Clinical trial NCT01792115
Conditions: Fatty Liver
Keywords: Fatty Liver Alpha-Tocopherol Vitamin E Non-Alcoholic Steatohepatitis
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date: 2013-05-01; Completion date: 2019-08-30
Enrollment: 22 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

* Non-alcoholic fatty liver disease (NAFLD) is an excess accumulation of fat in the liver cells. It is associated with obesity, high blood pressure, high cholesterol, and diabetes. Some people with NAFLD only have excess fat in the liver. However, other people may develop a worse form of NAFLD with liver injury and scarring. This form, called non-alcoholic steatohepatitis (NASH), can lead to liver failure, liver cancer, and death. Not much is known about why some people develop NASH and others do not.
* Lifestyle changes such as diet, exercise, and weight loss can decrease the liver damage in NAFLD. Some studies show that vitamin E can also help treat NAFLD. The dose of vitamin E used in these studies is almost 40 times the recommended amount of vitamin E intake from food. It is unclear whether a lower dose could achieve the same effect. Researchers also want to study how vitamin E works at different doses to treat NAFLD.

Objectives:

* To find out the most effective dose of vitamin E to treat NAFLD.
* To gain a better understanding of how NAFLD and NASH develop, and predict who will respond to treatment.

Eligibility:

\- Individuals at least 18 years of age with suggestion of non-alcoholic fatty liver disease.

Design:

* Participants will be screened with a physical exam and medical history. Blood and urine samples will be collected.
* For the first 12 weeks of the study, participants will meet with a nutritionist. They will have personalized diet and exercise plans. Treatment will be monitored with diaries and questionnaires to fill out at home. Participants will also wear a pedometer to measure physical activity.
* After the 12-week period, participants will have a full physical examination with the following tests:
* Blood tests
* Glucose tolerance tests
* Imaging studies (DEXA scan and magnetic resonance imaging)
* Liver and fatty tissue biopsy
* Two weeks after the tests, participants will start vitamin E treatment. They will take up to two pills a day, taken with fat-containing foods.
* 4 weeks after starting treatment they will have a repeat full evaluation with imaging tests, blood work, and liver and fat biopsies.
* Participants who are taking vitamin E will take it for up to 120 weeks. They will have monitoring visits every 8 to 12 weeks. At the end of 120 weeks, they will have another full evaluation, with imaging tests, blood work, and liver and fat biopsies.

Detailed description:
Non-alcoholic fatty liver disease (NAFLD) is the most common cause for liver test abnormalities in the western world, and an increasingly rising cause for liver-related morbidity and mortality. Vitamin E, a fat-soluble anti-oxidant was recently found to be an effective treatment for NAFLD; however, its mechanism of action is unclear. In a controlled clinical trial vitamin E treatment was shown to significantly reduce the hepatic fat burden, suggesting mechanisms other than reducing oxidative stress are involved. Furthermore, the optimal dose of vitamin E to treat NAFLD is unknown.

We propose a phase IIa study to determine the optimal dose of vitamin E and its mechanism and site of action. In this study we aim to enroll up to 90 patients with NAFLD. Initially, all patients will undergo 12 weeks of intensive lifestyle modification. Following that, all patients will be randomized to treatment with 3 different doses of natural vitamin E (rrr- -tocopherol at 200, 400 or 800 IU/d) for 24 weeks. The primary end points for efficacy are normalization of liver enzymes and reduction in liver fat contents by magnetic resonance spectroscopy. Patients will undergo liver and adipose tissue biopsies before vitamin E treatment and after 4 weeks of therapy, and the biopsy samples will be used to measure changes in gene expression and markers of oxidative stress. This will be coupled with extensive phenotyping before and after treatment using serological, radiological and dynamic endocrine testing and is aimed at finding the dose-response characteristics of vitamin E in NAFLD, and allowing us to understand the mechanism of its action.

After 24 weeks of randomized treatment, all patients will be switched to a dose of 800 IU/ml and will continue treatment for up to 30 months, at the end of which another liver biopsy will be performed. From this phase we will assess the effects of dose increase of vitamin E on liver enzymes and fat content, and will determine the effect of long-term treatment on histological outcome.

Arms: arm 1: Subjects randomized to vitamin E 200 IU/day for 24 weeks; invited to optional extension of open- label vitamin E 800 IU /day for up to 120 weeks following the initial 24 week period. arm 2: Subjects randomized to vitamin E 400 IU/day for 24 weeks; invited to optional extension of open- label vitamin E 800 IU/day for up to 120 weeks following the initial 24 week period. arm 3: Subjects randomized to vitamin E 800 IU /day for 24 weeks; invited to optional extension of open- label vitamin E 800 IU /day for up to 120 weeks following the initial 24 week period.
Interventions: intervention 1: Vitamin E 200 IU/d intervention 2: Vitamin E 400 IU/d intervention 3: Vitamin E 800 IU/d intervention 4: Diet and Exercise
Intervention descriptions: intervention 1: Supplement-low dose intervention 2: Supplement-intermediate dose intervention 3: Supplement High Dose intervention 4: Diet and Exercise for all Arms of the study at baseline

Protocol documents:
Prot_SAP_000.pdf: 
CLINICAL RESEARCH PROTOCOL
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
PROTOCOL NUMBER: 13DK0002
VERSION DATE: JULY 7, 2020
TITLE: TREATMENT OF NON-ALCOHOLIC FATTY LIVER WITH DIFFERENT DOSES OF 
VITAMIN E
IND: 117534, held by NIDDK
Short Title: Vitamin E for NAFLD
Identifying Words: Non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, 
steatosis, vitamin E, lifestyle intervention, diet
NIH Principal Investigator:
Yaron Rotman, MD
Liver Disease Branch
NIDDK Institute
Building 10, Rm 10N24
9000 Rockville Pike
Bethesda, MD 20892
Phone: 301-451-6553
E-mail: rotmany@niddk.nih.gov
Estimated Duration of Study: 7 years
Start Date: 8/1/2012
End Date: 8/1/2019
Number and Type of Patients: Up to 90 patients with non-alcoholic fatty liver disease, 
aged 18 years or above, both males and females. No healthy volunteers.
Project Uses Ionizing Radiation: Research indicated, RSC approval 2346-B, expiring 
June 2021
Project does not use durable power of attorney, is not a multi-site study and does not use 
an off-site location


Vitamin E for Non-Alcoholic Fatty Liver
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Précis
Non-alcoholic fatty liver disease (NAFLD) is the most common cause for liver test 
abnormalities in the western world, and an increasingly rising cause for liver-related 
morbidity and mortality. Vitamin E, a fat-soluble anti-oxidant was recently found to be an 
effective treatment for NAFLD; however, its mechanism of action is unclear. In a 
controlled clinical trial vitamin E treatment was shown to significantly reduce the hepatic 
fat burden, suggesting mechanisms other than reducing oxidative stress are involved. 
Furthermore, the optimal dose of vitamin E to treat NAFLD is unknown.
We propose a phase IIa study to determine the optimal dose of vitamin E and its mechanism 
and site of action. In this study we aim to enroll up to 90 patients with NAFLD. Initially, 
all patients will undergo 12 weeks of intensive lifestyle modification. Following that, all 
patients will be randomized to treatment with 3 different doses of natural vitamin E (rrr-α-
tocopherol at 200, 400 or 800 IU/d) for 24 weeks. The primary end points for efficacy are 
normalization of liver enzymes and reduction in liver fat contents by magnetic resonance 
spectroscopy. Patients will undergo liver and adipose tissue biopsies before vitamin E 
treatment and after 4 weeks of therapy, and the biopsy samples will be used to measure 
changes in gene expression and markers of oxidative stress. This will be coupled with 
extensive phenotyping before and after treatment using serological, radiological and 
dynamic endocrine testing and is aimed at finding the dose-response characteristics of 
vitamin E in NAFLD, and allowing us to understand the mechanism of its action. 
After 24 weeks of randomized treatment, all patients will be switched to a dose of 800 
IU/ml and will continue treatment for up to 30 months, at the end of which another liver 
biopsy will be performed. From this phase we will assess the effects of dose increase of 
vitamin E on liver enzymes and fat content, and will determine the effect of long-term 
treatment on histological outcome. 


Vitamin E for Non-Alcoholic Fatty Liver
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Table of Contents
Précis....................................................................................................................................3
Table of Contents.................................................................................................................4
Introduction..........................................................................................................................6
Non-alcoholic fatty liver disease .....................................................................................6
Vitamin E.........................................................................................................................7
Vitamin E for NAFLD.....................................................................................................8
Aims...................................................................................................................................10
Primary Aims:................................................................................................................10
Secondary aims:.............................................................................................................10
Study design and methods .................................................................................................11
Overall design (Figure 3)...............................................................................................11
Initial screening and enrollment ....................................................................................12
Run-in phase and lifestyle intervention .........................................................................12
Detailed in-patient assessment.......................................................................................14
Vitamin E Treatment .....................................................................................................15
Extended Treatment Phase ............................................................................................16
Details of procedures .........................................................................................................18
Adipose Tissue Biopsy ..................................................................................................18
Liver Biopsy ..................................................................................................................18
Magnetic Resonance Spectroscopy ...............................................................................19
Justification for study design.............................................................................................19
Run-in phase ..................................................................................................................19
Liver biopsies.................................................................................................................20
Absence of a control arm...............................................................................................21
Duration of treatment.....................................................................................................21
Inclusion and exclusion criteria.........................................................................................21
Inclusion ........................................................................................................................21
Exclusion criteria...........................................................................................................22
Monitoring subjects and criteria for withdrawal of subject from the study ......................24
Analysis of the study .........................................................................................................27
End points ......................................................................................................................27


Vitamin E for Non-Alcoholic Fatty Liver
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Primary end points.....................................................................................................27
Secondary end points.................................................................................................27
Basic scientific end points .........................................................................................27
Statistical analyses.........................................................................................................28
Sample size calculation..................................................................................................28
Human subject protections ................................................................................................28
Rationale for subject selection.......................................................................................28
Recruitment strategy......................................................................................................29
Informed consent process ..............................................................................................29
Evaluation of benefits and risks.....................................................................................30
Benefits......................................................................................................................30
Risks and Discomforts...............................................................................................30
Adverse event reporting and data monitoring ...................................................................33
Monitoring .................................................................................................................34
Collection and storage of human specimens or data .........................................................35
Remuneration/compensation .............................................................................................35
References..........................................................................................................................38


