MMTT visit is within the NIH Clinical Center guidelines of 9 ml/kg 
in 6-8 weeks and within Children’s Hospital Colorado’s institutional guidelines of 5 ml/kg. These 
studies involve sampling blood at multiple time points. Thus, an IV is needed, so as to avoid 
multiple needle sticks. These studies are focused on measured rates of change which 
necessitates the sampling of the same test over time. Proper sterile technique will be used with 
blood draws and IV placement to decrease the infection risk. EMLA cream will be offered if 
participant desires to minimize pain of IV. If the patient is being admitted that same day for 

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bariatric surgery this IV can be left in place, so that when patient is admitted for their bariatric 
surgery, a new IV does not need to be started. 
 
2. Mixed Meal Tolerance Test (MMTT): 
Description: An MMTT will be performed with multiple blood draws. The purpose of the MMTT is 
to provide a controlled oral stimulus to effect changes in glycemia, pancreatic and gut hormones 
and lipolysis.  
Risk: There are no known risks for an MMTT, but participants may not like the taste of the drink 
and may rarely experience nausea after consuming the drink.  
Justification/Minimization: Our team of investigators, CTRC pediatric research nursing staff and 
physicians are well experienced with similar blood draw procedures. A CTRC nurse will be 
available during our outpatient visits and patients will be distracted by TV or other similar means 
during the MMTT, to minimize queasiness. 
 
 
3. Magnetic Resonance Imaging (MRI) and MR elastography and MR spectroscopy 
Description: The MRI will usually be obtained the day of MMTT at the UCD Brain Imaging 
Center on the Fitzsimmons campus. A trained research radiographer who is supervised by Dr. 
Mark Brown, of UCD radiology, will perform an abdominal MRI to obtain hepatic, visceral and 
subcutaneous fat on a 3.0 T whole–body MRI scanner (Siemens MAGNETROM, Malverne, PA). 
Participants will lie supine while these measurements are obtained, need to hold reasonably still 
during the scans of approximately 1 hour and cannot weigh >550 lbs or have a largest body 
circumference > 200 cm. A standard research sequence to measure hepatic fat is performed ( 
approx. 10-15 min). A standard clinical MR elastography sequence to measure the amount of 
fibrosis (if any present) in the liver will be performed (approximately 5 min). A specialized 
phosphorus coil will be utilized to measure the concentration of 31P via MR Spectroscopy to 
assess mitochondrial function (approximately 20-30 min). 
Risks: Minimal. Participants may develop claustrophobia in the magnet. 
Minimizing Risk: The participant is provided with audio protection and optional television to help 
increase comfort. Some participants might feel claustrophobic while having an MRI and the 
scan will be stopped if it cannot be tolerated. In addition, any participants with implanted metal 
that is not cleared by the MRI technician may not be able to have the MRI due to the type of 
magnet involved. 
Justification/Minimization: MRI is a non-invasive and non-radiation method to assess body fat, 
and mitochondrial function. The risks are minimized by assuring patient comfort prior to starting 
the scan, placing eye goggles that plays movies on the participants. Further, per standard 
protocol, no patient will be placed into the scanner if they do not meet the rigorous safety 
standards for the MRI, including the absence of non-compatible implanted metal. 
 
4. Body Composition 
Description: Participants will also undergo body composition analysis by BodPod by standard 
methods. BodPod is a noninvasive air displacement plethysmograph which uses whole-body 
densitometry to determine fat and fat-free mass. Participants sit inside of the BodPod for 
approximately 2 minutes and rest while measurements are taken.  
Risk: Minimal. Participants may feel claustrophobic while in the BodPod. 
Justification/Minimization: There is a large glass window in which the participant can see out of 
and there is also a button located inside the chamber that can be pressed if the participant wishes 
for the procedure to stop and exit the BodPod. 
 
