Conditions: Nonalcoholic Fatty Liver Disease (NAFLD)
Keywords: Fructose NASH advanced fibrosis biomarker magnetic resonance
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 118
Healthy volunteers: yes

Brief summary:
This study will advance several goals of the NIH Action Plan: 1) establish a multidisciplinary team to develop quantitative methodologies and imaging protocols for liver, 2) validate diagnostic criteria and methodologies for imaging in liver in both a cross-sectional and a longitudinal dietary intervention study of patients with Nonalcoholic Fatty Liver Disease (NAFLD), 3) create a liver tissue bank with correlative imaging data, 4) develop reliable non-invasive MR markers to distinguish simple steatosis from Nonalcoholic Steatohepatitis (NASH), and 5) define the dynamic changes in metabolism, energy homeostasis, and MR biomarkers as they relate to fructose-related liver injury.

Detailed description:
Like obesity, NAFLD and NASH are closely linked to nutrition and the "Western diet" which is rich in saturated fats and refined sugars. Although fat consumption has remained relatively stable, the marked increase in dietary fructose consumption (more than doubling in the past 30 years alone) supports the role of fructose in NAFLD and the metabolic syndrome. Although the mechanism(s) for fructose-related liver injury is not yet well defined, fructose-related hepatic adenosine triphosphate (ATP) depletion may contribute to liver injury. Observations in animals suggest that fructose induces metabolic syndrome and NAFLD independent of energy intake. One key difference in fructose metabolism (as opposed to glucose) relates to ATP depletion and the necessity of adenosine monophosphate (AMP) kinase to replenish ATP stores. As opposed to glucose, initial fructose metabolism involves phosphorylation of fructose to fructose-1-phosphate by fructokinase (ketohexokinase, KHK) using the substrate ATP. Unlike glucokinase, the phosphorylation of fructose by KHK is specific for fructose and not rate limited. Replenishment of ATP stores requires phosphorylation of AMP back to ATP via AMP kinase (which is inhibited in insulin resistance (common in patients with NAFLD) or conversion to uric acid via xanthine dehydrogenase resulting in hyperuricemia. The high activity of KHK in phosphorylating fructose to fructose-1-phosphate in the liver, could result in hepatic ATP depletion with habitual fructose consumption.

Published animal and human studies support our hypothesis that fructose is a risk factor for NAFLD and NAFLD-related liver disease progression. In animal models, diets high in fructose induce features of the metabolic syndrome including weight gain, insulin resistance, hypertriglyceridemia, and hypertension. Similar effects are not observed with the administration of other simple sugars such as glucose. Fructose (or sucrose) administration to humans also causes features of metabolic syndrome which are quite typical of patients with NAFLD. Fructose is lipogenic, stimulates triglyceride synthesis and causes hepatic steatosis. As previously reported in animals, our group reported that increased fructose consumption (assessed as fructose-containing beverages only) is a risk factor of metabolic syndrome and biopsy-proven NAFLD and that patients with NAFLD consume 3-4 times more fructose than age, gender, and mass index (BMI) matched controls without liver disease.

In addition to increased fructose consumption being a risk factor for NAFLD, fructose has been implicated in NAFLD disease progression. The administration of a diet with 25% of total energy as sucrose (which contains 50% fructose) resulted in a rise in liver aminotransferase levels within 18 days. This study, performed nearly 25 years ago, is all the more alarming as current sugar intake of Americans is in this same range. In our study of 427 patients with biopsy-proven NAFLD, increased consumption of fructose-containing beverages was univariately associated with decreased age (P \< 0.0001), male sex (P \< 0.0001), hypertriglyceridemia (P \< 0.04), low high density lipoprotein (HDL) cholesterol (\<0.0001), decreased serum glucose (P \< 0.001), increased calorie intake (P \< 0.0001), and hyperuricemia (P \< 0.0001). After controlling for age, sex, BMI, and total calorie intake, daily fructose consumption was associated with lower steatosis grade and higher fibrosis stage (P \< 0.05 for each). Being that triglyceride synthesis requires ATP, we hypothesize that lower hepatic steatosis may reflect deceased ATP availability. Additionally, in older adults (age ≥ 48 years), daily fructose consumption was associated with increased hepatic inflammation (P \< 0.05), and hepatocyte ballooning (P\< 0.05). However, the mechanism(s) by which fructose causes liver injury remains unknown.

In support of our hypothesis that ATP depletion underlies liver injury in patients with NAFLD, our group has demonstrated that patients with biopsy-proven NAFLD have increased hepatic mRNA (messenger ribonucleic acid) expression of KHK compared to matched controls. Indeed, in human pilot studies, intravenous (IV) fructose administration is associated with hepatic ATP depletion which can be assessed by 31P magnetic resonance spectroscopy (MRS). Reduced hepatic ATP stores are more prevalent in overweight and obese subjects than in lean subjects. Furthermore, recovery from fructose-induced ATP depletion was found to be delayed in patients with NAFLD (n=8). However, a limitation to this existing work is the small sample size and the inability to assess a cause-effect relationship(s) between BMI, NAFLD, energy homeostasis, and histologic features of liver injury. In liver cells, ATP depletion could perpetuate chronic liver injury by making fatty hepatocytes less proliferative. Hepatic ATP depletion also encourages the expansion of liver progenitor populations, causes arrest in protein synthesis, induces inflammatory and prooxidative changes, increases endoplasmic reticulum stress, promotes activation of stress-related kinases, induces mitochondrial dysfunction, and increases apoptotic activity. This supporting data suggests that fructose may be associated with NAFLD, NASH, and progressive fibrosis. Further, a study by Loguercio et al. demonstrated that increased uric acid levels above the basal level after IV fructose infusion was significantly higher (p \< 0.01) in patients with cirrhosis (3 mg/dl) and NASH (1.9 mg/dl) than in healthy controls (1.2 mg/dl). This effect was completely reversed by fructose 1,6-diphosphate which could replenish the ability to resynthesize ATP (adenosine triphosphate) from ADP (adenosine diphosphate). Therefore, an IV fructose challenge could effectively differentiate healthy subjects, from chronic hepatitis, from cirrhosis.

