Randomized, Controlled Dietary Treatment Study of Pediatric NAFLD
Phase: NA
Conditions: Nonalcoholic Fatty Liver Disease
Sponsor: University of California, San Diego

i 
 
 
 
 
 
 
 
Randomized, Controlled Dietary Treatment Study of Pediatric NAFLD 
 
 
 
Funding Source: Nutrition Science Initiative  
Principal Investigators: Miriam Vos, MD, MSPH and Jeff Schwimmer, MD 
Draft or Version Number: 7.0 
 
 
 
 
February 10, 2016 
  

 
1 
 
TABLE OF CONTENTS   
TABLE OF CONTENTS .............................................................................................................. 1 
LIST OF ABBREVIATIONS ......................................................................................................... 3 
PROTOCOL SUMMARY ............................................................................................................. 4 
1  
KEY ROLES .................................................................................................................... 6 
2 
Introduction: Background Information and Scientific Rationale ........................................ 9 
2.1 
Background Information .................................................................................... 9 
2.2 
Justification ..................................................................................................... 11 
2.3 
Potential Risks and Benefits ........................................................................... 12 
2.3.1 
Potential Risks .....................................................................................12 
2.3.2 
Potential Benefits.................................................................................13 
2.3.3 
Alternative treatments and procedures ................................................13 
2.3.4 
Protection against risks .......................................................................13 
3 
OBJECTIVES ................................................................................................................ 14 
3.1 
General hypothesis ......................................................................................... 14 
3.2 
Specific Hypothesis ......................................................................................... 14 
3.3 
Implications ..................................................................................................... 14 
3.4 
Primary Outcome Measure ............................................................................. 14 
3.5 
Secondary Outcome Measures ....................................................................... 14 
4 
Study Design ................................................................................................................. 15 
4.1 
Overview ......................................................................................................... 15 
4.2 
Intervention ..................................................................................................... 15 
4.3 
Dietary Assessment ........................................................................................ 16 
4.4 
Sweetness Perception Testing ........................................................................ 17 
4.4.1 Participant preparation ...........................................................................17 
4.4.2 Sensory Ratings .....................................................................................17 
4.5 
Insulin Sensitivity Measurements .................................................................... 18 
4.6  
Study duration for participants......................................................................... 18 
4.7 
Study Timeline ................................................................................................ 18 
5 
Study Enrollment and Withdrawal .................................................................................. 19 
5.1 
Participant Inclusion Criteria ........................................................................... 19 
5.2 
Participant Exclusion Criteria .......................................................................... 19 
5.3 
Strategies for Recruitment .............................................................................. 20 
5.4 
Reasons for Withdrawal .................................................................................. 20 
5.4.1 
Handling of Withdrawal ........................................................................20 
5.4.2 
Termination of Study ...........................................................................20 
6 
Study Schedule ............................................................................................................. 22 
6.1 
Screening ....................................................................................................... 22 
6.2 
Day 0: Baseline Visit ....................................................................................... 23 
6.3 
Follow-up Visit Day 28 .................................................................................... 24 
6.4  
Follow-up Visit Day 56 .................................................................................... 24 
6.5 
End of study follow-up ..................................................................................... 24 
7 
Statistical Considerations .............................................................................................. 25 

 
2 
 
7.1 
Sample Size Considerations ........................................................................... 25 
8 
Ethics/Protection of Human Participants ........................................................................ 27 
8.1 
Ethical Standard ............................................................................................. 27 
8.2 
Institutional Review Board ............................................................................... 27 
8.3 
Informed Consent Process .............................................................................. 27 
8.4 
Informed Assent Process ................................................................................ 27 
8.5 
Participant Confidentiality ................................................................................ 27 
8.6 
Study Discontinuation ..................................................................................... 28 
8.7 
Data Safety Monitoring Plan ........................................................................... 28 
9 
Data Handling and Record Keeping .............................................................................. 29 
9.1 
Data Management Responsibilities ................................................................. 29 
9.2 
MRI Data Coordination.................................................................................... 29 
9.3 
Types of Data ................................................................................................. 29 
9.5 
Study Records Retention ................................................................................ 29 
10 
Literature References .................................................................................................... 30 
11 
Attachments 11.1 SCHEDULE OF EVENTS ................................................... 32 
11.2 BLOOD DRAW SCHEDULE .................................................................................. 33 

 
3 
 
 
LIST OF ABBREVIATIONS  
Adipo-IR 
Adipose Tissue Insulin Resistance 
AE 
Adverse Event  
ALT 
Alanine Transaminase 
AST 
Aspartate Aminotransferase 
BMI 
Body Mass Index 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
GCP 
Good Clinical Practice 
GGT 
Gamma-glutamyl Transpeptidase 
HIPAA 
Health Insurance Portability and Accountability Act 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
MRI 
Magnetic Resonance Imaging 
NDSR 
Nutrition Data System for Research 
NAFLD 
Nonalcoholic Fatty Liver Disease 
NASH 
Nonalcoholic Steatohepatitis 
NuSI 
Nutrition Science Initiative 
PI 
Principal Investigator 
RCT 
Randomized Control Trial 
SOM 
School of Medicine 
TPN 
Total Parenteral Nutrition 
 
 

 
4 
 
PROTOCOL SUMMARY 
Title: 
Randomized, Controlled Dietary Treatment Study of Pediatric NAFLD 
 
Summary: 
This is a multisite, randomized, controlled, 8-week outpatient feeding study at 
Emory University and University of California, San Diego. Participants will be 
non-diabetic male adolescents with biopsy-proved NAFLD. Two groups of 20 
participants will be followed for 8 weeks with hepatic fat content assessed by 
MRI PDFF at weeks 0, 4, and 8. One group will be a standard of care control to 
track the naturally-occurring changes in hepatic fat content in children over 
time. The other group will be provided with a low free sugars (<3% total daily 
calories) version of their habitual diets. The primary outcome is % change in 
MRI PDFF over time in the treatment group compared to the control group. 
Additional parameters of liver function and metabolic status (e.g., serum ALT) 
will also be assessed.
Objectives: 
 
To evaluate hepatic fat by MRI PDFF before and after 8 
weeks of a study-provided low free sugars diet (<3%) 
compared to a prospective, standard of care, control group 
receiving only imaging. 
Population: 
Males age 11 to 16 with a history of liver biopsy confirming 
NAFLD and hepatic fat by MRI PDFF ≥ 10% during 
screening. 
Type of Trial: 
Phase IIb, randomized clinical trial  
Study 
Duration: 
2 years 
Participant 
Participation 
Duration: 
Up to 16 weeks 
Total Number 
of 
Participants: 
40 complete (20 per site – 10 interventions and 10 controls) 
May enroll up to 60 across sites to account for screen fails 
and drop outs 

 
5 
 
SCHEMATIC OF STUDY DESIGN 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
6 
 
1  
KEY ROLES  
Principal Investigators: 
 
Jeffrey Schwimmer, MD 
University of California, San Diego 
UCSD Medical Center 
200 West Arbor Drive, MC 8450 
San Diego, CA 92103 
 
Miriam Vos, MD, MSPH 
Emory University School of Medicine 
1760 Haygood Drive, W450 
Atlanta, GA 30322 
Co-Investigators: 
 
Adina Alazraki, MD 
Children’s Healthcare of Atlanta 
 
Claude Sirlin, MD 
University of California, San Diego 
 
Jonathan Africa, MD 
University of California, San Diego 
 
Michael Middleton, MD, PhD 
University of California, San Diego 
 
Ariel Feinstein, MD 
Children Specialists of San Diego 
 
Joel Lavine, MD, PhD 
Columbia University 
 
Jean Welsh, PhD, RN, MPH 
Emory University School of Medicine 
 
Jay Freeman, MD 
Emory University School of Medicine 
 
Nitika Gupta, MD 
Emory University School of Medicine 
 
Saul Karpen, MD, PhD 
Emory University School of Medicine 
 
Rene Romero, MD 
Emory University School of Medicine 
 
 

 
7 
 
Dellys Soler Rodriguez, MD 
Emory University School of Medicine 
 
Nitika Gupta, MD 
Emory University School of Medicine 
 
Saul Karpen, MD, PhD 
Emory University School of Medicine 
 
Rene Romero, MD  
Emory University School of Medicine 
 
Dellys Soler Rodriguez, MD  
Emory University School of Medicine 
 
Institutions: 
Emory University and Children’s Healthcare of Atlanta 
 
University of California, San Diego and UCSD Medical Center  

 
8 
 
 
Study staff: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Institutional Review 
Boards: 
 
 
 
Project Management   
Rebecca Cleeton, MPH, CCRP 
Research Manager 
Emory University School of Medicine 
 
Maria C. Cordero 
Research Coordinator 
Emory University School of Medicine 
 
Janis Durelle, BS 
Project Manager 
University of California, San Diego 
 
Patricia Ugalde Nicalo, MD 
Lead Coordinator 
University of California, San Diego 
 
Jennifer Sanford, BA 
Research Coordiantor 
University of California, San Diego 
 
Nutritionists 
Juna Konomi, PhD 
Emory University School of Medicine 
 
Cynthia Knott, RD 
University of California, San Diego 
 
Statistician 
Courtney McCracken, PhD 
Emory University School of Medicine 
 
Emory University  IRB  
1599 Clifton Road NE, 5th Floor 
Atlanta, GA 30322 
 
University of California, San Diego 
Human Research Protections Program 
9500 Gilman Drive, Mail Code 0052 
La Jolla, California 92093 
 