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Introduction
Non-alcoholic fatty liver disease
Non-alcoholic fatty liver disease (NAFLD) is the most common cause for liver enzyme 
abnormalities in the western world. This disorder, consisting of abnormal accumulation of 
triglycerides in the liver, is estimated to affect approximately 30% of the US population1. 
NAFLD commonly exists with features of the metabolic syndrome (obesity, hypertension, 
diabetes mellitus and hypertriglyceridemia) and its rising prevalence seems to accompany 
the increasing rates of obesity. 
A subset of patients with NAFLD develop non-alcoholic steatohepatitis (NASH), where 
the fat accumulation is accompanied by hepatocellular injury, inflammation and fibrosis. 
NASH can be a progressive disease, leading to cirrhosis, liver failure and hepatocellular 
carcinoma. NASH and NAFLD are also associated with increased cardiovascular 
mortality, even in patients without other “classical” cardiovascular risk factors. 
The pathogenesis underlying NAFLD and NASH is not clearly understood. Insulin 
resistance is commonly, but not universally, seen in these patients but it is unclear whether 
it is causing the fat accumulation or is being caused by it. Triglyceride storage in the liver 
may have a physiological role as a protective mechanism in the face of increased free fatty 
acid (FFA) flow from adipose tissue. When the ability of the hepatocytes to store these 
fatty acids as non-toxic triglycerides is overwhelmed, hepatic lipotoxicity ensues, while 
peripheral lipotoxicity worsens muscle insulin resistance and pancreatic beta-cell 
function2. The accumulation of free fatty acids in hepatocytes causes endoplasmic 
reticulum (ER) stress, an increase in reactive oxygen species (ROS) and apoptosis. This is 
accompanied by inflammatory cell infiltration, an increase in inflammatory cytokines3, 
activation of macrophages and stellate cells and fibrosis. 
Innate immune cells such as natural killer T (NKT) cells and natural killer (NK) cells are 
thought to contribute to the disease pathogenesis. NKT cells, for example, get activated by 
lipids and, in a TIM-1-mediated fashion, by apoptotic hepatocytes4, produce cytokines that 
promote fibrosis and then undergo apoptosis themselves.  CD1d-deficient mice that lack 
NKT cells are protected from fibrosis5. NK cells are activated by cytokines and by altered 
surface marker expression by hepatocytes, and are able to exert rapid effector functions 
such as cytokine production and cytotoxicity. Both NKT and NK cells constitute the major 
lymphocyte population in the liver.
There is a marked heterogeneity in the presentation and phenotype of patients with 
NAFLD. Racial and ethnic variability is significant; for example, Asian patients tend to 
have NAFLD at lower BMIs than their Caucasian counterparts, African-Americans seem 
to be relatively protected and Hispanics are at increased risk. Genetic factors clearly play 
a role, with most data pointing towards variants in the PNPLA3 (adiopnutrin) gene as 
strongly associated with disease occurrence and severity6. However, it is still unclear why 
some patients develop NASH and progressive disease, while others remain in the relatively 
benign steatotic stage. To date, no test apart from a liver biopsy accurately differentiates 
NASH from steatosis. 


Vitamin E for Non-Alcoholic Fatty Liver
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Currently, there is no standard approved therapy for NASH. Life style modification is 
routinely advocated, although only recently it was actually shown to benefit patients7. Diet, 
weight loss and physical exercise improve insulin sensitivity not only at the hepatic level, 
and thus have an important role in reducing the release of FFA from adipose tissue8. For 
patients who are not able to gain benefit from lifestyle changes, a logical choice is 
pharmacological intervention. 
Vitamin E
Vitamin E is a fat-soluble antioxidant that is believed to be important in maintaining the 
integrity of lipid membranes and preventing oxidative injury to lipids, cholesterol and cell 
signaling molecules. Clinically apparent vitamin E deficiency is rare and is usually 
associated with severe malabsorption syndromes, advanced liver disease or inherited 
deficiencies in vitamin E transport proteins. In adults, vitamin E deficiency is associated 
with neuropathy and ataxia. In animals, vitamin E deficiency is associated with 
reproductive failure and with poor fetal neurological development.
The term “Vitamin E” is a collective name for several fat-soluble compounds; naturally 
occurring vitamin E exists in multiple chemical forms (α, β, γ, and δ-tocopherol and –
tocotrienol), which have variable levels of antioxidant activity and biologic effects in 
different cell systems. In humans, however, only α-tocopherol appears to have significant 
biologic effects9. There are 3 chiral centers in the α-tocopherol molecule, yielding 8 
possible racemic isomers; of these, RRR-α-tocopherol is the most active form and the one 
that is present in nature, while isomers with S chirality in the first chiral center are inactive 
and are not accumulated in tissues.
Vitamin E is present naturally in many foods and is absorbed in the intestine only in the 
presence of fats. The recommended daily intake for adults is 15 mg (22.4 IU) of d-alpha-
tocopherol. Even this low amount is actually more than the average American consumes 
daily; in fact, it is estimated that about 90% of Americans are marginally deficient in 
vitamin E intake from dietary sources10. Multiple vitamin E supplements are available and 
an estimated 11% of the adult US population takes vitamin E supplement daily (which 
usually have 400 IU). Vitamin E is also present in many multivitamins, but usually in lower 
amounts (40-50 IU). Vitamin E supplements are manufactured from different sources and 
by different techniques. Synthetic supplements are the least expensive and contain and all-
racemic mixture of all isomers, and thus have a potency of ~50% that of natural vitamin E, 
which contains mostly or purely RRR-α-tocopherol. Both formulations are available over 
the counter.
The absorption of vitamin E is dependent upon the presence of dietary fat. Thus, even if 
vitamin E supplements are taken, if not taken with a meal that has at least some fat, 
absorption will be minimal. Vitamin E from the intestines is taken up into chylomicrons, 
transported to the circulation via the thoracic duct and then rapidly and almost completely 
extracted from the post-prandial blood by the liver. In the liver, rrr-α-tocopherol is bound 
by the tocopherol transfer protein (TTP), which can deliver it into and across lipid 
membranes. The other forms of vitamin E (l-α-tocopherol and γ-tocopherol) for instance) 
are not bound to TTP and are metabolized by the liver and excreted in bile. Thus, the 
specificity of TTP for rrr-α-tocopherol is what determines that this form of vitamin E is the 


Vitamin E for Non-Alcoholic Fatty Liver
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biologically active form in humans. The hepatic TTP also allows for vitamin E to be 
secreted into the blood where it is taken up by HDL and LDL lipoprotein particles. These 
lipoprotein particles deliver vitamin E to other tissues. Inside of cells, vitamin E remains 
bound to lipids and can act to protect intracellular membranes from free radical damage 
probably by forming transient oxidative intermediates and delivering the free electrons to 
other anti-oxidants such as vitamin C11. The overall effects of vitamin E are multiple, 
perhaps because it protects membrane bound enzymatic and cell-signaling molecules non-
specifically which are involved in a multitude of activities. Vitamin E also appears to have 
its major effect in the presence of environmental oxidative stress, so that cells are more 
able to resist injury when vitamin E replete. 
The antioxidant activities of vitamin E and epidemiologic evidence linking vitamin E 
intake to several diseases led to extensive, large scale studies of vitamin E for prevention 
of cancer as well as cardiovascular and cerebrovascular disease. While controversial, 
overall these studies showed little or no effect of vitamin E supplementation on subsequent 
cancer or cardiovascular events.  Indeed, perhaps the most reproducible effect seen was a 
slight increase in hemorrhagic stroke12 that has been attributed to the anti-platelet effects 
of higher doses of vitamin E. For these reasons, vitamin E supplementation has become 
more controversial.  
Vitamin E for NAFLD
The anti-oxidant effect of vitamin E can potentially ameliorate the lipotoxicity and ROS-
induced damage in NASH and affect disease progression. Several treatment trials were 
conducted with inconclusive results seen in the smaller studies13. However, two recent 
studies have suggested that vitamin E may be beneficial in nonalcoholic fatty liver disease 
(NAFLD) and nonalcoholic steatohepatitis (NASH) and that a surrogate marker for its 
effect might be serum alanine aminotransferase (ALT) levels.
The PIVENS trial14 was a double-blind, double-dummy placebo-controlled trial conducted 
at 8 U.S. medical centers in 286 adults with liver biopsy proven NASH who took either 
pioglitazone (30 mg), vitamin E (800 IU of rrr-α-tocopherol) or placebo daily for 2 years 
(96 weeks) with regular monitoring of serum enzymes and liver biopsy done before and at 
the end of treatment. Follow up also occurred 24 weeks after stopping the interventions. 
The primary endpoint was improvement in liver histology which was statistically 
significant comparing placebo to vitamin E with decreases in steatosis, inflammation and 
cell injury scores, but little change in fibrosis (Figure 1, Panel A). In addition, there were 
marked differences in ALT levels during treatment and this effect was seen within 6 
months of starting therapy (Figure 2, Panel A). Overall, histological and biochemical 
response were achieved in 43% of patients treated with vitamin E in this study. 
The TONIC trial15 was a similar double-blind, double-dummy placebo-controlled trial 
conducted at the same 8 U.S. medical centers in 116 children or adolescents with biopsy-
proven NAFLD who took either metformin (1000 mg), vitamin E (800 IU of rrr-α-
tocopherol) or placebo daily for 2 years (96 weeks) with regular monitoring of serum 
enzymes and liver biopsy done before and at the end of treatment. The primary endpoint 
was improvement in ALT, even though liver histology was also obtained, largely because 
liver histologic changes had never been documented in children before this study. There 


Vitamin E for Non-Alcoholic Fatty Liver
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were statistically significant differences in ALT levels between vitamin E and placebo 
treated children at 6 months and one year, but the differences were no longer significant at 
2 years largely because of improvements in the placebo group (Figure 2, panel B). 
Importantly, there were improvements in liver histology in steatosis, inflammation and cell 
injury, although they were significant only for cell injury (Figure 1, panel B).
Figure 1 – Histological changes in vitamin E studies
-60
-40
-20
0
Change (U/L)
0
24
48
72
96
Weeks
Placebo
Vitamin E
PIVENS: ALT
-60
-40
-20
0
Change (U/L)
0
24
48
72
96
Weeks
Placebo
Vitamin E
TONIC: ALT
Figure 2 – Changes in serum ALT in vitamin E studies
While these two studies showed convincing evidence that vitamin E has an effect on 
NAFLD and NASH, they also raised many issues regarding optimal treatment and 