NAFLD lacks accurate and robust non-invasive biomarkers to grade and stage histologic disease activity. This is a critical barrier to understanding the influence of this important environmental risk factor (increased/habitual fructose consumption) on the pathogenesis and progression of NAFLD. Currently, reliable assessment NAFLD requires liver biopsy and interpretation of histology. Serum aminotransferase levels and conventional imaging methods can detect liver fat but cannot grade or stage NAFLD. Furthermore, current developments in biomarker are cross-sectional in nature and do not characterize the dynamic changes which underlie liver injury in patients with NAFLD. In vivo 31P MRS permits the evaluation of dynamic changes of individual phosphorus-containing metabolites in the liver parenchyma, such as phosphomonoester (PME), ATP, and inorganic phosphate (Pi). Intravenous fructose load alters phosphorus metabolites and allows assessment of liver function by 31P MRS. Other investigators have demonstrated that fructose loading could be used effectively as a tool to investigate change in metabolic steps of hepatic metabolism in humans with alcohol-related liver disease. Further, IV fructose loading causes significantly higher ATP degradation and uric acid production in cirrhotic patients than in healthy controls. The associations between fructose, increased uric acid, and hepatic ATP depletion has been previously described. Increased uric acid is an independent risk factor for NAFLD and in keeping with our hypothesis, hyperuricemia may be a surrogate marker of impaired hepatic energy homeostasis in patients with NAFLD. The proposed mechanism for fructose-related hepatic ATP depletion, NAFLD, NASH and the associated hyperuricemia is depicted in Figure 1 is novel, innovative, scientifically rigorous and address an important public health concern-the impact of fructose on the rising epidemic of NAFLD.

Arms:
arm 1: 70 subjects with biopsy-proven NAFLD; subjects will be challenged with a fructose infusion after a period for 12 hours fasting. arm 2: 15 healthy controls for comparison with NAFLD patients.The 15 subjects will be challenged with a fructose infusion after a period for 12 hours fasting.

Interventions: intervention 1: intravenous fructose challenge intervention 2: Blood Draw
Intervention descriptions: intervention 1: Patients will be admitted to our Duke Clinical Research Unit (DCRU) at least 12 hours prior to morning intravenous fructose challenge. All patients will have a "standard" meal in order to control for dietary composition and calorie intake prior to intravenous fructose challenge. Patients will be NPO (nothing by mouth) after midnight for morning IV fructose MR biomarker measures. Patients with suspected NAFLD will have had an historical standard of care liver biopsy in the past and will have IV fructose Magnetic Resonance biomarker measures in the morning. intervention 2: Fasting bloodwork will be obtained before and after the IV fructose challenge.

Protocol document:
Prot_SAP_000.pdf: 
Version Date: 25Jul2016 
 
 
 
 
 
 
Duke NAFLD Clinical Research Program 
 
 
IRB Protocol Summary 
Confidential 
 
Impact of Fructose on Metabolism,  
Energy Homeostasis and MR biomarkers in NAFLD 
 
 
 
 
Investigators / Role: 
 
Manal F. Abdelmalek, MD, MPH, Co-PI 
Division of Gastroenterology 
Brian J. Soher, PhD, Co-PI 
Dept of Radiology 
Mustafa Bashir, MD, Sub-investigator 
Dept of Radiology 
 
 
Cynthia Guy, MD, Sub-investigator 
Dept of Pathology 
Dawn Piercy, NP, CCRC, Sub-investigator 
Division of Gastroenterology 
Mariko Kopping, MS, RD, Coordinator 
Division of Gastroenterology 
Mary Cris Margrito, RN, Coordinator              Division of Gastroenterology 
 
 
 
Sponsor: NIH RO Application Federal Grant Identifier #10788882 
 
 
 
Protocol Revision 
 Version Date:  July 25, 2016 
 
NCT01930123


Fructose-MRI Protocol 
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1. Hypothesis and Principal Objective 
 
1.1 Primary hypothesis  
• Fructose is a risk factor for hepatic steatosis, NASH, and advanced fibrosis and that 
fructose exposure will worsen the metabolic, energy homeostatic and associated MR 
based biomarker associated features of nonalcoholic fatty liver disease (NAFLD).  
1.2 Principal objectives:   
• To define whether baseline and dynamic changes in hepatic ATP during a fructose 
challenge are associated with NAFLD and/or liver disease severity in patients with 
NAFLD. 
• To assess whether such factors can be measured using magnetic resonance (MR) 
biomarkers  
We will measure baseline and dynamic changes in metabolism, energy homeostasis and MR 
biomarkers in healthy volunteers and patients with biopsy-proven NAFLD.  The effect of acute 
fructose challenges on changes in metabolism and energy homeostasis will be assessed in a 
cohort of patients with NAFLD.   
 
2. Background and Significance 
 
2.1 Prevalence of NAFLD 
 
Nonalcoholic fatty liver disease (NAFLD) is now the most common liver disease in the United 
States and possibly worldwide (1). Nonalcoholic steatohepatitis (NASH), a more serious form of 
NAFLD, can progress to cirrhosis and even hepatocellular carcinoma (2). These liver diseases 
represent the hepatic component of the metabolic syndrome (3). The epidemic of NAFLD-
related chronic liver disease has paralleled the rise in obesity world-wide. 
 
2.2 Fructose as a Risk Factor for NAFLD 
 
Like obesity, NAFLD and NASH are closely linked to nutrition and the “Western diet” which is 
rich in saturated fats and refined sugars. Although fat consumption has remained relatively 
stable, the marked increase in dietary fructose consumption (more than doubling in the past 30 
years alone) supports the role of fructose in NAFLD and the metabolic syndrome (4-6). 
Although the mechanism(s) for fructose-related liver injury is not yet well defined, fructose-
related hepatic adenosine triphosphate (ATP) depletion may contribute to liver injury.  
Observations in animals suggest that fructose induces metabolic syndrome and NAFLD 
independent of energy intake (7, 8).  One key difference in fructose metabolism (as opposed to 
glucose) relates to ATP depletion and the necessity of adenosine monophosphate (AMP) kinase 
to replenish ATP stores.  As opposed to glucose, initial fructose metabolism involves 
phosphorylation of fructose to fructose-1-phosphate by fructokinase (ketohexokinase, KHK) 
using the substrate ATP. Unlike glucokinase, the phosphorylation of fructose by KHK is specific 
for fructose and not rate limited (9). Replenishment of ATP stores requires phosphorylation of 
AMP back to ATP via AMP kinase (which is inhibited in insulin resistance (common in patients 
with NAFLD) or conversion to uric acid via xanthine dehydrogenase resulting in hyperuricemia. 


