Genetic-specific Effects of Fructose on Liver Lipogenesis
Phase: NA
Conditions: NAFLD
Sponsor: University of North Carolina, Chapel Hill

 
Genetic-Specific Effects of Fructose on Liver Lipogenesis 
 
 
 
NCT number NCT03783195 
Document Date 05/15/2021 
 

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PROTOCOL TEMPLATE: INTERVENTIONAL STUDY 
Complete Title: Genetic-specific effects of fructose on liver lipogenesis  
 
Short Title: Fructose-Liver fat study (FLFS) 
Drug or Device Name(s): Sugar-sweetened drinks 
FDA IND/IDE (if applicable): 
Sponsor: Trans-NORC complementary pilot grant award 
Protocol Date: 05/15/21 
Amendment 1 Date: 
Amendment 2 Date:  
Amendment 3 Date: 
Amendment 4 Date:  
 
 
 
 

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PROTOCOL TITLE:  
Genetic-specific effects of fructose on liver lipogenesis  
Short Title:  
Fructose-Liver fat study (FLFS) 
Lead Investigator: 
Saroja Voruganti, PhD 
 University of North Carolina at Chapel Hill 
Protocol Version: 3 
Version Date: 05/15/2021 
 
I confirm that I have read this protocol and understand it.    
Principal Investigator Name:  
 
Saroja Voruganti 
Principal Investigator Signature:          
 
Date:   
 
 
 
05/15/2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
Table of Contents ................................................................................................................................ 3 
Abbreviations and Definitions of Terms ........................................................................................... 4 
Protocol Synopsis ............................................................................................................................... 5 
1 
BACKGROUND  AND RATIONALE ............................................................................................... 8 
2 
STUDY OBJECTIVES .................................................................................................................... 9 
3 
INVESTIGATIONAL PLAN ........................................................................................................... 10 
4 
STUDY PROCEDURES ............................................................................................................... 10 
5 
STUDY EVALUATIONS AND MEASUREMENTS ....................................................................... 13 
6 
STATISTICAL CONSIDERATIONS .............................................................................................. 13 
7 
STUDY INTERVENTION (DEVICE OR OTHER INTERVENTION) ............................................. 14 
8 
STUDY INTERVENTION ADMINISTRATION .............................................................................. 14 
9 
SAFETY MANAGEMENT ............................................................................................................. 14 
10  DATA COLLECTION AND MANAGEMENT ................................................................................. 14 
11 RECRUITMENT STRATEGY ....................................................................................................... 14 
12 CONSENT PROCESS .................................................................................................................. 14 
13 PUBLICATION .............................................................................................................................. 14 
14 REFERENCES.............................................................................................................................. 15 
Appendix .............................................................................................................................................. 0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
Insert and delete terms as relevant 
 
Abbreviation 
Definition 
NAFLD 
Nonalcoholic fatty liver disease 
PNPLA3 
phospholipase, domain-containing 3 
GCKR 
Glucokinase regulatory protein 
AST 
Aspartate transaminase 
ALT 
Alanine transaminase 
GGT 
Gamma Glutamyl transferase 
HDL 
High-density lipoprotein 
LDL 
Low-density lipoprotein 
TG 
Triglycerides 
VLDL-TG 
Very low-density lipoprotein-triglycerides 
MRI 
Magnetic resonance imaging 
DNL 
de novo lipogenesis 
SNP 
Single nucleotide polymorphism 
LCMS 
Liquid chromatography mass spectrometry 
 
 

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PROTOCOL SYNOPSIS 
 
Study Title 
Genetic-specific effects of fructose on liver lipogenesis  
Funder 
Trans-NORC Complementary pilot Grant Program 
Clinical Phase 
N/A 
Study Rationale 
Fructose consumption has dramatically increased over the past 20 years in the United 
States (US) and worldwide1. High fructose intake has been shown to increase serum 
triglycerides, upregulate de novo lipogenesis, reduce fatty acid oxidation and decrease 
the clearance of very low density lipoprotein triglycerides in liver leading to 
nonalcoholic fatty liver disease (NAFLD)1. Nonalcoholic fatty liver disease (NAFLD), 
the leading cause of chronic liver disease in the US, is characterized by fat 
accumulation in liver cells not caused by alcohol2. A group of disorders including 
steatosis, nonalcoholic steatohepatitis with fibrosis and cirrhosis, NAFLD has 
substantially risen in prevalence over the last two decades with recent estimated 
prevalence being 20% among US adults and 25% in young adults (18-39 years).2  
 