 

 
9 
 
2 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information  
The field of pediatric nonalcoholic fatty liver disease (NAFLD) has grown exponentially 
over the past decade in response to the growth of the disease and increased awareness 
of this important liver disease. NAFLD is now the most common liver disease for both 
adults and children in the US and is estimated to affect more than 20 million persons1, 2.  
It is a chronic liver disease closely associated with the metabolic syndrome, although its 
role as cause, effect or innocent bystander has yet to be defined3-9.  NAFLD is an 
umbrella term and it is histologically categorized into nonalcoholic fatty liver (NAFL), 
defined as hepatic steatosis (fat) without hepatocellular injury and nonalcoholic 
steatohepatitis (NASH), defined as hepatic fat plus hepatocellular injury with or without 
fibrosis10.   
In the US, Mexican-Americans are the group most affected by NAFLD and it is estimated 
that 1 of 4 Mexican American adults has NAFLD 1. Hispanic-Americans are 2.5 times 
more likely to have NAFLD compared to African Americans2, 11, 12.  Liver disease is the 
6th leading cause of death for Hispanic adults compared to 12th for the general 
population13, 14. NAFLD increases the risk of liver disease but also increases risk of type 
II diabetes, cardiovascular disease (CVD) and the metabolic syndrome15-20.  For 
example, a person with NAFLD and fibrosis has 2.5 – 3.5 times the risk of 
cardiovascular disease death and increased risk of type II diabetes compared to a 
similarly overweight person without NAFLD (reviewed in Armstrong et al21). An estimated 
1/3 of adults with nonalcoholic steatohepatitis (NASH), a more severe form of NAFLD, 
will go on to develop cirrhosis and liver cancer22 and it is the most rapidly increasing 
reason for liver transplants in adults. The morbidity and mortality of NAFLD, along with 
its high prevalence in a vulnerable population make NAFLD a public health priority23.   
In a healthy person, almost no fat (<5% of the liver by volume) is stored in the liver, 
despite the fact that the liver is a major site of metabolism for dietary fat, cholesterol, 
triglyceride, free fatty acids and more.  NAFLD develops when the balance of triglyceride 
metabolism in the liver becomes dysregulated in the setting of insulin resistance (IR).  
Triglyceride rich lipoproteins either originate in the intestine or liver and the sources of 
fatty acids used for lipoprotein assembly are derived either from de novo lipogenesis 
(DNL), the diet or from the plasma pool of nonesterified FA (NEFA)24.  Hepatic DNL 
appears to underlie the mechanism of excess hepatic storage of triglyceride during 
carbohydrate feeding in individuals with NAFLD24-26.  Compared to subjects with 
metabolic syndrome and no increased hepatic fat, subjects with NAFLD have 3 fold 
higher rates of DNL in addition to higher plasma levels of FFA during the nighttime25.  
The mechanisms of NASH, in which hepatic fat is associated with inflammation and 
hepatocellular injury are complex but likely involve lipotoxicity and increased oxidative 
stress.   

 
10 
 
Because of the high level of consumption and its metabolic fate, fructose has been 
studied in relation to many health problems, including NAFLD. Fructose was initially 
linked to steatosis and NAFLD in animal models but more recently, a number of human 
studies have demonstrated links. Fructose enters the diet primarily from sugars that are 
in processed food and beverages, often referred to as added sugars. The World Health 
Organization (WHO) has provided a draft of guidelines on sugar consumption and has 
clarified the term “free sugars” which includes all monosaccharides (fructose, glucose, 
galactose) and disaccharides (sucrose, maltose, trehalose) which are added to the foods 
by the manufacturer, the cook, or the consumer.  The “free sugars” category includes 
naturally occurring sugars in honey, fruit juices, and syrups and excludes sugars from 
fruits, vegetables or the lactose from dairy. Naturally occurring sources of fructose 
include fruit and vegetables, but only account for a small percentage of fructose in the 
diet27. In the U.S., added sugar consumption rose over most of the past century 27, 28 until 
the 2000’s when consumption of sugar sweetened beverages began to decline29. Recent 
estimates indicate a slightly declined consumption of total added sugar, fructose, and 
sugar sweetened beverages29-32. Even with this decline, the amount of added sugars, 
and thus the amount of fructose, in the typical US diet remains elevated at ~16% of total 
calories, which exceeds the upper recommendation of 5-10%33.   
 
Fructose is absorbed from the lower part of duodenum and jejunum both passively and 
actively primarily through GLUT5. After absorption across the brush border of the small 
intestine into the portal blood supply, fructose is cleared from the blood in the liver on the 
first pass and almost exclusively metabolized in hepatocytes. Most absorbed fructose is 
cleaved into glyceraldehyde and dihydroxyacetone phosphate, and these trioses further 
go to glycerol phosphate and pyruvate metabolic pathways, promoting gluconeogenesis 
and DNL, respectively. It is this stimulation of DNL that has been studied closely and 
may be a critical environmental contributor to NAFLD.  When compared with a isocaloric 
diet with the same macronutrient distribution, a fructose containing diet (25% of calories) 
was associated with both higher DNL and higher liver fat in a short term study of healthy 
adults34.   
 
NAFLD patients have been found to be high consumers of fructose.  In a cross-sectional 
analysis, Quyang et al. found a 2-3 fold increase of fructose consumption in patients with 
biopsy-proven NAFLD as compared to their sex, age, and BMI matched controls35. A 
longitudinal study in obese adolescents indicated that energy-adjusted fructose intake 
was independently associated with NAFLD during a 3-year follow up36.  And fructose 
may be associated with the severity of NAFLD. For example, work by Abdelmalek et al. 
reported a significant association between fructose intake and fibrosis severity in a 
cohort of 427 adults with histologically-confirmed NAFLD 37.   
 
There are a few small studies demonstrating improvement in features of NAFLD with 
fructose reduction but this is an area that deserves more research. In a randomized 
controlled trial, 4-week fructose restriction in children with NAFLD improved their 
adipose insulin sensitivity, high sensitivity C-reactive protein (hs-CRP), and LDL 

 
11 
 
oxidation; whereas their liver enzymes and intrahepatic fat remained unchanged 38. Over 
a longer intervention as of 6 months, improvement of liver enzyme was seen with 
lowering dietary component of fructose and glycemic load in NAFLD subjects 39. Finally, 
a recently reported 10 day study in children on a fructose-free diet demonstrated a 20 
percent drop in liver fat. 
 
Of note, while the studies above isolated fructose as the dietary component being 
evaluated, in real life, most fructose in the diet is from added sugars. Thus, while 
fructose is important to evaluate for the mechanism of effect, added sugars are a human 
health relevant environmental exposure that is being considered in this study.   
 
Another technology utilized in this protocol is magnetic resonance imaging-estimated 
liver proton density fat fraction (MRI-estimated liver PDFF) for the measurement of 
hepatic fat.  This is a research technology that is not yet clinically available, but has 
recently been validated for use in children and is highly beneficial because it can be 
used instead of a liver biopsy to measure hepatic fat40.  In the validation study, MRI-
estimated liver PDFF was used to measure hepatic steatosis and was compared this to 
histopathologic grading in 174 children with a mean age of 14 years (range 8 to 16 
years)41. All the children completed the MRI acquisition protocol without difficulty 
demonstrating the feasibility of MR-based steatosis measurements in young children.  
The correlation of the MRI-estimated liver PDFF with the pathology assigned steatosis 
grade was very good (0.725)41.  Further, it is a precise, low risk technology42. No MRI 
contrast is required and the scan time can be brief (~15 minutes) thus making MRI PDFF 
ideal for measuring hepatic fat in this research protocol. 
 
In summary, free sugars are associated with severity of NAFLD and may be an 
important environmental cause of increased DNL leading to hepatic steatosis. In this 
proposal, we will test the effect of a low “free sugars” (<3% total daily calories) diet in 
children with biopsy-proven NAFLD using innovative, low risk, non-invasive tools like 
MRI for hepatic fat measurement.   
2.2 
Justification   
Before designing a large RCT it is prudent to first test effect size of reducing sugar in 
male children with a history of NAFLD on hepatic fat, ALT, and other markers in a 
smaller, randomized, controlled study. 
Population studies demonstrate that NAFLD is more than twice as likely in boys 
compared to girls2, 11.  In the recently completed NIH sponsored NASH randomized, 
controlled trial, XX% of the subjects enrolled were boys, despite best efforts to increase 
recruitment of girls.  Puberty is a strong modifier of NAFLD severity43.  Girls undergo 
puberty earlier than boys.  Together, these effects could confound the study results 
because more girls would be in later stages of puberty compared to the boys.  
Increasing the sample size would allow for subgroup analysis and controlling for sex, 
however budget constraints did not allow for a larger cohort.  Thus, the study was 

