The Effect of Vitamin E and Docosahexaenoic Acid Ethyl Ester on Non-Alcoholic Fatty Liver Disease (NAFLD) - Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Clinical Trial (PUVENAFLD)
Phase: PHASE2
Conditions: Non-Alcoholic Fatty Liver Disease, Non-Alcoholic Fatty Liver, Non-Alcoholic Steatohepatitis
Sponsor: Naga P. Chalasani

 
 
 
PROTOCOL FOR CLINICAL STUDIES 
 
CONFIDENTIALITY STATEMENT 
The information contained in this document is proprietary and therefore is provided to 
you in confidence for review by you, your staff, an applicable Independent Ethics 
Committee/Institutional Review Board and regulatory authorities. It is understood that 
the information will not be disclosed to others without prior written approval from the 
PI, except to the extent necessary to obtain informed consent from those persons to 
whom the product may be administered. 
Confidential 
TITLE 
 
THE EFFECT OF VITAMIN E AND DOCOSAHEXAENOIC ACID ETHYL ESTER ON 
NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) - A RANDOMIZED, DOUBLE-
BLIND, PLACEBO-CONTROLLED, PARALLEL-GROUP CLINICAL TRIAL 
(PUVENAFLD) 
 
 
 
PROTOCOL NUMBER:  
2017-1088 
 
 
VERSION 3.0  
 
NCT/ IND #: 
NCT04198805/145428 
 
 
DATE: MAY 10, 2021 
 
 
 
 
IND Sponsor - Principal Investigator: Dr Naga Chalasani, Indiana University School of 
Medicine 
 
 
 
 
 
 
FUNDED BY 
DSM NUTRITIONAL PRODUCTS LTD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ROLES AND RESPONSIBILITIES  
 
 
IND SPONSOR- PRINCIPAL INVESTIGATOR 
 
NAGA CHALASANI, MD 
 
 
Title, Function 
David W. Crabb Professor & Director  
Division of Gastroenterology and Hepatology 
 
Institution 
Indiana University School of Medicine 
 
Address 
Rotary Building, Suite 225 
702 Rotary Cir 
Indianapolis IN 46202-5175 
USA 
Phone 
(317) 278-0414 
Fax 
(317) 278 1949 
E-mail 
nchalasa@iu.edu 
 
 
CONTRACT RESEARCH ORGANISATION 
 
BIOFORTIS, INC. 
CONTACT PERSON: LINDA DERRIG 
 
Title, Function: Director of Project and Data Management 
Address 800-A South Rohlwing Rd Addison, Ill 60101 
Phone 630-617-2000 
E-mail linda.derrig@mxns.com 
 
 
Central Lab 
ACM MEDICAL LABORATORY, INC. 
CONTACT PERSON: ALICIA VAN STRANDER 
 
Title, Function: Senior Project Manager 
Address 160 Elmgrove Park Rochester, NY 14624 
Phone 585-429-2229 
E-mail avanstrander@acmgloballab.com 
 
Central IRB 
Address Advarra 
6100 Merriweather Dr., Suite 600, Columbia, MD   20144 
Phone 410-884-2900 
E-mail https://advarra.com 
 
 
 

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Principal Investigator’s Signature 
 
I HAVE CAREFULLY READ THIS PROTOCOL ENTITLED THE EFFECT OF VITAMIN E AND DOCOSAHEXAENOIC ACID ETHYL ESTER ON AND 
NON-ALCOHOLIC FATTY LIVER DISEASE- A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, PARALLEL-GROUP CLINICAL 
TRIAL AND AGREE THAT THIS STUDY IS TO BE CONDUCTED IN ACCORDANCE WITH GOOD CLINICAL PRACTICE (GCP), THE ETHICAL 
PRINCIPLES THAT HAVE THEIR ORIGIN IN THE DECLARATION OF HELSINKI AND FURTHER DESCRIBED IN INTERNATIONAL CONFERENCE 
ON HARMONISATION (ICH-GCP) E6, TITLE 45 OF THE CODE OF FEDERAL REGULATIONS § 46, TITLE 21 OF THE CODE OF 
FEDERAL REGULATIONS §§ 50, 56, AND 312, AND EUROPEAN UNION DIRECTIVE 2001/20/EC FOR CLINICAL TRIALS AND 
2005/28/EC FOR GOOD CLINICAL PRACTICE. 
IN THAT IT CONTAINS ALL THE NECESSARY INFORMATION REQUIRED TO CONDUCT THE STUDY. I AGREE TO CONDUCT THIS STUDY 
AS OUTLINED IN THE PROTOCOL. 
I UNDERSTAND THAT THIS STUDY WILL NOT BE INITIATED WITHOUT APPROVAL OF THE APPROPRIATE INSTITUTIONAL REVIEW 
BOARD (IRB) /INDEPENDENT ETHICS COMMITTEE (IEC), AND THAT ALL ADMINISTRATIVE REQUIREMENTS OF THE GOVERNING BODY 
OF THE INSTITUTION WILL BE COMPLIED WITH FULLY. 
THE PROTOCOL, INFORMED CONSENT FORM(S), RECRUITMENT MATERIALS, AND ALL PARTICIPANT MATERIALS WILL BE SUBMITTED 
TO THE IRB FOR REVIEW AND APPROVAL.  APPROVAL OF BOTH THE PROTOCOL AND THE CONSENT FORM MUST BE OBTAINED 
BEFORE ANY PARTICIPANT IS ENROLLED.  ANY AMENDMENT TO THE PROTOCOL WILL REQUIRE REVIEW AND APPROVAL BY THE IRB 
BEFORE THE CHANGES ARE IMPLEMENTED TO THE STUDY.  ALL CHANGES TO THE CONSENT FORM WILL BE IRB APPROVED; A 
DETERMINATION WILL BE MADE REGARDING WHETHER A NEW CONSENT NEEDS TO BE OBTAINED FROM PARTICIPANTS WHO 
PROVIDED CONSENT, USING A PREVIOUSLY APPROVED CONSENT FORM 
INFORMED WRITTEN CONSENT WILL BE OBTAINED FROM ALL SUBJECTS/PATIENTS IN ACCORDANCE WITH THE INSTITUTIONAL 
REQUIREMENTS AS SET FORTH IN THE TERMS OF THE DECLARATION OF HELSINKI AS SPECIFIED IN THE GOOD CLINICAL PRACTICE 
CONSOLIDATION GUIDANCE ISSUED BY THE INTERNATIONAL CONFERENCE ON HARMONIZATION. 
I WILL ENROL SUBJECTS/PATIENTS WHO MEET THE PROTOCOL INCLUSION CRITERIA. 
I UNDERSTAND THAT MY SIGNATURE ON THE COMPLETED CASE REPORT FORM(S) INDICATES THAT I HAVE CAREFULLY REVIEWED 
EACH PAGE AND ACCEPT FULL RESPONSIBILITY FOR THE CONTENTS THEREOF. 
I UNDERSTAND THAT THE INFORMATION PRESENTED IN THIS STUDY PROTOCOL IS CONFIDENTIAL, AND I HEREBY ASSURE THAT NO 
INFORMATION BASED ON THE CONDUCT OF THE STUDY WILL BE RELEASED WITHOUT PRIOR CONSENT FROM SPONSOR UNLESS THE 
REQUIREMENT IS SUPERSEDED BY THE APPROPRIATE REGULATORY AUTHORITY. 
 
PRINCIPAL INVESTIGATOR AT 
SITE 
 
 
Signature 
 
 
Place and date 
 
 
 
 
IND SPONSOR-INVESTIGATOR APPROVALS AND SIGNATURES  
 
IND SPONSOR-
INVESTIGATOR  
Naga Chalasani, MD 
 
Signature 
 
 
Place and date 
 
 
 
 
 

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SYNOPSIS 
 
Title THE EFFECT OF VITAMIN E AND 
DOCOSAHEXAENOIC ACID ETHYL ESTER ON NON-
ALCOHOLIC FATTY LIVER DISEASE (NAFLD) - A 
RANDOMIZED, DOUBLE-BLIND, PLACEBO-
CONTROLLED, PARALLEL-GROUP CLINICAL TRIAL 
 
 
 
 
STUDY ACRONYM: PUVENAFLD 
 
PROTOCOL NUMBER:  2017-1088 
 
IND NO.145428 
 
VERSION 3.0  
 
DATE MAY 10, 2021 
 
IND SPONSOR-INVESTIGATOR: Naga Chalasani, MD 
 
Clinicaltrials.gov: 
NCT04198805 
STUDY PHASE/TYPE 
PHASE II /PROOF-OF-CONCEPT INTERVENTIONAL STUDY 
    
 
 
 
PLANNED START OF STUDY Q3 2019 
 
ESTIMATED STUDY 
DURATION: 3-4 YEARS 
 
PRINCIPAL INVESTIGATOR 
Dr. Naga Chalasani 
Indiana University 
Department of Medicine, Gastroenterology/Hepatology 
Division 
Rotary Building, Suite 225 
702 Rotary Cir 
Indianapolis IN 46202-5175 
USA 
 
STUDY SITES: US, MULTI - CENTER 
 
RESEARCH AREA/APPLICATION FIELD: NON-ALCOHOLIC 
FATTY LIVER DISEASE/ PREVENTION OF NON-ALCOHOLIC 
STEATOHEPATITIS (NASH) 
 
 
 
RATIONALE 
This study will examine the combination of Vitamin E 
and Docosahexaenoic Acid Ethyl Ester (DHA EE) to 
improve symptoms of non-alcoholic fatty liver disease 
(NAFLD). This combination has not previously been 
tested but may provide benefit to patients with NAFLD 
by antioxidant and anti-inflammatory mechanisms of 
action.    
 
PRIMARY OBJECTIVE 
To determine the effect of the combination of vitamin E 
and omega-3 fatty acid, DHA EE versus placebo on 
reducing liver fat content after 6 months of intervention 
in adults with NAFLD. 
 
 
 
SECONDARY OBJECTIVES 
 
 
 

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• 
To determine effect of Vitamin E alone on 
reducing liver fat content after 6 months of 
intervention in adults with NAFLD. 
• 
To determine effect of DHA EE alone on reducing 
liver fat content after 6 months of intervention 
in adults with NAFLD. 
• 
To determine the change after 6 months of 
Vitamin E and /or DHA EE intervention in 
anthropometric, metabolic, hepatologic, 
nutrient, and inflammatory parameters, and 
quality of life status of adults with NAFLD. 
 
STUDY DESIGN 
Multi-center, randomized, double-blind, placebo-
controlled, parallel, 4-group intervention study in adults 
with NAFLD 
 
 
 
STUDY POPULATION 
≥18 years old male or female subjects with presence of 
fatty liver (min. 12% liver fat content) by magnetic 
resonance imaging proton density fat fraction (MRI-
PDFF) 
 
 
 
 
SAMPLE SIZE 
Total number of subjects: 200 (n=65/65/35/35 for 
Combination/Placebo/DHA only/Vitamin E only groups) 
 
NUMBER OF SITES 
Estimated 16 US sites  
 
INVESTIGATIONAL DRUG PRODUCT 
1. Vitamin E [(all-rac)-α-tocopheryl acetate] 
administered orally via softgel capsules  
2. Omega-3 fatty acid (DHA EE) administered orally 
via softgel capsules  
3. Vitamin E and DHA EE combination administered 
orally via separate softgel capsules 
4. Matching placebo (soy oil) administered orally via 
softgel capsules 
 
 
 
DOSAGE AND REGIMEN 
Vitamin E [(all-rac)-α-tocopheryl acetate ]1000 IU/day 
(1000mg/d) 
DHA EE 1.89 g/day 
 
DURATION OF 
SUPPLEMENTATION 
6 months 
 
 
 
SAFETY PARAMETERS 
- 
Vital signs (e.g. blood pressure, heart rate) 
- 
Serum hematology and biochemistry  
- 
AE/SAE reporting  
 
 
 
 
 
 
SUBJECT SELECTION CRITERIA 
1.1.1 Inclusion criteria  
 
 
 
 

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1. Male or female gender 
2. ≥18 years of age 
3. A new diagnosis or reconfirmation of previously 
known fatty liver by imaging (ultrasound or CT or 
MRI), or by liver biopsy within ≤ 4 years 
4. Fibroscan Controlled Attenuation Parameter 
(CAP) score ≥ 300db 
5. Liver fat content (≥ 12%) measured by MRI-PDFF 
6. ALT ≥ 40 U/L 
7. eGFR/eCreatinine Clearance ≥ 60ml/min 
8. Participants with previously diagnosed Type 2 
diabetes (up to 50% of sample):  they must either 
be taking anti-diabetic medications, or their 
fasting (>10 hours) glucose must be ≥ 100 mg/dL 
at the time of screening 
9. Stable weight (±5%) for at least 3 months  
10. Subjects willing and able to give written 
informed consent and to understand, to 
participate and to comply with the clinical study 
requirements. 
1.1.2 Exclusion criteria 
 
1. Evidence of alternative causes of hepatic 
steatosis or other forms of chronic liver disease, 
e.g. Hepatitis B, Hepatitis C (with <3 years 
treatment) 
2. Evidence of acute Hepatitis A 
3. Serum ALT or AST ≥ 250 U/L 
4. Serum Alkaline Phosphatase > 2 ULN 
5. Total bilirubin > 2 ULN in the absence of Gilbert’s 
Syndrome [In patients with Gilbert’s Syndrome, 
direct bilirubin must not exceed 2 ULN] 
6. HbA1c > 9.5% 
7. Decompensated acute or chronic liver disease 
8. Clinical, imaging or histological evidence of 
cirrhosis 
9. Use of anti-NASH drugs (e.g. thiazolidinediones 
(TZD)) in the 3 months prior to randomization 
10. Use of a non-stable dose of statins or fibrates in 
the 3 months prior to randomization 
11. Use of fish oil, algal oil or Krill oil supplements, 
drugs or foods fortified with omega-3s in the 2 
months prior to randomization (>200 mg DHA/d 
and/or > 60mg EPA/d intake by FFQ) 
12. Known intolerance to vitamin E or DHA 
13. Malabsorption of Vit E (e.g. due to steatorrhea, 
chronic pancreatitis, severe cholestasis) 
14. Vitamin E supplementation of greater than 100 
IU/day in the 3 months prior to randomization 
15. History of bariatric surgery (jejunoileal bypass or 
gastric weight loss surgery) or currently 
undergoing evaluation for bariatric surgery 
16. History of biliary diversion 

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17. Known positivity for antibody to Human 
Immunodeficiency Virus 
18. Patients with coagulopathy (PT ≥ 3 sec. from 
ULN) or thrombocytopenia (<70K) 
19. Contraindication to MRI (implants, metal…) 
20. Active, serious medical disease or disease 
diagnosis of a life-expectancy less than 5 years 
21. Ongoing or recent alcohol consumption > 21 
drinks (1 drink= 12 oz regular beer, or 5 oz wine, 
or 1.5 oz distilled spirits) per week in men and > 
14 drinks per week in women as per subject self-
report as part of medical history. 
22. Active substance abuse, such as oral, inhaled or 
injected illicit drugs (except marijuana), in the 
year prior to screening 
23. Women of childbearing potential: positive 
pregnancy test during screening or at 
randomization or unwillingness to use an 
effective form of birth control during the trial 
24. Women who are breastfeeding 
25. Any other condition which, in the opinion of the 
investigator would impede compliance or hinder 
completion of the study 
26. Subjects who are enrolled in an interventional 
clinical study or have received an investigational 
new drug or product within the last 30 days prior 
to screening 
27. Participants diagnosed with type 1 diabetes 
 
STUDY PROCEDURES 
 
Study visit overview: 
 
The patient-related activities of the PUVENALFD trial 
are divided into 5 phases:  
1. Screening of eligibility for enrolment (Visit 0), 
2. 2nd Screening with MRI-PDFF (Visit 1)  
3. Randomization (Visit 2), 
4. Phone call at 1 month for compliance  
5. Visit at 3 months (Visit 3), and  
6. Visit at 6 months (Visit 4) 
7.  Screening of eligibility for enrollment (Visit 0): 
All participants undergo a Fibroscan first (≥300db) and 
must meet criteria for ALT ≥ 40 to assess the eligibility 
for enrolment into the trial.  
Alcohol consumption will be ascertained by subject self-
report through medical history. Participants are not 
allowed to use any prescription or over-the-counter 
medication, or herbal remedy taken with an intent to 
improve or treat fatty liver, liver disease, or obesity for 
the 3 months before randomization. Such agents include 
but are not limited to thiazolidinediones (TZDs). These 
agents are not to be used during screening nor for the 
duration of the trial. If a participant is using a statin or 

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fibrate medication to improve hyperlipidemia during 
screening, he/she is required to be on a stable dose in 
the 3 months before randomization. Participants using 
anti-diabetic medications must also be on a stable dose 
in the 3 months before randomization. Poorly controlled 
diabetic patients will be referred back to their 
diabetologist to manage hyperglycemia.  Use of fish oil, 
algal oil or Krill oil supplements, drugs, or foods 
fortified with omega-3s are not allowed for at least 2 
months before randomization and are prohibited after 
randomization and will be assessed by a Food Frequency 
Questionnaire (FFQ) at screening V0 with <200mg/d DHA 
and/or <60mg/d EPA intake to qualify for enrollment. 
 