5. Body fat distribution   

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Description: Height, weight, waist circumference, and hip circumference will be measured. Body 
fat distribution will be determined using the waist-to-hip ratio where the waist circumference is 
measured 1/2 the distance from the xiphoid process to the navel and the hip circumference is 
measured at the level of the greater trochanter. 
Risk: None 
Justification/Minimization: IR has been associated with central obesity. Whereas we are 
measuring central obesity with MRI, it is important to see if this simple non-invasive measure 
matches the MRI results, as it is a much simpler measure to follow clinically. 
 
6. Accelerometer:  
Description: Participants will be provided two accelerometers (GT3X BT by Actigraph and ActiWatch 
by Philips Respironics) to be worn for seven days to measure level of habitual physical activity, which 
affects insulin sensitivity, and sleep patterns. The accelerometers will be worn on the participant’s 
wrist. The wrist position has been validated to hip position actigraphy in this population. 
Risk: There is no risk involved with the accelerometer. 
Justification/Minimization: Accelerometers are effective tools for the objective measurement of 
physical activity 31 because they have the ability to continuously record physical activity data 
and such data can be used to estimate METs of activity. They provide more detailed information 
than pedometers, which only measure walking steps, and help get around the recall bias of 
questionnaires. We are currently using the GT3X BT Actigraph in adolescents in our other 
diabetes studies; therefore, we are familiar with their use in this population and have the 
necessary computer software and interpretation skills.  The Actiwatch is being used as a tool for 
objective measurement of sleep patterns. The Actiwatch is fitted with a LED monitor that 
records multiple spectrums of light to better assess sleep patterns in this population. 
 
7. Indirect Calorimetry with a metabolic cart: 
Description: The metabolic cart measures the amount of air that the participant breathes in and 
out. The machine attaches to the participant’s mouth through a tube, or a plastic bubble that is 
placed over the participant’s head. There is the potential for experiencing claustrophobia from 
having the plastic bubble over the participant’s head. A metabolic cart will be utilized during the 
MMTT study day to measure rates of oxygen consumption and carbon dioxide release. These 
rates can be utilized to calculate rates of carbohydrate and fat oxidation and resting energy 
expenditure. 
Risk: Minimal risk of claustrophobia. 
Justification/Minimization: These studies are well tolerated by youth, and involve placing a clear plastic 
hood over the participant’s head for approximately 20 minutes. This procedure has been well tolerated 
in the youths we have studied. 
 
8. Liver biopsy.  
Description: A liver biopsy will be collected at the time of bariatric surgery. This tissue will be 
used for pathology scoring, isotopomer analysis and for oroboros. 
Risk: The biopsy will only be collected as part of standard medical care and therefore no 
additional risk is posed by the newly proposed study. Risks of this procedure will be reviewed 
with participants by the surgical team. 
Justification: NALFD is high in severe obesity, and the liver biopsy is collected for clinical 
purposes. The amount of tissue required for oroboros is minimal, and does not change the 
biopsy procedure. 
 
9. Food Frequency Questionnaire (SEARCH FFQ) 
Description: Customary macronutrient pattern will be ascertained by diet interview at the time of 
admission using a SEARCH FFQ, modified to incorporate common food choices among 

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ethnically and regionally diverse youth aged 10-19 participating in another large childhood 
diabetes study, SEARCH (48).  The instrument is self-administered with staff support to provide 
instructions, answer questions, and to review the form after completion, and captures the last 
week of dietary intake.   
Risk: None 
Justification/Minimization: Several of the measurements being assed are affected by prior 
nutritional intake.  
 
10. 3DPar Questionnaire  
Description: A questionnaire (3DPAR) recalling the physical activity levels of the three previous 
days will be completed at visit 3. 
Risk: None 
Justification/Minimization: Physical activity can directly affect insulin sensitivity, our primary 
outcome measure. The 3DPar is a well validated measure to assess 3 days of physical activity in 
youth, and includes a variety of youth centric activities. 
 