 
12 
 
designed with boys alone, excluding girls.  There are limitations of including only boys 
including that it makes the study less generalizable.  This will hopefully be addressed by 
proceeding with a larger, longer trial including both sexes justified by the data of this 
initial study.   
2.3 
Potential Risks and Benefits 
2.3.1 Potential Risks 
There is minimal risk in this study. Potential risks are related to 1) the MRI PDFF 2) 
blood draws, 3) NPO status, 4) cumulative blood loss, 5) confidentiality and 6) diet 
low in free sugars.   
1) MRI PDFF – Some children may find the MRI examination to be a fear invoking one.  
These will be done at a research imaging facility where the staff is experienced in 
working with children. The MRI scanner also has movies for improving the 
experience.  
2) The placement of an intravenous catheter and drawing of blood specimens has 
minimal risk of discomfort, bruising, or bleeding. There is minimal risk of infection or 
extravasation. Experienced staff with pediatric expertise will place all catheters and 
draw blood. 
3) Patients will remain NPO from 8 pm the evening before the study until the fasting 
portion of the study is concluded. Some children may become agitated with NPO 
status. Blood sugar will be monitored at the beginning of the study and water will be 
encouraged to maintain hydration. Each participant will be asked to drink one glass 
of water at bedtime prior to the study and one glass in the morning before leaving 
home for the research center. Food and water will be provided immediately following 
the conclusion of fasting visits 
4) Blood loss: During the study visits, blood will be drawn from an indwelling IV at 19 
time-points. To minimize risk to the patient, we will limit amounts in accordance with 
the guidelines of our IRB and the NIH. The total blood volume drawn over the course 
of the study will be 97 mL. We expect all of our patients to be >37 kg making these 
amounts well within the NIH clinical center guidelines recommending less than 
3ml/kg at a single research visit and no more than 7ml/kg over any 6 week period.   
5) There is a small risk of loss of confidentiality.  We will follow all procedures required 
by respective institutions to protect participants. Efforts will be made to ensure that 
all personal information remains confidential. All data will be stored in locked offices 
and password-protected computers. Personal identity will be protected in any 
publication. 
6) Potential risks of a low “free sugars” diet: sugar is not a required nutrient but it is 
known to have some addictive qualities. Elimination of “free sugars” in the diet may 
at first result in cravings of sugar and this is expected to fade over 2-3 weeks.   

 
13 
 
2.3.2 Potential Benefits 
The proposed research has substantial potential benefits for children. Sugar is known to 
increase adiposity in children over time and even a short reduction of 8 weeks is likely to 
have benefits from a BMI and cardiovascular standpoint, although these may not be 
sustained after the trial ends. In the standard of care control group, families will receive 
gift cards to cover groceries and this may allow them to purchase healthier foods such 
as vegetables.   
Participants in both groups will benefit from the knowledge that they are helping to 
contribute to the knowledge of how diet changes can help treat NAFLD.   
In addition, at the conclusion of the study, all participants in the studies will receive 
copies of their liver transaminases, lipid results and baseline MRI PDFF results and 
these will be discussed with them by one of the investigators. Education will be provided 
as appropriate regarding the results and standard of care advice for diet and physical 
activity levels.  
2.3.3 Alternative treatments and procedures 
Patients that are approached about the study can choose not to participate. If they 
decide not to participate it will not impact their standard of care treatment. 
2.3.4 Protection against risks 
Risk will be minimized to the children by using nurses and staff experienced in pediatrics 
for the research studies. As discussed above, the risks associated with heavy water will 
be minimized by giving the first dose at the research center under supervision. Risk to 
confidentiality will be reduced by using non-identifying participant numbers and removing 
all identifiers as early as feasible. Only study personnel will have access to identifying 
data. Laboratory personnel will only have access to numeric identifiers and the key will 
be kept in a password protected database.   
Children will provide written assent (11-16 y). Children are able to decline to continue to 
participate at any time before and during the study or study visit. During study visits, if an 
IV fails, we will attempt to replace it. If the child does not wish to have it replaced, we 
encourage them to tell us this and the visit will be stopped immediately. Study stipends 
are provided at a standardized pro-rated level appropriate for the time/study procedures 
completed.  

 
14 
 
3 
OBJECTIVES 
What is the role of sugar consumption in the etiology and treatment of pediatric NALFD? 
3.1 
General hypothesis 
Sugar restriction will reduce hepatic fat content and reverse liver histopathology in 
children with NAFLD (and by extension, such foods/beverages that trigger the condition). 
3.2 
Specific Hypothesis 
Restricting sugar in beverages, in food, or in both will decrease hepatic fat content and 
reverse liver histopathology in children with NAFLD in relation to the type and/or degree 
of restriction (and by extension, such foods/beverages that trigger the condition).  
3.3 
Implications 
Improvement in NAFLD/NASH in children can be achieved by limiting all sugars, or by 
limiting a specific form of sugar (free sugars in beverages or in food) alone. Avoiding 
such foods will prevent development of NAFLD and NASH.  
3.4 
Primary Outcome Measure 
Change in hepatic steatosis (%) by MRI PDFF in the intervention group compared to 
change in the control group. 
3.5 
Secondary Outcome Measures  
ALT, AST, GGT, Adipo-IR, fasting glucose, insulin, serum lipids, sweetness perception 
testing, compliance (all baseline compared to 8 weeks, change in intervention group 
compared to control). 
 
 
 
 
 
 
 
 

 
15 
 
4 
STUDY DESIGN 
 
A study that tests nutritional interventions that clinicians currently prescribe, such as ad 
libitum weight-reducing diets, has the potential to directly and rapidly affect clinical 
practice, but does not necessarily provide significant insight into the dietary trigger of 
disease or the dietary mechanism driving any observed effects. Conversely, a highly 
controlled diet study may more clearly identify the nutritional trigger and mechanism 
(necessary for the successful prevention of the disease in public health campaigns) but 
may be dismissed by many clinicians as divorced from actual practice. Here we describe 
a trial that hopes to balance the need for establishing practical clinical guidelines for 
dietary treatment and experimental controls sufficient to identify the dietary trigger for 
NAFLD in a pediatric population. 
4.1 
Overview 
This is a 2 site, 8-week randomized, controlled, outpatient feeding study at Emory 
University and UCSD. Participants will be non-diabetic male adolescents with biopsy-
proven NAFLD. Two groups of 20 participants will be followed for 8 weeks with hepatic 
fat content assessed by MRI PDFF at weeks 0, 4, and 8. The participants will be 
randomized to either standard of care  control arm to track the naturally-occurring 
changes in hepatic fat content  over time, or replacement of habitual diet with a low “free 
sugars” (<3% of total daily calories) version, intervention arm. The primary outcome is 
percent change in liver fat content over time in the treatment group compared to the 
control  group. Additional parameters of liver function and metabolic status (e.g., serum 
ALT) will also be assessed.  
4.2 
Intervention 
The intervention is replacement of habitual diet with a low “free sugars” diet (<3% of total 
calories). The intervention will be applied by family and will aim to alter the diet by 
specifically targeting the foods that contain free sugars (WHO definition: glucose, 
fructose, galactose, sucrose, maltose, trehalose) added to food by consumer, cook, or 
manufacturer, while preserving the family’s other food group choices. At recruitment, 
families will be randomized to either the low “free sugars” diet (intervention) or remain on 
their regular diet (standard of care control). Following screening procedures and prior to 
study initiation, a home visit will be scheduled at which current food consumption, food 
preferences, and weekly food volume required for the family will be assessed and 
recorded. Additionally, common recipes used by the family will be collected. One day 
prior to study initiation (Day 0), the nutritionist/coordinator/staff will assist the parent or 
guardian in selecting and removing all sugar and sugar-containing products from the 
home. The items will be replaced with similar foods that contain no free sugars.  In 
general, artificial sweeteners will be avoided although in some instances it will be 
necessary to use them.   

 
16 
 
For each week of the study (8 weeks total), families will be able to choose meals from a 
list of foods similar to their usual diet including ready-to-eat foods, breakfast foods, lunch 
items, dinner entrees, fruits, and snacks. Food will be prepared by dieticians/nutritionists 
at the research metabolic kitchen and supplies for several days at a time will be 
delivered to the family by the study staff. Each child will be provided with a lunch bag 
and instructed to bring lunch to school to maintain the study diet.  
Families will have the opportunity to choose from a list of pre-prepared evening meals 
that are similar to what they consumed before study initiation but not containing any free 
sugars. Dinners will typically be fresh or frozen and re-heating in the oven or microwave 
may be required. The families will be instructed to not eat any food outside of assigned 
diet. For the duration of the study a water dispenser will be provided. Families will be 
instructed to avoid all fruit juice and sweet drinks. Fruit consumption will be allowed but 
restricted in amount (to 1-2 portions/ day per family member). 
Methods to improve compliance: Twice a week, a coordinator/study staff member will 
conduct home visits or phone calls to assess food satisfaction as well as diet compliance 
in the intervention arm. In the standard of care control arm, study staff will visit the home 
once a week to check on their compliance and provide them with any study-related items 
(water, gift cards etc.). To enhance compliance, the family will be allowed 1 meal “off” of 
the diet 4 times during the study.  These meals will be planned in coordination with the 
staff and may include holidays or special family events or other days that they select. 
These “off” meals may not be used during the last week of the intervention.  
Compliance will also be increased by informing participants that the investigators can 
assess compliance with the diet by measuring sugar in the blood work. If there is 
evidence of the participant not complying, the study coordinator or investigator will meet 
with the participant and family members and explain the importance of only consuming 
the study diet.  If the study coordinator/staff perceives evidence of persistent non-
compliance by the participant, families will be withdrawn from study and the diet will be 
stopped. 
4.3 
Dietary Assessment 
Baseline diet by 24-hour recalls will include 2 x 24-hour recalls to reflect diet during 
weekdays and 1 x 24-hour recall to reflect diet on a weekend day. Families will be asked 
to specify times that work best to conduct the 24-hour recalls and will be called at 
random, to minimize diet change. The 24-hour recalls will aid in getting a better sense of 
the food groups consumed by the participant and his family and in the preparation of a 
personalized, tailored diet. Following study participant randomization, two weeks prior to 
study initiation, a home visit will be scheduled where current food consumption, food 
preferences, and weekly food volume required for the family will be recorded. 
Additionally, common recipes will be collected. During the study, every week a 
coordinator/study staff member will conduct two home visits to assess food satisfaction 
and diet compliance. If the study coordinator/staff perceives non-compliance by the 