8. 2nd Screening with MRI-PDFF (Visit 1) 
For those participants who pass the first screening 
criteria, an assessment of liver fat percentage by MRI-
PDFF will occur to confirm ≥12% liver fat content for 
trial eligibility. A second ALT measure will be taken to 
confirm ALT ≥ 40 as well as second alkaline phosphatase, 
and bilirubin measures.  If results exceed more than 50% 
from first lab results, and are above the normal range, 
then a third test (unscheduled visit) will be repeated 
(only for the elevated test(s)) before V2 to determine 
the direction of the change. If the third test shows a 
continued increase greater than 25% higher than the 
second test in ALT, Alk-P or Bilirubin the subject will be 
disqualified.  For those that qualify, all values will be 
recorded on the CRF. 
 
9. Randomization (Visit 2): 
Subjects will be randomly assigned to one of the study 
interventions or placebo and stratified by Type II 
diabetes Dx (in no greater than 50% of sample). 
 
10. Phone call at one month for Compliance 
 
11. Visit at 3 months (Visit 3) and visit at 6 months 
(Visit 4):  
For details see Table for Scheduled Assessments 
 
STATISTICAL CONSIDERATIONS 
For a within-subject 3.6% absolute reduction in liver fat 
for the combination group vs. placebo and setting 
α=0.05 and 80% power, n=60/arm for the combination 
and placebo groups are needed. [1, 2] To estimate 
secondary outcomes of DHA alone and Vitamin E alone 
versus placebo with a confidence interval ±3% of liver 
fat, n=30/arm for the DHA only and Vitamin E only 
groups are needed. With 10% expected attrition, the 
total sample size is 200 subjects.  
Sample will be stratified by Diabetes Dx (no greater than 
50% of sample). 
 
 
 

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ABBREVIATIONS 
 
 
AE 
Adverse Event 
AHRQ 
Agency for Healthcare Research and Quality 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
ANCOVA 
Analysis of Covariance 
ANOVA 
Analysis of Variance 
AR 
Adverse Reaction 
AST 
aspartate transaminase 
AUDIT 
alcohol use disorders identification test 
BMI 
body mass index 
CAP 
Controlled Attenuation Parameter 
CK-18 
cytokeratin 18 
CRF 
Case Report Form 
CRO 
Clinical Research Organization 
CS 
Clinically significant 
DHA 
docosahexaenoic acid 
DNP 
DSM Nutritional Products 
DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
e.g. 
for example 
EPA 
eicosapentaenoic acid 
EE 
ethyl ester 
FFQ 
Food Frequency Questionnaire 
FIB-4 
Fibrosis -4 score 
GGT 
γ -glutamyl-transferase 
GCP 
Good Clinical Practice 
GRAS 
Generally Recognized as Safe 
HDL-C 
high-density lipoprotein cholesterol 
IEC/IRB 
Independent Ethics Committee/Institutional Review Board 
INR 
International Normalized Ratio 
IOM 
Institute of Medicine 
IP 
Investigational Product 
ISF 
Investigator Site File 
ITT 
Intent-to-Treat 
IWRS 
interactive-web-based response system 
LC-PUFA 
Long-chain polyunsaturated fatty acid 
LDL-C 
low density lipoprotein cholesterol 
MedDRA 
Medical Dictionary for Drug Regulatory Affairs 
MRI 
magnetic resonance imaging 
NAFLD 
Non-alcoholic fatty liver disease 
NASH 
Non-alcoholic steatohepatitis 
PBO 
placebo 
PDFF 
proton density fat fraction 
PI 
Principal Investigator 

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PP 
Per Protocol 
PPAR 
peroxisome proliferator activated receptors 
RBC 
red blood cell 
RCTs 
randomized controlled trials 
ROI 
regions of interest 
ROS 
reactive oxygen species 
SAE 
Serious Adverse Event 
SAP 
statistical analysis plan 
SAR 
Suspected Adverse Reaction 
SC 
Study Coordinator 
SF-36 
Short form 36 
SID 
subject identifier 
SN 
Study Nurse 
SOP 
Standard Operating Procedure  
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TE 
transient elastography 
TEAE 
treatment-emergent adverse events 
TGs 
triglycerides 
TMF 
Trial Master File 
TNFα 
tumor necrosis factor-α 
TZDs 
thiazolidinediones 
UDCA 
ursodeoxycholic acid 
UL 
Tolerable Upper Intake Level 
vs 
versus 
 
 
 

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TABLE OF CONTENTS 
1 
INTRODUCTION 
14 
1.1 
Background information 
14 
1.2 
Investigational products 
14 
1.2.1 
Vitamin E [(all-rac)-α-tocopheryl acetate] 
14 
1.2.2 
DHA Ethyl Ester 
15 
1.3 
Rationale for conducting the clinical study 
16 
2 
OBJECTIVES AND ENDPOINTS 
17 
2.1 
Primary objective and endpoint variable 
17 
2.1.1 
Primary objective 
17 
2.1.2 
Primary endpoint 
17 
2.2 
Secondary objectives and endpoint variables 
17 
2.2.1 
Secondary objectives 
17 
2.2.2 
Secondary endpoints 
17 
2.3 
Safety objectives and endpoint variables 
18 
2.3.1 
Safety objective(s) 
18 
2.3.2 
Safety endpoint variable(s) 
18 
3 
STUDY DESIGN 
18 
3.1 
Duration of clinical study 
18 
3.2 
Duration of clinical study per subject 
181 
4 
SUBJECTS AND SITE 
191 
4.1 
Number of subjects 
191 
4.2 
Clinical study population 
19 
4.2.1 
Inclusion criteria 
191 
4.2.2 
Exclusion criteria 
19 
4.3 
Randomization, blinding and treatment allocation 
20 
4.4 
Unblinding procedure 
20 
4.5 
Number of sites 
203 
5 
INVESTIGATIONAL DRUG PRODUCT AND REGIMEN 
203 
5.1 
Investigational product (IP) 
203 
5.1.1 
Investigational drug product names and formulations 
203 
5.1.2 
Other product name(s) and formulation(s) 
213 
5.1.3 
Packaging and labelling 
21 
5.1.4 
Handling and storage conditions 
21 
5.2 
Drug regimen 
214 
5.2.1 
Rationale for dose selection 
214 
5.2.2 
Dosage regimen and dose adjustment 
214 
5.2.3 
Route of administration 
224 
5.2.4 
Dosing duration 
22 
5.3 
Dispensing and accountability 
22 

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5.4 
Compliance 
225 
5.5 
Warnings and precautions 
225 
5.5.1      Summary of Known Potential Risks with Study Medication                            25 
5.6 
Concomitant treatments/supplements and restrictions 
258 
6 
STUDY PROCEDURES 
258 
6.1 
Recruitment procedures 
258 
6.2 
Schedule of assessments 
259 
6.3 
Screening and eligibility 
30 
6.3.1 
Obtaining informed consent 
30 
6.3.2 
Visit 0 – Screening examinations 
30 
6.3.3 
Visit 1- 2nd Screening 
30 
6.3.4 
Assessment of eligibility and randomization procedure 
30 
6.4 
Intervention phase 
31 
6.4.1 
Visit 2 Randomization 
31 
6.4.2 
Phone call at one month 
31 
6.4.3 
Visit 3 (3mos Visit) 
31 
6.4.4 
Visit 4 (6mos Visit) 
31 
7 
KEY MEASUREMENTS AND ASSESSMENTS 
32 
7.1 
Blood samples 
32 
7.1.1 
Blood sample collection 
32 
7.1.2 
Blood sample storage and shipment 
32 
7.1.3 
Blood sample assessment 
32 
7.2 
MRI-PDFF 
33 
7.2.1 
Assessment of liver fat percentage by MRI-PDFF 
33 
7.3 
Other key assessments 
33 
7.3.1 
Liver Fibroscan 
33 
7.3.2 
Liver Fibrosis score 
33 
7.3.3 
Anthropometrics 
33 
7.3.4 
Alcohol Consumption 
34 
7.3.5 
Urine sample collection and storage 
34 
7.3.6 
Questionnaires 
34 
8 
EARLY SUBJECT WITHDRAWAL 
34 
9 
DATA PROCESSING AND STATISTICAL CONSIDERATIONS 
35 
9.1 
Case Report Form 
35 
9.1.1 
Electronic Data Capture 
35 
9.2 
Data Management 
35 
9.3 
Statistical Analysis 
36 
9.3.1 
Statistical hypotheses and methods 
36 
9.3.2 
Primary endpoint analysis 
36 
9.3.3 
Secondary endpoint variable(s) and analyses 
36 
9.3.4 
Exploratory variables 
347 
9.3.5 
Safety variables 
347 
9.3.6 
Compliance Analysis 
348 
9.3.7 
Sample size: 
348 
9.3.8 
Study Populations 
358 

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9.3.9 
Poolability of Data 
358 
10 
SAFETY 
39 
10.1 
Definitions and Standards 
39 
10.2 
Adverse event assessment 
40 
10.3 
Safety Management 
41 
 10.3.1      Procedures  
       
 
 
 
             
        41 
10.3.2 
Treatment and Holding/Stopping Rules for Drug Induced Liver Injury 
42 
10.3.3 
Responsibilities 
45 
 
11 
ETHICAL CONSIDERATIONS 
46 
11.1 
Local Regulations and Declaration of Helsinki 
46 
11.2 
GCP Management Directive 
46 
11.3 
Informed Consent 
47 
11.4 
IRB/IEC approval 
47 
11.4.1 
Risk-benefit assessment 
47 
11.5 
Confidentiality 
47 
11.5.1 
Data 
47 
11.5.2 
Subject Anonymity 
47 
11.6 
Subjects’ compensation / remuneration 
48 
11.7 
Registration of study in a public clinical trial database 
48 
12 
STUDY DOCUMENTATION AND RECORD KEEPING 
48 
12.1 
Protocol amendments 
48 
12.2 
Investigator Site File 
48 
12.3 
Source document and source data verification 
49 
12.4 
Insurance 
459 
12.5 
Monitoring 
459 
12.6 
Quality assurance and quality control 
50 
12.7 
Final Study Report 
50 
12.8 
Archiving 
50 
12.9 
Publication 
50 
13 
CONDITIONS FOR TERMINATING THE STUDY 
50 
14 
REFERENCES 
51 
15 
APPENDICES 
53 
15.1 
Appendix 1 
53 
15.2 
Appendix 2 
55 

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Introduction 
1.2 Background information 
Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat accumulation in the 
liver and is defined by evidence of hepatic steatosis (via imaging or histology) and is not due to 
secondary liver fat accumulation from excessive alcohol consumption or hereditary disorders 
(e.g. Wilson’s disease) [3]. NAFLD is most commonly associated with metabolic syndrome, 
consisting of obesity, insulin resistance, elevated blood pressure and dyslipidemia. NAFLD is one 
of the most common causes of chronic liver disease, globally with a prevalence as high as 30% in 
Western countries [4]. It includes a spectrum of diseases from steatosis to non-alcoholic 
steatohepatitis (NASH), liver fibrosis, cirrhosis, and hepatocellular carcinoma [5]. Non-alcoholic 
fatty liver does not involve hepatocellular injury in the form of ballooning hepatocytes, whereas 
NASH is defined by steatosis, inflammation, and hepatocyte injury (ballooning) with or without 
fibrosis [3]. The causes of NAFLD are likely due to a combination of genetic and physiologic 
factors, namely those that promote oxidative stress and inflammation such as metabolic 
syndrome, visceral adiposity, and changes in intestinal microbiota [6]. NAFLD is significantly 
associated with increased risk of Type II Diabetes and cardiovascular disease, and increased 
overall mortality compared to age matched controls. There is currently no approved drug 
treatment for NAFLD or NASH. Dietary restriction for weight loss and increased physical activity 
are the recommended therapies albeit with limited success.  
1.3 Investigational products 
1.3.1 Vitamin E [(all-rac)-α-tocopheryl acetate] 
Vitamin E is a fat-soluble vitamin that is synthesized naturally in plants in four tocopheryl forms: 
α, β, γ and δ. All-rac-α-tocopheryl acetate has the highest biological activity in animal models 
[7] and it is the α-tocopheryl form that is used to prevent and treat Vitamin E deficiency in 
humans. Functionally, Vitamin E is an antioxidant and peroxyl radical scavenger. It is an inhibitor 
of lipid peroxidation and can also inhibit and modulate intracellular signaling molecules, e.g. 
protein kinase C, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [7]. α-
tocopheryl regulates gene expression of several intracellular enzymes such as 5-lipoxygenase and 
cyclooxygenase and has anti-inflammatory activity (i.e. decreasing cytokine release and plasma 
C reactive protein). It is also known to inhibit platelet adhesion and aggregation. 
 
In hepatocytes, Vitamin E suppresses expression of xanthine oxidase, a source of reactive oxygen 
species (ROS). It inhibits the propagation of peroxyl radicals by donating its phenolic hydrogen 
and converts the radical to a hydroperoxide, nonradical product [8]. Proliferation and 
enlargement of hepatic peroxisomes are seen in hepatic steatosis. In patients with NASH, 
increased fatty acid oxidation and decreased mitochondrial respiratory chain activity, including 
adenosine triphosphate (ATP) depletion is present. These patients also have higher cytochrome 
P450 activity which metabolizes long chain fatty acids producing increased ROS. Such increased 
ROS enhance lipid peroxidation and form aldehyde by-products such as malondialdehyde and 
increased cytokines e.g. tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-
β), markers of inflammation. These cytokines can damage mitochondrial DNA and function, 
leading to a continuous cycle of peroxidation, fatty acid oxidation, and inflammation that result 
in hepatocyte apoptosis and eventually fibrosis. Vitamin E’s antioxidant, anti-inflammatory, and 
inhibitory intracellular signaling functions prevent the propagation of free radicals and 
inflammatory processes. 
 
Vitamin E has been shown to have a beneficial effect on NAFLD in previous studies. In a meta-
analysis of 5 randomized controlled trials (RCTs), Sato [9] demonstrated that vitamin E 
significantly reduced liver enzymes: aspartate transaminase (AST), alanine aminotransferase 

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(ALT) and alkaline phosphatase (ALP), and significantly decreased steatosis, lobular 
inflammation, and hepatocellular ballooning compared to control or placebo. In this meta-
analysis, hepatic fibrosis was improved with Vitamin E but was not statistically significant 
(p=0.06). In the largest of these trials, Sanyal [10] found that 800 IU Vitamin E (as RRR-α-
tocopherol) /day for 96 weeks, administered to 84 non-diabetic adults with NASH significantly 
improved NASH, based on a composite score of histologic features, by 43% compared to 19% in 
the placebo group (n=83) (p=0.001). The rate of improvement of the comparator, 30 mg/d 
pioglitazone was only 34% and not significantly different from placebo. Significant reductions in 
hepatic steatosis and lobular inflammation were seen with both agents, but improvement in 
fibrosis was not significant. Other smaller studies in adult patients with NAFLD or NASH [11]  [12] 
[13] and [14], given 500-800 IU/day Vitamin E have also shown significant decreases in liver 
enzymes, steatosis [12] [13] [14],  inflammation [12] [13], and ballooning [13]. Two of the trials 
administered Vitamin E with background ursodeoxycholic acid (UDCA) but guidelines from the 
American Association of Study for Liver Disease considers UDCA ineffective for NASH 
treatment[15].  
The Institute of Medicine (IOM),[16] has established the Tolerable Upper Intake Level (UL) for 
Vitamin E as 1000 mg/d of α-tocopheryl (all stereoisomers), equivalent to 1000 IUs by US 
Pharmacopeia. Few adverse effects are seen below doses of 2100 mg according to IOM. The UK 
Expert Group on Vitamins and Minerals, [17] also established a safe upper level of consumption 
of 800 IU. Sanyal’s 2010 study [10] which used 800 IU Vitamin E found no differences between 
groups in adverse events including no difference in number of cardiovascular events. Although a 
study by Klein [18] of 400IU Vitamin E and Prostate cancer risk found a 17% increased risk in 
healthy men, other studies have found either no increased risk or decreased incidence in 
prostate cancer [19] [20] and improved prostate cancer survival [21]. In an 18 year follow up of 
the Alpha-Tocopheryl, Beta-Carotene (ATBC) Cancer Prevention study [22], α-tocopheryl had no 
effect on prostate cancer incidence (RR 0.97), no effect on overall mortality and decreased 
prostate cancer mortality (RR 0.84). Additionally, an Agency for Healthcare Research and Quality 
(AHRQ) report [23] concluded that trials of Vitamin E altogether had no overall effect on cancer. 
 