Although evidence has been presented for the positive relationship between 
fructose intake and NAFLD, the role of fructose in liver fat accumulation has been 
unclear. Individuals with specific genotypes seem to be more susceptible to the 
adverse metabolic effects of fructose. For example Hispanic children with rs738409 
(GG) single nucleotide polymorphism (SNP) of patatin-like phospholipase, domain-
containing 3 (PNPLA3) increased their liver fat by 23% more than CG/CC children 
exposed to the same amount of dietary fructose3. Similarly, adolescents with 
rs1260326 (TT) of glucokinase regulatory protein (GCKR) variant increased their de 
novo lipogenesis by 44% as compared to CC adolescents when given a sugar 
challenge4. Therefore, we hypothesize that when given the same amount of fructose 
individuals with specific set of genotypes will be more susceptible to increased de novo 
lipogenesis and liver fat accumulation than others.  
Study Objective(s) 
Our goal for this study is to generate preliminary data that is needed to support an 
application to NIH for funding of a larger, adequately-powered study to explore 
whether a set of genotypes, that increase the risk for nonalcoholic fatty liver disease 
(NAFLD), predispose individuals to increased de novo lipogenesis (DNL) and liver fat 
accumulation when exposed to fructose intake.  
Primary Objective: To determine the impact of acute and prolonged exposure of 
fructose on hepatic lipid accumulation in adolescents and yound adults (12-40 years) 
with high and low genetic risk for NAFLD using liver mass resonance imaging (MRI).   
Secondary Objective: To explore the genetic-specific effects of fructose on markers 
of liver fat accumulation (fibroscan5 and fatty liver index6) and serum biomarkers 
related to lipids, uric acid and liver function. 
Test Article(s) 
(If Applicable) 
 Sugar-Sweetened bevarages  
Study Design 
 
a. We will screen 200 Caucasian children and young adults (12-40 years) to obtain 
24 children and 24 young adults who are overweight/obese and drink <14 fructose 
drinks/week to participate in a 3-week fructose intervention study. The 200 
participants will be genotyped for 10 SNPs involved in lipid metabolism and 
associated with increased NAFLD risk in previously published studies7,8. We will 
select 12 children and young adults with 15 or more risk alleles and 12 children 
and 12 young adults with 6 or fewer risk alleles and provide them fructose + 
glucose9 drinks who will ingest one drink (equal to 2 soft drinks) per day for 3 
weeks;  
b. We will measure liver fat using Liver MRI9,10 and transient elastography 
(Fibroscan)5 at week 0 and 3; 

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c. Data will be analyzed using paired and unpaired students T test to differentiate the 
responses between the time points and between the two groups, respectively. 
Specific Aim2: Determine the impact of acute exposure of fructose on hepatic de novo 
lipogenesis  
a. Using the cohort described in Specific Aim1, we will conduct oral fructose 
tolerance test at week 0 and week 3; 
b. We will measure liver fat using Fibroscan 
c. Data will be analyzed using unpaired students T test and repeated measures 
ANOVA to differentiate the responses between and within the two groups, 
respectively. 
Specific Aim3: Determine the relationship between markers of DNL, liver fat 
accumulation and serum concentrations of lipids, uric acid and liver function markers 
before and after the fructose challenge 
a. We will measure serum concentrations of liver function markers, lipids and uric 
acid at week 0 and week 3; 
b. As in previous aims, data will be analyzed using students T test, and Pearsons 
correlations. 
 
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria 
1. Subjects between 12 and 40 years 
2. No history of alcohol abuse (> 7 drinks per week) 
3. History of fructose intake of < 14 drinks per week 
4. Caucasian ethnicity 
5. BMI > 25kg/m2 – 32kg/m2 or 85th -99th percentile but otherwise healthy 
Exclusion Criteria 
1. ages < 12 and > 40 years  
2. Pregnant/lactating 
3. known alcohol abuse or fructose intake > 14 drinks per week 
4. not of Caucasian ethnicity 
5. glucose levels > 100 mg/dl if fasting,  > 140mg/dl if within 2 hours post meal 
and > 200 mg/dl if random sample 
6. taking anti-hypertensive, anti-diabetic, uric acid and/or lipid-lowering 
medications 
7. known diagnosis of diabetes, fructose intolerance, chronic kidney disease, 
NAFLD or any liver-related disease, hypertriglyceridemia, polycystic overy 
syndrome, hypothyroidism, obstructive sleep apnea, hypopituitarism and 
hypogandism 
8. BMI < 25kg/m2 or > 32 kg/m2 or < 85th or > 99th percentile 
9. Liver fat fraction >5% as per baseline MRI scan 
Number Of Subjects  
 
200 for first phase and 48 for the second and third phases  
Study Duration 
The first phase will be about 30 minutes and the second and third phase will be 
divided into two visits. Both visits in both phases will be about 3.5 hours each.  
Study Phases 
Screening 
Study Treatment 
Follow-Up   
 (1) Screening: We will screen participants for eligibility to participate in the study 
based on the inclusionary/exclusionary criteria specified above. The 200 children and  
young adults selected to participate will provide saliva which will be used to extract 
DNA and then genotype 10 SNPs. These SNPs are selected based on their 
association with de novo lipogenesis (DNL) or liver fat accumulation in previously 
published studies in Caucasians. We will select 12 children and 12 young adults with 
more than 15 risk alleles and 12 children and 12 young adults with less than six 
number of risk alleles. 
 (2) Intervention: This part is divided into two phases. Each phase entails two visits. 
Phase2-visit 1: The 48 enrolled (24 in each of the high and low genetic risk group) will 

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visit the UNC NRI clinical suite run by the Human Research Core. They will be taken to 
the WakeForest imaging center for their baseline MRS scan.  
Visit 2: Acute fructose challenge:  The next day, they will have their initial 
anthropometrics and Fibroscan measurements. They will then  undergo an oral 
fructose challenge using a protocol used by Hudgins et al13. The participants will be 
provided with a drink consisting of fructose (0.75g/kg body weight) + glucose (0.45g/kg 
body weight) dissolved in 24 Oz of water (equal to 2 soda cans) to be consumed within 
15 minutes. Blood and urine will be sampled at baseline and 1 and 3 hours after sugar 
drink ingestion. During the challenge period, no other beverage (except water) or food 
will be allowed.  
 