Vitamin E for Non-Alcoholic Fatty Liver
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mechanism of action. Vitamin E was anticipated to affect inflammation and injury through 
its anti-oxidant effect. However, there was also marked and significant decrease in liver 
fat, which was not associated with improvement in insulin sensitivity, through an unclear 
mechanism. Theoretically, vitamin E could affect liver fat by modulating the expression of 
lipogenic or lipolytic enzymes, by altering the delivery of free fatty acids from adipose 
tissue to the liver or by affecting the release of triglycerides from the liver. Vitamin E was 
shown to activate the nuclear receptor pregnane X receptor (PXR)16, to induce the 
expression of an endogenous PPAR agonist in adipocytes17 and it was also suggested that 
vitamin E changes the expression of lipogenic enzymes in hepatocytes (Sanyal, A, 
unpublished results). 
In addition, the clinical studies of vitamin E in NAFLD did not address the important 
problems of vitamin E dosage. The preparation used was “natural” vitamin E (rrr-α-
tocopherol) and was >40 times the recommended daily allowance for vitamin E. This dose 
was higher than those used in almost all previous studies of vitamin E such as for cancer 
and heart disease prevention that typically used 100-400 IU of racemic, dl-α-tocopherol, 
equivalent to 10% to 25% of the dose used in PIVENS. The dose of vitamin E is particularly 
an issue in patients who might be at risk of hemorrhagic stroke such as those on anti-platelet 
medications or with hypertension and diabetes, features that are commonly found in 
patients with NASH and NAFLD. 
Finally, the studies did not identify markers of anti-oxidant status that might be correlated 
with effects of vitamin E on fatty liver disease. In preliminary studies of vitamin E levels 
and of serum markers of anti-oxidant status (including proteomics studies), no marker was 
identified that was different between vitamin E and placebo treated subjects. 
We propose to conduct a phase IIa study of natural vitamin E in patients with NAFLD, to 
determine the optimal dose of vitamin E, its mechanism of action, and to help understand 
the pathophysiology of the disease.
Aims
Primary Aims:
1. Primary clinical aim: Defining the optimal dose of vitamin E to treat NASH
2. Primary scientific aim: Determining the mechanism leading to fat loss with vitamin E 
treatment.
Secondary aims:
1. Will patients who do not respond to low-dose vitamin E benefit from a higher dose?
2. Is the vitamin E-induced fat loss due to reduction in hepatic de-novo lipogenesis, 
decrease in free fatty acid supply to the liver, increase in -oxidation or increase in 
VLDL secretion?
3. Is the vitamin E action mediated through its anti-oxidant effect or through direct effect 
on gene induction?
4. Is there a dose response curve for the anti-platelet effect of vitamin E?


Vitamin E for Non-Alcoholic Fatty Liver
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5. Liver fat has been suggested as a cause of peripheral and hepatic insulin resistance. 
Since vitamin E is not thought to affect insulin sensitivity directly, does the reduction 
in fat caused by vitamin E drive a change in liver and peripheral insulin sensitivity?
6. What is the role of liver-resident innate immune cells, especially NKT and NK cells, 
in NASH, and how are these affected by treatment with vitamin E?
7. What effect does genetic variability have on hepatic and adipose tissue physiology in 
NAFLD, and on the response to treatment?
Study design and methods
Overall design (Figure 3)
A prospective randomized trial. Eligible patients will be screened and enrolled. After 
enrollment, patients will undergo an initial period of 12 weeks of lifestyle modification. At 
the conclusion of that phase, the patients will be admitted to the clinical center for an 
extensive in-patient evaluation and a liver and adipose tissue baseline biopsy. Patients will 
then be randomized to receive vitamin E 200 IU/d, 400 IU/d or 800 IU/d for 24 weeks. 4 
weeks after starting vitamin E, the patients will be admitted again, to undergo a second 
liver and adipose tissue biopsy. At the end of the 24 week period, all patients will undergo 
an extensive in-patient evaluation identical to the baseline admission (but without the 
biopsies) and will then be offered the option to switch to treatment with open-label vitamin 
E 800 IU/d for up to 120 weeks more.
Figure 3 – Overall study design. Extended studies highlighted in squares, liver and adipose tissue biopsies 
in triangles.


Vitamin E for Non-Alcoholic Fatty Liver
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Initial screening and enrollment
Patients will be screened during one or more visits in the Outpatient Clinic with history, 
physical examination, review of outside medical records, routine blood tests and bleeding 
time. Alcohol history will be assessed in the medical interview and will be quantified using 
the AUDIT questionnaire, which has been validated as a screening tool for excessive 
alcohol consumption18. We will observe the total score (reflecting the risk for alcohol-
related problems) as well as quantify alcohol intake (using the first 3 items on the 
questionnaire). The initial screening visit to assess eligibility for the protocol will be done 
under protocol 91-DK-0214, “Evaluation of Patients with Liver Disease”; only eligible 
patients will then be enrolled into this protocol.  All patients will be asked to stop any 
medication being used for their liver disease, including herbal medications and vitamins. 
Patients meeting the eligibility criteria will be enrolled and consented. Enrolled patients 
will be asked to complete baseline dietary and exercise questionnaires and will be given 
food and exercise logs as well as a pedometer. Following enrollment, patients will undergo 
a magnetic resonance spectroscopy to quantitatively measure liver fat content (see below), 
to be performed at or before the day of their first appointment with the nutritionist.
Run-in phase and lifestyle intervention
One to four weeks after enrollment, patients will meet with a Clinical Center nutritionist 
for an individual counseling session. All patients will be instructed on a balanced diet, 
including decreased intake of saturated and trans fats and increased intake of fiber. In 
overweight patients (BMI >= 25 kg/m2), the dietary intervention will focus on a calorie-
restricted diet, aiming for a weight reduction of 5-8% from their baseline weight, preferably 
attained over a 6-month period. The calorie-restricted diets will be planned to create a 
deficit of 500-700 kcal/day and will be individualized for each study subject. Patients will 
be encouraged to achieve a regimen of moderate-intensity physical activity, with gradual 
increases in intensity or duration, aiming at eventually exercising for at least 150 minutes 
per week. Patients will be instructed to stop taking any vitamins or supplements and a 
standard dose multi-vitamin will be prescribed for the duration of the study.
After enrollment, the patients will follow every two weeks with the nutritionist. On the first 
follow-up visit (2 weeks after enrollment), there will be an individual session with the 
nutritionist during which progress, including keeping of food logs, will be reviewed and 
diet plans will be adjusted and reinforced as needed. On this visit and on subsequent visits 
throughout the run-in phase, patients will attend 30-45 minute educational/support group 
sessions. Sample topics to be covered during these group sessions will include heart healthy 
eating guidelines, food-label reading, appropriate portion sizes and healthy strategies for 
eating out. 
Baseline dietary intake will be assessed using the NCI Diet History Questionnaire II (DHQ 
II) utilizing the past month, with portion size version. Patients will be advised to complete 
the online version of the questionnaire at home, the day before coming to clinic, or on a 
computer in the clinic waiting room. Patients who do not have access to the internet, or 
who are not comfortable with using the internet version will fill out the paper version of 
the questionnaire in the clinic. Before enrollment, and again at the end of the 12 week 
run-in phase, patients will be directed to complete a 3-day food record which will serve as 


Vitamin E for Non-Alcoholic Fatty Liver
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the primary measure of change in food intake. Food records will be reviewed for 
completeness and accuracy and will be analyzed using Nutrition Data System for Research 
(NDS-R) software. Patients will also be instructed to track their intake in a daily food log, 
mainly for self-monitoring purposes. 
On enrollment, patients will be provided with pedometers and will be instructed to record 
their daily step-count in a diary. Physical activity levels will also be monitored using the 
International Physical Activity Questionnaire (IPAQ), which will be completed on 
enrollment and again at the end of the 12 week lifestyle modification program. Patients 
will be encouraged to achieve a regimen of moderate-intensity physical activity, with 
gradual increases in intensity or duration, aiming at eventually exercising at least 150 
minutes per week. 
During the randomized vitamin E treatment phase, patients will be continue to be seen by 
the nutritionist every 8 weeks for individual follow-up visits and to reinforce components 
covered during the initial 12 week lifestyle intervention. During the extended treatment 
phase, the patients will be seen by the nutritionist every 12 weeks. Patients will complete 
the 3-day food intake diary and IPAQ every 24 weeks during the randomized treatment 
and extended treatment phases. The data obtained from these diaries and questionnaires 
will be used to ensure that differences in outcomes observed between the different vitamin 
E groups are not due to differences in adherence to lifestyle changes. Although part of the 
effect of treatment may be due to lifestyle intervention, it should affect all treatment groups 
in a similar manner and the effect of vitamin E will be deduced from the dose response. 
Detailed in-patient assessment
At the beginning of the randomized-dose treatment phase, patients will be admitted to the 
Clinical center for a detailed histological, physiological and biochemical analysis, 
including:
1. History and physical examination: including vital signs, weight and 
anthropomorphic measurements. 
2. Fasting blood tests: chem-20, GGT, CBC, PT, PTT, serum insulin, c-peptide, lipid 
panel, apolipoproteins, total free fatty acids, hemoglobin A1c, alpha-fetoprotein, 
ferritin, iron, transferrin saturation, immunoglobulins, TSH, C-reactive protein, 
PSA and vitamin E levels
3. Research blood: 10 ml of serum will be separated from collected blood and stored 
in 2 separate aliquots in -80C for determining serum levels of hormones, cytokines 
and adipokines, injury markers (such as CK-18 fragments), and for archival storage. 
10 ml of plasma will be separated from collected blood and stored at -80ºC for 
analysis of oxidative stress measures.
4. NKT and NK cell analysis: 40 ml of blood will be drawn for analysis of NKT and 
NK cell phenotype and effector function. Additional 8 ml of serum tube (8 ml) will 
be used to provide autologous serum for in vitro functional assays. The frequency 
of performing these assays is detailed in table 1.
5. Urine tests: urinalysis, pregnancy test (for women of child-bearing potential) and 
24-hour urine collection for creatinine clearance and protein and uric acid 