1.3.2 DHA Ethyl Ester 
Long-chain polyunsaturated fatty acid (LC-PUFA), docosahexaenoic acid (DHA) is an important 
omega-3 fatty acid for brain, eye and cardiovascular development and health. It significantly 
reduces triglycerides (TGs), lowers heart rate [24], lowers blood pressure [25], and reduces the 
risk of cardiac death by an overall 8% [26]. Both DHA and eicosapentaenoic acid (EPA) have anti-
thrombotic, anti-inflammatory, and anti-oxidative properties [27]. As NAFLD patients are at 
significantly greater risk of cardiovascular disease and higher overall mortality, the 
cardioprotective effects of DHA are significant and may be beneficial in the NAFLD population.  
 
Potential mechanisms for DHA’s effects in NAFLD include reduction of TG synthesis via activation 
of peroxisome proliferator activated receptors (PPAR-α and γ) which accelerates fatty acid 
oxidation in liver mitochondria [5]. DHA is also known to have an integral role in maintaining and 
improving cell membrane fluidity, as a fatty acid that is incorporated into the phospholipids of 
the membrane, thereby optimizing surface receptors and signal transduction pathways in liver 
cells. The anti-inflammatory role of DHA in NAFLD may be mediated through activation of 
adiponectin secretion through the PPAR-γ path. Adiponectin improves lipid oxidation and 
reduces insulin resistance and inflammation [5] [28]. DHA also decreases expression of pro-
inflammatory cytokines and increases DHA-derived prostaglandins and leukotrienes which inhibit 
pro-inflammatory cytokines. DHA-derived resolvins, protectins, and maresins are also anti-
inflammatory by stimulating the resolution of inflammation [27]. Many of these mechanisms may 
contribute to the effects of DHA on metabolic syndrome and improve liver function in NAFLD. 
 
A meta-analysis by Guo [5] demonstrated decreased levels of DHA in NAFLD patients compared 
to case controls, and their analysis of 10 RCTs showed significant reductions in ALT, AST, TGs, 

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and liver fat with doses of 1-5 g/d omega-3 supplementation. They showed that with each one 
gram/day increment in DHA, decreases in liver enzymes, i.e. -7.42 U/L ALT, -5.3 U/L AST, and -
7.26% reduction in liver fat were achieved. Other meta-analyses of RCTs [[28] [29, 30] have also 
shown significant decreases in liver fat and positive results (or positive trends) in liver enzymes: 
decreases in ALT, AST, GGT, significant decreases in TGs, and increases in high-density 
lipoprotein cholesterol (HDL) with omega-3 supplementation in NAFLD patients. As reported by 
Parker et al [30] a 27% reduction of steatosis, measured by ultrasound, was seen across 5 trials 
with doses of 2-5g/d omega-3 treatment and may be considered clinically significant. In a RCT of 
omega-3 (DHA+EPA) ethyl esters (Lovaza®, 4g/d), Scorletti [2] demonstrated an overall trend for 
a decrease in liver fat (β= -3.64, p=0.1) but compliance and contamination in the placebo group 
limited the significance of the results. However, a regression analysis of DHA independently 
showed a significant 1.7% decrease in liver fat for each 1% increase in red blood cell (RBC) DHA 
(p=0.007); when adjusted for cofactors such as age, gender, body weight, each 1% increase in 
RBC DHA, was associated with a 3.3% decrease in liver fat (p=0.0001). This relationship was not 
seen with EPA. A larger RCT of EPA ethyl ester alone [31] in patients with biopsy-confirmed 
NASH found no significant differences in the NAFLD activity score, steatosis, inflammation, 
ballooning, or liver enzymes between 2 doses of active (1.8 g/d or 2.7 g/d) and placebo. 
Although this trial was conducted in patients at a later stage of disease and had a higher than 
expected placebo response rate, the results collectively may suggest that DHA is the key omega-
3 fatty acid to provide benefit for patients with fatty liver, i.e. NAFLD. 
 
 
1.4 Rationale for conducting the clinical study 
The combination of Vitamin E and DHA has not been tested in previous clinical trials of adults 
with NAFLD. This combination may provide optimal benefit for patients with NAFLD due to their 
associated mechanisms of action, namely Vitamin E’s antioxidant action, preventing lipid 
oxidation of long chain fatty acids such as DHA and thus preventing the propagation of free 
radicals and ROS. Vitamin E’s protection of LC-PUFA DHA therefore assists it in maintaining cell 
membrane stability and optimal signalling. Their combined anti-inflammatory effects (e.g. 
inhibiting pro-inflammatory cytokines, increasing adiponectin, and producing docosanoids to 
resolve inflammation) may also be efficacious for those with metabolic syndrome and NAFLD. 
The combination of Vitamin E and DHA will specifically be used in this study to determine if a 
reduction in liver fat occurs after 6 months of co-administration, using a magnetic resonance 
imaging (MRI) technique, proton density fat fraction (PDFF). PDFF imaging is non-invasive and 
highly sensitive to detect liver steatosis in patients with NAFLD. PDFF imaging is significantly 
more accurate than liver ultrasound-based transient elastography (TE) in detecting steatosis 
(AUROC 0.99 vs. 0.85 by TE-CAP) [32] [33]. It also significantly correlates with histology-
determined steatosis (I2=0.56, p<0.0001) in adults with NAFLD [34]. MRI-PDFF is also an 
appropriate technique to diagnose and stage disease in those with metabolic syndrome and 
NAFLD. 
The clinical trial is designed to test the combination of Vitamin E and DHA against placebo, to 
demonstrate efficacy and safety. It is not powered however, to compare the combination 
against each of the active products for superiority. Each active product will be included as 
additional treatment arms to gain further insight into their contribution to changes in NAFLD 
parameters and to collect additional safety data. 

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2 
OBJECTIVES AND ENDPOINTS 
2.1 Primary objective and endpoint variable 
2.1.1 Primary objective 
To determine the efficacy of the combination of vitamin E and omega-3 fatty acid (DHA EE) 
versus (vs) placebo on reducing liver fat content after 6 months of intervention in adults with 
NAFLD. 
2.1.2 Primary endpoint  
Decrease in hepatic fat fraction [%] relative to baseline between Vitamin E + DHA combination vs 
placebo, measured by MRI-PDFF after 6 months of intervention 
2.2 Secondary objectives and endpoint variables 
2.2.1 Secondary objectives  
• 
To determine effect of Vitamin E alone vs Placebo on reducing liver fat after 6 months of 
intervention in adults with NAFLD. 
• 
To determine effect of DHA alone vs Placebo on reducing liver fat after 6 months of 
intervention in adults with NAFLD. 
• 
To determine the change after 6 months of DHA EE and /or Vitamin E intervention in 
anthropometric, metabolic, hepatologic, nutrient, and inflammatory parameters, and quality 
of life status of adults with NAFLD. 
2.2.2 Secondary endpoints  
• 
Decrease in hepatic fat fraction [%] relative to baseline between Vitamin E vs placebo, 
measured by MRI-PDFF after 6 months of intervention 
• 
Decrease in hepatic fat fraction [%] relative to baseline between DHA vs placebo, measured 
by MRI-PDFF after 6 months of intervention 
• 
Change after 6 months of DHA EE and/ or Vitamin E intervention in: 
- 
Anthropometric measures (body weight, waist-to hip ratio, waist circumference, body mass 
index (BMI)  
- 
Insulin resistance  
- 
Liver enzymes  
- 
Fibrosis-4 (FIB-4) score  
- 
Plasma Vitamin E concentration  
- 
Plasma DHA concentration  
- 
Lipid profile (e.g. HDL-C, low density lipoprotein (LDL-C), TGs, oxidized LDL) 
- 
Health related quality of life score (Short form (SF-36)) 
- 
Dietary Intake Levels of LC-PUFA (i.e. DHA and EPA) as measured by the Food Frequency 
Questionnaire (FFQ) 
- 
Inflammatory markers (e.g. cytokeratin 18 (CK-18), TNFα, IL-1β) 
 
 
2.2.3 Exploratory Endpoints 
- 
Transcriptomic analysis of the PBMC gene expression 
- 
PNPLA3 genotype 
- 
HSD17b 
- 
Haptogloblin  
- 
LC-PUFA-derived oxylipins 
 

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2.3 Safety objectives and endpoint variables 
2.3.1 Safety objective(s)  
Safety objectives are to examine any treatment-emergent adverse events (TEAEs), and changes 
in clinical safety laboratory parameters 
2.3.2 Safety endpoint variable(s) 
- 
Vital signs 
- 
Serum hematology and biochemistry  
- 
AE/SAE reporting  
 
3 
STUDY DESIGN  
The study is a multi-center, randomized, double-blind, placebo-controlled, parallel, 4-group 
intervention study in adults with NAFLD. 
 
 
 
 
3.1 Duration of clinical study 
The project may take up to 3-4 years to complete, depending upon recruitment time. 
3.2 Duration of clinical study per subject 
Total duration of the study per subject is 6 months consisting of 2 screening visits, 
randomization/baseline visit, a one-month phone call, a visit at 3 months and a visit at 6 
months. 

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4 
SUBJECTS AND SITE 
4.1 Number of subjects 
Total number of subjects to be enrolled: 200 (unequal allocation: 65/65/35/35 for 
Combination/Placebo/DHA only/Vitamin E only groups, respectively). With an estimated 10% 
attrition, approximately 180 subjects are expected to complete the study. 
4.2 Clinical study population 
4.2.1 Inclusion criteria  
 
1. Male or female gender 
2. ≥18 years of age 
3. A new diagnosis or reconfirmation of previously known fatty liver by imaging (ultrasound 
or CT or MRI), or by liver biopsy within ≤ 4 years 
4. Fibroscan Controlled Attenuation Parameter (CAP) score ≥ 300db 
5. Liver fat content (≥ 12%) measured by MRI-PDFF 
6. ALT ≥ 40 U/L 
7. eGFR/Creatinine Clearance ≥ 60ml/min 
8. Participants with previously diagnosed Type 2 diabetes (up to 50% of sample):  they must 
either be taking anti-diabetic medications, or their fasting (>10 hours) glucose must be ≥ 
100 mg/dL at the time of screening 
9. Stable weight (±5%) for at least 3 months  
10. Subjects willing and able to give written informed consent and to understand, to 
participate and to comply with the clinical study requirements. 
4.2.2 Exclusion criteria 
 
1. Evidence of alternative causes of hepatic steatosis or other forms of chronic liver 
disease, e.g. Hepatitis B, Hepatitis C (with <3 years treatment) 
2. Evidence of acute Hepatitis A 
3. Serum ALT or AST ≥ 250 U/L 
4. Serum Alkaline Phosphatase > 2 ULN 
5. Total bilirubin > 2 ULN in the absence of Gilbert’s Syndrome [In patients with Gilbert’s 
Syndrome, direct bilirubin must not exceed 2 ULN] 
6. HbA1c > 9.5% 
7. Decompensated acute or chronic liver disease 
8. Clinical, imaging or histological evidence of cirrhosis 
9. Use of anti-NASH drugs (e.g. thiazolidinediones (TZD)) in the 3 months prior to 
randomization 
10. Use of a non-stable dose of statins or fibrates in the 3 months prior to randomization 
11. Use of fish oil, algal oil or Krill oil supplements, drugs or foods fortified with omega-3s in 
the 2 months prior to randomization (>200 mg DHA/d and/or > 60mg EPA/d intake by 
FFQ) 
12. Known intolerance to vitamin E or DHA 
13. Malabsorption of Vit E (e.g. due to steatorrhea, chronic pancreatitis, severe cholestasis) 
14. Vitamin E supplementation of greater than 100 IU/day in the 3 months prior to 
randomization 
15. History of bariatric surgery (jejunoileal bypass or gastric weight loss surgery) or currently 
undergoing evaluation for bariatric surgery 
16. History of biliary diversion 
17. Known positivity for antibody to Human Immunodeficiency Virus 

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18. Patients with coagulopathy (PT ≥ 3 sec. from ULN) or thrombocytopenia (<70K) 
19. Contraindication to MRI (implants, metal…) 
20. Active, serious medical disease or disease diagnosis of a life-expectancy less than 5 years 
21. Ongoing or recent alcohol consumption > 21 drinks (1 drink= 12 oz regular beer, or 5 oz 
wine, or 1.5 oz distilled spirits) per week in men and > 14 drinks per week in women as 
per subject self-report as part of medical history. 
22. Active substance abuse, such as oral, inhaled or injected illicit drugs (except marijuana), 
in the year prior to screening 
23. Women of childbearing potential: positive pregnancy test during screening or at 
randomization or unwillingness to use an effective form of birth control during the trial 
24. Women who are breastfeeding 
25. Any other condition which, in the opinion of the investigator would impede compliance or 
hinder completion of the study 
26. Subjects who are enrolled in an interventional clinical study or have received an 
investigational new drug or product within the last 30 days prior to screening 
27. Participants diagnosed with type 1 diabetes 
4.3 Randomization, blinding and treatment allocation 
Subjects will be randomly assigned to one of four treatment arms, either 1000 mg Vitamin E, 
1.89 g DHA EE, 1000 mg Vitamin E + 1.89 g DHA EE, or placebo (PBO) based on a computer-
generated randomization scheme, i.e. interactive-web-based response system (IWRS). Subjects 
will be stratified by Diabetes, Type II diagnosis (in no greater than 50% of sample). Enrolment 
will be competitive across study sites. 
 
4.4 Unblinding procedure  
One unblinded representative from the participating Clinical Research Organization (CRO) who is 
not participating in the study will maintain access to the computer- generated randomization 
code, in case unblinding is necessary. In an emergency situation where unblinding is needed, the 
site’s Principal Investigator 
(PI) is instructed to contact the CRO. Only the PI will become unblinded to that subject’s 
treatment; other site staff and study team are to remain blinded. 
4.5 Number of sites 
An estimated 16 sites in the US will be recruited for this study based on MRI capabilities and 
expertise in NAFLD. For a sample size of 200 subjects, and an approximate recruitment time of 
24 months, it is anticipated that each site will recruit an estimated 1-2 subjects/month.  
5 
INVESTIGATIONAL DRUG PRODUCT AND REGIMEN 
5.1 Investigational product (IP) 
5.1.1 Investigational drug product names and formulations 
1.Vitamin E [(all-rac)-α-tocopheryl acetate], 1000 mg [1000 IU/day], API grade, DMF #  030673 in 
a 1 g soft gel capsule containing also natural citrus flavoring agent, and shell colorant, 
manufactured by Catalent Pharma and supplied by DSM Nutritional Products Ltd. 
 
2.DHA EE, 947 mg API grade (derived from DHASCO® marine microalgae, Crypthecodinium cohnii) 
in a 1 g soft gel capsule containing, natural citrus flavoring agent, and shell colorant, 

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manufactured by Catalent Pharma and supplied by DSM Nutritional Products. Dose of DHA EE is 
1.89 g/d (2 capsules). 
5.1.2 Other product name(s) and formulation(s) 
The placebo comparator is Soybean oil in a 1 g soft gel capsule containing natural citrus 
flavoring agent, and shell colorant, manufactured by Catalent Pharma and supplied by DSM 
Nutritional Products. 
5.1.3 Packaging and labelling 
The IP is packaged in blister cards to enable blinding of the 4 arms of the trial. The combination 
of Vitamin E and DHA EE requires 3 softgel capsules to be taken /day. Thus, all other arms must 
have 3 capsules/day. Each blister card will contain a 2-week supply of capsules (42 softgels). 
Each subject kit will contain 13 blister cards plus an extra 1-week supply of capsules (one 1/2 
blister card) to account for any potential missed dose or loss. The subject kit will contain the 
following information on the label: 
 
• 
Limited by Federal law, for investigational use only 
• 
DSM Nutritional Products & address 
• 
Product name and instructions for use 
• 
Protocol number 
• 
kit number 
• 
Subject ID (to be filled in) 
• 
Randomization # (to be filled in) 
• 
Storage conditions / keep away from children 
5.1.4 Handling and storage conditions 
Store in a cool, dry place (60-77°F). Capsules should not be kept for prolonged periods at high 
temperatures. 
5.2 Drug regimen 
5.2.1 Rationale for dose selection 
Oral vitamin E intake at 1000 IU daily dose is safe, as confirmed by the US Food and Nutrition 
Board of the Institute of Medicine (IOM), that set the Tolerable Upper Limit (UL) at 1000 mg/d of 
α-tocopheryl in any supplemental form. This dose is within the range of vitamin E dosage that 
has been tested for the treatment of NAFLD and NASH in previous studies, and Practice Guidance 
from the American Association for the Study of Liver Diseases states that Vitamin E administered 
at a daily dose of 800 IU/day improves liver histology in nondiabetic adults with biopsy-proven 
NASH. The dose of DHA EE (1.89 g/d) is also within the range of doses tested for treatment of 
NAFLD in previous studies and is below the Generally Recognized as Safe (GRAS) affirmation 
level (FDA 21CFR 184, docket #86G-0289, 1997). As there is no proven pharmacologic therapy for 
NAFLD, using a placebo for comparative purposes is justified. 
 