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NAFLD Clinical Research Program 
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The high activity of KHK in phosphorylating fructose to fructose-1-phosphate in the liver, could 
result in hepatic ATP depletion (10) with habitual fructose consumption.  
Published animal and human studies support our hypothesis that fructose is a risk factor for 
NAFLD and NAFLD-related liver disease progression.  In animal models, diets high in fructose 
induce features of the metabolic syndrome including weight gain, insulin resistance, 
hypertriglyceridemia, and hypertension (11). Similar effects are not observed with the 
administration of other simple sugars such as glucose (8). Fructose (or sucrose) administration to 
humans also causes features of metabolic syndrome (10, 12, 13) which are quite typical of 
patients with NAFLD. Fructose is lipogenic, stimulates triglyceride synthesis (14) and causes 
hepatic steatosis (15, 16). As previously reported in animals, our group reported that increased 
fructose consumption (assessed as fructose-containing beverages only) is a risk factor of 
metabolic syndrome and biopsy-proven NAFLD and that patients with NAFLD consume 3-4 
times more fructose than age, gender, and mass index (BMI) matched controls without liver 
disease (17).  
In addition to increased fructose consumption being a risk factor for NAFLD (17), fructose has 
been implicated in NAFLD disease progression (18, 19). The administration of a diet with 25% 
of total energy as sucrose (which contains 50% fructose) resulted in a rise in liver 
aminotransferase levels within 18 days (18). This study, performed nearly 25 years ago, is all the 
more alarming as current sugar intake of Americans is in this same range (4, 5). In our study of 
427 patients with biopsy-proven NAFLD, increased consumption of fructose-containing 
beverages was univariately associated with decreased age (P < 0.0001), male sex (P < 0.0001), 
hypertriglyceridemia (P < 0.04), low high density lipoprotein (HDL) cholesterol (<0.0001), 
decreased serum glucose (P < 0.001), increased calorie intake (P < 0.0001), and hyperuricemia 
(P < 0.0001) (19). After controlling for age, sex, BMI, and total calorie intake, daily fructose 
consumption was associated with lower steatosis grade and higher fibrosis stage (P < 0.05 for 
each). Being that triglyceride synthesis requires ATP, we hypothesize that lower hepatic steatosis 
may reflect deceased ATP availability. Additionally, in older adults (age ≥ 48 years), daily 
fructose consumption was associated with increased hepatic inflammation (P < 0.05), and 
hepatocyte ballooning (P< 0.05) (19). However, the mechanism(s) by which fructose causes liver 
injury remains unknown. 
2.3 Fructose Alters Energy Homoeostasis and Causes Hyperuricemia 
 
In support of our hypothesis that ATP depletion underlies liver injury in patients with NAFLD, 
our group has demonstrated that patients with biopsy-proven NAFLD have increased hepatic 
mRNA expression of KHK compared to matched controls (17). Indeed, in human pilot studies, 
intravenous (IV) fructose administration is associated with hepatic ATP depletion (20, 21) which 
can be assessed by 31P magnetic resonance spectroscopy (MRS) (22).  Reduced hepatic ATP 
stores are more prevalent in overweight and obese subjects than in lean subjects (23).  
Furthermore, recovery from fructose-induced ATP depletion was found to be delayed in patients 
with NAFLD (n=8) (24). However, a limitation to this existing work is the small sample size and 
the inability to assess a cause-effect relationship(s) between BMI, NAFLD, energy homeostasis, 
and histologic features of liver injury.  In liver cells, ATP depletion could perpetuate chronic 
liver injury by making fatty hepatocytes less proliferative.  Hepatic ATP depletion also 
encourages the expansion of liver progenitor populations (25), causes arrest in protein synthesis, 
induces inflammatory and prooxidative changes (26, 27), increases endoplasmic reticulum stress, 
promotes activation of stress-related kinases, induces mitochondrial dysfunction, and increases 


Fructose-MRI Protocol 
NAFLD Clinical Research Program 
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apoptotic activity (28-30). This supporting data suggests that fructose may be associated with 
NAFLD, NASH, and progressive fibrosis.  Further, a study by Loguercio et al. demonstrated that 
increased uric acid levels above the basal level after IV fructose infusion was significantly higher 
(p < 0.01) in patients with cirrhosis (3 mg/dl) and NASH (1.9 mg/dl) than in healthy controls 
(1.2 mg/dl) (31). This effect was completely reversed by fructose 1,6-diphosphate which could 
replenish the ability to resynthesize ATP from ADP. Therefore, an IV fructose challenge could 
effectively differentiate healthy subjects, from chronic hepatitis, from cirrhosis (31).  
2.4 Hepatic ATP Depletion and Hyperuciemia as Potential Non-invasive Biomarkers for 
NAFLD 
 
NAFLD lacks accurate and robust non-invasive biomarkers to grade and stage histologic disease 
activity. This is a critical barrier to 
understanding the influence of this 
important environmental risk factor 
(increased/habitual 
fructose 
consumption) on the pathogenesis and 
progression of NAFLD. Currently, 
reliable assessment NAFLD requires 
liver biopsy and interpretation of 
histology. 
Serum 
aminotransferase 
levels 
and 
conventional 
imaging 
methods can detect liver fat but cannot 
grade 
or 
stage 
NAFLD 
(32-35). 
Furthermore, current developments in 
biomarker are cross-sectional in nature 
and do not characterize the dynamic 
changes which underlie liver injury in 
patients with NAFLD. In vivo 31P MRS 
permits the evaluation of dynamic 
changes of individual phosphorus-
containing metabolites in the liver 
parenchyma, such as phosphomonoester 
(PME), ATP, and inorganic phosphate 
(Pi). Intravenous fructose load alters 
phosphorus metabolites and allows 
assessment of liver function by 31P 
MRS (36). Other investigators have demonstrated that fructose loading could be used effectively 
as a tool to investigate change in metabolic steps of hepatic metabolism in humans with alcohol-
related liver disease (36). Further, IV fructose loading causes significantly higher ATP 
degradation and uric acid production in cirrhotic patients than in healthy controls (36). The 
associations between fructose, increased uric acid, and hepatic ATP depletion has been 
previously described (14, 17, 19, 31, 36). Increased uric acid is an independent risk factor for 
NAFLD (37-41) and in keeping with our hypothesis, hyperuricemia may be a surrogate marker 
of impaired hepatic energy homeostasis in patients with NAFLD. The proposed mechanism for 
fructose-related hepatic ATP depletion, NAFLD, NASH and the associated hyperuricemia is 
depicted in Figure 1 is novel, innovative, scientifically rigorous and address an important public 
health concern—the impact of fructose on the rising epidemic of NAFLD.  