3-week intervention: The 48 participants will be provided with sugar drinks for the three 
week period. The drinks that need to be taken per day will contain fructose (0.75g/kg 
body weight) + glucose (0.45g/kg body weight) dissolved in 24 Oz of water (equal to 2 
soda cans) which is similar to that provided during the acute fructose challenge7. 
Addition of glucose to fructose in the challenge and 3-week intervention induces the 
lipogenic effects of fructose, alleviates gastrointestinal effects of fructose and mimics 
the real-life situation14. They will visit at the end of each week to collect drinks for the 
next week. In addition, participants will be given standardized meals for the day prior to 
the baseline fructose challenge. They will also be instructed to avoid alcohol, juices 
and sodas the previous evening. 
Efficacy Evaluations 
Changes in liver fat accumulation through MRI scans and fibroscan measurements, 
and serum concentration of other lipids, liver function markers and uric acid. 
Pharmacokinetic 
Evaluations 
N/A 
Safety Evaluations 
At screening, to check for blood glucose values, we will conduct a spot glucose check 
using a glucometer (Accu-Chek, Roche Diagnostics, CH) to check that the glucose 
levels of the participants are in the normal range (fasting < 100mg/dl, post meal < 140 
mg/dl and random < 200 mg/dl). Secondly, we will have a physician who will be 
available if needed. We will also conduct pregnancy test if needed. 
Also, we will exclude participants for further participation if we find that the participant 
has high liver fat fraction (>5%) during our baseline MRI scan10. The participant will be 
given a report that they can show to their physician for treatment. 
Statistical And Analytic 
Plan 
Our main outcome measure is change in liver fat  via MRI scan, fatty liver index 
equation and through fibroscan measurements. We will also measure serum 
concentrations of other lipids, liver function markers and uric acid. Data will be 
assessed using descriptive measures and confidence intervals. Paired t-tests and 
repeated measures regression analysis will be performed to explore the significant 
differences between week 0 and week 3. 
DATA AND SAFETY 
MONITORING PLAN 
A data and safety monitoring plan will be instituted for this study that will ensure 
adherence to the protocol approved by UNC IRB. Dr. Voruganti (PI) and co-
investigators will review the phenotype and genotype data as they are generated. Any 
adverse event that is reported to either the principal investigator or her designated 
research associates by the subject or medical staff caring for the subject and which 
meets the criteria will be documented as such. Any event that is reported will then 
generate an adverse event report, which will be submitted to the IRB. The report will 
include a  description of the event, when and how it was reported, as well as any 
official chart records or documentation to corroborate the event or the reporting of the 
event 
 
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BACKGROUND AND RATIONALE 
NAFLD is a significant health burden in the US:  The leading cause of chronic liver disease in the US, NAFLD is 
characterized by fat accumulation in liver cells not caused by alcohol. NAFLD, representing a group of disorders 
including steatosis, nonalcoholic steatohepatitis with fibrosis, has substantially risen in prevalence over the last two 

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decades with the estimated prevalence being 20% among US adults and 25% in young adults (18-39 years)2. Over 64 
million individuals are believed to have NAFLD with annual medical costs rising to more $100 billion. More common in 
individuals who are obese or diabetic and/or have metabolic syndrome, NAFLD has been associated with increased 
cirrhosis, liver-related mortality and hepatocellular carcinoma2.   
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dietary factors spur the development of NAFLD: Unhealthy diet plays a major role in the development of NAFLD. Most 
of the attention has been on increased calorie intake. However, an important but underappreciated dietary change in US 
society for the past few decades has been increase in fructose intake. Contained in soft drinks, fruit juices and energy 
drinks, fructose affects many metabolic processes, foremost being an increase in fat accumulation in liver and 
contributing to the onset and progression of NAFLD (Figure 1). Fructose, which is almost entirely metabolized in liver, is 
rapidly phosphorylated to fructose 1 phosphate by fructokinase (ketohexokinase) with ATP depletion in parallel. The 
fructose 1 phosphate gets metabolized to dihydroxyacetone-phosphate and glyceraldehyde and finally to triglycerides15-
17 which gets deposited in liver and leads to NAFLD.  
 