Vitamin E for Non-Alcoholic Fatty Liver
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excretion. A 5 ml urine sample will be stored at -80ºC for oxidative stress 
measurements.
6. Imaging: 
a.
Magnetic resonance imaging (MRI):
i. MR spectroscopy to quantitatively measure liver fat content.
ii. Liver volume estimation
iii. Adipose tissue distribution: measurement of total abdominal 
adipose tissue, visceral adipose tissue and subcutaneous adipose 
tissue volumes.
b. Total body mass, total body fat mass and bone mineral density will be 
determined using dual energy x-ray absorptiometry (DEXA).
7. Insulin sensitivity:
a.
Insulin-modified frequently sampled intravenous glucose tolerance test 
(FSIVGTT)19  – after a 12-14 hour fast, intravenous access will be achieved 
in both arms. After 15 minutes of comfortable rest, blood samples will be 
drawn at, 10 and 1 minute before glucose injection. At time 0, 300mg/kg of 
glucose, in the form of dextrose 50% in water, will be injected over 1-2 
minutes. Blood samples will be drawn at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 
16 and 20 minutes. At 20 minutes, IV insulin will be given at a dose of 4 
mU/min*kg for 5 minutes. Blood sampling will continue at 22, 23, 24, 25, 
26, 27, 28, 30, 40, 50, 60, 70, 80, 100, 120, 150 and 180 minutes. All 
samples will be analyzed for plasma glucose, insulin and free fatty acid 
concentrations20. Glucose effectiveness and insulin sensitivity will be 
calculated according to the minimal model using the MINMOD software.
b. Hepatic insulin sensitivity will be estimated using the HOMA-IR2 model. 
c.
Adipose tissue insulin sensitivity will be estimated at the basal state by the 
HOMA-FFA model. 
8. Liver biopsy: a percutaneous liver biopsy will be performed under local anesthesia, 
with the patient in a fasting state. Tissue will be used for histological evaluation, 
RNA analysis, immunological studies, measurement of oxidative stress and 
archival storage.
9. Subcutaneous adipose tissue biopsy: subcutaneous adipose tissue will be removed 
from the abdominal region by needle aspiration under local anesthesia. The sample 
will be submitted for histological evaluation, RNA analysis and an additional 
fraction will be used to for ex vivo studies (see below).
10. Resting energy expenditure (REE) measurement will be performed at the patient 
bedside.
11. Studies under other protocols. Participation in the following protocols will be 
offered to all patients in the study if eligible. Refusal to enroll in these additional 
studies will not preclude patients from participating in this protocol.


Vitamin E for Non-Alcoholic Fatty Liver
Page 14 of 39
a.
Genetic analysis: peripheral blood mononuclear cells will be extracted from 
20 ml of blood and stored for DNA extraction and genotyping (one time 
only). This will be done under a separate protocol and consent (91-DK-
0214).
b. Cardiovascular imaging: imaging by CT scan and MRI of the heart. This 
will be done under a separate protocol and consent (11-DK-0168).
c.
Fatty acid oxidation will be assessed by measurement of 13C-palmitate 
oxidation with a breath test device to detect expired 13CO2. This will be 
performed under a separate future protocol and consent. 
12. Fetal hemoglobin (HbF) levels may be measured in some of the patients at baseline 
and during treatment, to assess the effects of vitamin E on HbF induction.  
Vitamin E Treatment
Patients will be evaluated again in the outpatient clinic 1-2 weeks after the admission and 
will be given the biopsy results. Patients whose liver biopsy did not show evidence of 
NAFLD will be excluded at this visit and will end the study. All other patients will be 
randomized to receive 200 IU/d, 400 IU/d or 800 IU/d of vitamin E and will receive the 
medication for a planned duration of 24 weeks. 
The vitamin E formulation will be “natural” RRR-α-tocopherol, provided by PharmaVite 
Inc. (Northridge, CA) under a clinical trial agreement. The medication will be supplied in 
200 IU and 400 IU softgel capsules on an annual basis and will be stored and dispensed by 
the NIH Clinical Center Pharmaceutical Development Section. As described earlier, RRR-
α-tocopherol is thought to be the predominantly biologically active metabolite in synthetic 
vitamin E formulations (that contain a mixture of racemic enantiomeres), due to the 
specificity of affinity to hepatic TTP. There is no data suggesting difference in toxicity of 
different vitamin E formulations that is separate from differences in efficacy. Depending 
on their group allocation, patients will take one 200 IU capsule, one 400 IU capsule or two 
400 IU capsules, once daily.
Randomization will be done by the Clinical Center pharmacy Protocol Support Service and 
will be stratified by the result of the liver biopsy (classified as definite steatohepatitis, 
steatosis without steatohepatitis, or borderline21, 22) and by the presence or absence of 
diabetes mellitus type 2 at enrollment. Patients will be monitored on weeks 1, 2, 3, 4, 8, 
12, 16, and 20 of treatment. On each visit, fasting blood tests for Chem-20, insulin, C-
peptide, free fatty acids and vitamin E levels will be drawn, vital signs including weight 
will be measured and a symptom questionnaire will be administered. CBC, lipid panel, 
apolipoproteins and urinalysis will be drawn every 4 weeks. A nurse will determine weight, 
vital signs and symptoms on every visit. A physician will evaluate the patients every 4 
weeks. A serum sample for research purposes will be drawn on each visit and stored in -
80ºC.
On week 4 of treatment, patients will be admitted for a second liver biopsy and adipose 
tissue biopsy, and will have a repeat of the MR spectroscopy and FSIVGTT.
On week 24 of treatment, all patients will be admitted for an exit work-up, identical to the 
detailed in-patient assessment described above, but without the biopsies. 


Vitamin E for Non-Alcoholic Fatty Liver
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During the treatment, patients will be recording medication intake on a diary that will be 
collected every visit and will be used to assess compliance. 
Extended Treatment Phase
On discharge, all patients will be offered to continue treatment with open-label vitamin E 
800 IU/d (two 400 IU capsules taken once daily) for up to 120 more weeks, after which, 
an exit liver biopsy will be performed. Enrollment into the extended treatment phase is 
optional and refusal will not exclude the patient from the study. The medication intake 
diary will be used in this phase as well.
After 24 weeks in the extended phase (week 48 of the study), patients will undergo an 
additional MR spectroscopy. Based on the results of the MRS and liver enzymes, the 
response will be assessed prior to deciding on treatment continuation. For this purpose, 
response is defined as a decline of >=25% in ALT or normal ALT or normal AST or an 
absolute decline of more than 2% in liver fat content by MRS. The response criteria are 
selected to be sensitive, but not necessarily specific, aiming to predominantly identify 
patients who are not responding to treatment and in which continuation is futile. Based on 
the response assessment there are three possible outcomes (Figure 4):


Vitamin E for Non-Alcoholic Fatty Liver
Page 16 of 39
 
Figure 4 – Stopping rule based on week 48 results. 1Response defined as  25% decrease in ALT, or normal 
ALT  or  AST, or  absolute decrease of >2% in liver fat by MRS.
1. Patients who did not meet any of the response criteria compared to baseline will 
discontinue treatment for futility.
2. Patients treated with 200 or 400 IU/d during the first 24 weeks, will be reverted 
after 48 weeks to their original dose if they had a response at week 48 compared to 
baseline, but no incremental response when switching to 800 IU/d at week 24 (i.e., 
did not meet any of the response criteria comparing week 48 to week 24). 


Vitamin E for Non-Alcoholic Fatty Liver
Page 17 of 39
3. Patients who were treated initially by 800 IU/d and had a response at week 48 or 
patients who were treated with a lower dose initially and had an improvement when 
switching at week 24 to the higher dose, will be offered to continue treatment with 
the 800 IU/d dose. 
Details of procedures
Adipose Tissue Biopsy
Subcutaneous adipose tissue (1-10 g) will be removed from the abdominal region by 
aspiration with a 2.5 or 3 mm blunt Spirotri cannula under local anesthesia (1% lidocaine). 
The tissue obtained will be used to obtain isolated adipocytes and organ culture using 
standard procedures23, as well as isolation of preadipocyte cell lines that will be expanded 
and stored in liquid nitrogen. The remaining tissue will be separated into aliquots of 
approximately 500 mg for RNA isolation and protein extraction, flash frozen in liquid 
nitrogen, and stored at -80 ºC. The relatively large sample of fat from each subject will 
ensure extraction of sufficient RNA analysis. 
Liver Biopsy
A percutaneous liver biopsy will be performed under conscious sedation and local 
anesthesia using standard technique with the Klatzkin biopsy needle or a spring-loaded 
cutting needle (“biopsy gun”). A bedside ultrasound examination will be used to confirm 
an optimal biopsy site before the procedure. Following the biopsy the patients will be 
monitored overnight for recovery from sedation and for the development of complications. 
Accurate interpretation of liver histology is significantly impacted by the size of the 
histological specimen24, 25. Because the initial study biopsy will be used for staging as well, 
it is critical that adequate tissue be submitted for histological evaluation. A specimen of at 
least 2 cm length will be reserved for histological evaluation; the remainder (0.5-1 cm) will 
be split to 2 or 3 aliquots, one to be stored in RNALater (Qiagen, Valencia, CA) or a similar 
preservative and used in later analyses, and one flash-frozen in liquid nitrogen at bedside 
for archiving and for measurement of oxidative stress. If a sufficient specimen was 
obtained allowing for a third aliquot, 3-5 mm of tissue will be transported in RPMI medium 
for extraction of liver-resident lymphocytes and immunological studies. 
If inadequate tissue is obtained for clinical purposes, a second or third pass may be 
attempted, as is in keeping with clinical practice. The risk of complication from liver biopsy 
increases only after 3 biopsy passes are made. If after a single pass inadequate tissue is 
available for the research specimen (0.5 cm), one additional pass will be performed. If two 
passes have already been made in order to obtain adequate clinical material, a third pass 
will not be permitted to obtain a research sample. No more than three passes will be made 
for clinical purposes, in keeping with current practice guidelines. The potential need for 
one additional pass is discussed in the consent form. 
Histological evaluation will be performed by an expert liver pathologist in a qualitative 
manner and the presence or absence of NASH will be determined. Biopsies will be 
classified as showing definite steatohepatitis, borderline or not steatohepatitis but with 
steatosis21, 22. The biopsy sample will be scored semi-quantitatively using the scoring 