11. Strengths and Difficulties Questionnaire: 
Description: This is a survey which identifies areas in a youth’s life that they believe they are strong 
or weak in dealing with, as a measure of coping skills. Low coping skills have been associated with 
the development of depression. 
Risk: None 
Justification/Minimization: This survey can help identify youth at risk for depression or anxiety, and 
identify poor coping skills. It does not directly assess depression or suicidality. 
 
12. Stable Isotope Studies Glycerol:  
Description: Oral stable isotope tracer of glycerol will be utilized to determine rates of 
intrahepatic substrate flux. These are substances normally present or produced in the body, and 
thus pose no more risk than typical glucose infusions. Measurements of these metabolic 
processes are only able to be made with the utilization of stable isotope tracers. 
Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing 
the percentage. We are only giving this medication orally. These are NOT radioactive 
substances. 
Justification/Minimization: Pyrogen-free ²C13 glycerol will be obtained from the manufacturer 
and delivered to CHCO IDS. The IDS pharmacist will deliver the tracer to the pediatric CTRC 
(inpatient unit or 3rd floor outpatient) once ordered by the physician. 
 
 
13. Violation of Privacy and Loss of Confidentiality  
Description: These are both risks to which research participants are exposed. The possibility of 
these risks increases when protected health information is collected. Every effort will be made to 
decrease this risk by limiting access to protected health information, storing this information in a 
password protected database, and identifying participants only by a unique identifier that is kept 
in a separate location in a locked container, traceable only by study personnel. All of the tests 
involve the risk of identifying asymptomatic abnormalities. The study may include risks that are 
unknown at this time. 
Justification/Minimization: Every effort will be made to decrease the risk of loss of confidentiality 
by limiting access to protected health information, storing this information in a password 
protected database, and de-identifying study specimens.   
 

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E. Benefits of the study: 
Benefits to Society: 
This study has several potential benefits to society. We will be comparing less reliable non-
invasive clinical measures of NAFLD or invasive clinical NAFLD measures with multiple 
research methods of assessing NAFLD. Whereas we are utilizing a bariatric population for this 
study to take advantage of the clinical indication for a liver biopsy, the would could potentially be 
translated into numerous other populations which experience higher rates of NAFLD, such as 
those with obesity, polycystic ovarian syndrome, obstructive sleep apnea, and of Hispanic 
heritage. We will also be obtaining data on the post-bariatric surgery meal response in youth, to 
better understand how this procedure affects multiple aspects of metabolism and potential 
diabetes risk. 
Knowledge to be gained: 
A better understanding of how NAFLD in severe obesity could lead to more effective treatment 
strategies. This understanding could ameliorate development of diabetes and heart disease for 
these individuals. Since severe obesity is becoming more common, improving severe obesity 
care could have major health implications. The data to be generated from this project will be 
utilized to inform R01 grant applications of treatment trials with new medications.  
Benefits to participant: 
The participant will undergo a metabolic cart which will determine basal metabolic rate. This 
information can be used by the medical team post-surgery to help determine the optimal caloric 
intake to assist with weight loss post-surgery. 
The participant will have a thorough evaluation of their hepatic function. If abnormalities are 
noted, we will let the post-bariatric surgery team know, as this could influence their post-bariatric 
surgical care.  
With the MMTT, we can identify if the participant has pre-diabetes. This will not occur otherwise 
clinically, and this again may change the post-surgical screening, and would be brought to the 
attention of the clinical team.  
 
Evidence of Direct Benefit 
We believe that this protocol is in the 405 risk category for pediatric research. Participants can 
benefit from the above study measurements.  
Adolescents with severe obesity are at increased risk for diabetes, reduced exercise capacity, 
cardiovascular dysfunction, and increased visceral and liver fat. Therefore, obese non- severe 
obesity participants may benefit from the results of body composition, basal metabolic rate 
testing, MRI testing, glucose testing, insulin resistance assessment during baseline and 1-year 
post bariatric surgery. The metabolic cart testing can detect the basal metabolic rate, which can 
determine a personalized goal for target caloric intake following surgery. The MRI of the liver 
can detect early evidence of fatty liver disease or fibrosis. The blood tests done for screening 
can detect alterations in blood glucose and prediabetes. The risk of all of these endpoints is 
increased in sedentary participants, especially those who are also obese, and if left untreated 
can increase long term health risk, thus the benefit of detecting any of these would directly 
impact both the health and the longevity of the individual participant. 
 