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2.5 Use of MRI/ MRS as a Non-invasive Biomarker of NAFLD 
 
Various MRI/MRS methods are used to investigate liver status (42-45). Tools used in this study 
will be extensions of these methods validated in other organs, and that we have adapted for use 
in the torso. All spectroscopy tools will use localized techniques, to provide volumetric hepatic 
metabolite measures. Hepatic energy homeostasis (ATP values) will be measured using an ultra-
short TE sequence with “echo times” as short as 70 μs, which increases the signal to noise ratio 
allowing shorter temporal sampling if needed. Hepatic lipid and water signals will be measured 
within a breath hold using 1H MRI sequences .  
We will adapt the MRS acquisitions to provide a known reference against which to calibrate 
hepatic metabolite values. By calibrating the metabolites values, we will know exactly which 
metabolite(s) change and by how much, both within our cohort and in response to our dietary 
intervention study. 
2.6.  Summary   
 
This study will advance several goals of the NIH Action Plan: 1) establish a multidisciplinary 
team to develop quantitative methodologies and imaging protocols for liver, 2) validate 
diagnostic criteria and methodologies for imaging in liver in both a cross-sectional  and a 
longitudinal dietary intervention study of patients with NAFLD, 3) create a liver tissue bank with 
correlative imaging data, 4) develop reliable non-invasive MR markers to distinguish simple 
steatosis from NASH, and 5) define the dynamic changes in metabolism, energy homeostasis, 
and MR biomarkers as they relate to fructose-related liver injury.  
 
3. Study design 
3.1 Design overview 
The proposed open label study is single-center randomized, phase II clinical trial designed to 
address the following specific aims: 
a)  To optimize an MR protocol to characterize the range and rates of baseline and 
dynamic measures of metabolism and energy homeostasis following an acute IV fructose 
challenge. Volumetric and quantitative measures of hepatic lipid deposition, energy levels, 
and water homeostasis will be characterized in normal volunteers to determine typical ranges 
and coefficients of variation for each technique. 
b)  To differentiate the baseline and dynamic changes of metabolic and energy 
homeostasis following an acute IV fructose challenge in patients with biopsy-proven 
NAFLD. MR-based measurements from the Aim 1 protocol will be correlated to clinical 
measures of metabolism, energy homeostasis and histologic disease activity before, during 
and at 4 and 24 hours following IV fructose challenge in regions co-localized and distant 
from biopsy. 
A cohort study of healthy volunteers (n=30) and patients with NAFLD (n=70) will be enrolled.  
Healthy volunteers will have no evidence of chronic liver disease (normal liver enzymes and 
normal liver imaging and/or a liver biopsy performed for clinical evaluation of liver disease but 
whose pathology results note no pathologic abnormalities). Patients with suspected NAFLD who 
will undergo a standard-of-care liver biopsy, or who have had a historical biopsy within 6 


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months of the Screening visit, will have both acute intravenous fructose challenge for assessing 
baseline and post-dietary intervention static and dynamic measures of metabolism and energy 
homeostasis.  Co-localization of liver biopsy with MRI to optimize / assess MRI measures of the 
histologic features of NAFLD will be performed to develop MRI as a surrogate “biomarker” for 
liver biopsy.  With the exception of the liver biopsy, no accurate non-invasive biomarker for 
disease activity currently exists. 
Screening for eligibility and collection of baseline data will span up to 8 weeks. Patients who 
have had a liver biopsy showing NAFLD within 6 months of the Screening visit or patients 
who will be undergoing a standard of care percutaneous liver biopsy for NAFLD will be 
consented.  
 Measures of acute changes in metabolism and energy homeostasis will be performed the 
morning of the liver biopsy.  The primary comparisons will be made using an intention-to-treat 
analysis of the change in the (a) metabolic and (b) energy homeostatic measures from baseline.  
(c) compartment volumes by MRI,   Secondary outcomes measures will include changes in 
clinical and histologic features of NAFLD as assessed by liver enzymes and the NAFLD Activity 
Score (NAS), as determined from standardized histologic scoring of liver biopsies.  This study 
will be conducted in compliance with the protocol, Good Clinical Practice (GCP), and all 
applicable regulatory requirements. 
3.2 Population 
Participants at least 18 years of age, both healthy controls and those with suspected NAFLD. 
3.4 Sample size: 30 healthy controls and 70 subjects with biopsy-proven NAFLD 
 
3.5 Type of trial 
Single-center, phase 1 exploratory study to evaluate the acute (baseline and dynamic) changes in 
metabolic, energy homeostatic and MR biomarkers in volunteers and patients with biopsy-
proven NAFLD measures following IV challenge and dietary fructose restriction. 
3.6 Outcome measures 
Acute Fructose Challenge and MR Biomarkers 
Primary:   The difference(s) in rate and extent of the dynamic changes in metabolism and energy 
and water homeostasis in patients with biopsy-proven NAFLD.  Liver histology will be the 
primary predictor of disease and the primary outcome variable to which secondary measures (i.e. 
MR-biomarkers, metabolic parameters etc) will be measured. 
 
4. MRI Biomarkers of NAFLD 
4.1 MR-based Measures of Hepatic Energy Homeostasis 
31P Ultra-short TE MRS pulse sequence used in the Duke Center for Advanced MR 
Development (CAMRD) for the dynamic measurement of phosphorus metabolites is very similar 
in design to the UTE-MRS method by Cortez-Pinto, et al. (24)  just without the localization 
gradients. With a hard pulse excite of 100 μs, we can acquire an FID signal as short as 70 μs 
from the hard pulse center as compared with a nominal echo time of 2 ms for the standard 
Siemens slice-select excitation sequence. This gives 2-3 times the SNR for the short T2 
metabolites in the 31P spectrum.  


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All study scans will be taken on a 3T Siemens TIM Trio-CT scanner in CAMRD. We will 
contract with Clinical MR Solutions (Dr. Ralph Hashoian, Brookfield, WI) to set the 
specifications for a transmit/receive (TX/RX) Helmholtz-style 31P tuned flex-coil to provide 
superior B1 homogeneity to our current  surface coil design. Raw data will be transferred off 
scanner and analyzed using a suite of MRS processing and fitting software written by Dr. Soher.  
Possible Problems and Alternative Strategies – If data quality of the UTE MRS is insufficient to 
characterize the signal changes observed in volunteers and patients, then we will investigate the 
use of a single dimension encoding method to attempt to exclude confounding signals from 
tissues surrounding the liver. If problems arise with the data processing and analysis, the jMRUI 
program can also be used. An additional small reference phantom mounted on the 31P coil will 
identify its spatial location on MRI. 
 