Vitamin E for Non-Alcoholic Fatty Liver
Page 18 of 39
system utilized by the NASH Clinical Research Network trials26. The NAFLD activity 
score (NAS) will be calculated as the arithmetic sum of the scores for steatosis, ballooning 
injury and lobular inflammation. 
Magnetic Resonance Spectroscopy
Experiments will be performed on an MRI scanner using TIM phased-array coils. B0 shim 
parameters are optimized with a breath hold B0 mapping method27. Navigator gated T2 
weighted (T2w) and breath-hold T1 weighted (T1w) images in coronal and axial 
orientations will be used to prescribe a 20x20x20 mm3 spectroscopy volume in an area 
with high signal to noise ratio, free of obvious fatty structures or blood vessels. After 
additional manual shimming, and optimization of transmit power and WS RF level 32 
signals will be averaged with a minimum repetition time (TR) of 3s and 35 ms echo time 
(TE) with WET water suppression (WS) followed by 4 signals averaged with WS RF 
power set to zero. Spectral data will be fitted with AMARES28 using a model of four 
resonances for the lipid signals on the WS spectra. Additionally, the apparent T2’s of the 
summed lipid CH2 and CH3 resonances lipid and the T2 of water will be measured with five 
quick expiration breath hold non-WS spectra averaging two signals each at TR 3s and TE 
24, 36, 48, 96 and 144 ms. These T2 values will be used to correct the water and total lipid 
signals for signal decay in the 35 ms echo delay. Fat fraction will be then calculated as 
from the T2 corrected signals Swater and Slipid as: fat fraction =  Slipid/(Swater+Slipid).
Justification for study design
Run-in phase
To date, most trials in NAFLD enrolled patients based on the results of a pre-enrollment 
liver biopsy, which was not necessarily performed immediately prior to enrollment. 
However, in clinical practice a decrease in liver enzymes is often seen in the first few 
months after a patient is given a diagnosis of NAFLD, probably reflecting a serious attempt 
at lifestyle modification. Similar effects were noted in placebo-treated patients7, 29. This is 
especially important in studies with a histological end-point, in which the true baseline 
status may thus be different than the one suggested by a “distant” enrollment biopsy. 
Furthermore, it may not be justified to suggest pharmacological interventions to patients 
who are controlling their disease adequately with dietary modification and physical 
activity. 
In this study, patients can be enrolled based on non-invasive studies that are suggestive of 
the presence of NAFLD but not definitive. All patients will undergo a standardized lifestyle 
intervention period with a biopsy immediately preceding pharmacological therapy. This 
will ensure that the biopsy will serve as a true baseline. Furthermore, this design allows us 
to evaluate physiological changes induced by lifestyle modification alone.
Liver biopsies
In this study, patients will undergo 2-3 liver biopsies. The first biopsy is done for clinical 
and research purposes. Clinically, the biopsy is indicated as the gold-standard for 
diagnosing fatty liver disease and to date, the only reliable method to distinguish NASH 


Vitamin E for Non-Alcoholic Fatty Liver
Page 19 of 39
from simple steatosis. Thus, the first biopsy will serve clinically to confirm the diagnosis 
of NAFLD and to assess severity of disease. This biopsy is also crucial for research 
purposes, as it will provide a baseline for the comparison to the on-treatment and exit 
biopsies. 
A few patients may have had a liver biopsy done before enrollment into the protocol, either 
by a referring physician or at the LDB (for example in a previous protocol). However, a 
previous biopsy can not serve as a true baseline clinically (because of possible changes in 
disease behavior or management over time), nor will it provide the tissue for research this 
protocol requires. For that reason, even patients with a previous biopsy will have to agree 
to undergo an entry biopsy in order to enroll. If a patient had a previous liver biopsy, the 
baseline biopsy may not provide a direct clinical benefit. In such a case, the fact that the 
procedure is purely for research purposes will be clearly explained to the patient during 
enrollment and consent and will be documented in the patient’s chart. 
Patients will be informed of the findings in the entry biopsy before being randomized. If 
the biopsy does not show any evidence of NAFLD, the patients will be withdrawn from 
the study. We plan to treat all patients with NAFLD in the study, whether they have NASH 
or steatosis only. However, the severity (or lack of) of liver disease in the biopsy will be 
conveyed to the patients in laymen terms and patients will be given the option to withdraw 
themselves from the treatment phase if they wish so.
The second biopsy will be performed 4 weeks after starting vitamin E. The goal of this 
procedure is to obtain tissue to observe the effect of vitamin E on hepatic and adipose tissue 
gene expression. The histological response to pharmacological agents is slow and we do 
not anticipate a marked decrease in liver fat during these 4 weeks. This biopsy will allow 
us to detect an effect of vitamin E on gene expression and fatty acid metabolism without 
being confounded by the effects on the transcriptome of the decrease in liver fat. This 
biopsy is a purely research procedure and is not anticipated to provide clinical benefit; this 
will be clarified to the patients on enrollment, and again before the procedure.
A third liver biopsy will be performed in patients who agree to continue with extended 
vitamin E therapy, after 3 years of treatment. This biopsy will allow determination of 
histological response, especially whether the beneficial effects of vitamin E extend to 
halting progression of fibrosis. Although repeating liver biopsies in patients with NAFLD 
is not standard clinical practice, this biopsy is anticipated to provide important clinical 
information regarding the individual patient response and restaging, as well as research 
data.   
At the liver diseases branch, we have acquired considerable experience in the performance 
of liver biopsies for clinical and research indications. Several LDB past and present studies, 
as well as studies in other institutions in viral hepatitis and in fatty liver diseases employed 
more than one biopsy, with good patient acceptance, safety track and important data 
acquired.
Absence of a control arm
A placebo-controlled arm could be considered in this study. However, by including a fourth 
arm we would markedly reduce our sample size in each arm and the statistical power to 
detect differences. Furthermore, we do not think it would be justified to perform a second 


Vitamin E for Non-Alcoholic Fatty Liver
Page 20 of 39
liver biopsy on patients treated with placebo. The lead-in phase should eliminate most of 
the placebo effect, and the use of paired biopsies for the analysis will allow us to control 
for inter-individual changes.
Duration of treatment
There is no clear accepted duration of treatment for NASH and similar to other metabolic 
conditions like diabetes or hypertension, treatment is probably required for life. The 
kinetics of biochemical response in the previous vitamin E trials (figure 2) show near-
plateauing at week 24, and suggest little incremental benefit will be achieved by extending 
treatment beyond that. 
Inclusion and exclusion criteria
Inclusion 
1. Clinical suspicion of NAFLD, defined by the presence of at least two of the 
following criteria:
a.
Suggestion of liver fat by an imaging study (ultrasound, CT scan, MRI or 
MR spectroscopy) performed in the 6 months prior to enrollment.
b. Elevated aminotransferase levels (ALT > 31 U/L for men or > 19 U/L for 
women, or AST > 30 U/L) on at least two occasions in the 6 months 
preceding enrollment.
c.
Presence of the metabolic syndrome, defined according to the modified 
AHA/NCEP criteria30 as the presence of at least three of:
i. Abdominal obesity, defined as waist circumference > 102 cm for 
men or > 88 cm for women
ii. Elevated triglycerides (> 150 mg/dL) or the use of medication to 
lower triglycerides
iii. Reduced HDL cholesterol (< 40 mg/DL for men or < 50 mg/dL for 
women)
iv. Elevated blood pressure (> 135/80 mmHg) or use of medication for 
hypertension
v. Elevated fasting glucose levels (> 100 mg/dL) or use of anti-diabetic 
medication
For the purpose of inclusion, the presence of overt diabetes mellitus type 2 
will be considered equivalent to the presence of the metabolic syndrome, 
even if the other criteria are absent.
2. Estimated average alcohol consumption < 30 g/d for men or < 20 g/d for women in 
the 6 months prior to enrollment and no binge-drinking behavior.
3. Age > 18 years at enrollment
4. Willingness to participate in the study


Vitamin E for Non-Alcoholic Fatty Liver
Page 21 of 39
Exclusion criteria
1. Chronic infection with hepatitis C virus (HCV) or hepatitis B virus (HBV). Patients 
who were treated successfully for HCV and achieved sustained virological response 
can be eligible for enrollment > 18 months after treatment cessation. Patients who 
are inactive carriers of HBV (HBV DNA < 1000 copies/mL, HBeAg negative, Anti 
HDV negative) for at least 12 months prior to enrollment are also eligible. Patients 
receiving antiviral therapy are ineligible.
2. Concomitant liver disease such as autoimmune hepatitis, primary biliary cirrhosis, 
primary sclerosing cholangitis, Wilson’s disease, alpha-1 antitrypsin deficiency.
3. Presence of definite or probable drug-induced liver injury. In the case of lipid-
lowering, anti-hypertensive or anti-diabetic medications that are suspected to cause 
elevation of aminotransferases, patients will be eligible if treatment is associated 
with stable enzyme levels for at least 6 months and inclusion criteria 1a. and 1c. are 
both present.
4. Treatment with medications known to cause fatty liver disease such as atypical 
neuroleptics, tetracycline, methotrexate or tamoxifen
5. Uncontrolled hypo- or hyperthyroidism.
6. Decompensated advanced liver disease, defined as direct bilirubin > 0.5 g/dL, PT 
> 18”, albumin < 3 g/dL, or history of ascites, encephalopathy, variceal bleeding, 
spontaneous bacterial peritonitis or liver transplant.
7. Active coronary artery disease, defined as persistent angina pectoris, reversible 
ischemia on cardiac stress test or imaging, or the presence of significant coronary 
artery disease on imaging or catheterization. Patients with coronary artery disease 
that was treated by angioplasty or bypass surgery may be eligible if they have no 
evidence of active disease >= 1 year after intervention, can safely stop antiplatelet 
and anticoagulant medications before the performance of invasive procedures, and 
have adequate ventricular function as assessed by echocardiography or cardiology 
consultation. These patients will require cardiology consultation and clearance 
prior to enrollment.
8. Congestive heart failure.
9. Chronic kidney disease, with creatinine clearance < 60 ml/min or eGFR < 
60/ml/min/sq. m.
10. Uncontrolled diabetes mellitus. Patients may be enrolled if they have been on stable 
therapy with any anti-diabetic agent for at least 3 months prior to enrollment, are 
not foreseen to require antidiabetic medication or dose changes during the trial, and 
have an HbA1c <= 7.5% on enrollment.
11. Treatment with vitamin E. Patients who are currently taking vitamin E as a 
supplement will be requested to stop for at least 3 months before becoming eligible 
for enrollment. Patients who are taking vitamin E for a medical indication other 
than NAFLD will not be eligible. 
12. Contraindication or inability to perform a liver biopsy. 