F. Alternative Treatment 

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The alternative is for participants to not participate in the study 
G. Consideration of Specific Participant Categories 
1. Inclusion of Women 
Every effort will be made to include a diverse gender distribution. 
2. Inclusion of Minorities 
Every effort will be made to include a diverse participant distribution. Severe obesity affects 
Caucasians, Hispanics and African Americans equally. 
3. Inclusion of Children 
All participants will be between ages 13 and 20. Insulin sensitivity needs to be studied in the 
adolescent age group as available is scarce in this age group and it is critical to understand the 
pathophysiology of NAFLD in its developing stages.  
IV. Potential Scientific Problems:   
Limitation of Method Development: 
The protocol as described includes an OSTT with an oral tracer will be conducted in some of the 
participants. The specific points that are to be evaluated are listed below: 
1) Measurement of Hepatic Substrate flux in youth during fasting. 
a. Purpose: Asses hepatic substrate flux in youth looking at shifts from pentose 
phosphate pathway (PEP), TCA cycle and fatty acid synthesis 
i. Rational: utilizing and oral glycerol model with NMR isotopmer analysis is a newer 
technique. We have validated the timing with an OSTT in youth. However, this relies 
on consistent OSTT responses, and in this study we will be using an MMTT. When 
we anticipate that the response to the MMTT may change pre/post surgery, and the 
timing and thus results would vary, and we are thus better off using a fasting model. 
This has been done in adults, but not in our youth. 
ii. Measurement: NMR isotopomer analysis with pathway flux calculations and modeling 
in the fasting state 
iii. Revision plan if model not correct: 1) increase the amount of tracer given 2) increase 
the frequency of blood draws in the later sampling period. 
 
Overall Project Limitations: 
1) Participant recruitment is always a potential concern, but is minimized by a streamlined 
recruitment system and experience with this population; the previous, more intensive 
study is well tolerated in youth and enrolled faster than projected. The bariatric surgery 
clinic is becoming established, and referrals are increasing rapidly, now more than 10 a 
month. Of the surgeries completed in the last 5 months since the start of the clinic, 15 of 
the youth would have potentially qualified for this protocol. 
2) Participant drop-out: We expect reasonably good completion rates due to the minimally-
invasive nature of the study visits and our low dropout rate in our previous studies in 
control and severe obesity adolescents using similar procedures. 
3) Underpowered: This is a pilot study and is likely underpowered to find associations 
between changes in metabolism and NAFLD status. However, the data generated from 
this protocol could then be used to apply for funding to continue the study in a larger 
cohort of youth, to fully answer some more of the mechanistic questions. If we can 
obtain additional funding we will revise the protocol to include more participants. 
 
V. Data Management and Security Plan 

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Data Entry   
Data will be entered from paper forms. Once forms are completed, verified and corrected for 
inconsistencies, they will be manually entered at our site using a computerized data 
management system (Redcap).  
Edit Checks    
Computerized data validation routines will be used to enhance data quality and verify the 
accuracy of data within predefined value ranges. These checks include, but are not limited to: 
(a) initial screening of data, using logic and range checks built into data entry screens; (b) cross-
form functional and consistency checks; and (c) edits assessing the serial integrity of data. 
Disaster Recovery    
Routine data backup will occur on data in conjunction with the Children’s Hospital secure server 
and Redcap. 
Security and Confidentiality   
All hard copy forms will be de-identified with a study number and filed in a locked in a place in 
which only the investigators and qualified study team will have access. Standard protection 
against computer hackers is implemented. Recovery from natural disasters (water, fire, or 
electrical) can occur through the ability to reconstruct both the database management system 
and the data from nightly backups.   
VI. 
Data and Safety Monitoring Plan 
 