5. 
Intravenous Fructose Challenge with MRS Measures of Energy 
Homeostasis   
IV fructose challenge with MRS measures of energy homeostasis will be performed in the 
morning after an overnight fast. For all subjects, a vein will be catheterized and a venous blood 
sample drawn for laboratory evaluation (uric acid, glucose, insulin, lipids, and free fatty acids). 
A slow infusion of isotonic sodium chloride solution will be started to maintain catheter patency. 
After baseline MR data sets are obtained, fructose (250 mg/kg of body weight) dissolved in 25 to 
50 mL of isotonic sodium chloride solution will be infused rapidly (over 30-60 seconds) through 
the indwelling venous catheter; further spectra will be collected for 1 hour. The slow saline 
infusion will be continued until the end of the study. At that time, another venous blood sample 
will be collected for laboratory evaluation (uric acid, glucose, insulin, lipids, and free fatty 
acids). 
 
6.   Standardized Life-Style and Dietary Research Questionnaires 
6.1  Alcohol Use Disorders Identification Test (AUDIT) is a 10-item questionnaire with a 
simple scoring scale that will be administered during screening visit 1. A 3- item interim 
drinking history (AUDIT-C) measuring consumption in the past 90 days will be obtained during 
follow-up visits at 3 and 6 months 
6.2  Health Related Quality of Life (HRQOL; SF-36) is a 36-item, self-report measure designed 
to assess the quality of life in patients that will be administered during screening.  
6.3  Automated Self-Administered 24 Hour Recall (ASA24™) is a 24 hour dietary recall tool 
which will be utilized for the purpose of assessing average fructose consumption.  The National 
Cancer Institute ASA24™ is web-based, allowing patients to access the web-site at their 
convenience. It has long been regarded as the best methodology to measure food intakes for 
dietary surveillance, nutritional epidemiology, clinical research, and intervention research 
because it provides the highest‐quality and least biased dietary data for a single day. This method 
allows collection of detailed intake and portion sizes, and because the data collection occurs after 
the consumption, this methodology does not affect what an individual chooses to eat on a given 
day. The close proximity in time to the intake day minimizes memory and cognitive issues that 
afflict other methodologies.  ASA24™ has been developed to permit unannounced, automated, 
and self‐administered 24HR, enabling the administration of multiple days of recalls in large‐scale 


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epidemiologic studies, surveillance studies, behavioral trials, or clinical research, thus enhancing 
researchers' ability to assess usual dietary intakes. The format and design of ASA24™ are based 
on a modified version of the interviewer‐administered Automated Multiple Pass Method 
(AMPM) 24HR developed by the U.S. Department of Agriculture (USDA).  ASA24™ consists 
of a respondent Web site and a Researcher Web site. The Respondent Web site allows study 
participants to report their intake for the previous day. The Researcher Web site allows 
researchers to register a study and its participants, set study parameters (e.g., number of recalls, 
number of attempts per recall, time to complete a recall), manage study logistics, and obtain 
analysis files. The investigators and study coordinators will review this instruction document 
completely and also to become familiar with the Respondent Web site in order to effectively and 
successfully use ASA24™. An overview of the Respondent Web site is available online at 
http://riskfactor.cancer.gov/tools/instruments/asa24/info.html.  The ASA24™ allows for 
scheduled and non-scheduled responder recall nutritional data collection, would accommodate 
the number of patients in the proposed study, will allow for detailed analysis of nutritional data, 
and is free tool to researchers. 
6.4  NCI Dietary Health Questionnaire (DHQ*Web) is a web-based version identical in content 
to the original DHQ. By automating the DHQ and providing it on the Web for public use, 
researchers have another tool to collect and analyze food frequency questionnaire data. 
DHQ*Web takes advantage of what automated and electronic questionnaires are able to do -- 
respondents follow automated skip patterns, are queried to complete all questions before 
proceeding to the next, navigate within the instrument to correct or modify responses, and can 
log in at any time to complete the questionnaire, starting where they left off. DHQ*Web provides 
efficiency with respect to data quality because respondents cannot complete the questionnaire 
with missing or inconsistent responses.  Patient will be asked to complete the DHQ at entry..  An 
overview 
of 
the 
respondent 
web 
site 
is 
available 
online 
at 
http://riskfactor.cancer.gov/DHQ/webquest/index.html. 
7.  Liver Histology 
Liver histology is the “gold standard” by which to define disease severity for NAFLD.  MRI 
biomarkers as well as the metabolic and energy homeostatic measures associated with histologic 
features of NAFLD.   
8. Patient Selection 
8.1 Recruitment:  Thirty healthy controls and 70 patients with NAFLD will be recruited from 
the Duke Liver Clinics.  Eligible patients will be identified and recruited by the principle 
investigator and/or sub-investigator.  Recruitment into the study will be performed without 
regard to gender or ethnicity as any patient with suspected NAFLD will be considered eligible if 
all inclusion and no exclusion criteria are met.  
8.2 Inclusion criteria 
Patients must satisfy all of the following criteria to be eligible for enrollment: 
• Age > 18 years as of the initial screening interview and provision of consent 
• Healthy control as defined by normal liver aminotransferases AND no evidence of 
NAFLD on radiologic imaging studies OR liver biopsy (if one had been historically 
performed for evaluation of suspected liver disease). 
OR 


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• 
Patients who have had a liver biopsy showing NAFLD within 6 months of the Screening 
visit or patients who will be undergoing a standard of care percutaneous liver biopsy for 
NAFLD will be consented. Additionally, anyone with a prior diagnosis of NAFLD or 
NASH-related cirrhosis (histologically or clinically) may also enroll without having had a 
recent historical biopsy.   
 