 
17 
 
family at any point of the study, the intervention will be stopped and families will be 
withdrawn from the study. 
4.4 
Sweetness Perception Testing 
 
4.4.1 Participant preparation 
Participants will be asked to undergo sweetness taste perception testing at three points 
in the study: baseline (study initiation, week 0), follow-up visit 1 (week 4), and follow up 
visit 2 (study end, week 8). Participants will arrive at the clinic after an overnight fast to 
conduct the sweetness perception testing and MRI. Prior to each study visit, participants 
will be reminded not to eat or drink anything except for water after 8 pm the night before 
the visit. At each visit height, weight, and vital signs will be collected.  
 
4.4.2 Sensory Ratings 
Sweetness perception will be assessed in a test session of about 1 hour. Participants 
will rate both the intensity and pleasantness of model beverages that vary widely in 
sucrose concentration. The model beverages will be formulated using unsweetened 
Kool-Aid ™ drink mix at a fixed concentration. Concentrations of sucrose (ranging from 
barely sweet to very sweet) will be added to this fixed concentration of drink mix. Model 
beverages will be presented chilled. Over the course of a 1-hour session, participants 
will consume about 120 ml of model beverage, or the equivalent of about 1/3rd  of a 12 
oz can of soda. 
 
The first 10-15 minutes of each session will be devoted to instructions and practice 
ratings. Participants will rate perceived sweetness intensity using visual analog scales 
(i.e., vertical lines with the anchor labels “Tasteless” on the bottom and “Extremely 
Strong” on the top). Such scales are easy for participants to understand and use. 
Participants will practice by rating the sweetness of 8 sucrose solutions (different 
concentrations of Kool-Aid) that cover the range of sucrose concentrations to be using in 
sensory testing. Participants will rate pleasantness using a standard category scale 
ranging from “Dislike Extremely” (-4), to “Neither Like Nor Dislike” (0), to “Like Extremely” 
(+4). Again, the use of such scales is fairly intuitive.  
 
After scaling practice, participants will be instructed to rate the sweetness and 
pleasantness of 16 model drink samples. The 16 samples will be presented in 5 ml 
aliquots (in160 ml disposable plastic medicine cups). The 16 samples will be presented 
in two blocks of 8 trials. Each block will include all 8 sucrose concentrations, presented 
in random order. Prior to the first trial of each block, participants will be asked to rinse 
their mouth four times with water (same water used to make test beverage) and spit it 
out. Next, participants will sample each of the 8 cups by sipping, rolling the liquid sample 

 
18 
 
around in the mouth for several seconds, and then swallowing the sample. After 
swallowing, participants will rate both the sweet taste intensity and pleasantness of the 
sample. After each trial, the participant will rinse the mouth a few times with water (same 
water used to make test beverage), spit, and wait ~45 seconds before tasting the next 
sample. Each block (all 8 concentrations, once each, in randomized order) will be 
followed by a five minute break. The first block will be used as practice and will not be 
included in analysis. The second and third blocks (replicate ratings for each of the 8 
concentrations) will be used for analysis.   
4.5  
Study duration for participants 
• 
Screening MRI must occur within 14 days of baseline (Day 0) 
• 
56 days of treatment  
• 
14 days post-treatment assessment (by phone) 
 
4.6 
Study Timeline 
• 
Study initiation phase: 3 months (IRB, meal testing, etc.) 
• 
Recruitment phase: 4 – 8 months depending on enrollment pace 
• 
Follow-up phase: 10 months 
• 
Close out phase: 2 months (data analysis etc.) 
• 
Expected recruitment is 10 intervention participants and 10 control participants per 
clinical center, approximately 1 of each type per month per center 

 
19 
 
5 
STUDY ENROLLMENT AND WITHDRAWAL 
5.1 
Participant Inclusion Criteria 
• 
Boys age 11-16 
• 
Standard of care liver biopsy  
• 
Clinical history consistent with NAFLD 
• 
Definite NAFLD based on liver histology 
• 
Hepatic fat by MRI PDFF ≥ 10% on baseline MRI 
• 
Serum ALT ≥ 45 u/L 
• 
Written informed consent from parent or legal guardian 
• 
Written informed assent from the child 
• 
Currently consumes ≥ 2 eight ounce sugar drinks (or juice) per week 
5.2 
Participant Exclusion Criteria 
• 
Participants with a history of health issues that make it unsafe for them to participate in 
the opinion of the investigators 
• 
History of significant alcohol intake (AUDIT questionnaire) or inability to quantify alcohol 
consumption 
• 
Chronic use (more than 2 consecutive weeks) of medications known to cause hepatic 
steatosis or steatohepatitis (systemic glucocorticoids, tetracycline, anabolic steroids, 
valproic acid, salicylates, tamoxifen) in the past year. 
• 
The use of other known hepatotoxins within 90 days of liver biopsy or within 120 of 
baseline 
• 
New medications or vitamins/supplements started within the past 6 months intended to 
treat NAFLD/NASH 
• 
History of total parenteral nutrition (TPN) use in the year prior to screening 
• 
History of bariatric surgery or planning to undergo bariatric surgery during the study 
duration 
• 
Significant depression 
• 
Non-compensated liver disease with any one of the following hematologic, biochemical, 
and serological criteria on entry into protocol: 
a. Hemoglobin < 10 g/dL 
b. White blood cell < 3,500 cells/mm 
c. Neutrophil count < 1,500 cells/mm3 of blood 
d. Platelets < 130,000 cells/mm3 of blood 
e. Direct bilirubin > 1.0 mg/dL 
f. Total bilirubin > 3 mg/dL 
g. Albumin < 3.2 g/dL 
h. International normalized ratio (INR) > 1.4 
• 
Diabetes 
• 
Evidence of other chronic liver disease 

 
20 
 
• 
Children who are currently enrolled in a clinical trial or who received an investigational 
study drug within the past 60 days 
• 
Participants who are not able or willing to comply with the diet protocol or have any other 
condition that would impede compliance or hinder completion of the study, in the opinion 
of the investigator 
• 
Unable to have an MRI due to metal device, claustrophobia or other reason 
• 
Failure to give informed consent 
• 
Recipient of a liver transplant 
5.3 
Strategies for Recruitment  
Recruitment will be through discussions of the study in the pediatric clinics, community 
gastroenterology offices and through recruitment of eligible previous research 
participants who have previously consented to be contacted regarding future studies. 
5.4 
Reasons for Withdrawal 
Participants are free to withdraw from participation in the study at any time. They can 
notify the study coordinator that they would like to discontinue from the study for any 
reason during any part of the study. They may also mail in the letter of revocation given 
along with the consent form if they desire. 
5.4.1 Handling of Withdrawal 
Collected data for participants who ask to be withdrawn from the study will be 
maintained in the database and will be used for future studies unless the participant 
specifically requests that data be destroyed. The participant’s file will be flagged to 
ensure that no further contact is made.  
5.4.2 Termination of Study  
This study may be prematurely terminated if, in the opinion of the investigator or the 
sponsor, there is sufficient reasonable cause.  Written notification, documenting the 
reason for study termination, will be provided to the investigator or sponsor by the 
terminating party. 
Circumstances that may warrant termination include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants. 
• 
Insufficient adherence to protocol requirements. 
• 
Data that is not sufficiently complete and/or evaluable.  
If the study is prematurely terminated or suspended, the sponsor will promptly inform the 
investigator/institution, of the termination or suspension and the reason(s) for the 
termination or suspension. The IRB will also be informed promptly and provided the 

 
21 
 
reason(s) for the termination or suspension by the sponsor or by the 
investigator/institution, as specified by the applicable regulatory requirement(s). 

 
22 
 
6 
STUDY SCHEDULE 
The schedule of events (Section 11.1) summarizes the procedures to be done at each 
visit. The visit windows are the goal for the study and serve as a guideline for the clinical 
sites. These visit windows are not strictly set; conducting a study visit outside of the visit 
window will not be considered a protocol deviation. Visit windows are outlines in the 
Schedule of Events.  
6.1 
Screening 
Screening visits/procedures may be combined or changed to accommodate the sub-
study schedule. 
 