Fructose is a more potent stimulator of DNL than glucose:  In population studies, it has been shown that fructose, and 
not glucose, is associated with increased visceral adiposity, insulin resistance and hyperuricemia18-20. Fructose is 
unique in its effects that it stimulates KHK and thus potentiates its own metabolism. Secondly, the rate of 
phosphorylation of fructose by KHK is 10 times higher than the phosphorylation of glucose by glucokinase. Third, 
fructose is directly absorbed into portal vein and delivered to liver without entering the systemic circulation. Liver, thus, 
is exposed to a much higher fructose load than rest of the tissues. Fourth, fructose activates the lipogenic transcriptional 
factors, SREBP1c and ChREBP in the liver, promoting DNL21. And finally, fructose tends to deplete liver ATP levels and 
one of the outcomes is generation of more AMP which get converted to uric acid. Uric acid has been shown to stimulate 
fat synthesis in the hepatocyte thus pointing to an additional pathway through which fructose can increase liver fat22,23.   
Genes and gene by nutrient interactions play a major role in the regulation of NAFLD: Both genetic and environmental 
factors contribute to the onset and progression of NAFLD. NAFLD is a complex and heritable phenotype. Family-based 
studies have reported heritability estimates for NAFLD to be between 20% and 70%7,8.  Genome-wide and candidate 
gene studies have identified several genes associated with NAFLD8. Representative genes that were implicated in lipid 
metabolism and in the development and progression of NAFLD are shown in Table1. Few studies have shown that 
fructose affects lipogenesis in liver in a genotype-specific manner. Davis and colleagues found that Hispanic children 
with GG genotypes of PNPLA3 SNP rs738409 were more inclined to accumulate fat in the liver as compared to children 
with CC or CG genotypes3. Similarly, another study investigating the effects of added sugars on liver fat found that 
individuals with TT of rs1260326 of GCKR increased their de novo lipogenesis by 44% during an oral fructose + glucose 
challenge4. However, none of the studies have shown the cumulative effects of SNPs on change in liver fat when 
exposed to fructose.  
Gene 
SNP 
Major/Minor MAF (%) 
PNPLA3 
rs738409 
C/G 
23 
GCKR 
rs1260326 
C/T 
45 
TMS6F2 
rs58542926 T/C 
07 
APOC3 
rs2854116 
T/C 
39 
EFCAB4B rs887304 
G/T 
49 
NCAN 
rs2228603 
C/T 
07 
COL13A1 
rs1227756 
A/G 
48 
FDFT1 
rs2645424 
G/A 
42 
LYPLAL1 
rs12137855 C/T 
21 
PPP1R3B rs4240624 
A/G 
7 
Fructose-1-Phosphate 
KHK, GCKR 
Uric acid 
ATP     AMP 
Glyceraldehyde 
Citrate 
Triglycerides 
Potential genes (italicized and bold) which modulate the effect of fructose on lipogenesis in the liver. Figure 1 shows 
genes in the fructose-induced lipogenic pathway. Table 1 shows the genes and variants that have been associated with 
liver lipogenesis through genome-wide association studies. PNPLA3- patatin-like phospholipase, domain-containing 3, 
GCKR- glucokinase regulatory protein, APOB and APOCIII - apolipoprotein B and CIII (APOB and APOC3), TM6SF2 -
Ttransmembrane 6 superfamily member 2, EFCAB4B - EF-hand calcium-binding domain-containing protein 4B, 
COL13A1 - Collagen type XIII alpha 1chain, FDFT1 - farnesyl-diphosphate farnesyltransferase 1, NCAN- Neurocan, 
LYPLAL1-Lysophospholipase like1,  PPP1R3B- Protein phosphatase 1 regulatory subunit 3B. 
Glyceraldehyde-3-
phosphate 
Export from liver 
as VLDL 
Steatosis 
APOC3, TMS6F2 
Malonyl CoA 
Aldolase B 
PNPLA3 
ECAB4B, 
COL13A1 
Inflammation 
FDFT1, LYPLAL1, 
PPP1R3B 
Fructos

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The genetic-specific effects of fructose on liver lipogenesis need to be studied as a first step towards individualized 
nutrition:  This study assumes significance given that fructose affects liver lipogenesis differentially based on genotypes. 
It also paves the way towards individualized or personalized nutrition. Previous studies investigating the metabolic 
effects of fructose have been controversial. Some have shown significant effects of fructose on insulin sensitivity and de 
novo lipogenesis and triglycerides while others have shown no effects23-27. The range of these studies is wide, with the 
duration of the intervention ranging from 7 to 665 days, sample size ranging between 8 and 64 per group and amount of 
fructose per day ranging from 40 to 250g/d. For this exploratory and feasibility study we have selected the duration of 21 
days, sample size as 12 per group and amount of fructose a ~0.75g/kg/day (equivalent to two sodas/per day). 
 