Vitamin E for Non-Alcoholic Fatty Liver
Page 22 of 39
13. Patients who had a liver biopsy performed <= 2 years before enrollment, unless 
they are willing to undergo all of the trial biopsies, knowing that these biopsies are 
purely for research and are not clinically indicated. This will be clearly documented 
in the patients’ charts prior to enrollment. 
14. Patients with coagulopathy (PT/PTT values that are prolonged >= 3 seconds from 
the upper limit of the normal, including treatment with oral and parenteral 
anticoagulants), thrombocytopenia (<70,000), abnormal bleeding time or platelet 
dysfunction will not be enrolled because of potential increase in risk of bleeding 
with vitamin E treatment. Antiplatelet agents taken for cardiovascular prevention 
will not exclude patients, unless they cannot be stopped safely for the performance 
of a liver biopsy.
15. Maldigestion or malabsorption that can interfere with absorption of vitamin E 
including: steatorrhea of all causes, chronic pancreatitis, cystic fibrosis, short bowel 
syndrome, severe cholestasis, orlistat treatment and similar conditions
16. Inability to swallow vitamin E capsules
17. Allergy to vitamin E 
18. Alcohol or substance abuse within the past 12 months. Patients will be required to 
have an AUDIT score of 7 or less18, and drink no more than 14 drinks/week (for 
men) or 7 drinks/week (for women). 
19. For women of childbearing age, pregnancy or inability (or unwillingness) to 
practice contraception for the duration of the study or breast feeding.
20. Hemoglobin level < 11 g/dL
21. History of past or present significant gastrointestinal bleeding, unless treated 
definitively (i.e. resection, eradication of H. pylori with documentation of ulcer 
healing, etc). 
22. Known history of past or present condition that according poses a significant risk 
for significant gastrointestinal bleeding (i.e. peptic ulcer) unless definitively 
treated.
23. Inability to understand and give informed consent for participation
Monitoring subjects and criteria for withdrawal of 
subject from the study
Patients will be monitored every 2 weeks during the run-in phase. At this stage, the only 
intervention is lifestyle modification and no related serious adverse events are anticipated. 
At this stage, visits that are within 1 week before or after the planned visit date will not be 
considered a protocol violation.
After the run-in phase, patients will be admitted for up to 5 days, for the initial biopsies 
and extended tests as detailed above. At the LDB, it is our standard practice to keep all 
patients for observation overnight after a biopsy, and to perform a routine complete blood 
count 6 hours after the procedure. Following discharge, patients will be seen in the 


Vitamin E for Non-Alcoholic Fatty Liver
Page 23 of 39
outpatient clinic for the first 3 weeks of treatment, and will be subsequently admitted during 
the 4th week of treatment, for the second biopsy. At this stage, visits that are within 2 days 
before or after the scheduled visit date will not be considered a protocol violation. 
Following the second admission, the patients will be followed in clinic every 4 weeks. 
From this time point onwards, visits that are within 1 week before or after the planned visit 
date will not be considered a protocol violation. Patients will be admitted again at the end 
of the 24 week period for another extensive evaluation. During the extended treatment 
phase, patients will be seen every 12 weeks.
Patients will also be monitored as appropriate for patients with liver disease. Specifically, 
patients with liver cirrhosis or high-risk for hepatocellular carcinoma (HCC) will undergo 
surveillance for HCC by ultrasound or cross sectional imaging every 6-12 months, or 
according to most current guidelines. Patients with cirrhosis will be monitored by 
endoscopy as appropriate for the development of esophageal varices.
Patients who become pregnant during the study will be withdrawn, as pregnancy and 
associated hormonal and metabolic changes may affect hepatic fat metabolism and will 
confound the analysis. Patients who do not respond to treatment by week 48 will be 
withdrawn for futility. There are no other pre-specified criteria for withdrawal of patients 
from the study. Patients will be withdrawn if, according to the best judgment of the treating 
physician or the principal investigator, continuing treatment poses an unacceptable risk to 
the patient. 
Patients with hemoglobin < 10 g/dL will not undergo the FSIVGTT test and/or collection 
of blood for NK/NKT cells. Patients with hemoglobin < 10 g/dL will not undergo the first 
(baseline) liver biopsy. That biopsy may be deferred for up to 4 weeks if the anemia can 
be treated sufficiently by that time. Patients who cannot undergo the baseline liver biopsy 
will be withdrawn from the study. Patients with hemoglobin < 10 g/dL will not undergo 
the second or third liver biopsy; these biopsies will not be deferred but the patients may 
continue on the study.


Vitamin E for Non-Alcoholic Fatty Liver
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Table 1 – Monitoring and investigation schedule
Phase
Visit 
week
Vit E dose
Nurse1
MD
Nutritionist2
Routine 
labs3
Extended 
labs4
NK/NKT 
cell studies5
Liver & Adipose 
Tissue Biopsy
FSIVGTT6
Imaging
Breath Test
Questionaires
Enrollment
(-4) – (-1)
+
+
+
DHQ II, IPAQ, 3-day food
Run-in
0
+
++
+
+
+
MRS, MRI
Symptom, quality of life
2
++, +
4
+
+
+
6
+
8
+
+
+
10
+
Admission
12
+
+
++
+
+
+
+
+
MRS, MRI, 
DEXA
+
3-day food, IPAQ, 
symptom, quality of life
Randomized
Rx 
0 (1-2 wk 
later)
200/400/800
+
+
+
1
200/400/800
+
+
2
200/400/800
+
+
3
200/400/800
+
+
2nd 
Admission
4
200/400/800
+
+
+
+
+
+
MRS
8
200/400/800
+
+
++
+
12
200/400/800
+
+
+
symptom
16
200/400/800
+
+
++
+
20
200/400/800
+
+
3rd 
Admission
24
800
+
+
++
+
+
+
+
MRS, MRI, 
DEXA
+
3-day food, IPAQ, 
symptom, quality of life
Extended Rx
36 
800
+
+
++
+
48
800 
(or 
lower7)
+
+
++
+
+
MRS
3-day food, IPAQ, 
symptom, quality of life
60-132 
(q12 wks)
800 
(or 
lower7)
+
+
++
+
+ 
(q24 
wks)
3-day food, IPAQ, 
symptom, quality of life 
(q 24 wks)


Vitamin E for Non-Alcoholic Fatty Liver
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Exit 
Admission
144
Stop
+
+
++
+
+
+5
+
+
MRS, MRI, 
DEXA, 
+
3-day food, IPAQ, 
symptom, quality of life 
1 Nursing visit – includes vital signs, weight, list of current study and non-study medications and nursing assessment
2 Nutrition visit - + marks group session, ++ marks individual session
3 Routine labs – complete blood count, Chem-20, serum insulin, c-peptide, free fatty acids, lipid panel, vitamin E level, research blood 
(total volume 31.5 ml) 
4 Extended labs – GGT, PT, PTT, HbA1c, CRP, Thyroid panel, apolipoproteins, ferritin, iron, transferrin saturation, immunoglobulins, 
PSA, research plasma (total volume 52.5 ml with Routine labs), urinalysis
5 NK/NKT cell analyses – On Run-in phase, first admission and week 144 will be drawn for all patients. On weeks 4 and 24 these will 
be performed only on the 200 IU/d and 800 IU/d arms (total volume 48 ml). On week 144 only 30 ml will be obtained. Will not be 
performed if Hb < 10 g/dL.
6 FSIVGTT – total blood volume drawn 120-150 ml. Will not be performed if Hb < 10 g/dL.
7 Possible lowering of vitamin E dose based on week 48 results (see Figure 4).


Vitamin E for Non-Alcoholic Fatty Liver
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Analysis of the study 
End points
Primary end points
There are two primary clinical end points in the study. The primary biochemical response 
is defined in a binary manner as an end-of-treatment AST <=32 or ALT <=35 U/L. The 
selection of these cut-off values is based on a pilot analysis by us that examined in an 
unbiased manner several rules for biochemical response and found this to be the one most 
strongly associated with histological response to treatment, irrespective of treatment 
modality31. We will also analyze the absolute change in ALT and AST from pre-treatment 
to end-of treatment as continuous parameters of the effect of treatment on liver enzymes. 
Patients who at the time of starting vitamin E treatment have normal liver enzymes will be 
excluded from this analysis. The primary physiological response is the absolute change in 
liver fat content as determined by comparing the 1H-MRS results from pre-treatment to end 
of treatment.
Secondary end points 
Secondary end points include: absolute and percent decrease in liver enzymes, decrease in 
GGT levels, percent reduction in liver fat.
A secondary analysis will analyze the effect of the lifestyle modification by comparing the 
end points between enrollment and the end of the lifestyle modification phase. Another 
analysis will determine the effect of increasing the dose of vitamin E by comparing the end 
points between the end of the randomized phase (week 24) to weeks 24 and 144 of the 
extended phase.
The kinetics of response will be determined by analyzing sequential ALT and AST 
measurements. Current data suggest biphasic response pattern to vitamin E and 
mathematical modeling will be applied to the results and will be compared between doses. 
Similar modeling will be applied to changes in liver fat content by 1H-MRS. 
Basic scientific end points
Gene expression in liver and adipose tissue will be analyzed using microarray and 
quantitative PCR (qPCR) of tissue samples. Treatment-induced changes or comparisons 
between groups of patient (i.e. comparison of baseline expression between responders and 
non-responders) will be expressed as fold-induction. 
NK and NKT cell changes in the serum and liver tissue will be analyzed using phenotyping 
and functional assays.
Oxidative stress will be measured in the serum, urine, liver and adipose tissue using 
TBARS assay, F2-isoprostanes, 9-HODE, 13-HODE, SOD activity or similar measures. In 
women of child-bearing age, the phase of the menstrual cycle will be recorded on extended 
visits, as F2-isoprostane levels can be affected by it.