Day -60 Screening visit: Participants will provide verbal consent or in-person written 
consent indicating that they wish to participate in the study so that a screening visit may 
be scheduled to assess eligibility. Informed consent will be obtained prior to initiating any 
research procedures. The screening visit may be conducted at a clinical visit if that is 
convenient for the participant. The ALT for eligibility must be drawn at or within 7 days 
before this visit.   
Vital signs, height, and weight, waist and hip circumference measures will be obtained at 
the screening visit as well as a physical exam. Alcohol use will be assessed using the 
Alcohol Use Disorders Identification Test (AUDIT) and baseline beverage consumption 
will be assessed using the Bev-Q beverage questionnaire. A background medical history 
will be obtained and concomitant medications will be reviewed as well. 
The following labs will be drawn at the screening visit: 
• 
CMP 
• 
CBC 
• 
Direct bilirubin 
• 
PT/INR 
• 
HbA1c 
• 
Plasma and serum for storage 
Day -25  Dietary Assessment: After the screening visit, the baseline usual diet will be 
assessed using three 24-hour, interviewer assisted, dietary recalls (1 weekend day and 
2 week days). These will be administered by telephone or in person by a trained study 
nutritionist or coordinator after the screening visit/enrollment and prior to Day 0.  
Day -14 MRI,: MRI will be used to assess level of liver fat within 10 days of Day 0 (diet 
initiation). At the Emory site, both the usual MRI based quantification of hepatic fat and a 
10-minute HISTO protocol will be collected during a single 30 minute MRI. The HISTO 

 
23 
 
protocol is a highly precise measurement of hepatic fat and will be used to determine 
eligibility for the study.  If the hepatic fat is >10% by MRI PDFF, the participant will be 
enrolled.  The % hepatic fat for the outcome measurements will be determined by the 
UCSD center’s hepatic PDFF MRI protocol. The MRI may be repeated if images are 
inadequate and the MRI may be used to measure visceral fat. 
Re-screening: Participants that screenfail for any reason may re-screen after a period of 
6 months. 
Day -9   Randomization: Randomization will be assigned after the initial screening visit 
and the participant will be informed of their assignment after the 24-hour dietary recalls 
but before Day 0.  
Day -7 Home Visit Food Assessment:  A study coordinator and nutritionist will visit the 
home of intervention-arm participants and complete the assessment of the family’s usual 
consumption patterns, recipes and the initial menus.   
6.2 
Day 0: Baseline Visit 
 
Once the screening assessments are complete and the participant is deemed eligible to 
participate in the study the participant will be scheduled for their Day 0 visit. This visit 
may be scheduled up to 60 days after the screening visit.  
Height, weight, waist and hip circumference measurements and vital signs will be 
collected at this visit. Any adverse events and changes to any concomitant medications 
will be reviewed as well.  A sweetness perception test will be administered at this visit. 
For the intervention arm, the nutritionist will meet with the family during this visit to 
review the details of the intervention. The standard of care participants will have diet 
assessment but no instructions on diet. Instructions will be given for compliance and the 
nutritionist will arrange a time to meet with the family in the home. 
The following labs will also be drawn at this visit: 
• 
CMP 
• 
GGT 
• 
Lipid Profile 
• 
Insulin 
• 
Plasma and serum for storage 
• 
Stool sample collection 
After the research visit, the food for the first several days of the study will be delivered to the 
home of the intervention participants and the sugar-containing non-perishable foods will be 
boxed up and stored at the home (or another location at the discretion of the family).  
Perishable sugar-containing foods will be disposed of and replaced with no free sugars 
containing versions.   
 

 
24 
 
6.3 
Follow-up Visit Day 28 
The Day 28 visit will take place 28 days (4 weeks) after baseline. All assessments and 
testing done at the baseline visit will be repeated at this visits with the exception of the 
nutrition counseling, and stool collection. 
6.4  
Follow-up Visit Day 56 
Up to 6 weeks prior to the Day 56 visit three 24-hour, interviewer assisted, dietary recalls 
(1 weekend day and 2 week days) will be performed. These will be administered by 
telephone or in person by a trained study nutritionist or coordinator at least 1 week prior 
to the Day 56 visit. This visit will take place 56 days (8 weeks) after baseline. All 
assessments and testing done at the baseline visit will be repeated at this visit with the 
exception of the nutrition counseling. 
6.5 
End of study follow-up 
A phone visit will take place two weeks after the Day 56 visit. The purpose of this call is 
to discuss feedback about the study with the participant and his or her family. We will 
ask for details about what they liked or disliked about study participation and ask for any 
suggestions for future studies. 

 
25 
 
7 
STATISTICAL CONSIDERATIONS 
7.1 
Sample Size Considerations 
Initial analyses will be undertaken to inspect data for errors, inconsistencies, and 
incomplete information. This will include examining the data with simple frequency tables 
and dot plots for univariate data and scatter plots and multi-way dot plots with bivariate 
and multivariate data. Data anomalies and outliers will be examined and corrected if 
necessary. To summarize bivariate relationships between predictors and hepatic fat 
percent, Spearman’s rank correlation coefficient, rs, will be used. For reporting inferential 
statistics, such as differences in means, 95 percent confidence intervals will be used 
extensively to quantify degree of clinical efficacy. For any models, appropriate 
assumptions and model conditions will be verified prior to analysis.  
Analyses will include descriptive statistics at baseline and for each treatment group for 
all outcome variables, plots of longitudinal data over time, and examination of 
distributions within groups at important nodal points (e.g., Baseline, 4 weeks and 8 
weeks).  All longitudinal models will include baseline measurements as a covariate to 
adjust for potential differences between treatment groups at baseline. All efficacy 
analysis will follow the intention-to-treat convention (inclusion of all randomized 
participants in the analysis). Participants that drop out or are lost to follow-up will be 
compared to those that remain in the study to assess for bias and generalizability of the 
results.  All analyses will be conducted using SAS v9.3 for Windows (Cary, NC, USA).  
Power Analysis 
The primary outcome is change in MRI PDFF from baseline to 8 weeks in the 
intervention group compared to the standard of care group. Participants will be screened 
at baseline and confirmed that their hepatic fat is < 10%. Given 40 completed 
participants, (20 randomized to the diet low in free sugars and 20 randomized to usual 
diet), our goal is show a 25% improvement in MRI PDFF with intervention over control.  
For example:  
 
Intervention: 15% hepatic fat baseline – (30% change = 4.5%) = Hepatic fat of 10.5% at 
8 weeks 
 
Control: 15% hepatic fat at baseline – (5% change = 0.75%) = Hepatic fat of 14.25% at 8 
weeks 
 
A two sample, two-tailed t test with an overall sample size of 40 participants (20 per 
group) achieves greater than 90% power to detect a true difference of means of 4% 
when the sigma is 3%. Power was calculated assuming that 20% of patients in each 
group will be lost to follow-up with a 0.05 significance level.  
 
 

 
26 
 
Plan for missing data:  
We note that attrition, participant compliance and systematic data collection are 
fundamental requirements for this study. Successful coordination of participants and 
management of data are important prerequisites for a subsequent trial. Thus, we focus 
appropriate attention to missing data. Prevention is the first line for controlling bias and 
loss of power from missing data. Upon entry, alternative contacts will be identified for all 
participants to minimize loss-to follow-up.  Consistent with the intent to treat principle, we 
will follow-up with all randomized participants regardless of the actual treatment received 
(we will invite families who miss visits to return for assessments later if possible). 
Participants who express intent to dropout completely will be asked to attend an early 
termination visit to collect endpoint measures. Timely data entry combined with monthly 
missing data reports will prompt tracking down missing outcome assessments. Despite 
these prevention efforts, missing data will occur. Our primary analysis is valid under the 
assumption that missing data are missing at random (MAR).  To evaluate this 
assumption, we will examine the extent of missing data and missing data patterns, and 
determine the reasons and time of dropout.  
 

 
27 
 
8 
ETHICS/PROTECTION OF HUMAN PARTICIPANTS 
8.1 
Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report: Ethical Principles and Guidelines for the 
Protection of Human Participants of Research, as drafted by the US National 
Commission for the Protection of Human Participants of Biomedical and Behavioral 
Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the ICH E6; 62 Federal 
Regulations 25691 (1997).   
8.2 
Institutional Review Board 
Permission to perform this study will be sought from the Emory University IRB and the 
USCD IRB. All future modifications of the study or changes in the protocol will receive 
IRB approval. 
8.3 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continues throughout the individual’s study participation. 
Informed consents and HIPAA waivers will be obtained prior to initiating any study 
procedure. Participants and their parents or legal guardian will be approached to 
participate in the study. The research coordinators and PI/Co-I will discuss the study with 
them and give them all the information listed above in language understandable at the 
level of the parent/guardian and all information needed to make an informed choice 
about participation, including information about NAFLD, the study intervention, possible 
risks to participation, study procedures, study visits/contacts and potential benefits to the 
participant. Consent will be documented by signature of the parent/guardian. 
8.4 
Informed Assent Process 
All children must provide written assent (11-16 y) in a language appropriate for the age 
of the child. Written assent will be documented by signature in age 11-16 yrs. Assent will 
be documented by signature of the child participant. 
8.5 
Participant Confidentiality 
Participant confidentiality is strictly held in trust by the participating investigators, their 
staff, and the sponsor(s) and their agents. The study protocol, documentation, data, and 
all other information generated will be held in strict confidence. No information 
concerning the study or the data will be released to any third party without prior approval 
of the participant. 