The principal investigator and study coordinator will monitor the protocol and the safety of the 
research participants.  The PI will review all laboratory data and report any abnormal values to 
the patient and guardian and instruct the participant to follow-up with their PCP.  If an abnormal 
result from a research procedure exists, the PI will notify the family, the bariatric surgery team 
and their PCP and refer the participant to the appropriate clinic for further evaluation. The PI 
may also share research results in a reasonable and prudent manner with appropriate medical 
professionals if the participant was seriously injured as a result of a procedure or if follow-up of 
the result of the procedure is in the best interest of the participant’s health as determined by a 
medical professional. If immediate medical follow-up of participant required, the PI will share the 
research results via EPIC when clinically relevant. The PI will report adverse events, and any 
decision to suspend or halt the protocol to CTRC and COMIRB immediately.  The PI will also 
prepare a written report for the yearly continuing review required by COMIRB and the CTRC.  
There are no other entities that require notification about this protocol.   
 
No protected health information will be collected until the appropriate HIPAA forms are 
completed. The protected health information that will be collected will include: Name and phone 
number, demographic information (DOB, sex, ethnicity, address, etc.), diagnosis(es), history 
and physical, laboratory or tissue studies, radiology studies, procedure results, 
survey/questionnaire results, research visit records, and portions of previous Medical Records 
that are relevant to this study. This information will be accessible only by the study investigators, 
Federal agencies overseeing human participant research, the Colorado Multiple Institutional 
Review Board, regulatory officials from the institution where the research is being conducted to 
monitor safety and compliance with policies. 
 
 
A. Adverse Events (AE)  

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The MMTT is a standard procedure used in a large number of research studies and settings.  
Adverse events are uncommon when the procedure is done by experienced personnel in an 
appropriate setting.   
 
MRI procedures are considered safe as long as the participant does not have implanted metal. 
All participants undergoing screening by trained MR personnel prior to any scanning 
procedures. 
 
1. Adverse Event Definition 
For the purposes of this study, an Adverse Event (AE) is defined as any significantly abnormal 
physical finding identified on examination and any significantly abnormal laboratory result 
obtained on the patient between visits or at the time of the visit.  Questions answered YES and 
any new abnormal physical findings are pursued by the study staff in order to determine the 
seriousness of the event and the need for further evaluation, follow-up, or referral. If follow-up or 
referral of abnormal research results or procedure is required, the PI will place a referral in 
EPIC, update the PCP on patient condition and reason for referral and contact family to discuss 
follow-up treatment options. 
a) Adverse Event Reporting 
AEs are reported on a standard form that is completed by the study staff at each regular follow-
up visit and phone interview.  Adverse events reported or ascertained between clinic visits are 
captured and reported at the time of the next scheduled visit. 
 
Pre-existing conditions (that is, conditions present prior to study enrollment) are not considered 
or recorded as AEs or SAEs unless the condition worsens in intensity or frequency after 
enrollment.  Likewise, continuing adverse events are not reported as AEs at subsequent visits 
unless they increase in severity or frequency between the visits, they result in criteria for an 
SAE, and/or they resolve between visits. 
B. Serious Adverse Events (SAE) 
1. Serious Adverse Event Definition 
Events are divided into those that are not serious (AE) and those that are serious (SAE).  The 
distinction between an SAE and an AE is a regulatory definition established by the FDA, not a 
clinical definition.  The definition of SAE is not always related to clinical severity of the event.  
For the purposes of this study an AE is considered a Serious (SAE) when it satisfies any one of 
the following criteria: 
 
• 
The event results in an inpatient hospitalization (any overnight stay associated with an 
admission). 
• 
The event results in the prolongation of a hospital stay. 
• 
The event results in permanent or severe disability. 
• 
The event results in death. 
• 
The event is life-threatening. 
• 
Treatment is required to prevent a serious event. 
 