1.1 
Introduction 
This study is a combination of acute and long-term investigation of the effects of fructose on liver lipogenesis and 
whether genetics modify this relationship. It is similar to a case-control study based on the number of alleles that 
increase the risk for liver lipogenesis which further increases the risk for NAFLD. There is an increasing concern about 
the effects of fructose on metabolic diseases. Some studies, as shown in the background section, have also suggested 
that effects of fructose may be modified by some genotpes. However, all these studies have focused on only one gene 
or genotype. We fill the gap by conducting the study with a set of genotypes.  
1.2 
Name and Description of Investigational Product or Intervention  
Acute fructose challenge: We will be using a fructose drink which will be prepared by dissolving 0.75g/kg fructose + 
0.45g/kg glucose (NOW Foods, Bloomingdale IL), in 24oz water. This composition of fructose and glucose mimics the 
composition of fructose and glucose in 2 regular sodas28 based on an approximate adolescent/adult body weight of 
~65kg.  
3-week intervention: We will provide each participant the same drink as described in the acute fructose challenge to be 
taken one per day. 
1.3 
Non-Clinical and Clinical Study Findings 
There are no immediate benefits. In long-term, we can provide genetic risk-guided diet plans to individuals who may be 
at high risk. 
 
Risks: We don’t anticipate major risk since we will exclude individuals who are diabetic, pre-diabetic, have high liver fat 
at baseline and are at high risk for these and other metabolic diseases. The only risk can be weight gain. We will 
advise on cutting calories elsewhere in their daily diet. We will also advise the participants to avoid fruit juices and 
other sodas for the duration of 3 weeks. 
 
1.4 
Relevant Literature and Data 
The studies similar to that being conducted here have been conducted before (Table 1, Figure 1). The only difference 
is that we are using a set of 10 genotypes instead of individual genotypes that were used in studies before. 
2 
STUDY OBJECTIVE  
2.1 
Our goal for this study is to generate preliminary data that is needed to support an application to NIH for funding of 
a larger, adequately-powered study to explore whether a set of genotypes, that increase the risk for nonalcoholic 
fatty liver disease (NAFLD), predispose individuals to increased de novo lipogenesis (DNL) and liver fat 
accumulation when exposed to fructose intake.  
 
2.2 
Primary Objective: To explore the genetic-specific effects of fructose on liver fat accumulation  using MRI  
2.3 
Secondary Objective: To explore the genetic-specific effects of fructose on markers of liver fat accumulation 
(fibroscan5 and fatty liver index6) and serum biomarkers related to lipids, uric acid and liver function. 
 
3 
INVESTIGATIONAL PLAN (brief overview) 
3.1 
Study Design 
- 
This exploratory and feasibility study is an intervention study assessing the effects of fructose on liver fat 
accumulation. The study design is described below. 
3.2 
Allocation to Treatment Groups and Blinding (if applicable) 

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- 
We wil allocate participants to high and low genetic risk groups 
3.3 
Study Duration, Enrollment and Number of Subjects 
- 
The study has three phases. First phase will have 200 participants who will visit Human Research Core clinical 
suite and spend about 30 minutes providing saliva sample and completing a brief questionnaire. 
- 
Second and third phases will have 48 participants. They will first undergo a MRI scan and next day a 3 hour 
acute fructose challenge (total time 3.5 hours) and then will consume sugar drink as given to them. They will 
visit again at the end of week 3 for further biomarker, MRI scan and fibroscan measurements. 
3.4 
Study Population 
- 
Inclusion criteria: Causasian children and young adults, overweight/obese, ages between 12 and 40 years and 
no history of alcohol abuse (> 7 drinks per week for a year) and no history of excessive fructose intake (> 14 
drinks per week for a year). We will conduct a spot glucose check using a glucometer (Accu-Chek, Roche 
Diagnostics, CH) to check that the glucose levels of the participants are in the normal range (fasting < 
100mg/dl, post meal < 140 mg/dl and random < 200 mg/dl).  
- 
Exclusion criteria includes ages < 12 and > 40 years, healthy weight or morbidly obese, known alcohol abuse, 
not of Caucasian ethnicity, pregnancy, lactation, glucose levels > 100 mg/dl if fasting,  > 140mg/dl if within 2 
hours post meal and > 200 mg/dl if random, baseline MRI scan of liver fat fraction > 5%27, and known 
diagnosis of diabetes, fructose intolerance and chronic kidney disease, use of lipid or uric acid lowering 
medications.  
 
4 
STUDY PROCEDURES (what will be done) 
4.1 
Screening/Baseline Visit procedures 
Phase 1: Recruitment and screening of participants:  
Screening: We will recruit 200 Caucasian children and young adults fulfilling our inclusionary criteria: ages 
between 12 and 40 years, overweight or obese, no history of fructose intake > 14 drinks per week and alcohol 
abuse (> 7 drinks per week for a year). Height will be measured to the nearest 0.1 cm by a stadiometer situated 
against the wall in an upright standing position. Height and weight recorded will be used to calculate BMI as kg/m2. 
We will match all participants for BMI ( 25kg/m2 -32kg/m2 or 85th-99th percentile). 
 
We will conduct a spot glucose check using a glucometer (Accu-Chek, Roche Diagnostics, CH) to check that the 
glucose levels of the participants are in the normal range (fasting < 100mg/dl, post meal < 140 mg/dl and random < 
200 mg/dl). The exclusion criteria includes ages < 12 and > 19 years, known alcohol abuse or fructose intake > 14 
drinks per week, not of Caucasian ethnicity, glucose levels > 100 mg/dl if fasting,  > 140mg/dl if within 2 hours post 
meal and > 200 mg/dl if random, and known diagnosis of diabetes, fructose intolerance and chronic kidney 
disease.  
 