Vitamin E for Non-Alcoholic Fatty Liver
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Statistical analyses
Parameters of response to treatment will be analyzed by matched-pair comparison between 
pre-treatment and end of treatment values. Student’s t-test will be used for values that are 
distributed normally while Wilcoxon’s sign-ranked test will be used for non-parametric 
values. Categorical comparisons will be performed using Χ2 or Fisher’s exact test, as 
appropriate. The Cochran-Armitage test for trend will be used to test for a dose effect on 
the primary clinical end points.
To determine the effect of genetic variants, genotype-based analyses will be preferred to 
allelic analyses, with an additive model used primarily, unless other (dominant or 
recessive) models seem more appropriate. Linear or ordinal logistic regression will be used.
All analyses will be done with and without stratification for the presence of diabetes 
mellitus.
An α≤0.05 will be used to determine statistical significance.
Sample size calculation
The main goal of the study is to understand and characterize the mechanism of response to 
vitamin E. An important goal of the study is to simultaneously collect liver and adipose 
tissue to determine the site of action of the vitamin E and the relative contribution of each 
tissue to the disease. For most of the planned end points, including changes in liver fat 
content by 1H-MRS and the basic scientific end points, there is not enough pre-existing 
data to allow for accurate sample size calculation. 
For the comparison of effectiveness between different doses of vitamin E (primary aim 1), 
the primary biochemical end point will be used. We assume a response rate of 45% with 
the 800 IU/d dose (similar to that seen at the PIVENS trial), a 10% response rate to the 200 
mg/d dose and a 20% response to the 400 mg/d dose. Under these assumptions, a sample 
size of 25 patients per group will give a power of 80% to detect a linear trend with statistical 
significance of 5% (Cochran-Armitage test). To account for possible drop-out and loss to 
follow-up, we are aiming to recruit 30 patients per group or an overall of 90 patients in the 
study. 
As the sample size calculation is limited by the absence of published data, we will re-assess 
our calculations after 10 patients have been enrolled in each arm, and again after 20 have 
been enrolled. 
Human subject protections
Rationale for subject selection
The study is designed to enroll patients with NAFLD that can be potentially benefit from 
treatment. Currently, there are no reliable non-invasive markers that accurately distinguish 
NASH from steatosis only, although the latter seems to carry a more favorable prognosis. 
For patients with steatosis only, it is unclear whether treatment can improve their disease 
and prevent progression to NASH. In most clinical trials with histological end points, 
patients with steatosis only were excluded as they cannot show significant histological 


Vitamin E for Non-Alcoholic Fatty Liver
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improvement. In this study, with a primarily scientific outcome, we are interested in 
studying the effect of vitamin E on these patients as well and we consider the intervention 
to be low-risk enough to allow us to recruit all patients with NAFLD. 
The exclusion criteria for the study are mainly geared towards limiting enrollment to 
patients for whom treatment is considered safe and that have no confounders that would 
limit the analysis of the results. There are no limitations based on race or ethnicity. 
However, we anticipate that very few patients of African ancestry will be enrolled, because 
of the scarcity of NAFLD in these patients and their apparent “protection” from liver fat 
accumulation.
We intend to enroll women as well as men and do not foresee a marked difference by 
gender. We exclude pregnant women and require stable contraception from participants, 
because of the interventions, blood draws and imaging using radiation. Furthermore, 
pregnancy has an effect on metabolic parameters and fat handling and will not allow 
analysis of the data. Similarly, we choose not to enroll breast-feeding women as the dietary 
recommendations for them are different from those given to the general population and 
will limit the ability to analyze the data.
Enrollment in this study is limited to adult patients as the pediatric form of NAFLD has 
unique clinical and histological features and the choice of therapies may be different.
Recruitment strategy
Patients with NAFLD who have been followed at the LDB clinic will be offered 
participation based on eligibility. New patients are continuously evaluated at the clinic and 
will be enrolled as well. We will inform local primary care providers, gastroenterologists, 
hepatologists and free clinics of the availability of the protocol and eligibility criteria. 
Patients seen by the Liver Service as consult cases will be offered participation in the 
protocol if deemed eligible and only with the agreement of the referring physician and the 
principal investigator for the referring protocol. We aim to recruit 90 patients for the study 
within 3 years.
Informed consent process
The initial screening visit to assess eligibility for the protocol will be done under protocol 
91-DK-0214, “Evaluation of Patients with Liver Disease”; only eligible patients will then 
be consented into this protocol. Written informed consent will be obtained from the 
participant prior to any study procedures or treatments. The Principal Investigator or other 
designated qualified protocol investigators will explain the study in language 
understandable to the subject. Sufficient time and opportunity will be given for discussion 
of the research as well as to answer any questions they may have, taking care to minimize 
or eliminate the perception of coercion or undue influence. The participant and the 
investigator will sign the current IRB-approved informed consent documentA copy of the 
consent will be given to the subject for future reference. The signed documents will be sent 
to the Medical Records Department for placement in the subject's permanent CC medical 
record. The consent process will additionally be documented in the electronic medical 
record (CRIS).


Vitamin E for Non-Alcoholic Fatty Liver
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We anticipate the enrollment of Spanish speaking subjects into this study for which English 
consents have been fully translated. 
Evaluation of benefits and risks
Benefits
Most patients with NAFLD are overweight and have some degree of insulin resistance. For 
these patients lifestyle modification with dietary consultation aiming at weight loss and a 
moderate exercise regimen is of proven benefit, is considered standard medical care32 and 
conforms to the recommendations for the population as a whole33, 34. The target level of 
exercise, 150 minutes of moderate intensity per week, is the minimum recommended for 
all adults.
Currently, there are no reliable non-invasive markers that accurately distinguish NASH 
from steatosis only, which carries a much more favorable prognosis. Even the presence of 
elevated liver enzymes in patients with liver fat predicts the presence of NASH in only 
55%35 to 62% (NASH-CRN data, unpublished) of cases. Thus, a liver biopsy is essential 
for proper staging, as well as to provide tissue for the scientific studies.
Patients with NAFLD who did not previously have a liver biopsy can benefit from one; 
however, a liver biopsy is not considered essential in routine management of NAFLD. 
Repeat liver biopsies are not routinely performed on patients with fatty liver disease outside 
of the research context but in this study are crucial to define the histology and to allow 
evaluation of response mechanism.
Vitamin E therapy for NASH has been shown to improve fat deposition in the liver, 
decrease liver enzymes and reduce histological evidence of cellular injury, without 
affecting insulin sensitivity3. Thus, although it is not yet approved as standard medical 
treatment for NASH, there is clearly a potential benefit to the patients. 
Risks and Discomforts
Physical exercise. Adults who exercise are inherently at a higher risk for exercise-induced 
musculoskeletal injury, but lower risk for non-leisure time injuries; however, the risk of 
injury with a supervised program and moderate intensity is not much different for that of 
sedentary adults36. The risk of a major cardiac event during exercise is small with 
moderate-intensity activity and there is no clear evidence to suggest a need for screening 
of asymptomatic individuals. In this study, we adopt the screening recommendations of the 
American Heart Association for diabetic patients who start an exercise program37 and apply 
them to the whole study population. 
Exercise induced hypoglycemia can occur in diabetic patients treated by insulin or insulin 
secretagogues, especially those maintaining tight glycemic control. An endocrine consult 
will be requested on these patients to assess the need for dose modification and the patients 
will be encouraged to monitor their blood sugar levels routinely. 
Diet. The dietary intervention in this study is aimed at achieving a balanced diet with mild 
caloric deficit and gradual weight reduction, and conforms with the general 


Vitamin E for Non-Alcoholic Fatty Liver
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recommendations for obese or diabetic patients. No significant complications or risks are 
anticipated from this diet.
Liver biopsy. Patients will undergo 3 liver biopsies in this protocol; one after the lifestyle 
modification phase, a second after 4 weeks of vitamin E therapy, and an exit biopsy at the 
end of the study. The liver biopsy will be performed when the patients are admitted as 
inpatients to the Clinical Center for the extensive evaluation detailed above. 
The major side effects of liver biopsy are pain, bacteremia, puncture of another organ and 
bleeding38. Local pain and discomfort at the liver biopsy site occur in about 20% of persons 
undergoing percutaneous liver biopsies. This is transient (lasting one to twelve hours) and 
usually mild, rarely requiring analgesics. Bacteremia occurs in 1-2% of persons undergoing 
liver biopsy. In the absence of bile duct obstruction, this is almost always self-limited and 
is rarely symptomatic. Inadvertent puncture of another organ such as lung, colon, gall 
bladder, kidney and adrenal gland can occur during liver biopsy. All biopsies will be 
performed after a bedside ultrasound examination by the performing physician to identify 
internal organs and select and optimal site and direction for the biopsy. Significant bleeding 
after liver biopsy is the most serious side effect of this procedure. In the absence of blood 
coagulation defect or hepatic malignancy, significant bleeding is rare, occurring in less 
than one in a thousand cases of liver biopsy. Death due to bleeding after liver biopsy has 
been reported to occur in less than 1/10,000 cases. At the NIH Clinical Center, the Liver 
Diseases Branch has performed approximately 150 liver biopsies each year for the past 25 
years. During this time, two patients have died as a result of liver biopsy. One patient had 
cirrhosis and advanced hepatocellular carcinoma. The second patient had Gaucher’s 
disease with hepatic involvement and severe coagulopathy. Both patients died after 
surgical attempts to stop the bleeding were unsuccessful.
Adipose Tissue Biopsy. The major risks/discomforts are represented by pain, bruising, 
hematoma, infection, scarring, and localized lipodystrophy. The procedure will be 
performed under sterile technique to minimize the chances of infection. Ice will be applied 
to the site immediately after the procedure to limit bruising, swelling and tenderness. After 
subcutaneous tissue biopsy, patients will be monitored by nurses. Post-biopsy, the needle 
site will be cleaned and covered with gauze and translucent dressing tape. Study 
participants will be instructed to report to the clinical staff any changes at the biopsy site 
including bleeding, secretion, erythema, pain, and signs and symptoms of infection. Study 
participants will be instructed to self-monitor the puncture site after discharge from the 
Clinical Center.
Frequent phlebotomy. To document the baseline and to monitor the effects and toxicities 
of treatment, frequent blood sampling will be required. Patients will have 15-20 
venipunctures during the period of evaluation and the first 6 months of therapy and 4 
venipunctures per year in the extended treatment phase. Each venipuncture will be for 32 
to 150 ml of blood. However, no more than 550 ml or 10.5 ml per kg (the lower of the two) 
will be drawn from any one person during any eight-week period.
Imaging studies. 
1) DEXA scan. Patients will undergo 2 DEXA scans in the first year of treatment 
and another test at the end of the extended treatment phase, to assess whole 
body composition. The effective dose of radiation that patients will receive 