8.3 Exclusion criteria 
Patients who satisfy any of the following exclusion criteria will be ineligible for enrollment: 
• Current or history of significant alcohol consumption for a period of more than 3 
consecutive months within 1 year prior to screening (significant alcohol consumption is 
defined as more than 20 g/day in females and more than 30 g/day in males, on average) 
• Inability to reliably quantify alcohol consumption based upon local study physician 
judgment 
• Prior or planned (during the study period) bariatric surgery  
• Uncontrolled diabetes defined as HbA1c 9.5% or higher within 60 days prior to 
enrollment 
• A platelet count below 90,000/mm3 
• Clinical evidence of hepatic decompensation as defined by the presence of any of the 
following abnormalities: 
 Serum albumin less than 3.2 g/dL 
 INR greater than 1.3 
 Direct bilirubin greater than 2.0  mg/dL 
 History of esophageal varices, ascites or hepatic encephalopathy 
• Evidence of other forms of chronic liver disease (with the exception of a prior diagnosis 
of NAFLD or NASH-related cirrhosis)  
 Hepatitis B as defined by presence of hepatitis B surface antigen (HBsAg) 
 Hepatitis C as defined by presence of hepatitis C virus (HCV) RNA or positive 
hepatitis C antibody (anti-HCV) 
 Evidence of ongoing autoimmune liver disease as defined by consistent liver 
histology 
 Primary biliary cirrhosis as defined by the presence of at least 2 of these criteria 
(i) 
Biochemical evidence of cholestasis based mainly on alkaline phosphatase 
elevation 
(ii) 
Presence of anti-mitochondrial antibody  (AMA) 
(iii) 
Histologic evidence of nonsuppurative destructive cholangitis and 
destruction of interlobular bile ducts 
 Primary sclerosing cholangitis 
 Wilson’s disease as defined by ceruloplasmin below the limits of normal and 
consistent liver histology 
 Alpha-1-antitrypsin(A1AT) deficiency as defined by diagnostic features in liver 
histology (confirmed by alpha-1 antitrypsin level less than normal; exclusion at the 
discretion of the study physician) 
 History of hemochromatosis or iron overload as defined by presence of 3+ or 4+ 
stainable iron on liver biopsy 
 Drug-induced liver disease as defined on the basis of typical exposure and history 
 Known bile duct obstruction 


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 Suspected or proven liver cancer 
 Any other type of liver disease other than NASH 
• Serum alanine aminotransferase (ALT) greater than 300 U/L 
• Serum creatinine of 2.0 mg/dL or greater 
• Unstable therapy for components of the metabolic syndrome (ie. recent starting or 
stopping of insulin sensitizing agent, lipid lowering agent, and/or antioxidant therapy).   
Recent starting or stopping (for more than 7 days)  the use of a thiazolidinedione 
(pioglitazone or rosiglitazone) 90 days before the entry biopsy or anytime thereafter 
• Use of any prescription or over-the-counter medication or herbal remedy that are believed 
to improve or treat NASH or liver disease or obesity for the 90 days prior to baseline 
liver biopsy or prior to randomization 
 Patients must not take any other agent to treat NASH except the treatment assigned 
after randomization. 
• Inability to safely obtain a liver biopsy 
• Active substance abuse including inhaled or injection drugs in the year prior to screening 
• Pregnancy, planned pregnancy, potential for pregnancy and unwillingness to use effective 
birth control during the trial, breast feeding 
• Any other condition which, in the opinion of the investigator, would impede compliance 
or hinder completion of the study 
• A contraindication to MRI examinations 
• Extreme claustrophobia 
• Weight or girth exceeds the scanner capabilities 
• Any condition or circumstance that, in the opinion of the site investigator, would interfere 
with completion of MR examinations  
• Failure to give informed consent 
 
9.  Trial protocol—Study Visits and Events 
 
9.1. Visit schedule overview  
 
Healthy controls will ONLY undergo: 
• Screening for eligibility for enrollment (1-2 visits over a maximum of 8 weeks) 
• Acute intravenous fructose challenge with measure of metabolism and energy 
homeostasis by magnetic resonance spectroscopy (MRS) (baseline and assessment of 
change over 60 minutes.  
• MRI of the liver 
Data collected from healthy controls will be used as reference and optimization values for which 
study will assess metabolic and energy homeostatic measures in those with NAFLD. 
 
Patients with NAFLD will undergo the following related activities study phases: 
• Screening for eligibility for enrollment (1-2 visits over a maximum of 8 weeks) 
• Acute intravenous fructose challenge with measure of metabolism and energy 
homeostatasis by magnetic resonance spectroscopy (MRS) (baseline and assessment of 
change over 60 minutes. MRI of the liver 


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• Co-localization of non-invasive MR biomarkers with liver biopsy procedure. 
 
9.2. Screening and Baseline Data Collection 
Patients who appear to be eligible after chart review and have completed of standard of care tests 
and procedures for NAFLD or suspected NAFLD will be invited to undergo screening. 
Recording of screening data may not start until the patient has signed the consent statement. 
Screening and baseline data collection procedures will include questionnaires, physical 
examination, measurement of fasting serum glucose and insulin, routine liver, lipid and 
metabolic tests, uric acid and urine analysis for pregnancy testing.  Standard therapy for NAFLD 
will be reviewed.  Risks, potential benefits and alternatives to study participation will be 
reviewed in detail.  
Patients suspected of having NAFLD, requiring a standard of care biopsy to establish their 
diagnosis and guide their clinical management, will be approached to participate in this study. 
Patients with a historical biopsy confirming NAFLD within 6 months of the Screening visit will 
also be approached to participate in this study. 
All participants who sign the consent statement will be registered in the trial database. Patient 
charts will be reviewed for historical information. Each participant who starts screening will be 
accounted for at the end of screening, as either screening success (enrolling in the trial) or a 
screening failure. A screening failure is defined as a participant who signed the consent form, but 
is found to be ineligible prior to randomization; patients who meet medical eligibility criteria but 
who refuse enrollment in the trial; and patients whose liver biopsies do not meet entry criteria. 
Reasons for screening failure will be recorded in the trial database.   
The patient will sign the consent at screening visit to obtain any tests and procedures needed to 
finalize eligibility after chart review and will undergo a history and physical examination to 
identify other abnormalities and contraindications for participation. Anthropomorphic 
assessments (body weight [kg], body height [m], body mass index [BMI], waist circumference 
[cm], hip circumference [cm] and waist-to-hip ratio; vital signs (systolic and diastolic blood 
pressure, heart rate, respiratory rate, body temperature); and general physical findings, including 
hepatosplenomegaly, peripheral manifestations of liver disease, ascites, wasting or fetor, will be 
collected and recorded.  Use of anti-NASH, antidiabetic, statin and fibrate medications in the 90 
days prior to the biopsy will be obtained and recorded.  Laboratory test results that need to be 
recorded from chart review or obtained as part of screening visit 1 include hepatitis B (HBsAg) 
and hepatitis C (anti-HCV), antinuclear antibody (ANA), anti-smooth muscle antibody (ASMA), 
anti-mitochondrial antibody (AMA), ceruloplasmin (if patient is less than 40 years old), A1AT 
concentration, iron and transferrin saturation, immunoglobulins (IgA, IgG, IGM), fasting 
glucose, insulin, and free fatty acids; CBC (Hgb, WBC, platelets, MCV, hematocrit), 
prothrombin time and INR, complete metabolic panel (sodium, potassium, chloride, bicarbonate, 
calcium, phosphate, BUN, creatinine, uric acid, albumin, total protein), GGT and hepatic panel 
(ALT, AST, alkaline phosphatase, total bilirubin, direct bilirubin), fasting lipid profile (total 
cholesterol, triglyceride, LDL, HDL), and hemoglobin A1c (HbA1c). Frequency and amount of 
alcohol intake will be obtained. Women of childbearing potential must have a negative 
pregnancy test. 
 