We will collect saliva from the eligible 200 children and young adults, extract DNA and genotype 10 SNPs shown in 
Table1.  
 
Genotyping: SNPs will be genotyped from DNA extracted from saliva. Genotyping will be conducted in Voruganti 
lab using TaqManSNP genotyping assays (Applied Biosystems, Foster City, CA). The minor allele frequencies 
(MAFs) of these SNPs range between 7 and 49% based on Caucasian ethnic background. Therefore we are 
confident about finding 48 individuals with all 10 SNPs. If we don’t find individuals with the 10 SNPs, we will mine 
the literature and find more SNPs that have a role in liver lipogenesis.  
 
4.2 
Intervention/Treatment procedures (by visits) 
Phase2: Selection of 48 participants for the acute fructose challenge and 3-week intervention study: 
The risk allele will be defined as the allele that is associated with higher levels of liver lipogenesis or fat as per 
previously published studies in Caucasians.The number of risk alleles will range from 0 to 20. The SNP will get a 
score of 2 with two risk alleles, 1 with one risk allele and 0 with no risk alleles. All the score will be added to get a 
cumulative score between 0 and 20 for 10 SNPs. We will select participants  having > 15 risk alleles and < 6 risk 
alleles for our acute challenge and 3-week intervention study. 
 
Phase 2&3- visit 1- MRI scan: The 48 individuals enrolled will visit the UNC NRI clinical suite run by the Human 
Research Core. After obtaining consent, they will be taken to the WakeForest Imaging center for baseline MRI 
scan. Studies have shown that 5-6% of liver fat is considered normal and anything more than that can reflect 
increased risk for fatty liver. If the MRI report of the participants shows liver fat > 6%, we will consult with the 
participant (if > 18years) and/or their parents in confidence and explain why they (or their child) cannot continue to 

11 
 
participate and that they should consult with their primary care physician. We will also advise them on diet and 
lifestyle that they can follow to minimize the risk. If non-liver adverse findings are reported during MRI, we will 
provide the participant with a letter that they can show to their physician. 
 
Phase 2&3- visit 2- Oral fructose challenge:. After their initial anthropometrics and Fibroscan measurements, 
they will undergo an oral fructose challenge using a protocol used by Hudgins et al13. The participants will be 
provided with a drink consisting of 0.75g/kg fructose + 0.45g/kg glucose dissolved in 24 Oz of water to be 
consumed within 15 minutes. An average overweight adolescent of 65 kg will consume 48.75g (65 x 0.75g/kg) 
fructose and 29.25g (65 x 0.45g/kg) of glucose which is similar equivalent to the sugars in two soda cans28.  
 
Blood and urine will be sampled at baseline and 1 and 3 hours after fructose ingestion. During the challenge 
period, no other beverage (except water) or food will be allowed.  
 
Anthropometric measurements: Height will be measured using a portable stadiometer. Body composition and 
weight will be measured by bioelectric impedence technology using the Tanita Dual Frequency total Body 
composition analyzer (Tanita Inc., Arlington Heights, IL). Measurements will be conducted in a standing position, 
with subjects wearing light clothing and without shoes. Height will be measured to the nearest 0.1 cm by a 
stadiometer situated against the wall in an upright standing position. Height and weight recorded will be used to 
calculate BMI as kg/m2. Waist circumference (WC) will be measured using a stretch-resistance tape at the 
midpoint between the lower margin of the least rib and the top of the iliac crest to the nearest 0.1 inch. Waist and 
hip circumferences will be measured to the nearest cm in a standing position. Sitting blood pressure (systolic and 
diastolic blood pressure) will be measured three times using the Omron digital blood pressure monitor 
(HEM907XL, Omron Healthcare Inc, Lake Forest, IL). The means of these measurements will be used for 
analysis. Well trained personnel in the UNC NRI Human Research Core will administer questionnaires as well as 
measure anthropometrics, by one staff member to minimize measurement variation and margin of error. 
 
Questionnaires: The following six sets of questionnaires will be administered to the study participants. 
• 
Personal Interview Form: Information related to demographics 
• 
Medical History: Information related to medical conditions, procedures and surgeries 
• 
Modifiable activity form: Information related to physical activity, sedentary time and job activities 
• 
Medications: Information related to prescription, non-prescription and traditional medications 
• 
Dietary data: Combination of diet recall and food frequency questionnaire will be used to obtain detailed 
dietary data. 
 
Sample preparation: A total of 24 mL of blood will be drawn at fasting at baseline, 1 hour, 3 hours, at weeks 0 
and 3 by a trained phlebotomist through venous puncture. After centrifugation, the serum and plasma fractions of 
the blood samples will be aliquoted and stored at -80°C to measure the biomarkers listed below using standard 
reference procedures. All samples will be labeled and inventoried as per standard protocols. Urine will be 
collected at the same points as blood samples collected at baseline, 3 hour at weeks 0 and 3 will be stored at  -
80°C. The Human Research Core will assist with phlebotomy services 
 
Meals: All participants will be provided standardized meal for the evening prior to participation of part 1 of phase 
2.  
 