 
28 
 
Authorized representatives of the sponsor may inspect all documents and records 
required to be maintained by the investigator.   
8.6 
Study Discontinuation 
In the event that the study is discontinued, participants will be notified of the date of 
discontinuation.  
8.7 
Data Safety Monitoring Plan 
Data Management: Data for each subject will be collected in individual folders kept in a 
locked filing cabinet in a secure office. Data will be entered into a secure RedCap 
database within the same division. The study statistician will run routine reports for 
completeness and send to the site PIs. 
Adverse Events: The PI at each site will monitor adverse events throughout the course 
of the study. Any adverse events occurring during the study will be documented and 
reported according to applicable IRB policies and procedures.  AE’s will be entered into 
RedCap in a timely fashion. The study statistician will run quarterly reports and will send 
out pooled analyses to the PI’s at both sites. A status report will be provided to the IRB 
at the time of continuing review.  
Serious Adverse Events: SAE’s are not anticipated in this study, however, any SAE that 
occurs will be reported to the other site, respective IRBs, and sponsor within 10 business 
days of first knowledge of the event. 
Monitoring: A representative from the sponsor will conduct a site monitoring visit after 
the 1st patient is randomized and then again approximately every 6 months. This site 
visit will review the informed consent process, eligibility, CRFs, and AE reporting. A 
monitoring report will be provided to the PIs within 10 business days of review. The PI 
will review the monitoring report and follow up on any corrective actions on the site 
monitoring log and will notify the respective IRB according to applicable policies and 
procedures.  

 
29 
 
9 
DATA HANDLING AND RECORD KEEPING 
All data will be entered, stored and processed within REDCap, a secure, web-based 
application for managing databases. All access to office space containing paper source 
documents requires badged entry. All computer files are stored on secure servers.  
Data management staff members are required to complete and pass an on-line HIPAA 
course and other confidentiality certification procedures upon employment.  All computer 
systems and programs are password protected, and all web-based electronic 
communications of study information is encrypted. Good computer security practices 
(restricting physical access to computers, prohibition of password sharing, timing out of 
system access interfaces, etc.) is required. Virus protection software is installed on all 
study machines. The virus protection tools are used, maintained, and updated as 
necessary on all computers and pathways into the system. 
Only study personnel (PI, co-Is, study coordinators, research assistants, and 
nutritionists) will have access to identifiable data. Data will be de-identified before 
analysis and will be stored in locked research material cabinets. 
9.1 
Data Management Responsibilities 
Emory University will serve as the data coordinating center for this study and will be 
responsible for data management, quality review, analysis, and reporting of the study 
data.  The statistician will review data completion monthly during the active portion of the 
study.   
9.2 
MRI Data Coordination 
UCSD will serve as the radiology coordinating center for this study. Anonymized CD’s 
will be shipped to UCSD and all MRI’s will be read and interpreted at this site. The 
screening MRI will be transferred to UCSD via a secure cloud-based network for a faster 
read turnaround for eligibility. 
9.3 
Types of Data 
Data for this study will include self-report patient data, nutritional data from NDSR, 
laboratory values, medical records, and imaging data. 
9.5 
Study Records Retention 
Study documents will be retained for the minimum number of years per Emory University 
and UCSD requirements. No records will be destroyed without the written consent of the 
sponsor. It is the responsibility of the sponsor to inform the investigator when these 
documents no longer need to be retained. Electronic data, stripped of identifiers, will be 
stored on a secure server and will be kept indefinitely. 
 

 
30 
 
10 
LITERATURE REFERENCES 
 
1. 
Lazo M, Hernaez R, Eberhardt MS, Bonekamp S, Kamel I, Guallar E, Koteish A, 
Brancati FL and Clark JM. Prevalence of nonalcoholic fatty liver disease in the United 
States: the Third National Health and Nutrition Examination Survey, 1988-1994. Am J 
Epidemiol. 2013;178:38-45. 
2. 
Welsh JA, Karpen S and Vos MB. Increasing prevalence of nonalcoholic fatty 
liver disease among United States adolescents, 1988-1994 to 2007-2010. J Peds. 
2013;162:496-500. 
3. 
Fishbein MH, Miner M, Mogren C and Chalekson J. The spectrum of fatty liver in 
obese children and the relationship of serum aminotransferases to severity of steatosis. 
J Pediatr Gastroenterol Nutr. 2003;36:54-61. 
4. 
Lavine JE and Schwimmer JB. Nonalcoholic fatty liver disease in the pediatric 
population. Clinics in liver disease. 2004;8:549-58, viii-ix. 
5. 
Louthan MV, Barve S, McClain CJ and Joshi-Barve S. Decreased serum 
adiponectin: an early event in pediatric nonalcoholic fatty liver disease. J Pediatr. 
2005;147:835-8. 
6. 
Louthan MV, Theriot JA, Zimmerman E, Stutts JT and McClain CJ. Decreased 
prevalence of nonalcoholic fatty liver disease in black obese children. J Pediatr 
Gastroenterol Nutr. 2005;41:426-9. 
7. 
Schwimmer JB, Deutsch R, Rauch JB, Behling C, Newbury R and Lavine JE. 
Obesity, insulin resistance, and other clinicopathological correlates of pediatric 
nonalcoholic fatty liver disease. The Journal of pediatrics. 2003;143:500-5. 
8. 
Schwimmer JB, McGreal N, Deutsch R, Finegold MJ and Lavine JE. Influence of 
gender, race, and ethnicity on suspected fatty liver in obese adolescents. Pediatrics. 
2005;115:e561-5. 
9. 
Schwimmer JB, Pardee PE, Lavine JE, Blumkin AK and Cook S. Cardiovascular 
risk factors and the metabolic syndrome in pediatric nonalcoholic fatty liver disease. 
Circulation. 2008;118:277-83. 
10. 
Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M 
and Sanyal AJ. The diagnosis and management of non-alcoholic fatty liver disease: 
practice Guideline by the American Association for the Study of Liver Diseases, 
American College of Gastroenterology, and the American Gastroenterological 
Association. Hepatology (Baltimore, Md). 2012;55:2005-23. 
11. 
Schwimmer JA, McGreal N, Deutch R, Finegold MJ and Lavine JE. Influence of 
gender, race and ethnicity on suspected fatty liver in obese adolescents Pediatrics. 
2005;115:e561-e565. 
12. 
Wagenknecht LE, Scherzinger AL, Stamm ER, Hanley AJ, Norris JM, Chen YD, 
Bryer-Ash M, Haffner SM and Rotter JI. Correlates and heritability of nonalcoholic fatty 
liver disease in a minority cohort. Obesity (Silver Spring, Md). 2009;17:1240-6. 
13. 
Hoyert D and Xu J. Deaths: Preliminary Data for 2011. National Vital Statistics 
Reports Centers for Disease Control and Prevention. 2012;61. 
14. 
Heron M. Deaths: Leading Causes for 2009. National Vital Statistics Reports 
Center for Disease Control and Prevention. 2012;61. 

 
31 
 
15. 
Adams L, Lymp J, Sauver J, Sanderson S, Lindor K, Feldstein A and Angulo P. 
The natural history of nonalcoholic fatty liver disease: a population based cohort study. 
Gastroenterology. 2005;129:113-121. 
16. 
Ramilli S, Pretolani S, Muscari A, Pacelli B and Arienti V. Carotid lesions in 
outpatients with nonalcoholic fatty liver disease. World J Gastroenterol. 2009;15:4770-4. 
17. 
Targher G, Chonchol M, Bertolini L, Rodella S, Zenari L, Lippi G, Franchini M, 
Zoppini G and Muggeo M. Increased risk of CKD among type 2 diabetics with 
nonalcoholic fatty liver disease. J Am Soc Nephrol. 2008;19:1564-70. 
18. 
Kontush A and Chapman M. Functionally defective high-density lipoprotein: a 
new therapeutic target at the crossroads of dyslipidemia, inflammation, and 
atherosclerosis. Pharmacol Rev. 2006;58:342-74. 
19. 
Hanley AJ, Williams K, Festa A, Wagenknecht LE, D'Agostino RB, Jr., Kempf J, 
Zinman B and Haffner SM. Elevations in markers of liver injury and risk of type 2 
diabetes: the insulin resistance atherosclerosis study. Diabetes. 2004;53:2623-32. 
20. 
Hanley AJ, Williams K, Festa A, Wagenknecht LE, D'Agostino RB, Jr. and 
Haffner SM. Liver markers and development of the metabolic syndrome: the insulin 
resistance atherosclerosis study. Diabetes. 2005;54:3140-7. 
21. 
Armstrong MJ, Adams LA, Canbay A and Syn WK. Extrahepatic complications of 
nonalcoholic fatty liver disease. Hepatology (Baltimore, Md). 2014;59:1174-97. 
22. 
Soderberg C, Stal P, Askling J, Glaumann H, Lindberg G, Marmur J and 
Hultcrantz R. Decreased survival of subjects with elevated liver function tests during a 
28-year follow-up. Hepatology. 2009. 
23. 
Fielding J. Disparities in Deaths from Chronic Liver Disease and Cirrhosis. Office 
of Health Assessment and Epidemiology 2012;June. 
24. 
Jacome-Sosa MM and Parks EJ. Fatty acid sources and their fluxes as they 
contribute to plasma triglyceride concentrations and fatty liver in humans. Curr Opin 
Lipidol. 2014;25:213-20. 
25. 
Lambert JE, Ramos-Roman MA, Browning JD and Parks EJ. Increased de novo 
lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease. 
Gastroenterology. 2014;146:726-35. 
26. 
Sevastianova K, Santos A, Kotronen A, Hakkarainen A, Makkonen J, Silander K, 
Peltonen M, Romeo S, Lundbom J, Lundbom N, Olkkonen VM, Gylling H, Fielding BA, 
Rissanen A, Yki-Jarvinen HCINAJCNO and Pmid. Effect of short-term carbohydrate 
overfeeding and long-term weight loss on liver fat in overweight humans. The American 
journal of clinical nutrition. 2012;96:727-34. 
27. 
Vos MB, Kimmons JE, Gillespie C, Welsh J and Blanck HM. Dietary fructose 
consumption among US children and adults: the Third National Health and Nutrition 
Examination Survey. Medscape J Med. 2008;10:160. 
28. 
Bray GA and Popkin BM. Calorie-sweetened beverages and fructose: what have 
we learned 10 years later. Pediatric obesity. 2013;8:242-8. 
29. 
Welsh JA, Sharma AJ, Grellinger L and Vos MB. Consumption of added sugars 
is decreasing in the United States. The American journal of clinical nutrition. 
2011;94:726-34. 
30. 
Makarem N, Scott M, Quatromoni P, Jacques P and Parekh N. Trends in dietary 
carbohydrate consumption from 1991 to 2008 in the Framingham Heart Study Offspring 
Cohort. The British journal of nutrition. 2014:1-14. 