There have been no SAE’s in the research groups experience in the Pediatric CTRC.  We do 
not anticipate encountering SAE’s; however, we have identified the following as possible SAE’s 
for the purposes of monitoring: 
• 
Infection related to blood draw or IV placement 
 

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a) Serious Adverse Event Reporting 
Study patients are instructed to contact the clinic with any serious adverse event meeting the 
above criteria.  Each SAE is recorded on the study form and the PI is informed as soon as 
possible after they occur and preferably within 24 hours of the notification of the clinic staff.  This 
notification should occur even if data are incomplete.  Additional data and follow-up information 
are documented and sent subsequently as an update to the original report.  The PI immediately 
forwards SAE reports to COMIRB and any other required institutional monitoring committee.   
            
C. Participant Discontinuation Criteria 
If a participant experiences any of the following, the participant will be withdrawn from the study. 
1. Inability to complete study procedures 
2. Abnormal clinical labs (HbA1C>6.4%, Hg <10 mg/dL) 
3. Participant becomes pregnant during study 
 
D. Protocol Stopping Criteria 
If one or more participants experience any of the SAE’s listed above, the PI will consult with the 
study staff prior to continuing study visits with participants.  The PI and RSA will consult about 
the significance of the SAE’s and make a recommendation about participant continuation in the 
study.  
 
VII. Data Analysis Plan:   
 
Mathematical plans: 
     
MMTT: The metabolic response to the MMTT will be measured for 4 hours. Blood will be 
collected for glucose, insulin, C-peptide, GLP-1 and glucagon at -10, 0, 10, 20, 30, 45, 60, 90, 
120, 150,180 and 240 min after the start of the liquid meal ingestion. β-cell function will be 
quantitated by mathematical modeling of the plasma C-peptide response to the MTTT 32,33. The 
MMTT allows for calculating an estimate of the principal parameters of β-cell function after a 
physiological stimulus and is better tolerated than oral glucose post-bariatric surgery. The main 
parameter of the model is β-cell glucose sensitivity (ß-GS), which is calculated as the mean 
slope of the dose-response function (i.e., the relationship between insulin secretion and plasma 
glucose concentrations during corresponding times of the MMT). The model also yields a 
measure of total insulin release over the 3 hr 34. Intestinal GLP-1 and α-cell glucagon peak 
responses and area under the curve will also be calculated. The primary model outcome is β-
cell glucose sensitivity (β-GS), a measure of β cell function calculated as the mean slope of the 
dose-response function i.e., relationship between insulin secretion rates and glucose 
concentration during the MMTT. The model also provides an estimate of glucose rate sensitivity 
(insulin secretory response to rate of change in glucose), and total insulin output (total amount 
of insulin released over the 4 h of the MMTT)34. The metabolic response to the MMTT can also 
be calculated using the using the trapezoid method for plasma glucose, insulin, and C-peptide 
AUCs35.  
 
Overall Project Statistical Plan: 
 
SA1: Changes in hepatic fat will be compared to changes in insulin resistance, beta cell function 
and 31P-MRS and metabolomics score. Results will be adjusted for absolute and percent 
weight loss.  
SA3: Pearson correlations between 31P-MRS rate of ATP recovery, metabolomics outcomes, 
hepatic fat fraction, MRE, Oroboros outcomes and biopsy scores will be performed to see which 

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minimally invasive measures relate most to biopsy markers. Liver measures will also be 
compared to insulin sensitivity and insulin secretion from the MMT. 
 
Overall Power analysis:  
SA1: The data collected for SA3 is to be preliminary data to provide background data for future 
additional studies in youth.  
SA2/SA3: Relation of metabolic outcomes for this type of protocol typically requires 6-8 
participants, especially with such a homogenous participant group. However, as it is known that 
African American tend to have different hepatic fat metabolism patterns compared to Hispanics, 
we have allowed for 10 participants. 
 
VIII. Summarize Knowledge to be Gained:  
 
Overall Expected Results:  We anticipate that we will identify several minimally invasive 
biomarkers in severely obese patients that can be used to predict NAFLD status. We also 
anticipate that we will generate preliminary data as to the relationship between type 2 diabetes 
risk and liver health in these patients, and how these both change following bariatric surgery.  
This data would then provide the background evidence for additional post-bariatric mechanistic 
studies and future therapeutic trials.  
 