5.2.2 Dosage regimen and dose adjustment 
Subjects will be provided blister cards of capsules with a 2-week supply per blister card and will 
be instructed to take 3 capsules/day. All capsules will look identical to maintain blinding. 
Subjects assigned to Vitamin E will take 1 active and 2 PBO capsules. Subjects assigned to DHA 
EE will take 2 active and 1 PBO capsules. Subjects assigned to the combination will take 1 
Vitamin E and 2 DHA EE capsules. Subjects assigned to PBO will take 3 PBO (soybean oil) 
capsules.  

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Subjects will be supplied with 13 cards, plus one extra ½ card (in case of a lost/misplaced card 
or late visit) in a blinded kit. Each study site will receive approximately 12 kits initially, and will 
be resupplied, depending upon enrolment. Kits will be labelled with a unique identifier (kit #) 
linked to the randomization (IWRS). 
  
5.2.3 Route of administration 
Three softgels per day are to be taken by mouth with food at the same time each day. 
5.2.4 Dosing duration 
The duration of IP administration in the trial is 180 +3 days. IP will not be provided after the 
subject completes the study. 
5.3 Dispensing and accountability 
The study sites will receive a Product Dispensing and Return Log to account for investigational 
product dispensed and returned to the site. Individual Product Disposition Sheets must be 
completed for each subject and must be kept up to date. It lists the subject ID, the amount of IP 
and date dispensed to the subject and the amount of product and date returned. Product 
inventory will also be tracked via the IWRS. Subjects will be instructed to return all empty and 
unused blister cards to their clinic at the 3 month visit and at the end of the study. 
 
The monitor will inspect the amount of IP dispensed, returned and on stock at the site (including 
empty kits, partly used, and used kits) during monitoring visits.  The investigator must discard 
all unused product at the end of the study, according to site policy and as agreed with the 
Sponsor.  Unused means undispensed kits.  Therefore, study sites may discard any previously 
dispensed kits (containing both empty blister packs and unused returned product) once final 
product accountability has been conducted by the study monitor.  Document the destruction of 
the returned kits (documentation must include the kit number, date of destruction and clinic 
staff member responsible for product disposal).    
 
5.4 Compliance 
Compliance will be determined by capsule counts and by DHA and α-tocopheryl blood levels. 
Subjects must take a minimum of 80% of capsules in order to be compliant. DHA and α-
tocopheryl blood levels will be used to confirm accuracy of the capsule counts. A subject may be 
withdrawn if non-compliant. Non-compliant subjects will not be replaced and may be removed 
from the per-protocol analysis. 
5.5 Warnings and precautions 
Subjects must be eligible for MRI scanning in this study. 
 
5.5.1 Summary of Known Potential Risks with Study Medication 
 
Vitamin E (study drug) is an essential nutrient found in highest quantities in sunflowers, 
almonds, hazelnuts, and peanuts. It is essential for the body’s cells to function.  
 
To our knowledge, there are two published studies with 800 IU/day of a-tocopherol, both in 
NAFLD and NASH (PIVENS and TONIC) 
 

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In the PIVENS trial, 84 adults with biopsy proven NASH were administered 800 IU/d of a-
tocopherol for 96 weeks (Sanyal AJ, Chalasani N, Kowdley KV, et al.  Pioglitazone, vitamin E, or 
placebo for nonalcoholic steatohepatitis. N Engl J Med 2010; 362:1675-1685  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In the TONIC trial (Lavine, 2011), 58 children with biopsy proven NAFLD received 800 IU/day of 
a-tocopherol for 96 weeks.  
 
AEs by 
severity 
Placebo 
(N=58) 
Attribution to Study 
Drug 
Vitamin E 
(N=58) 
 
Mild  
 
6 
Not Related 
 
10 
Moderate 
12 
Not Related 
7 
Severe 
9 
Not Related 
3 (Bronchospasm 1, 
elevated blood glucose 1, 
hepatotoxicity 1); Note:  No 
specific details of this 
Hepatotoxicity SAE are 
available from the 
publication. In this paper 
(supplemental table 
eTable2), it was reported 
that there was only one 
patient with ALT > 2 ULN in 
vitamin E group as opposed 
to 5 in placebo group 
(p=0.09). No cases of 
bilirubin > 3 mg/day in 
either group.  
 
Vitamin E administered prophylactically or therapeutically within the range of the tolerable 
upper limit (i.e., <1000 mg) established for adults by the Institute of Medicine, has evidenced 
very low toxicity in humans.  Summaries of the adverse events reported by double-blind 
randomized-controlled trials of vitamin E, including dl-α-tocopheryl acetate, are included in 
tables 5.5 and 5.6 of the dl- Alpha- Tocopherol Acetate Investigator’s Brochure. Overall, oral 
AEs by severity Placebo 
(N=83) 
Attribution to Study 
Drug 
Vitamin E 
(N=84) 
 
Mild  
 
20 
Not Related 
 
24 
Moderate 
20 
Not Related 
19 
Severe 
10 
Not Related 
7 
Gastroenteritis 1; 
Hyperglycemia 1; Pain 3; 
Syncope 1; Urticaria 1 
Life 
threatening 
0 
Not Related 
2  
Cardiac ischemia/infarction 
1 
Liver dysfunction 1 
Fatality 
0 
Not Related 
1 (Cause of death: 
Pneumonia and liver failure 
secondary to sepsis from 
gram negative bacteremia. 
Deemed unrelated to the 
study drug).  

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administration of therapeutic levels of vitamin E, including use for up to one year in NAFLD 
patients at dose levels of 800mg/d, has shown very few AEs.  Additionally, Section 5.2.1 of the 
Investigator’s Brochure discusses potential adverse effects involving blood coagulation from 
oral intake of high-dose vitamin E and aspirin, anti-coagulant drugs and/or vitamin K deficiency. 
 
When given in tablets, vitamin E may cause some, all or none of the side-effects listed below at 
the dose and duration used in the study. 
 
Side effects 
Frequent 
>20% of 
participants 
Occasional 
2 - 20% of 
participants 
Rare 
Less than 2% 
of participants 
Serious 
 
 
 
Less Serious 
 
 
blood 
coagulation 
with 
medications 
Minor 
 
 
 Fatigue, 
headache, 
flatulence, 
gastrointestin
al disorders 
 
 
 
DHA EE (study drug) is an essential nutrient found in highest quantities in salmon, mackerel, 
oysters and anchovies. It is essential for the body’s cells to function.  
 
The Investigator Brochure for DHA EE in Tables 5-1 and 5-2 lists published studies in healthy 
adults and in those with various medical conditions with DHA and include dose, duration and 
safety outcomes. The maximal dose tested was 12g/d DHA in a prostate cancer study of 6 
months duration; one SAE of urinary obstruction in one subject was reported and considered 
unrelated to DHA administration. There have been no deaths associated with the use of DHA in 
these studies. Table 5-5 in the IB provides a frequency table of reported AEs by SOC and PT in 
556 subjects (DHA n=379; PBO n=220); the most commonly reported AEs attributed to DHA are 
gastrointestinal disorders. Additionally, Table 5-6 provides a frequency table of SAEs by SOC and 
PT across the same studies. A total of 9 subjects who received DHA reported 15 SAEs with the 
most frequent SOC, infections and infestations.  Scorletti, et al, 2014 reported no SAEs 
attributed to omega-3 administration (Omacor® 4g/d: 1.52g DHA EE and 1.84g EPA EE) over 15-18 
months in NAFLD patients. 
 
Literature citations for toxicology studies with DHA are found in Module 2, 24-Nonclinical 
overview, section 2.4.6 and cited publications can be found in Module 4, 43-lit-ref. 
 
When given in tablets, DHA EE may cause some, all or none of the side-effects listed below at 
the dose and duration used in the study. 
 
 
 
 
 Side Effects 
Frequent 
>20% of 
participants 
Occasional 
2 - 20% of 
participants 
Rare 
Less than 2% of 
participants 
Serious 
  
 
 
Less Serious 
  
 
 

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Minor 
 
 
Eructation, Gas, 
Bloating 
 
 
5.6 Concomitant treatments/supplements and restrictions 
 
Participants are not allowed to use any prescription or over-the-counter medication, or herbal 
remedy taken with an intent to improve or treat fatty liver, liver disease or obesity for the 3 
months before randomization. Such agents include but are not limited to, TZDs. These agents 
are not to be used during screening nor for the duration of the trial. If a participant is using a 
statin or fibrate medication to improve hyperlipidemia during screening, he/she is required 
to be on a stable dose in the 3 months before randomization. Additionally, participants are 
allowed to continue on prescription anti-hyperlipidemic agents, if they are on a stable dose. 
Participants on antidiabetic medication are allowed in the trial if they are on a stable dose in 
the 3 months prior to randomization. Use of fish oil, algal oil or Krill oil supplements, drugs, 
e.g. Lovaza® or foods fortified with omega-3s are not allowed for at least 2 months before 
randomization and are prohibited after randomization. Fatty fish intake (e.g. salmon, tuna, 
herring, etc) is restricted to <2 fish meals/week. Participants are interviewed with a FFQ for LC-
omega-3s at screening, randomization, and at every visit to document the quantity of LC-omega-
3s consumed. Vitamin E supplementation of greater than 100 IU/day are not allowed in the 3 
months prior to randomization and are prohibited after randomization.  
 
6 
STUDY PROCEDURES 
6.1 Recruitment procedures 
Subjects will be recruited from hepatology clinics that are experienced in diagnosing and 
treating NAFLD and NASH and that have qualified MRI facilities. It is anticipated that sites will 
pre-screen subjects from their existing databases to identify potential subjects for this study. It 
is expected that an advertising and/or social media campaign will be needed also to assist with 
recruitment. Each site will create a recruitment plan in order to meet study recruitment goals. 
6.2 Schedule of assessments 
Visits 
V0 Screening+ 
2nd Screening 
V1+ 
V2 Randomization 
Phone call at 1 
mos3 
V3 3mos 
V4 6mos 
Day** 
-28 to -14 
-21 to -5 
 
30±3 
90 -7 to +3 
180 -7 to +3 
Informed Consent 
X 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
Physical Examination 
X 
 
 
 
X 
 
LIVER FIBROSCAN 
X 
 
 
 
 
 
MRI-PDFF for Liver 
fat 
 
X 
 
 
 
X 
Clinical chemistry* 
X 
ALK-P, AST, 
ALT, Bili only 
 
 
X 
X 
HbA1c and Insulin 
X 
 
 
 
 
X 
Hep A (HAVIgM)# 
X 
 
 
 
 
 

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Hematology* 
X 
 
 
 
CBC 
X 
Coagulation: INR 
X 
 
 
 
 
 
Urine pregnancy test 
(DipStick) 
X 
 
X 
 
 
 
Skinner 
questionnaire2 
X 
 
 
 
 
 
AUDIT questionnaire^ 
X 
 
 
X 
X 
X 
Vital signs 
X 
 
X 
 
X 
X 
Lipid panel* including 
oxidized LDL 
 
X 
 
 
 
X 
Plasma/serum for 
inflammatory 
markers 
 
X 
 
 
 
X 
Anthropometrics 
X 
 
 
 
X 
X 
RANDOMIZATION 
 
 
X 
 
 
 
DHA & α-tocopheryl 
determination and 
oxylipins1 
 
X 
 
 
X 
X 
Product dispense 
 
 
X 
 
 
 
Compliance-Pill 
count 
 
 
 
X 
X 
X 
Adverse event 
reporting 
 
X 
X 
X 
X 
X 
ConMeds 
 
 
X 
X 
X 
X 
FFQ 
X 
 
X 
X 
X 
X 
SF-36 questionnaire 
 
 
X 
 
 
X 
Biosample collection 
–for exploratory 
analyses and future 
research use¶  
 
 
 
X 
 
 
 
X 
* fasted blood draw required and ALT, AST, Alk-P, bilirubin is measured 2x;  
+values obtained at screening (except vital signs and FFQ) will be considered ‘baseline’ values;  
#HAVIgM to be performed unless negative results in past 3 years;  
1oxylipins measured only at V1 and V4. 
2Skinner Lifetime Drinking Questionnaire and AUDIT given at V0 Screening  
3 Reminder phone calls at months 2,4 and 5are advised to assist with retention and check on subject compliance, no 
diet fluctuations, ConMeds, and AEs. Only deviations, ConMeds, and AEs are to be recorded in the eCRF from such 
phone calls. 
** Additional time may be allowed between study visits with approval and subsequent protocol deviation. 
^ Audit questionnaire - Self-report version is collected at V0, V3 and V4 and the interview is only conducted during the 1-
month phone call 
¶ Refer to lab manual for sample collection table - Exploratory analyses include gene expression at V1 and V4 in a sub-
set of 40 subjects, and PNPLA3, HSD17b, haptoglobin genotyping at V1.  
 
6.3 Screening and eligibility 
6.3.1 Obtaining informed consent 
Subjects who are foreseen to fulfil the inclusion / exclusion criteria for enrolment into the 
clinical study will be asked to give informed consent in writing prior to any clinical study specific 
procedures at the screening visit. Both the subject (or legal representative) and investigator will 
sign and date the informed consent form. All subjects who have signed the informed consent 
form will be listed on the Subject Screening and Enrolment Log. 

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6.3.2 Visit 0 – Screening examinations 
The following assessments are done at the screening visit (up to 28 days prior to randomization): 
1. Inclusion/Exclusion criteria 
2. Informed Consent 
3. Medical History 
4. Physical Exam  
5. Liver Fibroscan  
6. Fasted Blood draws (>10 hours) for Clinical Chemistry (including HbA1c, insulin, and 
eGFR/eCreatinine Clearance), Hematology (including Protime and International 
Normalized Ratio (INR)) and Hep A (HAVIgM) test unless negative results obtained in past 
3 years 
7. Skinner Lifetime Drinking Questionnaire 
8. AUDIT Questionnaire – Self-Reported Version  
9. Urine Pregnancy test (for females of child-bearing potential who are not post-menopausal 
or surgically sterilized) 
10. Vital signs (heart rate, blood pressure) 
11. Anthropometrics: body weight [kg], waist-to hip ratio, waist circumference [cm or 
inches],], body mass index (BMI)[kg/m2] 
12. FFQ (<200mg/d DHA and/or<60mg/d EPA to qualify) see Appendix 1 
13. FIB-4 score calculated 
 
6.3.3 Visit 1- 2nd Screening 
A second fasted blood draw (>10 hours) for ALT, AST, Alkaline phosphatase, and Bilirubin will be 
taken to confirm the first lab results at V0. If results exceed more than 50% from first lab 
results, and are above the normal range, then a third test (unscheduled visit) will be repeated 
(only for the elevated test(s)) before V2 to determine the direction of the change. If the third 
test shows a continued increase greater than 25% higher than the second test in ALT Alk-P or 
Bilirubin the subject will be disqualified.  For those that qualify, all values will be recorded on 
the CRF.   
 
All participants will undergo a Fibroscan (≥300db) at the first screening visit, V0 and then, after 
liver enzymes and all other labs, are confirmed to be within eligibility cut-offs, (V1) an 
assessment of liver fat percentage (min. 12%) by MRI-PDFF examination will occur. MRI scans will 
be sent to a Central reader to assess the eligibility for enrolment into the trial. 
 
Fasted blood draws (>10 hours) for Fatty Acid, oxylipins and Vitamin E analyses, Lipid panel, 
PBMC gene expression (in a sub-set of 40 subjects), and Inflammatory markers will occur at V1. 
Blood analysis of PNPLA3, HSD17b and haptoglobin will also occur at Visit 1. 
6.3.4 Assessment of eligibility and randomization procedure 
Based on screening examinations, imaging and lab results, the investigator will assess the 
subject’s eligibility to be entered into the clinical study. On the Subject Screening and 
Enrolment Log, the decision whether to randomize the subject into the clinical study or not is 
documented. For all eligible subjects, values obtained at screening (except vital signs and FFQ) 
will be considered ‘baseline’ values. The vital signs measured at randomization will be 
considered baseline values. The SF-36 and FFQ at randomization will be considered baseline 
values. 
 