9.3.   Visit Day -1 


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All patients will be provided with a “standard” meal the day prior to the fructose challenge MRI 
in order to control for dietary composition and calorie intake prior to intravenous fructose 
challenge. Patients will be NPO after midnight for morning IV fructose MR biomarker measures.   
9. 4.  Visit Day 0:   
Patients with suspected NAFLD, who have not had a biopsy in the prior 6 months, will have a 
standard of care liver biopsy followed by co-localization of liver biopsy with MR.   
Patients will be escorted to the Department of Radiology for baseline MRI/MRS imaging (MRI 
study protocol and data acquisition sequences detailed in Appendix A).  Time 0 uric acid, 
insulin, glucose, lipid panel, and free fatty acids will be obtained.  IV Fructose will be 
administered via heplock and dynamic measures of hepatic ATP and other metabolite measures 
will be obtained over 1 hour.  At that time, another venous blood sample will be collected for 
laboratory evaluation (uric acid, glucose, insulin, lipids, and free fatty acids). 
After acquisition of these late hepatic recover ATP measures, for the patients who have not had a 
historical biopsy in the 6 months prior to screening, a percutaneous liver biopsy will be 
performed as standard of care.  After standard of care procedures and recovery are completed for 
the percutaneous liver biopsy, a very limited MRI will be performed to co-localize the site of 
liver biopsy with the exact site of MRI for modeling of non-invasive MRI biomarkers to features 
of chronic liver disease as characterized on liver biopsy.    Subjects with suspected NAFLD may 
have two MRI’s (one for IV fructose challenge and one for co-localization with liver biopsy 
site).  For subjects with NAFLD, study participation will end after completion of the second 
MRI. 
9.5 Adverse event reporting and Data Safety Monitoring 
The investigators and staff will monitor and report adverse events to ensure patient safety.   Any 
unanticipated or adverse events will be reported to IRB and NIH (Sponsor) in compliance with 
government regulations of event reporting in research studies.  Dr. Anna Mae Diehl, Chief of the 
Division of Gastroenterology and researcher with prior experience in the conduct of IV fructose 
challenge and MRS will serve as a data-safety monitor is any adverse events arise in the context 
of this study. 
9.5.1.   Definitions 
 
Adverse event: An adverse event is any untoward medical occurrence that may present 
itself during treatment or administration with a pharmaceutical product or clinical procedure 
and which may or may not have a causal relationship with the treatment. Adverse events 
include any unanticipated problems involving risks to participants, or breaches of protocol 
which might entail risk to participants. The term "unanticipated problem" includes both new 
risks and increased rates of anticipated problems. 
 
 
Serious adverse event: A serious adverse event (SAE) is an adverse event occurring at 
any time during the study that results in death, life-threatening adverse drug experience, 
inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant 
disability/incapacity, or a congenital anomaly/birth defect. Other events may also be 
considered an SAE if, based on medical judgment, the event jeopardized the patient to the 
point of requiring medical or surgical intervention to prevent the occurrence of any of the 
conditions for an SAE listed above. 


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Unexpected adverse event: An unexpected adverse event is any adverse event with 
specificity or severity that is not consistent with the risk information in the study protocol, 
current investigator brochure, or current package insert. 
 
 
Adverse Events associated with the use of the intervention (i.e. fructose administration 
or restriction): Any unexpected adverse event for which there is reasonable possibility that 
the adverse experience may have been caused by the administration of intravenous fructose 
and/or restriction of dietary fructose. 
 
9.5.2. Monitoring for adverse events 
Adverse events will be recorded on study data forms whether or not they are thought to be 
associated with the study. Adverse events may be discovered during regularly scheduled visits or 
through unscheduled patient contacts between visits. Summary data on adverse events will be 
promptly assessed by investigators if they occur. Analyses or listings of adverse events will not 
be provided to the IRBs; however, adverse events involving unanticipated problems involving 
risks to participants, or breaches of protocol which might entail risk to participants will be 
reported to the IRB as soon as possible after they are discovered. 
 
10.  Statistical design and analysis 
Coefficients of variance (CV) will be used to evaluate reproducibility of MRS/MRI baseline 
measures. Intra-class correlations (ICC) will be used to measure absolute test-retest baseline 
measure reliability for the test-retest subjects. ICCs will be calculated with a model that separates 
subject, voxel, measurement and error variances using an analysis of variance (ANOVA) 
method. We will include a number of multi-factorial random effects models (66, 67) which use a 
restricted maximum likelihood (REML) algorithm to prevent negative variance terms. The 
ANOVA model for metabolite estimates across all subjects will be Mijk = μ + Si + Vij + Nk + eijk 
where μ is the average across all voxels, Si = subject, Vij = voxel and Nk = scan random effects, 
respectively and eijk is residual error. For ICC in individual subjects, we leave out the Si term. 
ICCs for test-retest measures will be based on random-effects analysis of the variance models 
and will be computed as follows: ICC = σ2B / (σ 2B+σ 2W+σ 2e) where σ2B is generalized between-
subject variance of metabolites, σ 2W is within-subject variance between scans 1 and 2 and σ 2e is 
variance due to random noise (68). CVs for test-retest reproducibility will be calculated using 
CV = sqrt(σ 2w+σ 2e) / μ. Similarly we will obtain CVs from the first 20 subjects in Aim2 and will 
compare these to the normal population. 
Dynamic changes will be characterized in a more ad hoc manner. From the metabolite estimates 
at time t (Mijkt), the following paramerters will be measured for each individual (subscript i): 
baseline (Bi = mean of Mjkt, where t = prior to fructose challenge), range of change from baseline 
(Di=Bi-min(Mjkt) if any), time to maximal change (Ti = time to reach min(Mijkt)), time to baseline 
recovery (Ri, if applicable). A 95% confidence interval of baseline will be used to test if the 
metabolite level is recovered to the baseline. If it does not reach the baseline, we will estimate 
the baseline based on the assumption of a linear recovery rate. Since metabolite levels will be 
measured at discrete time points, spline smoothing will be used to fit the curve. These values will 
be averaged across the cohort of volunteers and simple measures of variance calculated.  