Significance: Recent evidence in adults with severe obesity indicates that 60-70% have 
NAFLD37,38. And youth may have a similar prevalence. NAFLD has been described as the 
primary driver of worsening metabolic syndrome and CVD in obesity across populations, and 
can progress to cirrhosis and liver failure39,40. Thus, understanding the relative contributions of 
NAFLD to metabolic disease critical to designing strategies to improve the long-term health of 
these youth and reduce their risk of T2D, CVD and liver failure. Further as bariatric surgery is 
now being advocated for treatment of NAFLD, T2D and severe obesity, understanding the 
effects and mechanism on metabolic disease are critical. 
 
 
 
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Marchesini G, Brizi M, Bianchi G, et al. Nonalcoholic fatty liver disease: a feature of the metabolic 
syndrome. Diabetes 2001;50:1844-50. 
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Korenblat KM, Fabbrini E, Mohammed BS, Klein S. Liver, muscle, and adipose tissue insulin 
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Alisi A, Locatelli M, Nobili V. Nonalcoholic fatty liver disease in children. Current Opinion in 
Clinical Nutrition & Metabolic Care 2010;13:397-402. 
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Fabbrini E, Sullivan S, Klein S. Obesity and nonalcoholic fatty liver disease: biochemical, 
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Crespo M, Lappe S, Feldstein AE, Alkhouri N. Similarities and differences between pediatric and 
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Samuel VT, Shulman GI. Nonalcoholic Fatty Liver Disease as a Nexus of Metabolic and Hepatic 
Diseases. Cell metabolism 2018;27:22-41. 
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Cree-Green M, Bergman BC, Coe GV, et al. Hepatic Steatosis is Common in Adolescents with 
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10. 
Sunny NE, Parks EJ, Browning JD, Burgess SC. Excessive hepatic mitochondrial TCA cycle and 
gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab 2011;14:804-10. 
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Satapati S, Kucejova B, Duarte JA, et al. Mitochondrial metabolism mediates oxidative stress and 
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Parks EJ, Skokan LE, Timlin MT, Dingfelder CS. Dietary sugars stimulate fatty acid synthesis in 
adults. The Journal of nutrition 2008;138:1039-46. 
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Owen OE, Kalhan SC, Hanson RW. The key role of anaplerosis and cataplerosis for citric acid 
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Laufs A, Livingstone R, Nowotny B, et al. Quantitative liver 31P magnetic resonance 
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Fritsch M, Koliaki C, Livingstone R, et al. Time course of postprandial hepatic phosphorus 
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Leij-Halfwerk S, van den Berg JW, Sijens PE, Wilson JH, Oudkerk M, Dagnelie PC. Altered 
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Ren J, Yang B, Sherry AD, Malloy CR. Exchange kinetics by inversion transfer: integrated 
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Abrigo JM, Shen J, Wong VW, et al. Non-alcoholic fatty liver disease: spectral patterns observed 
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Manco M, Mosca A, De Peppo F, et al. The Benefit of Sleeve Gastrectomy in Obese Adolescents 
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Feldstein AE, Charatcharoenwitthaya P, Treeprasertsuk S, Benson JT, Enders FB, Angulo P. The 
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Fishbein MH, Gardner KG, Potter CJ, Schmalbrock P, Smith MA. Introduction of fast MR imaging 
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Fishbein MH, Mogren C, Gleason T, Stevens WR. Relationship of hepatic steatosis to adipose 
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Cree MG, Newcomer BR, Katsanos CS, et al. Intramuscular and liver triglycerides are increased 
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Perseghin G, Scifo P, De Cobelli F, et al. Intramyocellular triglyceride content is a determinant of 
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Nadeau KJ, Zeitler PS, Bauer TA, et al. Insulin resistance in adolescents with type 2 diabetes is 
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