The study staff will utilize a computer-generated IWRS to randomize all eligible subjects. 
Subjects will be stratified by Type 2 Diabetes diagnosis (up to 50% of sample). Subject 
information (e.g. visit date, gender, diabetes) will be entered into the IWRS and the subject 

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identifier (SID) and a randomization code will be generated and recorded in the subject source 
documents and the electronic Case Report Form (eCRF). 
6.4 Intervention phase 
6.4.1 Visit 2 Randomization  
The following assessments are done during Randomization: 
1. Urine Pregnancy test (females of child-bearing potential who are not post-menopausal or 
surgically sterilized) 
2. Vital signs 
3. FFQ (<200mg/d DHA and/or <60mg/d EPA to qualify) see Appendix 1 
4. SF-36 Questionnaire (Quality of Life) see Appendix 2 
5. Product dispensation 
6. AEs 
7. Concomitant Medications   
6.4.2 Phone call at one month 
The following assessments are done at the one-month phone call: 
1. AUDIT Questionnaire – Interview Version  
2. Compliance (pill count check, can do by Skype or request photo of blister cards 
consumed) 
3. Adverse Events (including questions related to bleeding and infections) 
4. Con Meds 
5. FFQ 
 
Reminder phone calls at months 2,4 and 5 are advised to assist with retention and check on 
subject compliance, no diet fluctuations, ConMeds, and AEs. Only deviations, ConMeds, and AEs 
are to be recorded in the eCRF from such phone calls. 
6.4.3 Visit 3 (3mos Visit) 
The following assessments are done during the 3 mos Visit: 
1. Physical exam 
2. Vital signs (heart rate, blood pressure) 
3. Anthropometrics 
4. FFQ (see Appendix 1) 
5. Fasted Blood draws (>10 hours) for Clinical chemistry, CBC, Fatty Acid (DHA) and Vitamin 
E analyses 
6. AUDIT Questionnaire – Self- Report Version 
7. AEs 
8. Concomitant Medications   
9. Compliance (pill count- subjects to return all used and unused cards in the kit) 
6.4.4 Visit 4 (6mos Visit) 
The following assessments are done during the 6 mos Visit: 
1. MRI-PDFF 
2. Fasted Blood draws (>10 hours) for Clinical Chemistry (including HbA1c and insulin), 
Hematology, Fatty Acid, oxylipins and Vitamin E analyses, Lipid panel, gene expression 
(sub-set of 40 subjects), and Inflammatory markers 
3. Vital signs (heart rate, blood pressure) 
4. Anthropometrics 
5. FFQ (Appendix 1) 
6. FIB-4 score (calculated) 

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7. AUDIT Questionnaire – Self- Report Version 
8. AEs 
9. Concomitant Medications 
10. Compliance (pill counts- subjects to return all used and unused cards in the kit) 
11. SF-36 Questionnaire (Appendix 2) 
 
7 
KEY MEASUREMENTS AND ASSESSMENTS 
7.1 Blood samples 
7.1.1 Blood sample collection 
Fasted (>10 hours) plasma and serum samples will be collected from each subject at Screening 
V0, second Screening V1, Visit 3, and Visit 4. 
7.1.2 Blood sample storage and shipment 
Whole blood (between 10-25 ml, depending on the visit) will be drawn and processed at the 
clinical site, according to a central lab manual. Plasma and serum or whole blood will be shipped 
either ambient or frozen on dry ice to the central lab the day it is drawn and should be received 
within 24 hours at the central lab. 
7.1.3 Blood sample assessment 
Clinical chemistry: Glucose, HbA1c, ALT, AST, GGT, ALK-P, eGFR/creatinine clearance, 
creatinine, insulin, bilirubin, (note: for ALT and AST reference ranges for HH flags are to be 
used to determine clinical significance (CS)) 
Lipid panel: total cholesterol, HDL-cholesterol, LDL-cholesterol, oxidized LDL, TGs; 
Hematology: standard panel including complete blood count with automated differential 
including platelets, and coagulation panel INR, and Hep A (HAVIgM) test at screening, and 
Inflammatory markers (e.g. CK-18, TNFα, IL-6) will be analysed by a central lab. 
 
Fatty acid analyses and analyses of LC-PUFA-derived oxylipins will be performed using plasma 
samples at the DSM Clinical Laboratory, 6480 Dobbin Rd. Columbia MD 21045. Approximately 1.5 
mL of plasma/subject/time point (V1, V3, V4) will be aliquoted and shipped frozen on dry ice in 
batches from the central lab to DSM. 
 
500uL plasma for α-tocopheryl measurement (from V1, V3, V4) will be shipped frozen on dry ice 
in batches from the central lab to DSM Nutritional Products: 
 
DNP R&D Analytics, NIC-RD/A 
Sample Registration Office, 
c/o Warenannahme, Bldg. 214 
Wurmisweg 576 
CH-4303 Kaiseraugst / Switzerland 
Phone: + 41 (0) 61 815 8629 
Fax: + 41 (0) 61 815 7441 
 
Transcriptome analysis of Gene Expression: in a subset of 40 subjects (~10 subjects/treatment 
arm), whole blood (~2ml) will be collected in PAXgene blood RNA tubes. Samples will be 
collected at Screening V1 and at Visit 4 and will be shipped frozen using dry ice along with other 
blood samples to the central lab the day it is drawn. Samples will be batched shipped frozen 
from the central lab to DSM Nutritional Products, Kaiseraugst, Switzerland for analysis. 

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Genotype analyses 
An aliquot of whole blood (0.5ml) from each subject will be used for DNA extraction to identify 
those with single-nucleotide polymorphisms (SNP) i.e. PNPLA3, HSD17b and haptoglobin. Samples 
will be collected at Screening V1 and will be shipped frozen using dry ice along with other blood 
samples to the central lab the day it is drawn. Samples will be batched shipped frozen from the 
central lab to Indiana University for analysis. 
 
An additional 1 ml plasma (aliquoted into [2] 0.5ml tubes) will also be collected at V1 and V4, 
shipped frozen on dry ice to central lab and stored for future NAFLD biomarker use. 
7.2 MRI-PDFF 
7.2.1 Assessment of liver fat percentage by MRI-PDFF  
All subjects will undergo MRI-PDFF to determine eligibility for the trial, after all the V0 and V1 
screening procedures have been completed to confirm eligibility. A minimum 12% liver fat is 
required for trial eligibility. One standard MRI protocol will be used at all sites and one blinded 
central MRI reader will analyse all MRI assessments. A study-specific MRI-PDFF protocol will be 
developed for all participating sites. Changes in liver fat % assessed by MRI-PDFF will be 
measured in localized regions of interest (ROI) within each of the liver segments. MRI-PDFF will 
also be performed at Visit 4 (6 mos) using the same procedures. 
 
7.3 Other key assessments 
7.3.1 Liver Fibroscan 
Transient elastography (Fibroscan), a rapid measure of liver tissue stiffness, will be used at Visit 
0 to assess for eligibility in the trial and will be performed prior to MRI-PDFF. Qualifying cut off 
score on Fibroscan is CAP ≥300db. If the subject meets this cut off, then the MRI-PDFF is 
performed at the second screening visit.  
7.3.2 Liver Fibrosis score 
Fibrosis-4 score (FIB-4) predicts changes in fibrosis with high specificity. The FIB-4 score is 
calculated as Age x AST/platelet count x √ALT. The FIB-4 score will be calculated in the eCRF at 
Visit 0 and Visit 4. 
7.3.3 Anthropometrics 
Height, body weight, waist and hip circumference will be measured at Visit 0, 3 and 4 at the 
site. Waist to hip ratio and BMI will be calculated. Sites are advised to counsel the subjects to 
avoid major fluctuations in diet and exercise during the study. 
 
7.3.4 Alcohol Consumption 
At Visit 0, subjects will provide their typical weekly alcohol consumption as part of medical 
history to confirm eligibility requirements (>21 drinks per week in men and >14 drinks per week 
in women is exclusionary).  Additionally, the Skinner lifetime drinking history questionnaire will 
be administered via a structured interview and subjects will be provided the AUDIT test – Self 
Report Version questionnaire to complete (with cards to remind study participants of drink 
equivalents) in order to determine baseline alcohol consumption history/patterns.    
 

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Following randomization, the AUDIT test will be administered at 3 additional time points during 
the study to monitor alcohol consumption patterns.  During the one-month phone call, the AUDIT 
test – Interview Version will be administered by study staff.  At Visits 3 and 4, the AUDIT test – 
Self Report Version will be provided to the subject to complete in-clinic. 
 
Data from the AUDIT questionnaire will be used to assess whether alcohol consumption modified 
the treatment response. 
 
7.3.5 Urine sample collection and storage 
For all females of child-bearing potential, a urine pregnancy test (dipstick) will be conducted at 
the site at both Screening (Visit 0) and Randomization (Visit 2) to determine eligibility in the 
trial. A positive pregnancy test during screening or at randomization is exclusionary for trial 
eligibility. Negative results will be recorded in the eCRF. 
7.3.6 Questionnaires 
The Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36), a quality of life 
assessment will be administered to subjects at Visit 2 and 4. See Appendix 2. 
 
A validated FFQ [35] to measure LC-PUFA dietary intake (namely DHA and EPA) will be 
administered at visits V0, V2, phone call at 1mos, V3 and V4. See Appendix 1. 
 
8 
EARLY SUBJECT WITHDRAWAL 
Subjects have the right to withdraw from the study at any time for any reason, without being 
obliged to give reasons and without penalty or loss of benefits to which they are entitled. The 
investigator also has the right to withdraw subjects from the study if this is in the best interest 
of the subject. 
 
Any of the following conditions leads to premature withdrawal: 
• 
request by subject to discontinue for any reason during the study (withdrawal of consent) 
• 
erroneously included / randomized subject 
• 
adverse event or concurrent illness that, in the opinion of the investigator, warrants the 
subject’s withdrawal from intervention 
• 
intake of concomitant medications/dietary supplements prohibited by the protocol 
• 
volunteers who do not follow the requirements of the investigator, especially those 
concerning safety and/or if the subject, after his enrolment is uncooperative or not 
willing to comply with the protocol (non-compliant) 
• 
failure to return (lost to follow-up) 
• 
request by the Sponsor or regulatory agencies for termination of supplementation of an 
individual patient or all patients under this protocol 
• 
pregnancy, i.e. a protocol exclusion criterion 
 
The circumstances of any discontinuation must be documented in detail in the corresponding 
study disposition form in the eCRF. Whenever a subject is withdrawn from the study for 
whatever reason, every effort will be made to perform a final assessment to include standard 
medical and laboratory tests (including a final MRI, if possible) which would have been done 
upon normal completion of the study, i.e. V4. The data of the withdrawn subjects will be 
documented in the clinical study report and may be used in statistical analyses. 
 
Note: Subjects who withdraw prematurely from the study will not be replaced. 

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9 
DATA PROCESSING AND STATISTICAL CONSIDERATIONS 
9.1 Case Report Form 
9.1.1 Electronic Data Capture 
 
It is the responsibility of the investigator(s) to ensure the completeness and accuracy of the 
eCRF. One eCRF must be compiled for each subject participating in the study.  
 
Data required for the analysis will be acquired and transferred electronically to a central 
database by means of an Electronic Data Capture system (the “EDC-tool”). The system operates 
over the Internet according to the principle of online data capture. Data entered by 
investigators or data managers into local computers are directly transferred over the Internet to 
a central database, without any permanent local storage. 
 
High security standards for the transfer and storage of study data are guaranteed by the use of 
technologies, such as encrypted data transfer, firewalls and periodic backup to protect centrally 
stored data. Writing access to the system will be limited to authorized personnel and the system 
will automatically keep an audit trail of all entries and corrections to the eCRF. Training in the 
use of the EDC-tool will be provided by the CRO and local monitor. 
 
The EDC-tool will comprise an eCRF, designed specifically for the present study.  
 
The EDC-tool is operated under an electronic signature, consisting of the combination of an 
identification code and a password. In relation to the present study, passwords and electronic 
signatures will be distributed to users of the EDC tool, and used for entering, modifying, or 
viewing study data. 
 
It is the responsibility of individuals who receive an electronic signature to keep their password 
secret, i.e., not to reveal it to third parties, and to access the EDC-tool, to enter or modify 
study data using the EDC-tool only under their personal identification code and password. 
9.2 Data Management 
The CRO is responsible for the data management of the study. The CRO will design the eCRF 
with the PI, conduct quality control and review of the eCRF, and create a Data Management 
Plan, including programming and validation of the database, user training, data handling and 
edit checks, data cleaning and query generation and processing, medical coding, SAE 
reconciliation and database closure. Key site and CRO personnel will be trained on the eCRF and 
the EDC system.  
 
9.3 Statistical Analysis 
 
9.3.1 Statistical hypotheses and methods 
The primary hypothesis of the study is that Vitamin E and Omega-3 fatty acid (DHA EE) in 
combination will reduce hepatic fat fraction greater than placebo after 6 months of intervention 
in adults with NAFLD. 
 

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In general, continuous variables will be summarized with the mean, standard deviation, median 
and range. Categorical variables will be summarized with counts and percentages. SAS® version 
9.4 or higher will be used to analyse the data. 
 
9.3.2 Primary endpoint analysis 
Decrease in hepatic fat fraction [%] relative to baseline between Vitamin E + DHA EE 
combination vs. placebo, measured by MRI-PDFF after 6 months of intervention.  The decrease 
will be analysed with a linear mixed model with the hepatic fat fraction in % at 6 months as 
response variable. The hepatic fat fraction in % at baseline, treatment group and diabetes status 
will be included as fixed effects. Site will be included as random effect. This representation is 
formally identical to an analysis that uses the change in hepatic fat fraction in % (6 mos vs. 
baseline) as response variable and the hepatic fat fraction in % at baseline, treatment group and 
diabetes status as fixed effects and site as random effect [Laird, N. Further comparative 
analyses of pre-test post-test research designs. The American Statistician 1983, 37: 329-330; 
O’Connell, N. Methods for Analysis of Pre-Post Data in Clinical Research: A Comparison of Five 
Common Methods. Journal of Biometrics and Biostatistics, 2017. 8(1):1-8.]. However, this 
representation is chosen since it allows for easier variance-stabilizing transformations if 
necessary. 
 
  
Statistical tests of significance will be 2-tailed at a 5% significance level. 
With blinded data, the residuals of the model will be analysed visually (QQ-plots, residual plots) 
to determine if the model assumptions (normality, variance, homogeneity) are met. If 
necessary, variance-stabilizing transformations will be considered. 
 
9.3.3 Secondary endpoint variable(s) and analyses 
o Differences in hepatic fat fraction [%], measured by MRI-PDFF after 6 months of intervention, 
between DHA and placebo will be estimated with the same model as the primary endpoint. 
The estimated difference will be displayed with its 95% confidence interval. 
o Differences in hepatic fat fraction [%], measured by MRI-PDFF after 6 months of intervention, 
between Vitamin E and placebo will be estimated with the same model as the primary 
endpoint. The estimated difference will be displayed with its 95% confidence interval.  
o Between group differences in anthropometric measures, after 3 and 6 months of DHA EE 
and/or Vitamin E intervention will be analysed with the same approach as the primary 
endpoint, using the relevant anthropometric measure at baseline as the respective baseline 
covariate and gender as a categorical covariate.  
o Between group differences in inflammatory markers after 6 months of intervention and liver 
enzymes after 3 and 6 months of DHA EE and/or Vitamin E intervention will be analysed with 
the same approach as above and diabetes status as a categorical covariate included in the 
model. 
o Between group differences in the FIB-4 score after 6 months of DHA EE and/or Vitamin E 
intervention will be analysed with the same approach as above. 
o Between group differences in plasma DHA and plasma α-tocopheryl after 3 and 6 months of 
intervention will be analysed with the same approach as above.  
o Between group differences in SF-36 quality of life score after 6 months of DHA EE and/or 
Vitamin E intervention will be analyzed by ANCOVA with baseline score included in the 
model. 
o Between group differences in dietary intake of LC-PUFA (i.e. DHA and EPA) will be assessed 
by the FFQ and analysed over time by repeated measures ANOVA with baseline level and site 
included in the model. 
 

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With blinded data, the residuals of the models will be analysed visually (QQ-plots, residual 
plots) to determine if the model assumptions (normality, variance homogeneity) are met. If 
necessary, variance-stabilizing transformations or non-parametric methods will be 
considered. 
 
9.3.4 Exploratory variables 
Transcriptome analysis of gene expression will be performed in peripheral blood mononuclear 
cells in a subset of (n=40) subjects (~10 subjects/ treatment arm). Changes in gene expression 
from baseline to end of treatment (6 months) will be examined. The analyses will be conducted 
by DSM. Briefly, differentially expressed genes will be analysed by ANOVA. The false discovery 
rate (q values) will be used to correct for multiple testing. Using the global gene expression 
profiling results, an extensive pathway analysis (e.g. MetaCore program and/or Ingenuity 
Pathway Analysis) will be conducted to better understand gene regulation, mechanisms of action 
and activated pathways by the various treatments in this NAFLD population. 
 