Vitamin E for Non-Alcoholic Fatty Liver
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from a single study is 0.5 mrem; thus the total effective dose from DEXA for 
the duration of the study is 1.5 mrem.
2) MRI scans. Patients will undergo 5 MRI scans in the first year of treatment and 
another test at the end of the extended treatment phase, to determine the amount 
of liver fat, liver volume and subcutaneous and visceral fat. While serial MRI 
scanning is thought to be safe, the procedure may cause anxiety in some patients 
since current equipment used at the Clinical Center uses a closed tube. Patients 
will be offered sedatives such as Valium if they express worry about being in a 
closed space.
All female patients with childbearing potential will have a pregnancy test prior to any 
imaging test involving ionizing radiation.
IVGTT. Patients will undergo 3 IVGTT tests in the first year of the study and another one 
at the end of the extended treatment phase. Patients will have two intravenous catheters 
placed for the procedure, one for the infusion of glucose and insulin and the other for blood 
drawing. Patients may experience pain from intravenous catheter insertion or may feel a 
burning sensation during the rapid injection of glucose. There is a small risk of 
hypoglycemia from the short period of insulin infusion, although it is extremely rare and 
blood glucose will be monitored frequently during the test. With mildly low blood sugars 
(<50 mg/dl), subjects may get hungry, may become drowsy or sweaty. With blood sugars 
below 30 mg/dl, subjects may be difficult to arouse or have seizures. We do not anticipate 
significant hypoglycemia as most or all of the patients are predicted to have some degree 
of insulin resistance. Nevertheless, a physician or nurse will be present during this test for 
the first hour of the test (including the first thirty minutes following insulin administration) 
to monitor for signs of significant hypoglycemia. If significant hypoglycemia occurs it will 
be treated with intravenous dextrose or glucagon according to clinical evaluation. The total 
amount of blood that will be drawn during this test will be 120 to 150 ml. 
Vitamin E treatment. The dose of vitamin E used in this study is lower than the 
recommended upper limit of supplement intake (1500 IU/d)9. Some studies of vitamin E 
supplementation for primary or secondary prevention of disease suggested increased 
mortality or risk of hemorrhagic stroke12. Two meta-analyses39, 40 suggested slight 
increased mortality in patients treated with high-dose vitamin E. However, these works 
were criticized for several reasons and the studies selected for the meta-analyses included 
mostly patients with chronic diseases. A recent meta-analysis using all trials found no 
excess mortality risk41. Furthermore, these studies focused on the use of vitamin E for 
disease prevention (a scenario where little or no risk would be tolerable), whereas our trial 
uses vitamin E as a proven treatment for an existing disease. In the PIVENS trial3, no excess 
adverse events were noted in the vitamin E in comparison to the placebo arm. 
As some studies suggest an effect of vitamin E on platelet aggregation42, and given the 
possible association with hemorrhagic stroke, we exclude patients with high bleeding risk, 
such as those with coagulopathies, use of anticoagulants, thrombocytopenia or  known 
platelet dysfunction. Furthermore, due to the possibility of increased risk of gastrointestinal 
bleeding, patients with a pre-existing history of iron deficiency anemia secondary to 
gastrointestinal sources are excluded. On the other hand, vitamin E supplementation is not 
associated with changes in standard parameters of bleeding tendency, including PT, PTT, 


Vitamin E for Non-Alcoholic Fatty Liver
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platelet count and bleeding time43, 44, rendering these useless as a tool for safety monitoring 
during vitamin E treatment. However, if during the study the platelet count or PT/PTT 
cross the thresholds for study inclusion (i.e. platelets <70,000, PT/PTT prolonged by >= 3 
seconds), treatment will be stopped and patients withdrawn from the study.
Recently, data from extended follow-up of the SELECT trial45 suggested increased risk for 
the diagnosis of prostate cancer in patients treated with vitamin E. This is in contrast to the 
findings of two other similar studies46, 47 which did not show such an effect. Our patients 
are anticipated to be significantly younger than the study population in SELECT (median 
age 62); furthermore, risk for prostate cancer appeared to increase numerically only after 
more than 3 years of treatment. We will monitor PSA levels every 12 months on male 
patients in our study. Screening for prostate cancer may lead to further evaluation of 
abnormal results, including referrals, blood tests and possible invasive procedures. There 
is also a risk of detecting prostate cancers that are not destined to become clinically 
significant. The risks and benefits of screening with annual PSA testing will be discussed 
with all male participants prior to enrollment and patients will have the option to elect not 
to be screened. 
Adverse event reporting and data monitoring
Reportable events will be tracked and submitted to the IRB as outlined in Policy 801.
The PI will immediately report SAEs to the Sponsor according to the requirements of 21 
CFR 312.64(b) and as agreed upon with the sponsor.  The PI will record nonserious AEs 
and report them to the Sponsor according to the timetable for reporting specified in the 
protocol (21 CFR 312.64(b).
All serious, unexpected, suspected adverse reactions (SUSAR) will be reported to the 
sponsor within 15 calendar days.  All unexpected suspected adverse reactions resulting in 
death or life-threatening events will be reported to the sponsor within 7 calendar days. The 
investigator may also be responsible for completing an FDA MedWatch 3500 form and 
additional information for a follow-up SAE report as information becomes available.
Summary of adverse events and efficacy data will be provided in annual reports to the 
FDA.
Monitoring
Safety monitoring will be maintained by weekly meetings of the investigators to review 
clinical data on patients seen in the outpatient clinic and maintenance of individual flow 
sheets on each patient. Laboratory and imaging test results will be reviewed routinely by 
the treating physician, the principal investigator and the study nurse. 
As required by FDA 21 CFR 312.50, this study will be monitored to ensure compliance 
with the protocol and applicable regulatory requirements. Monitoring results will be 
reported to the Principal Investigator/Sponsor for further reporting to the FDA consistent 
with applicable regulations. The specific monitoring plan will be developed with the 
Principal Investigator and frequency of monitoring visits will be determined by such 
factors as study enrollment, data collection status and regulatory obligations. Study 


Vitamin E for Non-Alcoholic Fatty Liver
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documents and pertinent hospital or clinical records will be reviewed to verify that the 
conduct of the study is consistent with the protocol plan.
Collection and storage of human specimens or data
Patients will have serum stored from selected time points during this study. These 
specimens will be used for repeat testing (in case of missing samples) or for special tests 
as needed (such as for serum cytokines or adipokines). Serum samples will be stored at the 
NIDDK clinical core laboratory inside locked freezers in locked rooms and will be labeled 
by coded unique identifiers without personally identifiable information. The code to these 
samples will be maintained by the clinical core personnel. 
Liver and adipose tissue biopsy samples may be stored if residual tissue is available after 
routine samples are taken for routine histological staining and evaluation. PBMCs will be 
stored until they are used for NK/NKT cell and gene expression analysis. RNA or protein 
that is extracted from these samples will be stored as well. The samples will be stored in a 
locked freezer in a locked room at the LDB laboratory and will be labeled by a coded 
unique identifier without personally identifiable information. The code to these samples 
and their location will be maintained by the primary investigator and the laboratory 
manager. 
Access to stored samples and the identifiers will only be provided to the principal or 
associate investigators, and only with the written approval of the principal investigator and 
the branch chief. If samples are to be evaluated by outside collaborators in the future, they 
will be coded and all identifying data removed from the samples. The code to identify 
samples and associate them with patient identity will be maintained by the principal 
investigator and not shared with outside collaborators. 
Research records and data as well as liver biopsy slides, biopsy reports, liver tissue and 
sera will be stored indefinitely in our locked offices and freezers, the medical record 
department and the pathology department. These materials will be protected and tracked 
by standard operating procedures in the medical record and pathology departments as well 
as a compulsive filing system in our locked offices and freezers. There will be redundant 
storage of clinical information in the medical record department and our offices. Likewise, 
there will be redundant storage of biopsy information and materials in the pathology 
department and our offices. This should minimize the risk of loss or destruction of 
information and specimens. If that were to occur we would report it to the IRB. We do not 
plan to destroy this personal medical information or the liver biopsy specimens or research 
subject sera after completion of the study because it may be critically important for 
physicians (here or elsewhere) to have access to this information when caring for these 
patients in the future. 


Vitamin E for Non-Alcoholic Fatty Liver
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Remuneration/compensation
Compensation will be provided to subjects for participation in some of the research 
procedures and tests in the study, according to the following schedule:
For the baseline (week 0 of run-in phase) MRI …………………$ 60.00
For the 1st inpatient hospital admission including:
DEXA scan……………………….$ 10.00
MRI……………………………….$ 60.00
FSIVGTT ………………..……….$ 70.00
Adipose tissue biopsy……………..$ 50.00
Resting Energy Expenditure……....$ 25.00
Time ………………………………$ 120.00 (3 nights/$40.00 per night)
Total compensation for the admission……………………
$ 335.00
For the 2nd  (week 4) inpatient hospital admission including:
MRI……………………………….$  60.00
FSIVGTT………………………….$ 70.00
Liver Biopsy……………………….$ 280.00
Adipose tissue biopsy………………$ 50.00
Resting Energy Expenditure………..$ 25.00
Time ………………………………..$ 80.00 (2 nights/$ 40.00 per night)
Total compensation for the admission …………………… $ 565.00
For the 3rd  (week 24) inpatient hospital admission including:
DEXA scan………………………….$ 10.00
MRI………………………………….$ 60.00
FSIVGTT ………………..………….$ 70.00
Resting Energy Expenditure…………$ 25.00
Time ……………………………….. $ 40.00 (1 overnight/$40.00 per night)
Total compensation for the admission……………………
$ 205.00
For the final (Week 144) inpatient hospital admission including:
DEXA scan …………………………………$ 10.00
MRI ………………………………………... $ 60.00
FSIVGTT ……………………………
$ 70.00
Liver biopsy ……………………………… $ 280.00


Vitamin E for Non-Alcoholic Fatty Liver
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Adipose Tissue biopsy …………………… $ 50.00
Resting Energy Expenditure……………… $ 25.00
Time ……………………………………… $ 120.00 (3 nights/$ 40.00 per night)
Total compensation for the admission ………………………$ 615.00
Total overall (maximal) study compensation……………………….$ 1,780.00
No remuneration is offered for taking vitamin E, outpatient visits, blood tests, nutritional 
consults and study questionnaires.
Patients may also be eligible for reimbursement of travel expenses based on standard 
Clinical Center and LDB criteria.


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