Assessment of liver fat accumulation and stiffness: Magnetic resonance imaging (MRI): We will assess liver 
fat accumulation at week 0 and week 3 using the 2012 Siemens MAGNETOM Skyra 3T MRI system available at 
the WakeForest Translational Imaging program. If the liver fat fraction >5%10 then we will exclude the participant 
from further participation in the nutrient challenge. 
 
Transient elastography: We will use transient elastography to ascertain the changes in the amount of fat in the 
liver. Transient elastography using a Fibroscan® is a non-invasive method of assessing fat accumulation in liver 
and liver stiffness and works on the principle of vibration controlled transient elastography5. A vibration of mild 
amplitude and low frequency is transmitted through the intercostal space using a vibrator at the skin surface. The 
vibration induces an elastic shear that propagates through the hepatic tissue. Using pulse echo ultrasound 
acquisition, the velocity of the shear wave can be determined. The shear wave, directly related to tissue stiffness 
and expressed in kilopascal (kPa), can be a helpful aid to clinically manage patients with liver disease. The UNC 
Human Research Core houses a Fibroscan and its trained technicians will help with its use for this study.  
 

12 
 
Medical care: According to the CDC report (cdc.gov/nutrition/data&statistics), ~51% of Americans drink at least 
one soda or other type of sugar-sweetened beverages on a given day. Higher prevalences have been found in 
Southern states and in adolescents. Therefore, we don’t anticipate any medical issues as three sugar drinks per 
day is conforming to the present frequency. However, we will refer to our resident clinician in case any participant 
wants to consult or has concerns. The participant will be free to withdraw anytime during the study with no 
repercussions.  
 
Dietary compliance: Dietary compliance will be assessed by the use of frequency questionnaires and three 24-
hour diet recalls (Phases1,2 and 3). 
 
Dietary guidance: Since each participant will taking daily ~500 Kcal more than their normal daily dietary Kcal, we 
will provide guidance and options on how to cut down ~500 Kcal from elsewhere in their diet, especially in their 
consumption of sugary drinks. 
 
Biochemical phenotyping: Liver function markers (alanine transaminase (ALT), aspartate transaminase (AST), 
gamma glutamyl transferase (GGT)), uric acid and lipids (triglycerides and total cholesterol, high and low density 
lipoprotein (HDL and LDL) cholesterol will be measured in serum using standard reference methods via clinical 
chemistry analyzer. Serum lipids, uric acid and liver function markers will be measured in Voruganti lab.  
 
A combination of transient elastography (Fibroscan) and serum liver function markers will be used which 
will increase the accuracy of non-invasive measurement of fat accumulation in NAFLD; and  
 
Fatty Liver Index (FLI) is a surrogate measure for liver fat estimation. The FLI will be computed based on the 
following equation which was observed to be 84% accurate in liver fat estimation6  
 
Fatty Liver Index (FLI) = (e 0.953*loge (triglycerides) + 0.139*BMI + 0.718*loge (ggt) + 0.053*waist circumference - 15.745) / (1 + e 0.953*loge 
(triglycerides) + 0.139*BMI + 0.718*loge (ggt) + 0.053*waist circumference - 15.745) * 1006 
 
4.3 
Follow- up procedures (by visits) 
There is no follow-up 
 
4.4 
Unscheduled visits 
- 
If the participant walks-in or plans to visit due to a health concern, we will refer the participant to our resident 
clinician or to a healthcare provider 
4.5 
Concomitant Medication documentation 
- 
Sugary drinks consumption is very common, especially in this age group so we don’t anticipate adverse 
reactions and need for medication. However, if needed, our resident clinician will be available to assist. 
- 
All concomitant  medication will be documented in our questionnaire and incorporated as covariates in our data 
analysis 
4.6 
Rescue medication administration ( if applicable) 
4.7 
Subject Completion/ Withdrawal procedures 
- 
Participants can voluntarily withdraw anytime during the study if they feel that they cannot continue to consume 
the drinks. They will be paid according to completion of phases of the study.  
4.8 
Screen failure procedures 
- 
 
5 
STUDY EVALUATIONS AND MEASUREMENTS (how measurements will be made)   
- 
At baseline and end of week3, we will measure body weight, body composition, blood pressure, waist 
circumference, liver fat by transient elastography, MRI scan and serum and urinary biomarkers. Blood and 
urinary biomarkers will also be measured at one and 3 hour timepoints during the acute fructose challenge.  
- 
Body weight and composition (Body weight, body fat, etc) will be measured through a body fat analyzer (Tanita 
Corporation of America Inc.  Arlington Heights, IL) 
- 
Waist circumference (WC) will be measured using a stretch-resistance tape at the midpoint between the lower 
margin of the least rib and the top of the iliac crest to the nearest 0.1 inch. Waist and hip circumferences will be 
measured to the nearest cm in a standing position 
- 
Sitting blood pressure and pulse rate will be measured using the Omron digital blood pressure monitor 
(HEM907XL, Omron Healthcare Inc. Lake Forest, IL) 

13 
 
- 
Liver fat will be measured using MRI at the Wakeforest Imaging Center and transient elastography (Fibroscan, 
Echosens, Paris, FR) 
Quality control: All phenotypes will be checked for distribution and accuracy of values. Phenotype data collection 
will be performed on the same equipment with strict quality control regulations. Assay quality is assessed by 
tracking coefficients of variation for quality control samples that are run with each assay. Appropriate internal 
standards will be used wherever necessary.  
 