Between group differences in plasma LC-PUFA-derived oxylipins will be analysed by ANCOVA 
using the relevant baseline oxylipin level as a covariate. 
 
Sub-set analyses of PNPLA3, HSD17b and haptoglobin SNPs will be conducted on the Primary 
endpoint to assess potential genotypic responses to treatment. 
 
9.3.5 Safety variables 
All SAEs and AEs will be summarized by group. The number of subjects experiencing any events 
will be examined and the frequency of occurrence by system organ class will be tabulated using 
Medical Dictionary for Drug Regulatory Affairs (MedDRA) codes. 
 
Vital signs, Hematology, Lipid panel, and Clinical Chemistry data will be summarized using 
descriptive statistics by group per timepoint including change from baseline at 6 months with 
group differences analysed using Least Squares Means and 95% Confidence Intervals. 
 
A sub-set analysis will be performed in those with elevated baseline TGs to assess changes with 
the intervention over 6 months. 
 
 
9.3.6 Compliance Analysis 
A compliance analysis will be performed on all randomized participants who completed the 
study (6 mos) and ingested at least 80% of the study product using plasma DHA levels (µg/ml) 
and plasma α-tocopheryl (µmol/L) to confirm accuracy of the pill counts. ANOVA models will be 
employed with treatment as a factor. Frequency counts and percentages will also be presented 
for subjects who were at least 80% compliant by treatment group. Differences in this categorical 
outcome will be assessed using Fisher's Exact Test. 
 
9.3.7 Sample size:  
Le et al.[1] report that the variability (standard deviation) of the within-subject change in liver 
fat [%] for a period of 24 months in a similar population and with the same measurement method 
(MRI-PDFF) is 7%. Expecting a within-subject 3.64% absolute reduction in liver fat for the 
combination group vs. placebo [1, 2] and setting α=0.05 and 80% power, n=60/arm for the 
combination and placebo groups are needed (G*power 3.1.9.2, two-sample t-test).  
 

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To estimate the secondary endpoints (effect vs. placebo of the components (DHA EE alone and 
Vitamin E alone)) with a given precision (confidence interval width) of ±3% of liver fat, we need 
n=30/arm for the DHA EE only and Vitamin E only groups, leading to a necessary sample size of 
180. With 10% expected attrition, the total sample size is 200 subjects. 
 
Subjects will be stratified by Type II Diabetes diagnosis (no greater than 50% of sample).  
Subjects who withdraw prematurely from the study will not be replaced. 
 
 
9.3.8 Study Populations 
o The Intent-to-Treat (ITT) population will include all randomized subjects who receive at 
least one dose of study product, according to the product to which they were randomized. 
The primary efficacy analysis will be on the ITT population.  
o  The Per-Protocol (PP) population will include subjects who completed the study according to 
the protocol. The subject’s data will be excluded from the per protocol analyses if the 
subject fails to meet one or more of the inclusion criteria at enrollment or meets one or 
more of the exclusion criteria. Data will be excluded if the subject has other significant 
protocol deviations or significant non-compliance with taking the study product (to be 
determined at a blinded data review meeting). 
o  The Safety population will include all subjects randomized into the study who received at 
least one dose of study product, according to the product they actually received. 
 
9.3.9 Poolability of Data 
It is considered that data will be pooled across sites for all analyses, unless significant deviations 
or imbalance in one or more sites is identified. Such discrepancies will be reviewed at the 
blinded data review meeting. Heterogeneity of the variability across sites will be investigated, 
and assumptions regarding the homogeneity of the variability across sites will be relaxed in the 
applied statistical model if necessary. 
 
A statistical analysis plan (SAP) will be written by the statistician of the trial and will be 
finalized prior to database closure. The SAP will detail the handling of missing data and will 
outline all analyses to be conducted.  
10 
SAFETY  
10.1 
Definitions and Standards 
 
Adverse Event (AE): 
Any untoward medical occurrence in a subject involved in a clinical study administered an 
investigational product, whether or not related to this product.  
 
An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory 
finding for example), subjective and objective symptom, or disease temporally associated with 
the use of a product, accidents, and whether or not considered related to the product or study-
related procedure and reported by the subject or observed by the investigator. 
 
Adverse Reaction (AR): 

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All noxious and unintended responses to a product related to any dose should be considered as 
AR. ARs are a subset of all suspected AEs for which there is reason to conclude that the 
investigational product caused the event. 
 
Suspected Adverse Reaction (SAR) 
SAR means any AE for which there is a reasonable possibility that the product or clinical trial 
material caused the AE. A SAR implies a lesser degree of certainty about causality than an AR, 
which means any AE caused by an investigational product. 
 
Serious Adverse Event (SAE): 
Any AE that at any dose fulfils at least one of the following criteria: 
 
• 
Is fatal (results in death)  
(note: death is an outcome, not an event) 
• 
is life-threatening  
(note: the term “life-threatening” refers to an event in which the subject was at risk of 
death at the time of the event; it does not refer to an event which could hypothetically 
have caused death had it been more severe) 
• 
requires inpatient hospitalization or prolongation of existing hospitalization  
(note: “inpatient hospitalization” refers to an unplanned, overnight hospitalization) 
• 
results in persistent or significant disability/incapacity 
(note: the term means substantial disruption of one’s ability to conduct normal life 
function) 
• 
is a congenital anomaly/birth defect 
(note: congenital anomaly/birth defect in offspring of subject taking the product 
regardless of time to diagnosis) 
• 
is medically significant  
(note: Medical and scientific judgement should be exercised in deciding whether 
expedited reporting is appropriate in other situations, such as important medical events 
that may not be immediately life-threatening or result in death or hospitalisation but 
may jeopardise the subject or may require intervention to prevent one of the other 
outcomes listed in the definitions above). 
 
Suspected Unexpected Serious Adverse Reaction (SUSAR): 
All suspected adverse reactions that are both unexpected and serious.  
 
Unexpected Adverse Reaction: 
An adverse reaction, the nature, or severity or incidence of which is not consistent with the 
applicable product information (e.g. Investigator’s Brochure). 
10.2 
Adverse event assessment 
Expectedness: 
An unexpected AE is an event of which the nature or severity is not consistent with the 
applicable product information. 
 
 
 
Causality Assessment: 
The causality assessment of an AE to the investigational product will be rated as follows: 
 

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 No (Not related): The temporal relationship of the clinical event to product 
administration makes a causal relationship unlikely, or other 
drugs, therapeutic interventions or underlying conditions 
provide a sufficient explanation for the observed event. 
 Yes (Related): 
The temporal relationship of the clinical event to product 
administration makes a causal relationship possible, and other 
drugs, therapeutic interventions or underlying conditions do not 
provide a sufficient explanation for the observed event. 
 Suspected 
There is a reasonable possibility that the investigational 
product caused the observed event. Suspected implies a lesser 
degree of certainty about the causality of the observed event 
 
Severity / Intensity: 
The severity / intensity of AEs will be graded on a five-point-scale: 
- 
Mild or Grade 1: discomfort noted, but no disruption to normal daily activities. 
- 
Moderate or Grade 2: discomfort sufficient to reduce or affect normal daily activities. 
- 
Severe or Grade 3: inability to work or perform normal daily activities. 
- 
Life threatening or Grade 4  
- 
Death or Grade 5 
 
Outcome of event: 
The outcome of an event will be classified as follows: 
- 
Recovered 
- 
Recovered with sequelae 
- 
Ongoing 
- 
Fatal 
- 
Unknown / Lost to follow-up 
10.3 
Safety Management 
 
10.3.1 Procedures 
All AEs occurring during clinical studies are recorded in the eCRF and are classified according to 
the Common Terminology for Adverse Events (CTCAE) system. AEs should be captured from the 
screening visit (V1) to the end of the study.  Refer to the 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_R
eference_8.5x11.pdf and https://livertox.nih.gov/Severity.html, for additional information.  
 
CTCAE Terms  
An Adverse Event (AE) is any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medical treatment or 
procedure that may or may not be considered related to the medical treatment or procedure. An 
AE is a term that is a unique representation of a specific event used for medical documentation 
and scientific analyses. Each CTCAE v4.0 term is a MedDRA LLT (Lowest Level Term). 
 
CTCAE Grades  
Grade refers to the severity of the AE. The CTCAE displays Grades 1 through 5 with unique 
clinical descriptions of severity for each AE based on this general guideline:  
 

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Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
 
Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental ADL*.  
 
Grade 3 Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL**.  
 
Grade 4 Life-threatening consequences; urgent intervention indicated.  
 
Grade 5 Death related to AE. Grade 5 (Death) is not appropriate for some AEs and 
therefore is not an option. 
 
A Semi-colon indicates ‘or’ within the description of the grade.  
A single dash (-) indicates a Grade is not available. Not all Grades are appropriate for all 
AEs. Therefore, some AEs are listed with fewer than five options for Grade selection. 
 
Definitions  
A brief Definition is provided to clarify the meaning of each AE term. A single dash (-) 
indicates a Definition is not available.  
 
Navigational Notes  
A Navigational Note is used to assist the reporter in choosing a correct AE. It may list 
other AEs that should be considered in addition to or in place of the AE in question. A 
single dash (-) indicates a Navigational Note has not been defined for the AE term.  
 
Activities of Daily Living (ADL)  
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc.  
**Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, 
taking medications, and not bedridden. 
 
SAEs are reported in the eCRF and processed according to the applicable FDA requirements 
governing the conduct of clinical studies. All SAEs will be followed until resolution or at the 
latest 30 days after the subject’s completion in the study. 
 
SUSARs are reported and processed according to the FDA requirements governing the conduct of 
clinical studies.  
 
When an unexpected SAE not previously listed in the Investigator’s Brochure (IB) occurs in a 
multi-center clinical study, all investigational sites currently involved in the study should be 
notified by the study Sponsor or medical monitor within one week of receipt of the unexpected 
SAE. 
 
SAE and SUSARs are also reported to a third-party vendor (referenced as Pharmacovigilance 
Vendor (UBC)) per flowchart below.  
 
SAEs and SUSARs must be recorded on the electronic SAE Form. Following case processing, a US 
FDA MedWatch Form 3500A will be generated from the Pharmacovigilance Vendor for SUSARs. 
The event is entered into the PV safety database for filing and further expedited reporting by 
IND Sponsor-Principal Investigator. 
 

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The directions for completing the SAE Form are detailed in the “CRF Guidelines”. This guideline 
will be available to all sites in the study. 
 
For SUSARs, study treatment may be unblinded prior to reporting the case to the applicable 
regulatory authorities and IRB/IEC. Please see section 4.4 for unblinding procedures. 
 
A Data Safety Monitoring Board (DSMB) will be arranged to review the safety information in the 
trial on a bi-annual basis. Details of the DSMB’s responsibilities will be outlined in a DSMB plan. 
 
SAEs will be reviewed by the safety officer of the study and the DSMB. 
 
During or at the end of each study, an SAE reconciliation between data entered in the study 
database and the data entered in the pharmacovigilance database will be performed if any SAE 
has occurred in the study. All discrepancies found during the reconciliation are documented in an 
SAE reconciliation report and corresponding queries are sent to the investigators for clarification. 
 
Since pregnancy is an exclusion criterion in the study, then for any case of pregnancy occurring 
during the conduct of the study, the course of the pregnancy and the outcome of both the 
mother and the offspring have to be documented in the eCRF and on the Pregnancy Form. This 
form will be provided to sites in the study, if needed. 
 
The directions for completing the Pregnancy Form are detailed in the “Guidelines for Completing 
the Pregnancy Form”. This guideline will be provided to sites in the study when needed. 
 
 
 
 
 
10.3.2 Treatment and Holding/Stopping Rules for Drug Induced Liver Injury (DILI) 
 
  
See the chart below for DILI monitoring and stopping guidelines: 
 

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Treatment Emergent 
ALT Elevation 
Treatment 
Emergent Total 
Bilirubin 
Liver 
Related 
Symptoms 
Management 
Recommendation 
Normal/near normal 
baseline: 
  ALT ≥ 5 ×ULN 
 
Elevated baseline: 
 ALT >3x baseline or 
>300 U/L 
 
whichever occurs first 
Normal  
 
Patients with Gilbert's 
syndrome: 
   No change in 
baseline total bilirubin 
None 
• 
Repeat ALT, AST, 
ALP, TBL, in 2-5 
days.  
• 
Follow-up for 
symptoms 
• 
Initiate evaluation for 
other etiologies of 
abnormal liver tests  
Normal/near normal 
baseline: 
  ALT ≥ 3 ×ULN 
 
Elevated baseline: 
  ALT >2x baseline or 
>300 U/L 
 
whichever occurs first 
Normal  
 
Patients with Gilbert's 
syndrome: 
     No change in 
baseline total bilirubin 
Severe 
fatigue, 
nausea, 
vomiting, 
right upper 
quadrant 
pain, 
tenderness, 
fever, rash, 
and/or 
eosinophilia 
(> 5%) 
• 
Repeat ALT, AST, 
ALP, TBL, in 2-5 
days.  
• 
Follow-up for 
symptoms 
• 
Initiate evaluation for 
other etiologies of 
abnormal liver tests 
Normal/near normal 
baseline: 
  ALT ≥ ×8 ULN 
 
Elevated baseline: 
  ALT >5x baseline or     
>500 U/L 
 
whichever occurs first 
Normal  
 
Patients with Gilbert's 
syndrome: 
     No change in 
baseline total   
bilirubin 
None 
• 
Interrupt study drug 
• 
Initiate close 
monitoring and 
workup for 
competing etiologies 
• 
Study drug can 
be restarted only 
if another etiology 
is identified and 
liver enzymes 
return to baseline.  
 
Normal/near normal 
baseline: 
  ALT ≥ ×3 ULN 
 
Elevated baseline: 
  ALT >2x baseline or 
>300 U/L 
 
whichever occurs first 
TBL >2x ULN 
 
Patients with 
Gilberts syndrome: 
   Doubling of direct 
bilirubin or    
increased INR to 
>1.5 
Severe 
fatigue, 
nausea, 
vomiting, 
right upper 
quadrant 
pain, 
tenderness, 
fever, rash, 
and/or 
eosinophilia 
(> 5%) 
• 
Interrupt study drug 
• 
Initiate close 
monitoring and 
workup for 
competing etiologies 
• 
Study drug can 
be restarted only 
if another etiology 
is identified and 
abnormalities 
return to baseline.  
 
Normal/near normal 
baseline: 
  ALT ≥ ×5 ULN 
 
Elevated baseline: 
  ALT >3x baseline or 
>300 U/L 
Normal or Elevated 
Severe 
fatigue, 
nausea, 
vomiting, 
right upper 
quadrant 
• 
Interrupt study drug 
• 
Initiate close 
monitoring and 
workup for 
competing etiologies 
• 
Study drug can 
be restarted only 

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(whichever occurs 
first) 
pain, 
tenderness, 
fever, rash, 
and/or 
eosinophilia 
(> 5%) 
if another etiology 
is identified and 
abnormalities 
return to baseline.  
 
Source: Modified from Table 1 in REGEV et al, 2019 [36] 
 
 
* If a patent lives in a remote area, laboratory testing can be performed locally, and the 
results should be promptly communicated to the investigator site 
 
 
Study Stopping Rules: 
 
The DSMB will review clinical safety data on an ongoing basis to identify potential safety issues 
arising during the conduct of the study. Including but not limited to the following: 
 
1. 1 subject receiving the active treatment experiences a grade 5 CTCAE during the 
clinical trial that it is likely attributable to the study drug(s), OR 
2. 2 or more subjects receiving the active treatment experience a grade 4 CTCAEs 
 likely attributable to the study drug(s), OR 
3. 3 or more subjects receiving the active treatment experience a grade 3 CTCAEs 
 likely attributable to the study drug(s). 
 
10.3.3 Responsibilities 
 
• The Principal Investigator (PI) ensures that SAE reporting procedures outlined in the study 
protocol are adhered to and that all required documentation is up-to-date and that regulatory 
and IRB/IEC SAE reporting procedures are followed. 
• All study personnel at the study sites (Study Coordinator, Study Nurse, PI, or designee) who 
are in contact with clinical trial subjects are responsible for collecting AE information from 
the subject at each scheduled site visit or during telephone calls with the subject. Therefore, 
clinical research personnel or delegates (e.g. CRO) who initiate and monitor the study are 
responsible for explaining the procedures for reporting and evaluating AEs to the PI, and all 
study personnel who will be in contact with the subjects/patients.  
• During the course of the study complete reports of all AEs should be entered in the 
subject’s/patient’s site source documents, and on the appropriate study case report forms 
(CRFs). 
• A physician is responsible for: identifying and evaluating the severity (mild, moderate, or 
severe) and clinical importance of the AE, taking appropriate medical action(s), and for 
notifying a clinical research monitor, CRO and PV vendor immediately of an SAE, as specified 
in the protocol, and also for notifying the IRB/IEC. A copy of the source documents and 
related records should be supplied with the SAE report to the PV Vendor. 
• Likewise, a physician indicates the causality (relationship) of the AE to the study product. 
• For any laboratory abnormality, the PI will make a judgement as to its clinical significance. If 
the laboratory value is thought to be clinically significant, the IND Sponsor or medical monitor 
may be consulted, and an assessment will be made by the PI, as to its relationship to 
investigational product administration, and it will be documented on the AE page of the 
eCRF.  