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10.1 Sample size and Statistical Power Consideration: Assuming 20% change in hepatic ATP 
and uric acid (i.e. before~ Normal(1.2, σ2) and after ~ Normal(1, σ2)), one-side test with alpha = 
0.15, the power would be i) if σ2 = 1, power =0.523 ii) if σ2 = 0.8, power = 0.629, iii) if σ2 = 0.6, 
power = 0.784, iv) if σ2 = 0.5, power = 0.875. 
10.2  Analytical and Statistical Approach: We will test if the acute fructose challenge alters 
hepatic ATP in healthy volunteers compared to those with NAFLD and whether  changes MR 
parameters and/or energy homeostasis can predict the histologic severity of NAFLD.  
10.3 Management of Missing data: The occurrence of missing data in this trial is expected to 
be low. We estimate that careful selection of patients during the screening phase and the consent 
process should result in no more than 10% missing data from patients who drop out before 
completing the study.   
10.4 Justification of sample size:  A total of 67 patients will be included to compare the 
efficacy of acute fructose challenge on the metabolic and energy homeostasis changes in patients 
with NAFLD.   
 
11.0 Human Subject Issues 
11.1 Regulatory Compliance 
The study protocol, questionnaires, and consent forms will be submitted to IRB for approval.  All 
study personnel must complete training in the Protection of Human Subjects per NIH guidelines. 
The proposed study will require eligible patients without regard for gender or ethnicity. The 
patient must sign the consent to be eligible for the trial. The consent form will describe the 
purpose of the trial, the procedures to be followed, and the risks and benefits of participation. 
Copies of the signed consent forms will be given to the patient, and this fact will be documented 
in the patient’s study record. 
11.2 Risk to Human Subjects 
11.2.1 Intravenous Fructose Challenge Fructose has been recommended as an 
Fructose has been recommended as an intravenous energy source during parenteral nutrition for 
patients with hepatic disease, uncontrolled diabetes mellitus, and in the postoperative state. 
Fructose is metabolised in the liver where it causes increased lactate formation, high-energy-
phosphate depletion, increased uric-acid formation, and inhibition of protein synthesis. This 
results in increased concentrations of blood-lactate and serum-uric-acid. In hepatic disease 
fructose infusion may theoretically lead to lactic acidosis.  
 
11.2.2. MRI/MRS 
Standard safety precautions: screening; exclusion of potential participants with contraindications 
to MR. MR is a minimal risk procedure if standard precautions and practice exercised. Standard 
precautions for MR procedures include: claustrophobia, anxiety, discomfort from lying supine 
for 30-60 minutes, hearing loss, and heating of metal in the body. Patients with metal will be 
screened by questionnaire and excluded when the presence of metal is known or suspected. 
Sources include the presence of a cardiac pacemaker or defibrillator;  metal fragments in eyes, 
skin, body;  heart valve replacement, brain clips, venous umbrella;  being a sheet-metal worker 
or welder;  aneurysm surgery, intracranial bypass, renal, aortic clips;  prosthetic devices such as 
middle ear, eye, joint or penile implants, joint replacements;  hearing aid, neurostimulator, 


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insulin pump; intrauterine device;  shunts/stents, metal mesh/coil implants;  metal 
plate/pin/screws/wires, or any other metal implants;  permanent eyeliner, eyebrows. 
11.2.3. Patient privacy 
It is the investigator’s responsibility to conduct the protocol under the current version of 
Declaration of Helsinki, Good Clinical Practice, and rules of local IRBs. The investigator must 
ensure that the patient’s anonymity be maintained during all aspects of their study participation. 
Patients will be identified only by an identification code but not by their name, SSN, or hospital 
medical record number.  All laboratory specimens, study forms, reports, and other records that 
are part of the study data collection materials will be identified by coded number to maintain 
patient confidentiality. All records will be kept in locked office. All electronic records of study 
data will be identified by coded number. Clinical information will not be released without 
written permission of the patient, except as necessary for monitoring by the IRB. Consent 
procedures and forms, and the communication, transmission and storage of patient data will 
comply with individual site IRB and NIH requirements for compliance with The Health 
Insurance Portability and Accountability Act (HIPAA). Investigators will maintain a separate 
confidential enrollment log which matches identifying codes with the patients’ names and 
addresses (i.e., available only to local clinic staff). All study material will be maintained in strict 
confidence. 
 
11.2.5 Sample Biobanking 
It is anticipated that serum, plasma, DNA, and liver tissue from the participants will be stored for 
future studies related to NASH and possibly other liver/metabolic diseases in our Duke NAFLD 
Clinical Database and Biorepository. These samples will be stored in a central repository.  
Specific IRB approval will be as per specific institutional  guidelines addressing the issues such 
as (a) obtaining a separate informed consent, (b) storage, (c) transportation of the material, (d) 
who will have access to the material, and (e) what investigations will be conducted. 


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Appendix A:   
 
MRI/MRS Protocols for Assessment of Energy Homeostasis and MR 
Biomarkers for NAFLD 
 
In all cases no patient repositioning will need to occur between data acquisitions for different 
MR nuclei. Primary measures are indicated in blue and include 1H MRI/MRS for water and fat 
measures and 31P UTE MRS for fructose measures. In all cases, the multiple acquisitions 
acquired prior to the injection of fructose will serve as test-retest cases for confirming 
measurement variance in the presence of pathology. Interleaved measures for experimental 
hypotheses may be moved or removed in order to ensure sufficient primary measures within the 
allotted MR time slot. 
Only MR Slot 1 and Slot 2 protocols shown below will be used on the healthy volunteers for 
Aim 1 in order to optimize these protocols. The full MR protocol shown below will be 
performed for NAFLD subjects. 
MR Slot 1 (Time 0) 
MR Slot 2 (Post biopsy) 
1H  MRI - anatomical 
1H MRI/MRS – water/fat 
31P UTE MRS – fructose 
baseline (multiple acquire) 
Fructose injection 
31P UTE MRS – fructose 
dynamic  (multiple acquire) 
1H EPSI – water/fat 
31P UTE MRS – fructose 
dynamic (multiple acquire) 
 
1H  MRI - anatomical for 
biopsy localization 
31P UTE MRS – fructose 
recovery  (multiple acquire) 
1H MRI/MRS – water/fat