5.1 
Efficacy Evaluation ( if applicable) 
5.2 
Pharmacokinetic Evaluation ( if applicable) 
5.3 
Safety Evaluations 
 
6 
STATISTICAL CONSIDERATION    
6.1 
Primary Endpoint 
Our main outcome measure is liver fat estimation through MRI scans, transient elastography (Fibroscan) and fatty 
liver index. 
6.2 
Secondary Endpoint 
Other serum biomarkers such as liver function markers, lipids and uric acid 
 
6.3 
Statistical Methods 
Each subject will be given sugar drink containing fructose and liver MRI scan with fibroscan measurements will be 
taken at baseline and end of 3 weeks.  Data will be assessed using descriptive measures and confidence intervals. 
Paired t-tests and repeated measures regression analysis will be performed to explore the significant differences 
between week 0 and week 3. Also, for each subject, the area under the curve (AUC)13 will be computed and a 
linear regression with AUC as the response witll be performed during the acute challenge.  
 
6.4 
Sample Size and Power 
Although our sample of 48 may not have the power to detect significant effects of fructose on liver fat, this group of 
48 well-characterized individuals, focusing only on one ethnicity and one body weight group and specific 
genotypes will reduce variability and will generate meaningful data. We expect to apply these approaches and use 
these data to estimate sample size for larger studies with significant power to detect the effects of gene-nutrients 
interactions on NAFLD and other metabolic disorders. 
 
6.5 
Interim Analysis 
N/A 
 
7 
STUDY INTERVENTION (drug, device or other intervention details) 
- 
Phase 2 &3- visit 2: Acute fructose challenge – each participant will be given a drink made of 24 oz water and 
fructose (0.75g/kg) and gluose (0.45g/kg). 
- 
3-week intervention study- each participant will be given a drink to be taken one daily for three weeks starting 
the next day after acute challenge. These sugar drinks contain the same amount of sugars that are provided 
as part of the acute fructose challenge 
 
8 
STUDY INTERVENTION ADMINISTRATION(if applicable)  
N/A 
9 
SAFETY MANAGEMENT 
Data and Safety monitoring plan will be instituted for the project. As per the plan, PI, co-investigators and 
physician will ensure that the proper protocol is being followed. Any adverse event that is reported to either 
the principal investigator or her designated research associates by the subject or medical staff caring for the 
subject and which meets the criteria will be documented as such. Any event that is reported will then generate an 
adverse event report, which will be submitted to the IRB. The report will include a description of the event, when 
and how it was reported, as well as any official chart records or documentation to corroborate the event or the 

14 
 
reporting of the event. All adverse events will be graded on a scale from 0 to 5. Any severe and/or unanticipated 
adverse event will be immediately reported to the safety officer and IRB. All other adverse events will be reported 
in a timely fashion to IRB, preferably within 2 weeks of the date of the event. All adverse events will be 
summarized annually and submitted to the IRB. The annual report will address: (1) whether adverse event rates 
are consistent with pre-study assumptions; (2) reason for dropouts from the study; (3) whether all participants met 
entry criteria; (4) whether continuation of the study is justified on the basis that additional data are needed to 
accomplish the stated aims of the study; and (5) conditions whereby the study might be terminated prematurely. 
10 
DATA COLLECTION AND MANAGMENT  
All personnel with access to data collected for the study are required to sign a Confidentiality Pledge form which 
states that they understand the sensitive and confidential nature of the data and that divulgence of any information 
will result in disciplinary action. The pledge is co-signed and kept by the PI.  Only authorized staff members have 
the key to the office and access to the data forms. Passwords and/or encryption keys will be known only to 
authorized personnel safeguard data on computers at UNC-NRI. 
Any adverse event that is reported to either the principal investigator or her designated research 
associates by the subject or medical staff caring for the subject and which meets the criteria will be documented as 
such. Any event that is reported will then generate an adverse event report, which will be submitted to the IRB. The 
report will include a description of the event, when and how it was reported, as well as any official chart records or 
documentation to corroborate the event or the reporting of the event 
11 
RECRUITMENT STRATEGY 
- 
Subjects will be recruited through our website https://www.uncnri.or/index.php/volunteer/ and other community 
announcements and events 
12 
CONSENT PROCESS 
- 
All participants will provide informed consent. Each participant will be consented in private. The PI and/or other 
project investigators will explain the study to the participant and answer questions that the participant might 
have. If the participant agrees to participate after reviewing the risks/benefits, they will be asked to sign the 
consent form. For participants who are not adults, their parents permission will also be obtained. We will try our 
best to explain in a lay person’s language so that the participant is completely aware of the risks associated 
with participation.  
13 
PLANS FOR PUBLICATION 
- 
We do plan to publish at least one manuscript based on the results of this study. 
14 
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