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• The PI should comply with applicable regulatory requirement(s) related to the reporting of 
SAEs to the regulatory authority(ies) and the IRB /IEC. 
• At completion of the study, the final lists of AEs and SAEs will be MedDRA coded by the CRO 
or PV vendor. 
• The Study Protocol defines AEs (including SAEs) and instructs the PI and study personnel to 
evaluate and record the occurrence of all AEs during the study. The AEs should be recorded in 
the patients’ site source documents and on the appropriate AE page of the eCRF. The 
protocol defines the circumstances under which the blind may be broken and by whom when 
unblinding is deemed necessary for evaluating and managing a particular SAE or SUSAR. The 
Study Protocol also defines the follow-up period for SAEs. 
• The Monitor(s) should review all completed CRF data and should compare CRF entries with 
information recorded in the source documents. Any discrepancies or omissions in either data 
source should be discussed with the site personnel who should make the appropriate 
corrections to the documents. Any recorded SAE or SUSAR which has not been reported to 
CRO or PV Vendor should be discussed with the PI and should be reported immediately to the 
CRO or PV Vendor specified in the protocol, and also should be reported by the PI to the 
IRB/IEC.  
 
11 
ETHICAL CONSIDERATIONS 
11.1 
Local Regulations and Declaration of Helsinki 
This clinical study will be conducted in compliance with the ethical principles that have their 
origin in the Declaration of Helsinki, Title 21 of the Code of Federal Regulations §§ 50, 54, 56, 
and 312, and International Conference on Harmonisation E6, Good Clinical Practice. 
  
11.2 
GCP Management Directive  
 
The Principal Investigator will ensure that this clinical study is conducted in conformance with 
the principles of GCP, as outlined below:  
 
1. Clinical studies should be conducted in accordance with the ethical principles that have 
their origin in the Declaration of Helsinki, and that are consistent with the principles of 
GCP (where applicable) and the applicable laws and regulatory requirements governing 
the conduct of human studies;  
2. Foreseeable risks and inconveniences should be weighed against the anticipated benefit 
for the individual trial subject and the society; 
3. The rights, safety, and well-being of the subjects involved in clinical studies are the most 
important considerations and should prevail over interests of science and society; 
4. The available nonclinical and clinical information on an investigational product should be 
adequate to support the proposed clinical study; 
5. Clinical studies should be scientifically sound, and described in a clear, detailed protocol; 
6. Clinical studies should be conducted in compliance with the protocol that has received 
prior institutional review board (IRB)/independent ethics committee (IEC) 
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7. The medical care given to, and medical decisions made on behalf of, subjects involved in 
clinical studies should always be the responsibility of a qualified physician; 
8. Each individual involved in conducting clinical studies should be qualified by education, 
training, and experience to perform his or her respective task(s); 
9. Freely given written informed consent should be obtained from every subject prior to 
participation in a clinical study; 
10. All information from clinical studies should be recorded, handled, and stored in a way 
that allows its accurate reporting, interpretation and verification;  
11. The confidentiality of records that could identify subjects should be protected, 
respecting the privacy and confidentiality rules in accordance with the applicable laws 
and regulatory requirements; 
12. Investigational products should be manufactured, handled, and stored in accordance with 
DNP quality standards, or as applicable, good manufacturing practice (GMP); IP(s) should 
be used in accordance with the approved protocol; 
13. Systems with processes and procedures (such as SOPs) that assure the quality of every 
aspect of clinical studies should be implemented. 
11.3 
Informed Consent 
It is the responsibility of the investigator, or a person designated by the investigator (if 
acceptable by local regulations), to obtain written informed consent from each subject 
participating in this clinical study, or from the subject’s legally acceptable representative, after 
adequate explanation of the aims, methods, anticipated benefits, and potential hazards of the 
study. No study procedures may be performed prior to obtaining the informed consent. The 
investigator or designee must also explain that the subjects are completely free to refuse to 
enter the study or to withdraw from it at any time, for any reason.  
11.4 
IRB/IEC approval 
This protocol and any accompanying material provided to the subject (such as subject 
information sheets or descriptions of the study used to obtain informed consent) as well as any 
advertising or compensation given to the subjects, will be submitted by the investigators to an 
IRB/IEC. Approval from the IRB/IEC will be obtained before starting the study and will be 
documented in a letter to the investigators specifying the date on which the IRB/IEC met and 
granted the approval. 
 
Any modifications (amendments) made to the protocol after receipt of the IRB/IEC approval 
must also be submitted by the investigators to the IRB/IEC in accordance with local procedures 
and regulatory requirements. 
 
11.4.1 Risk-benefit assessment 
Oral vitamin E intake at the dose in the study is safe, as confirmed by the US Food and Nutrition 
Board of the Institute of Medicine (IOM). The DHA dose is also safe and below the GRAS 
affirmation level. There are no approved treatments for NAFLD. Minimal risks such as bruising at 
the blood draw site or gastrointestinal effects (e.g. gassiness, reflux) are anticipated in this 
study. 

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11.5 
Confidentiality 
11.5.1 Data 
All information regarding the nature of the proposed investigation provided by the Sponsor or 
CRO to the Investigator (with the exception of information required by law or regulations to be 
disclosed to the IRB/IEC, the subject, or the appropriate regulatory authority) must be kept in 
confidence by the Investigator in accordance with all applicable laws and regulations as 
specified in the Clinical Study Agreement.  
11.5.2 Subject Anonymity 
The anonymity of participating subjects must be maintained. Subjects will be identified by an 
assigned subject number (and optionally including their initials) on CRFs and other documents 
submitted to the CRO. Documents that will not be submitted to the CRO and that identify the 
subject (e.g., the signed informed consent document) must be maintained in strict confidence 
by the Investigator, except to the extent necessary to allow auditing by the appropriate 
regulatory authority, the Sponsor or its designee and in accordance with applicable regulatory 
requirements. 
11.6 
Subjects’ compensation / remuneration 
Minimal compensation for transportation costs or a meal may be provided to the subjects to 
facilitate attending the scheduled study visits and a small stipend may be awarded upon 
completion of scheduled study visits.  
11.7 
Registration of study in a public clinical trial database 
This study will be registered at http://clinicaltrials.gov - 
https://clinicaltrials.gov/ct2/show/NCT04198805 
12 
STUDY DOCUMENTATION AND RECORD KEEPING  
12.1 
Protocol amendments 
Any modification to the agreed protocol must be approved in writing by the IND Sponsor-
Principal Investigator. Amendments will be submitted to the IRB/IEC and FDA, as required. 
These procedures must be fulfilled before any modification is put into effect.  
Note: Sites must update the Protocol Amendments Tracking Form Log for each amendment. 
12.2 
Investigator Site File 
The Investigators must maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These documents 
should be classified into two different separate categories (1) Investigator's Site File (ISF), and 
(2) source documents. 
 
1. The ISF will contain the protocol/amendments, CRFs and Query Forms, IRB/IEC and 
governmental approval with correspondence, sample informed consent, drug records, 
staff curriculum vitae and authorization forms and other appropriate 
documents/correspondence, etc. 
 
2. Source documents (usually defined by the project in advance to record key 
efficacy/safety parameters independent of the CRFs) would include subjects’ records, 

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physician's, nurse's and research assistant’s notes, appointment book, original laboratory 
reports, X-rays, pathology, and special assessment reports, signed informed consent 
forms, consultant letters, and subject screening and enrolment logs.  
 
The Investigator should retain the study documents of the ISF for the retention time specified in 
their contract.  
 
Should the Investigator wish to assign the study records to another party or move them to 
another location, CRO must be notified in advance. 
 
If the Investigator cannot guarantee this archiving requirement at the investigational site for any 
or all of the documents, special arrangements must be made with the Investigator and CRO to 
store these in a sealed container(s) outside of the site so that they can be returned sealed to the 
Investigator in case of a regulatory audit. Where source documents are required for the 
continued care of the subject, appropriate copies should be made for storing outside the site. 
12.3 
Source document and source data verification 
According to the standards of the data protection law, all data obtained in the course of a 
clinical study must be treated with discretion in order to guarantee the rights of the subject's 
privacy. The Investigator should agree to allow the monitor/auditor/inspector to have access to 
any or all of the clinical study materials needed for source data verification and proper review 
of the clinical study progress.  
12.4 
Insurance and Funding 
Liabilities, in connection with the clinical study will be covered by an insurance policy with each 
clinical site in the event of a subject suffering any significant injury, which is proven as being a 
direct result of study participation (as specified in the Clinical Study Agreement). Funding for 
this trial is provided by DNP. 
12.5 
Monitoring 
It is understood that the responsible monitor will contact and visit the Investigator regularly and 
will be allowed, on request, to review the various records of the trial (CRFs and other pertinent 
data) provided that subject confidentiality is maintained in accordance with local requirements 
and as specified in the contract. 
The Study Coordinator (SC) or Study Nurse (SN) at the site of the investigation will be instructed 
by the monitor to ensure that the following responsibilities and tasks are carried out: 
• 
Monitoring the study supplies and, if requested, returning all undispensed supplies to 
manufacturer or CRO at completion of the study.  
• 
Maintaining all records of the study. 
• 
Checking source documents for legibility and completion at the time they are received 
from the investigator. After review of study data with the investigator, the SC/SN will 
complete the formal eCRFs. 
• 
If required by the study, removing tear-off labels on receipt of double-blind labelled 
investigational product prior to its being dispensed to the subject/patient.  
• 
Contacting patients to remind them of their scheduled visits and obtaining a final 
disposition for every subject/patient who is entered in the study. 

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• 
Checking for reasonableness and completeness of source documents and of recorded CRF 
data before CRFs are reviewed by a monitor at each routine monitoring visit. 
It will be the monitor's responsibility to review the study documents (e.g. CRFs) at regular 
intervals throughout the study, to verify the adherence to the protocol and the legibility, 
completeness, consistency, and accuracy of the data being entered on them. The monitor must 
have access to laboratory test reports and other subject records needed to verify the entries on 
the CRF.  
The investigator (or his/her deputy) facilitates the monitoring tasks including the source 
document verification and agrees to cooperate with the monitor to ensure that any issues 
detected in the course of these monitoring visits are resolved. 
12.6 
Quality assurance and quality control 
All IP used in clinical studies are subjected to quality control. Quality assurance audits may be 
performed by any health authority during the course of the clinical study or after its completion 
(see Archiving).  
 
The Investigator agrees to comply with regulatory requirements in terms of auditing of the 
clinical study. This includes access to the source documents for source data verification. 
12.7 
Final Study Report 
When all completed CRFs have been collected and data have been analysed, the results of this 
clinical study are to be documented in a comprehensive study report and manuscript. 
12.8 
Archiving 
 
The study documents will be retained for a period of 2 years following the date a marketing 
application is approved for the drug for the indication for which it is being investigated; or if no 
application is to be filed or if the application is not approved for such indication, until 2 years 
after the investigation is discontinued and FDA is notified.  
 
The Trial Master File (TMF) shall be retained for a longer period, where so required by other 
applicable legal or regulatory requirements and/or as specified in the contract of the 
Investigator. 
 
The ISF will be retained according to Section 12.2 
12.9 
Publication 
 
INSTITUTION and SPONSOR-INVESTIGATOR agree that the first publication, lecture, manuscript, 
poster presentation or other disclosure or dissemination of the data or results of this study 
(collectively, “PUBLICATION”) shall be made in conjunction with the presentation of a joint 
multi-centre publication of the results with the investigators and institutions from all STUDY 
sites contributing data (as specified in the Clinical Study Agreement). In this case, a coordinating 
investigator and authorship will be designated by mutual agreement.  
 

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13 
CONDITIONS FOR TERMINATING THE STUDY 
Both the IND Sponsor-Investigator and the Site Investigator reserve the right to terminate the 
clinical study at any time. Should this be necessary, both parties will arrange the procedures on 
an individual project basis after review and consultation. In terminating the project, IND 
Sponsor-Investigator, DNP and the Site Investigator will assure that adequate consideration is 
given to the protection of the subject’s interests (as specified in the contract). 
14 
REFERENCES 
1. 
Le, T.A., et al., Effect of colesevelam on liver fat quantified by magnetic resonance in 
nonalcoholic steatohepatitis: a randomized controlled trial. Hepatology, 2012. 56(3): p. 
922-32. 
2. 
Scorletti, E., et al., Effects of purified eicosapentaenoic and docosahexaenoic acids in 
nonalcoholic fatty liver disease: results from the Welcome* study. Hepatology, 2014. 
60(4): p. 1211-21. 
3. 
Chalasani, N., et al., The diagnosis and management of non-alcoholic fatty liver disease: 
practice guideline by the American Gastroenterological Association, American 
Association for the Study of Liver Diseases, and American College of Gastroenterology. 
Gastroenterology, 2012. 142(7): p. 1592-609. 
4. 
Lazo, M., et al., Non-alcoholic fatty liver disease and mortality among US adults: 
prospective cohort study. BMJ. 2011. 343: p. d6891. 
5. 
Guo, X.F., et al., Fatty acid and non-alcoholic fatty liver disease: Meta-analyses of case-
control and randomized controlled trials. Clin Nutr, 2017. 
http://dx.doi.org/10.1016/j.clnu.2017.01.003. 
6. 
Diehl, A.M. and C. Day, Cause, Pathogenesis, and Treatment of Nonalcoholic 
Steatohepatitis. N Engl J Med, 2017. 377(21): p. 2063-2072. 
7. 
Azzi, A., Many tocopherols, one vitamin E. Mol Aspects Med, 2017. 
http://dx.doi.org/10.1016/j.mam.2017.06.004. 
8. 
Pacana, T. and A.J. Sanyal, Vitamin E and nonalcoholic fatty liver disease. Curr Opin Clin 
Nutr Metab Care, 2012. 15(6): p. 641-8. 
9. 
Sato, K., et al., Vitamin E has a beneficial effect on nonalcoholic fatty liver disease: a 
meta-analysis of randomized controlled trials. Nutrition, 2015. 31(7-8): p. 923-30. 
10. 
Sanyal, A.J., et al., Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. 
N Engl J Med. 2010. 362(18): p. 1675-85. 
11. 
Bugianesi, E., et al., A randomized controlled trial of metformin versus vitamin E or 
prescriptive diet in nonalcoholic fatty liver disease. Am J Gastroenterol, 2005. 100(5): 
p. 1082-90. 
12. 
Hasegawa, T., et al., Plasma transforming growth factor-b1 level and effcacy 
of a-tocopherol in patients with non-alcoholic steatohepatitis: a pilot study. Aliment 
Pharmacol Ther. 2001. 15: 1667-1672. 
13. 
Pietu, F., et al., Ursodeoxycholic acid with vitamin E in patients with nonalcoholic 
steatohepatitis: long-term results. Clin Res Hepatol Gastroenterol, 2012. 36(2): p. 146-
55. 
14. 
Dufour, J.F., et al., Randomized placebo-controlled trial of ursodeoxycholic acid with 
vitamin e in nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol, 2006. 4(12): p. 
1537-43. 
15. 
Chalasani, N., et al., The diagnosis and management of nonalcoholic fatty liver disease: 
Practice guidance from the American Association for the Study of Liver Diseases. 
Hepatology, 2018. 67(1): p. 328-357. 
16. 
Compounds, I.o.M.U.P.o.D.A.a.R., Dietary Reference Intakes for Vitamin C, Vitamin E, 
Selenium, and Carotenoids. Washington National Academies Press, 2000: p. 1-530. 
17. 
Report on Safe Upper Levels for Vitamins and Minerals. Expert Group on Vitamins and 
Minerals, Food Standards Agency, 2003. ISBN 1-904026-11-7, p.1-360. 