 
32 
 
31. 
Ng SW, Slining MM and Popkin BM. Turning point for US diets? Recessionary 
effects or behavioral shifts in foods purchased and consumed. The American journal of 
clinical nutrition. 2014;99:609-16. 
32. 
Kit BK, Fakhouri TH, Park S, Nielsen SJ and Ogden CL. Trends in sugar-
sweetened beverage consumption among youth and adults in the United States: 1999-
2010. The American journal of clinical nutrition. 2013;98:180-8. 
33. 
Bray GA and Popkin BM. Dietary sugar and body weight: have we reached a 
crisis in the epidemic of obesity and diabetes?: health be damned! Pour on the sugar. 
Diabetes care. 2014;37:950-6. 
34. 
Schwarz JM, Noworolski SM, Wen MJ, Dyachenko A, Prior JL, Weinberg ME, 
Herraiz LA, Tai VW, Bergeron N, Bersot TP, Rao MN, Schambelan M and Mulligan K. 
Effect of a High-Fructose Weight-Maintaining Diet on Lipogenesis and Liver Fat. J Clin 
Endocrinol Metab. 2015:jc20143678. 
35. 
Ouyang X, Cirillo P, Sautin Y, McCall S, Bruchette JL, Diehl AM, Johnson RJ and 
Abdelmalek MF. Fructose consumption as a risk factor for non-alcoholic fatty liver 
disease. J Hepatol. 2008;48:993-9. 
36. 
O'Sullivan TA, Oddy WH, Bremner AP, Sherriff JL, Ayonrinde OT, Olynyk JK, 
Beilin LJ, Mori TA and Adams LA. Lower fructose intake may help protect against 
development of nonalcoholic fatty liver in adolescents with obesity. Journal of pediatric 
gastroenterology and nutrition. 2014;58:624-31. 
37. 
Abdelmalek MF, Suzuki A, Guy C, Unalp-Arida A, Colvin R, Johnson RJ and 
Diehl AM. Increased fructose consumption is associated with fibrosis severity in patients 
with nonalcoholic fatty liver disease. Hepatology. 2010;51:1961-71. 
38. 
Jin R, Welsh JA, Le NA, Holzberg J, Sharma P, Martin DR and Vos MB. Dietary 
fructose reduction improves markers of cardiovascular disease risk in Hispanic-
American adolescents with NAFLD. Nutrients. 2014;6:3187-201. 
39. 
Mager DR, Iniguez IR, Gilmour S and Yap J. The Effect of a Low Fructose and 
Low Glycemic Index/Load (FRAGILE) Dietary Intervention on Indices of Liver Function, 
Cardiometabolic Risk Factors, and Body Composition in Children and Adolescents With 
Nonalcoholic Fatty Liver Disease (NAFLD). JPEN J Parenter Enteral Nutr. 2015;39:73-
84. 
40. 
Schwimmer JB, Middleton MS, Behling C, Newton KP, Awai HI, Paiz MN, Lam J, 
Hooker JC, Hamilton G, Fontanesi J and Sirlin CB. Magnetic resonance imaging and 
liver histology as biomarkers of hepatic steatosis in children with nonalcoholic fatty liver 
disease. Hepatology. 2014. 
41. 
Zand KA, Shah A, Heba E, Wolfson T, Hamilton G, Lam J, Chen J, Hooker JC, 
Gamst AC, Middleton MS, Schwimmer JB and Sirlin CB. Accuracy of multiecho 
magnitude-based MRI (M-MRI) for estimation of hepatic proton density fat fraction 
(PDFF) in children. J Magn Reson Imaging. 2015. 
42. 
Artz NS, Haufe WM, Hooker CA, Hamilton G, Wolfson T, Campos GM, Gamst 
AC, Schwimmer JB, Sirlin CB and Reeder SB. Reproducibility of MR-based liver fat 
quantification across field strength: Same-day comparison between 1.5T and 3T in 
obese subjects. J Magn Reson Imaging. 2015. 
43. 
Suzuki A, Abdelmalek MF, Schwimmer JB, Lavine JE, Scheimann AO, Unalp-
Arida A, Yates KP, Sanyal AJ, Guy CD, Diehl AM and Nonalcoholic Steatohepatitis 

 
33 
 
Clinical Research N. Association between puberty and features of nonalcoholic fatty 
liver disease. Clin Gastroenterol Hepatol. 2012;10:786-94. 
44. 
Uwaifo GI, Parikh SJ, Keil M, Elberg J, Chin J and Yanovski JA. Comparison of 
insulin sensitivity, clearance, and secretion estimates using euglycemic and 
hyperglycemic clamps in children. The Journal of clinical endocrinology and 
metabolism. 2002;87:2899-905. 
45. 
Gungor N, Saad R, Janosky J and Arslanian S. Validation of surrogate estimates 
of insulin sensitivity and insulin secretion in children and adolescents. J Pediatr. 
2004;144:47-55. 
46. 
Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF and Turner RC. 
Homeostasis model assessment: insulin resistance and beta-cell function from fasting 
plasma glucose and insulin concentrations in man. Diabetologia. 1985;28:412-9. 
47. 
Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G and Quon MJ. 
Quantitative insulin sensitivity check index: a simple, accurate method for assessing 
insulin sensitivity in humans. The Journal of clinical endocrinology and metabolism. 
2000;85:2402-10. 
48. 
Mather KJ, Hunt AE, Steinberg HO, Paradisi G, Hook G, Katz A, Quon MJ and 
Baron AD. Repeatability characteristics of simple indices of insulin resistance: 
implications for research applications. The Journal of clinical endocrinology and 
metabolism. 2001;86:5457-64. 
49. 
Yokoyama H, Emoto M, Fujiwara S, Motoyama K, Morioka T, Komatsu M, 
Tahara H, Shoji T, Okuno Y and Nishizawa Y. Quantitative insulin sensitivity check 
index and the reciprocal index of homeostasis model assessment in normal range 
weight and moderately obese type 2 diabetic patients. Diabetes Care. 2003;26:2426-32. 
50. 
Uwaifo GI, Fallon EM, Chin J, Elberg J, Parikh SJ and Yanovski JA. Indices of 
insulin action, disposal, and secretion derived from fasting samples and clamps in 
normal glucose-tolerant black and white children. Diabetes Care. 2002;25:2081-7. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
32 
 
11 
Attachments 
11.1 SCHEDULE OF EVENTS 
*unless participating in DNL substudy, then see diagram 
Study Procedure 
Screening     
(up to -60 Days) 
-14 Days* 
(±1 week) 
-9 Days 
(±3 Days) 
-7 Days 
(±3 Days) 
Baseline 
(Day 0) 
Day 28       
(±5 Days) 
Day 56 
(±3 Days) 
Follow-up 
Day 68 
(±3 Days) 
Informed Consent and HIPAA Authorization 
X 
 
 
 
 
 
 
 
Review Inclusion/Exclusion Criteria 
X 
 
X 
 
 
 
 
 
Randomization 
 
 
X 
 
 
 
 
 
Demographics & Medical History 
X 
 
 
 
 
 
 
 
Vital Signs 
X 
X 
 
 
X 
X 
X 
 
Height and Weight 
X 
 
 
 
X 
X 
X 
 
Waist and Hip 
X 
 
 
 
X 
X 
X 
 
Physical Exam 
X 
 
 
 
 
 
 
 
MRI 
 
X 
 
 
 
X 
X 
 
AUDIT Questionnaire 
X 
 
 
 
 
 
 
 
3 x 24-hour food recalls (NDS-R) 
X 
 
 
 
 
 
X 
 
Beverage Questionnaire (Bev-Q) 
X 
 
 
 
 
 
 
 
Instructions for Compliance 
X 
 
 
 
X 
 
 
 
Home Visit Food Assessment 
 
 
 
X 
 
 
 
 
Sweet Taste Test 
 
 
 
 
X 
X 
X 
 
Adverse Events Review 
 
 
 
 
X 
X 
X 
 
Concomitant Medication Review 
X 
 
 
 