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Version: 
3 
 
Date: 
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18. 
Klein, E., et al Vitamin E and the risk of Prostate Cancer. JAMA, 2011. 306(14): p. 1549-
1556. 
19. 
Heinonen O.P. The Effect of Vitamin E and Beta Carotene on the incidence of Lung 
Cancer and other Cancers in male smokers,  NEJM,1994. 330: p.1029-35. 
20. 
Gaziano, J.M., et al., Vitamins E and C in the prevention of prostate and total cancer in 
men: the Physicians' Health Study II randomized controlled trial. JAMA, 2009. 301(1): p. 
52-62. 
21. 
Watters, J.L., et al., Associations between alpha-tocopherol, beta-carotene, and retinol 
and prostate cancer survival. Cancer Res, 2009. 69(9): p. 3833-41. 
22. 
Virtamo, J., et al., Effects of alpha-tocopherol and beta-carotene supplementation on 
cancer incidence and mortality: 18-year postintervention follow-up of the Alpha-
tocopherol, Beta-carotene Cancer Prevention Study. Int J Cancer, 2014. 135(1): p. 178-
85. 
23. 
Fortmann, S.P. Vitamin, Mineral, and Multivitamin Supplements for the Primary 
Prevention of Cardiovascular Disease and Cancer: A Systematic Evidence Review for the 
U.S. Preventive Services Task Force. Evidence Report 108, 2013. AHRQ Publication 14-
05199-EF-1. 
24. 
Hidayat, K., et al., Effect of omega-3 long-chain polyunsaturated fatty acid 
supplementation on heart rate: a meta-analysis of randomized controlled trials. Eur J 
Clin Nutr, 2017. https://doi.org/10.1038/s41430-017-0052-3. 
25. 
Ryan, A.S., et al., The hypolipidemic effect of an ethyl ester of algal-docosahexaenoic 
Acid in rats fed a high-fructose diet. Lipids, 2009. 44(9): p. 817-26. 
26. 
Maki, K.C., et al., Use of supplemental long-chain omega-3 fatty acids and risk for 
cardiac death: An updated meta-analysis and review of research gaps. J Clin Lipidol, 
2017. 11(5): p. 1152-1160 e2. 
27. 
Mori, T.A., Marine OMEGA-3 fatty acids in the prevention of cardiovascular disease. 
Fitoterapia, 2017. 123: p. 51-58. 
28. 
He, X.X., et al., Effectiveness of Omega-3 Polyunsaturated Fatty Acids in Non-Alcoholic 
Fatty Liver Disease: A Meta-Analysis of Randomized Controlled Trials. PLoS One, 2016. 
11(10): p. e0162368. 
29. 
Lu, W., et al., Effects of Omega-3 Fatty Acid in Nonalcoholic Fatty Liver Disease: A 
Meta-Analysis. Gastroenterol Res Pract, 2016. http://dx.doi.org/10.1155/2016/1459790. 
30. 
Parker, H.M., et al., Omega-3 Supplementation and Non-Alcoholic Fatty Liver Disease: A 
Systematic Review and Meta-analysis. J Hepatol. 2011. doi: 10.1016/j.jhep.2011.08.018. 
31. 
Sanyal, A.J., et al., No significant effects of ethyl-eicosapentanoic acid on histologic 
features of nonalcoholic steatohepatitis in a phase 2 trial. Gastroenterology, 2014. 
147(2): p. 377-84 e1. 
32. 
Park, C.C., et al., Magnetic Resonance Elastography vs Transient Elastography in 
Detection of Fibrosis and Noninvasive Measurement of Steatosis in Patients With Biopsy-
Proven Nonalcoholic Fatty Liver Disease. Gastroenterology, 2017. 152(3): p. 598-607 e2. 
33. 
Imajo, K., et al., Magnetic Resonance Imaging More Accurately Classifies Steatosis and 
Fibrosis in Patients With Nonalcoholic Fatty Liver Disease Than Transient Elastography. 
Gastroenterology, 2016. 150(3): p. 626-637 e7. 
34. 
Permutt, Z., et al., Correlation between liver histology and novel magnetic resonance 
imaging in adult patients with non-alcoholic fatty liver disease - MRI accurately 
quantifies hepatic steatosis in NAFLD. Aliment Pharmacol Ther, 2012. 36(1): p. 22-9. 
35. 
Kuratko, C., Food-frequency questionnaire for assessing long-chain omega-3 fatty-acid 
intake: Re: Assessing long-chain omega-3 polyunsaturated fatty acids: a tailored food-
frequency questionnaire is better. Nutrition, 2013. 29(5): p. 807-8. 
36. 
Regev, A ., et al., Consensus: guidelines: best practices for detection, assessment and 
management of suspected acute drug-induced liver injury during clinical trials in 
patients with nonalcoholic steatohepatitis. Alimentary Pharmacology & Therapeutics, 
2019. 49: p. 702-713. 
 
 

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Date: 
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15 
APPENDICES 
15.1 
Appendix 1 
 
 
DHA Food Frequency Questionnaire 
Subject ID #: 
 
Initials: 
 
 
 
 
 
Date: 
 
 
 
 
 
 
F 
 
M 
 
L 
 
MM 
DD 
YY 
 
Estimate your consumption over the past 2 months of the following foods.  Use the food model forms to help estimate 
portion sizes. 
 
 
 
 
 
 
 
Servings 
DHA 
 
EPA 
 
 
 
 
 
 
 
 
 
 
 
 
1. 
How many 3oz servings of the following fish do you eat monthly? 
 
 
x 22 
 
 
x 14 
 
 
bluefish 
herring 
sardines 
 
 
 
 
 
 
 
 
blue fin tuna 
mackerel 
salmon 
 
 
 
 
 
 
 
 
cisco, smoked 
pollock 
whitefish 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2. 
How many 3oz servings of the following fish do you eat monthly? 
 
 
x 10 
 
 
x 5 
 
 
bass 
mussels 
squid 
 
 
 
 
 
 
 
 
calamari 
perch 
swordfish 
 
 
 
 
 
 
 
 
catfish 
redfish 
trout 
 
 
 
 
 
 
 
 
drumfish 
rockfish 
tuna, canned (6oz can) 
 
 
 
 
 
 
 
 
flounder 
shark 
whiting 
 
 
 
 
 
 
 
 
grouper  
snapper 
 
 
 
 
 
 
 
 
 
halibut 
sole 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3. 
How many 3oz servings of the following fish/shellfish do you eat 
monthly?  
 
 
x 5 
 
 
x 6 
 
 
carp 
fish sticks 
pompano 
 
 
 
 
 
 
 
 
clams 
haddock 
scallops 
 
 
 
 
 
 
 
 
cod 
lobster 
shrimp (14 med.) 
 
 
 
 
 
 
 
 
crab 
mullet  
sturgeon 
 
 
 
 
 
 
 
 
crayfish 
oysters 
 
 
 
 
 
 
 
 
 
fish 
patties/squares  
pike 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4. 
How many 3oz servings of liver (chicken, turkey or beef) do you 
eat monthly? 
 
 
x 7 
 
 
x 2 
 
 
 
 
 
 
 
 
 
5. 
How many egg yolks do you eat weekly (including eggs yolks used 
in cooking)? 
 
 
x 3 
 
 
x 0.25 
 
 
 
 
 
 
 
 
 
6. 
How many 3oz servings of chicken, turkey or other poultry (not 
including livers) do you eat weekly?  
 
 
x 5 
 
 
x 3 
 
 
 
 
 
 
 
 
 
7. 
Any omega-3 dietary supplements or functional foods (i.e., flax, 
fish oil, Neuromins, DHA Gold, high DHA eggs)? 
 
 
 
 
 
 
 
 
amount or strength  ______     frequency  ______  
 
 
mg/
d 
 
 
mg/
d 
 
Sub-Total: 
 
 
 
 
 
 
 
TOTAL= Rounded to Nearest Whole #: 
 
DHA (mg/d) 
 
EPA (mg/d) 
 
 
 
DHA (mg/d) 
 
EPA (mg/d) 
 
© Martek Biosciences Corporation, now DSM Nutritional Products 
 
 
 

<  P U V E N A F L D  >  
 
 
Pr ot o c ol n o.:  2 0 1 7 -
1 0 8 8  
 
V ersi o n:  
3 
 
D at e:  
5 / 1 0 / 2 0 2 1  
 
P a g e 5 0  of 5 3  
 
C o nfi d e nti al  
1 5. 2  A p p e n di x 2  
 
 
S F -3 6(t m) H e alt h S ur v e y  
 
I nstr u cti o ns f or c o m pl eti n g t h e q u esti o n n air e: Pl e as e a ns w er e v er y q u esti o n. S o m e q u esti o ns m a y l o o k li k e ot h ers, b ut 
e a c h  
o n e is diff er e nt. Pl e as e t a k e t h e ti m e t o r e a d a n d a ns w er e a c h q u esti o n c ar ef ull y b y filli n g i n t h e b u b bl e t h at b est 
r e pr es e nts 
y o ur r es p o ns e.  
P ati e nt N a m e: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
SI D #: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D at e: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
P ers o n h eli n g t o c o m pl et e t his f or m: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
1. I n g e n er al, w o ul d y o u s a y y o ur h e alt h is:  
E x c ell e nt  
V er y g o o d  
G o o d  
F air  
P o or  
2 . C o m p ar e d t o o n e y e ar a g o, h o w w o ul d y o u r at e y o ur h e alt h i n g e n er al n o w? 
M u c h b ett er n o w t h a n a y e ar a g o  
S o m e w h at b ett er n o w t h a n a y e ar a g o  
A b o ut t h e s a m e as o n e y e ar a g o  
S o m e w h at w ors e n o w t h a n o n e y e ar a g o  
M u c h w ors e n o w t h a n o n e y e ar a g o  
3 . T h e f oll o wi n g it e ms ar e a b o ut a cti viti es y o u mi g ht d o d uri n g a t y pi c al d a y. D o es y o ur h e alt h n o w li mit y o u i n t h es e 
a cti viti es? If s o, h o w m u c h?  
a. Vi g or o us a cti viti es, s u c h as r u n ni n g, lifti n g h e a v y o bj e cts, p arti ci p ati n g i n str e n u o us s p orts.  
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
b . M o d er at e a cti viti es, s u c h as m o vi n g a t a bl e, p us hi n g a v a c u u m cl e a n er, b o wli n g, or pl a yi n g g olf? 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
c.  Lifti n g or c arr yi n g gr o c eri es. 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
d . Cli m bi n g s e v er al fli g hts of st airs. 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
e . Cli m bi n g o n e fli g ht of st airs.  
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
f. B e n di n g, k n e eli n g or st o o pi n g. 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
S F -3 6 2  
g. W al ki n g m or e t h a n o n e mil e.  
Y es,  li mit e d a l ot. 
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
h . W al ki n g s e v er al bl o c ks. 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
i. W al ki n g o n e bl o c k. 
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all. 
j. Bat hi n g or dr essi n g y o urs elf.  

<  P U V E N A F L D  >  
 
 
Pr ot o c ol n o.:  2 0 1 7 -
1 0 8 8  
 
V ersi o n:  
3 
 
D at e:  
5 / 1 0 / 2 0 2 1  
 
P a g e 5 1  of 5 3  
 
C o nfi d e nti al  
Y es, li mit e d a l ot.  
Y es, li mit e d a littl e.  
N o, n ot li mit e d at all.  
4 . D uri n g t h e p ast 4 w e e ks, h a v e y o u h a d a n y of t h e f oll o wi n g pr o bl e ms wit h y o ur w or k or ot h er r e g ul ar d ail y a cti viti es 
as a r es ult of  y o ur p h ysi c al h e alt h?  
a. C ut d o w n t h e a m o u nt of ti m e y o u s p e nt o n w or k or ot h er a cti viti es?  
Y es N o  
b.  A c c o m plis h e d l ess t h a n y o u w o ul d li k e? 
Y es N o  
c . W er e li mit e d i n t h e ki n d of w or k or ot h er a cti viti es 
Y es N o  
d.  H a d diffi c ult y p erf or mi n g t h e w or k or ot h er a cti viti es (f or e x a m pl e, it t o o k e xtr a ti m e)  
Y es N o  
5 . D uri n g t h e p ast 4 w e e ks, h a v e y o u h a d a n y of t h e f oll o wi n g pr o bl e ms wit h y o ur w or k or ot h er r e g ul ar d ail y a cti viti es 
as a r es ult of a n y e m oti o n al pr o bl e ms (s u c h as f e eli n g d e pr ess e d or a n xi o us)?  
a. C ut d o w n t h e a m o u nt of ti m e y o u s p e nt o n w or k or ot h er a cti viti es?  
Y es N o  
b.  A c c o m plis h e d l ess t h a n y o u w o ul d li k e 
Y es N o  
c.  Di d n't d o w or k or ot h er a cti viti es as c ar ef ull y as us u al 
Y es N o  
6 . D uri n g t h e p ast 4 w e e ks, t o w h at e xt e nt h as y o ur p h ysi c al h e alt h or e m oti o n al pr o bl e ms i nt erf er e d wit h y o ur 
n or m al s o ci al a cti viti es wit h f a mil y, fri e n ds, n ei g h b ors, or gr o u ps?  
N ot at all  
Sli g htl y  
M o d er at el y  
Q uit e a bit  
E xtr e m el y  
7 . H o w m u c h b o dil y p ai n h a v e y o u h a d d uri n g t h e p ast 4 w e e ks? 
N ot at all  
Sli g htl y  
M o d er at el y  
Q uit e a bit  
E xtr e m el y  
S F -3 6 3  
8. D uri n g t h e p ast 4 w e e ks, h o w m u c h di d p ai n i nt erf er e wit h y o ur n or m al w or k (i n cl u di n g b ot h w or k o utsi d e t h e h o m e 
a n d h o us e w or k)?  
N ot at all  
Sli g htl y  
M o d er at el y  
Q uit e a bit  
E xtr e m el y  
9 . T h es e q u esti o ns ar e a b o ut h o w y o u f e el a n d h o w t hi n gs h a v e b e e n wit h y o u d uri n g t h e p ast 4 w e e ks. F or e a c h 
q u esti o n, pl e as e gi v e t h e o n e a ns w er t h at c o m es cl os est t o t h e w a y y o u h a v e b e e n f e eli n g. H o w m u c h of t h e ti m e 
d uri n g t h e p ast 4 w e e ks  
a. di d y o u f e el f ull of p e p?  
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
b . h a v e y o u b e e n a v er y n er v o us p ers o n? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
c . h a v e y o u f elt s o d o w n i n t h e d u m ps n ot hi n g c o ul d c h e er y o u u p? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e 
N o n e of t h e ti m e  

<  P U V E N A F L D  >  
 
 
Pr ot o c ol n o.:  2 0 1 7 -
1 0 8 8  
 
V ersi o n:  
3 
 
D at e:  
5 / 1 0 / 2 0 2 1  
 
P a g e 5 2  of 5 3  
 
C o nfi d e nti al  
d. h a v e y o u f elt c al m a n d p e a c ef ul?  
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
e . di d y o u h a v e a l ot of e n er g y? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
f. h a v e y o u f elt d o w n h e art e d a n d bl u e? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
S F -3 6 4  
g. di d y o u f e el w or n o ut?  
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
h . h a v e y o u b e e n a h a p p y p ers o n? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
i. di d y o u f e el tir e d? 
All of t h e ti m e  
M ost of t h e ti m e  
A g o o d bit of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
1 0. D uri n g t h e p ast 4 w e e ks, h o w m u c h of t h e ti m e h as y o ur p h ysi c al h e alt h or e m oti o n al pr o bl e ms i nt erf er e d wit h 
y o ur s o ci al a cti viti es (li k e visiti n g fri e n ds, r el ati v es, et c.)?  
All of t h e ti m e  
M ost of t h e ti m e  
S o m e of t h e ti m e  
A littl e of t h e ti m e  
N o n e of t h e ti m e  
1 1. H o w T R U E or F A L S E is e a c h of t h e f oll o wi n g st at e m e nts f or y o u? 
a. I s e e m t o g et si c k a littl e e asi er t h a n ot h er p e o pl e  
D efi nit el y tr u e  
M ostl y tr u e  
D o n't k n o w  
M ostl y f als e  
D efi nit el y f als e  
b . I a m as h e alt h y as a n y b o d y I k n o w 
D efi nit el y tr u e  
M ostl y tr u e  
D o n't k n o w  
M os tl y f als e 
D efi nit el y f als e  
c.  I e x p e ct m y h e alt h t o g et w ors e 
D efi nit el y tr u e  
M ostl y tr u e  

<  P U V E N A F L D  >  
 
 
Pr ot o c ol n o.:  2 0 1 7 -
1 0 8 8  
 
V ersi o n:  
3 
 
D at e:  
5 / 1 0 / 2 0 2 1  
 
P a g e 5 3  of 5 3  
 
C o nfi d e nti al  
D o n't k n o w  
M ostl y f als e  
D efi nit el y f als e  
d . M y h e alt h is e x c ell e nt 
D efi nit el y tr u e  
M ostl y tr u e  
D o n't k n o w  
M ostl y f als e  
D efi nit el y f als e