X 
X 
X 
 
Stool Collection 
 
 
 
 
X 
 
X 
 
Initiate Heavy Water (30 min Observation) 
 
X 
 
 
 
 
 
 
Blood Draw 
X 
X 
 
 
X 
X 
X 
 
End of study phone interview 
 
 
 
 
 
 
 
X 

 
33 
 
11.2 BLOOD DRAW SCHEDULE 
 
 
Blood Test 
Screening        
(-60 days) 
Baseline 
 (Day 0) 
Follow-up 1 
Day 28        
Follow-up 2 
Day 56 
Total 
Volume 
Complete Blood Count 
1 
- 
- 
- 
1 
Comprehensive Metabolic Panel  
1 
1 
1 
1 
4 
HbA1c 
3 
- 
- 
- 
3 
Lipid Profile  
- 
3 
3 
3 
9 
Direct bilirubin 
1 
- 
- 
- 
1 
GGT 
- 
1 
1 
1 
3 
Insulin 
- 
1 
- 
- 
1 
Serum 
6 
6 
6 
6 
24 
Plasma 
12 
12 
12 
12 
48 
PT/INR 
3 
- 
- 
- 
3 
Total 
27 mL 
24 mL 
23 mL 
23 mL 
97 mL 
 

 
32 
 
Optional Sub-study Amendment: 
 
De novo lipogenesis sub-study for the protocol “Randomized, Controlled Dietary 
Treatment of Pediatric NAFLD” 
 
Background: 
The diet treatment study is an 8 week, randomized, controlled clinical trial of a low sugar 
diet as a treatment for NAFLD.  In the main study, children will be evaluated at baseline, 
4 weeks and 8 weeks using magnetic resonance spectroscopy and labs.  Because 
carbohydrates and especially simple sugars stimulate de novo lipogenesis (DNL – new 
fat synthesis in the liver), an important secondary outcome of the study is change in 
DNL.  In this optional sub-study, we propose to add the use of heavy water (2H2O) 
metabolite measurement as a tool for determining DNL and markers of fibrogenesis at 
baseline and at 8 weeks.   
 
Heavy water is a form of water that is enriched for the hydrogen isotope deuterium 
compared to the common hydrogen-1 isotope that makes up most hydrogen in water. 
Heavy water is not radioactive and is physically and chemically similar to regular water. 
The human body naturally contains some deuterium. Stable isotopes have a long 
history as a safe, effective tracer for measuring synthesis of molecules in humans 40. 
Small oral doses of 2H20 are routinely used in human metabolic experiments and there 
are no known health risks with small amounts.   
Utilizing in vivo heavy water labeling and a small amount of plasma, multiple pathways 
related to protein metabolism (fibrogenesis) and lipid metabolism can be quantified in a 
minimally invasive fashion in human subjects. Recently, new developments in stable 
isotope labeling techniques and advances in mass spectrometry have made in vivo 
kinetic measurement of slow metabolic processes possible. Through the use of 2H2O 
(heavy water) as the source of labeling, our collaborators and others have measured T-
cell proliferation 41, mammary epithelial cell proliferation 42, prostate epithelial cell 
proliferation 43, triglyceride synthesis 44and protein synthesis 45 in humans. DNL as well 
as hepatic TG assembly and cholesterogenesis are easily measured in plasma from 
patients consuming 2H2O, after 5-14 days of labeling the plasma DNL reaches a steady 
state and reflects hepatic DNL rates.    
Both Emory and UCSD research entities and IRBs have previous experience with heavy 
water based research studies and this sub-study is minimal risk to participants.  
 
Hypothesis:  We hypothesize that DNL will significantly decrease in the children with 
NAFLD randomized to the low sugar diet.   

 
33 
 
 
Risks: 
7) Heavy water – 2H2O Intake — Participants will be given heavy water (deuterated 
water, 2H2O) for up to 28 days over the course of the study. There are no known 
medical risks of heavy water (2H2O) intake.  2H2O is not radioactive. Rarely, 
individuals (<3%) may experience temporary light-headedness, dizziness, or vertigo 
(room spinning) when they take the first doses of 2H2O.  These symptoms are self-
limited and resolve within 2-3 hours. Participants will also be instructed to avoid 
driving or performing other potentially hazardous activities for one hour after drinking 
the initial dose of 2H2O in case they become dizzy. Participants will be instructed on 
the intake of 2H2O to ensure a slow rise in initial body water enrichment and thereby 
avoid these symptoms. This is achieved by drinking 50 mL of 70% 2H2O twice a day. 
This labeling protocol has been shown in >100 human participants in studies to 
produce a steady rise to plateau body water enrichments of 1.0-2.0% 2H2O  49.  
 
Potential Benefits: 
There are no additional benefits to the participants from this additional study measure.   
 
Protection Against Risks: 
Participants will be given their first dose of heavy water in the pediatric research unit 
and observed for at least 90 minutes afterwards.  The heavy water dose is divided into 
twice daily doses so that the dose is small at each consumption. No large loading dose 
(sometimes used in studies) is being used to improve safety for the children.   
 
Outcomes: 
Results of this sub-study are secondary outcomes for the main study.  Outcomes 
include baseline rates of DNL, fibrogenesis markers, change in DNL from baseline, and 
other metabolites of interest.  
 
Inclusion and Exclusion Criteria: 
Same as main study. 
 
Consenting and Assenting Process: 
After the main study is explained, the sub-study will also be explained.  Participants will 
be provided ample time to ask questions about the sub-study.  There is no penalty for 
not participating in the sub-study.   
 
 

 
34 
 
Sub-Study Schedule and Procedures 
The heavy water labeling will occur in two time periods – one at the beginning of the 
study and one at the end.   
 
Day -7 to -5: For all screening subjects participating in the sub-study, heavy water 
labeling will be initiated at a study visit in the pediatric research unit. Subjects will be 
monitored for 30 minutes and vitals will be obtained. Instructions and heavy water doses 
for 7 days will be provided to the participants to take home. The heavy water will be 
provided in sealed single dose bottles and participants will be asked to consume one in 
the morning and one any time after dinner.  
 
If subjects do not enroll in the full study because of their screening MRI results they will 
be instructed to dispose of the water at home and will not have any further visits. 
   
Day 0:  6 mL of fasting plasma will be collected along with a blood spot during their day 
0 visit at the research center.   
 
Day 47 to 53: Saliva sample and a blood spot will be collected during a home visit.  
After the specimen collection, the participant will resume heavy water twice a day for up 
to 7 days (completing the evening before the final study visit).   
 
Day 56: At the final study visit, 6 mL of fasting plasma and saliva will be collected from 
the participants. A blood spot on filter paper will also be collected at this visit.  
 
Sample Handling and Data Transfers: 
The saliva and plasma samples will be shipped to KineMed for measurement of the 
sub-study markers.  A second reserved plasma sample will be retained at each study 
site as back up in case of issues with the first sample.   
 
All samples will be de-identified and will be provided to KineMed collated and in random 
order in batches over the course of the study.  Saliva and plasma samples will be batch-
shipped to Kinemed from each site. These samples will be used to assess the methods 
and sample quality. Technicians at KineMed will be blinded to study assignment 
(intervention or control).   
 
The results of the analysis of the saliva and plasma will be entered into the study 
Redcap database through coordination between the study statistical team and KineMed 
using secure systems.   
 
 
 

 
35 
 
Power Analysis and Statistical Considerations: 
There are no previous studies reported in children with NAFLD evaluating de novo 
lipogenesis and thus the effect size is unknown.  Consistent with the intent of the main 
study to provide pilot data for a larger RCT, this sub-study will provide important, novel 
information on the rates of DNL and response to diet change in a cohort of children.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
36 
 
Sub-Study References: 
 
1. 
Jones, P.J., et al., Human cholesterol synthesis measurement using deuterated water. 
Theoretical and procedural considerations. Arterioscler Thromb, 1993. 13(2): p. 247-53. 
2. 
Hellerstein, M., et al., Directly measured kinetics of circulating T lymphocytes in normal 
and HIV-1-infected humans. Nat Med, 1999. 5(1): p. 83-9. 
3. 
Misell, L.M., et al., Development of a novel method for measuring in vivo breast 
epithelial cell proliferation in humans. Breast Cancer Reseach and Treatment, In Press. 
4. 
Misell, L., et al., Development of an in vivo stable isotope-mass spectrometric method for 
measuring proliferation of prostate epithelial cells (PEC) from prostate tissue and 
ejaculated seminal. Proceedings of the American Association for Cancer Research, 2004. 
45: p. 1032-1033. 
5. 
Turner, S.M., et al., Measurement of TG synthesis and turnover in vivo by 2H2O 
incorporation into the glycerol moiety and application of MIDA. Am J Physiol Endocrinol 
Metab, 2003. 285(4): p. E790-803. 
6. 
Hellerstein M, N.R., Kim Y-K, Schade- and C.M. Serin V, Measurement of synthesis rates 
of slow-turnover proteins from 2H2O incorporation into non-essential amino acids 
(NEAA) and application of mass isotopomer distribution analysis (MIDA). FASEB J., 2002. 
16: p. A256 (abstract). 
7. 
Neese, R.A., et al., Measurement in vivo of proliferation rates of slow turnover cells by 
2H2O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci U S A, 2002. 99(24): 
p. 15345-50.