Dietary Linoleic Acid Regulation of Omega-3 HUFA Metabolism: Satiety and Body Composition Among Overweight Female Subjects
Phase: NA
Conditions: Weight Gain, Healthy Subjects, Obesity, Women
Sponsor: National Institute on Alcohol Abuse and Alcoholism (NIAAA)

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 1 of 68 
 
1. Title Page 
Protocol Title:  Dietary linoleic acid regulation of omega-3 HUFA metabolism; 
satiety and body composition among overweight female subjects 
 
Protocol Number: 11-AA-0028 
Date of This Submission/Version:    03 January 2018/v9.1 
 
Principal Investigator 
Name, Degree 
 
Christopher Ramsden, MD 
Branch/Institute 
 
LMBB/NIAAA 
 
Total requested accrual 
  (0) Patients  (separately describe patient groups)              
  
  (180) Volunteers  
 
  
 
Project Uses Ionizing Radiation:   No  
X Yes (attach RSC/RDSC documentation) 
  
 
 Medically-indicated only 
  
 
X Research-related only (DEXA scans) 
  
 
 Both 
 
IND/IDE 
 
 
X No  
 
Yes (attach FDA documentation) 
  
Drug/Device/#_____________________ 
  
Sponsor: _________________________ 
 
Durable Power of Attorney  
X No  
 
 Yes  
 
Multi-institutional Project   X No  
 
 Yes  
 
 
 
 
 
Institution_________________    FWA #_________  
Date of IRB approval_________ (attach IRB documentation) 
 
Data and Safety Monitoring Board  
X No  
 
 Yes  
 
Technology Transfer Agreement  
X No  
 
 Yes  
Agreement type and number __________________Expiration Date_____________ 
 
Confidential Disclosure Agreement     
X No 
 
 Yes 
 
Samples are being stored  
 
 
 No   
         X Yes  
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 2 of 68 
2.  Table of Contents  (Main protocol)  
 
 
 
 
3 – 35, 52- 66 
Table of Contents  (Infusion Substudy) 
 
 
 
 
      36 – 52 
 
Main Protocol 
3.   Precis 
 
 
 
 
 
 
 
 
 
6 
3.1 Objective 
 
 
 
 
 
 
 
 
 
 
 
3.2 Study Population 
 
 
 
 
 
 
 
 
 
 
3.3.1 Three Group Comparative Analysis 
 
 
 
 
 
 
 
 
3.3.2 Continuation Phase Sub Study 
 
 
 
 
 
 
 
3.4 Outcome Measures  
 
 
 
 
 
 
 
 
3.4A Primary Dependent Variables 
 
 
 
 
 
 
 
3.4B Secondary Dependent Variables 
 
 
 
 
 
 
 
 
 
4. Introduction/ Scientific Rationale    
 
 
 
 
 
7 
4.1 Role of endocannabinoids in craving and satiety 
 
 
 
 
 
4.2 HUFA as precursors to endocannabinoids 
 
 
 
 
 
 
4.3 Translation from rats to humans  
 
 
 
 
 
 
           
 
 
 
 
 
 
 
 
 
 
 
 
5. Study objectives and hypotheses – Main protocol 
 
 
 
 
11 
5.1 Study objective 
 
 
 
 
 
 
 
 
 
 
5.2 Specific hypotheses   
 
 
 
 
 
 
 
 
5.2.1A Primary aim  
 
 
 
 
 
 
 
 
 
5.2.1B Primary hypothesis 
 
 
 
 
 
 
 
 
5.2.2A Secondary aims  
 
 
 
 
 
 
 
 
5.2.2B Secondary hypotheses 
 
 
 
 
 
 
 
 
 
 
 
6. Subjects 
 
 
 
 
 
 
 
 
 
12 
6.1 Description of study population 
 
 
 
 
 
 
 
6.2 Inclusion criteria (Eligibility Checklist as attachment) 
 
 
 
 
6.3 Exclusion criteria    
 
 
 
 
 
 
 
 
 
7. Study Design and Methods:  
 
 
 
 
 
 
13 
7.1 Study Overview and Design  
 
 
 
 
 
 
 
7.2 Recruitment  
 
 
 
 
 
 
 
 
 
7.3 Screening Methods  
 
 
 
 
 
 
 
 
7.3.1 Pre-screening 
 
 
 
 
 
 
 
 
 
7.3.2 Screening visit 
 
 
 
 
 
 
 
 
 
7.4 Study Design 
 
 
 
 
 
 
 
 
 
7.5 Study Procedures Overview  
 
 
 
 
 
 
   
7.6 Study Visit Procedure Specifics  
 
 
 
 
 
 
 
7.6.1 Food Records  
 
 
 
 
 
 
 
 
 
7.6.2 Randomization and Masking 
 
 
 
 
 
 
 
7.6.3 Specimen Collection for Biomarker Testing 
 
 
 
 
 
7.6.4 Insulin Sensitivity  
 
 
 
 
 
 
 
7.6.5 Functional Satiety Assessment 
 
 
 
 
 
 
 
7.6.6 Psychometric Assessments 
 
 
 
 
 
  
7.6.7 Body Composition 
 
 
 
 
 
 
 
  

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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7.6.8 Assessment of Dietary Adherence  
 
 
 
 
 
 
7.6.9 Follow-up/ termination procedures  
 
 
 
 
 
7.6.10 Radiation exposure 
 
 
 
 
 
 
 
 
7.6.11 Medications and devices requiring IND/IDE 
 
 
 
 
 
7.6.12 Storage of research samples and data 
 
 
 
 
 
 
 
8 Risks and Discomforts 
 
 
 
 
 
 
 
23 
8.1 Procedures with No Medical Risk and Minimal Discomfort   
 
 
 
8.2 Study Diets  
 
 
 
 
 
 
 
 
 
8.3 Specimen Collection for Biomarker Testing  
 
 
 
 
  
8.4 Muscle and Adipose Tissue Biopsies 
 
 
 
 
 
 
8.5 Questionnaires to Assess Mood 
 
 
 
 
 
 
 
8.6 Body Composition  
 
 
 
 
 
 
 
 
  
8.7 Radiation from DEXA Scans 
 
 
 
 
 
 
 
 
 
9.  Subject Monitoring, adverse event grading and withdrawal 
 
 
27 
9.1 Parameters to be monitored  
 
 
 
 
 
 
 
 
10. Outcome Measures  
 
 
 
 
 
 
 
27 
10A. Outcome Measures Main Protocol  
 
 
 
 
 
 
 
 
 
 
11. Statistical Analyses 
 
 
 
 
 
 
 
27 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12. Human Subjects protection 
 
 
 
 
 
 
29 
12.1 Regulatory and Ethical Considerations 
 
 
 
 
 
 
12.2 Equitable Inclusion 
 
 
 
 
 
 
 
 
12.3 Inclusion of children or other vulnerable subjects 
 
 
 
 
 
12.4 Justification of sensitive procedures 
 
 
 
 
 
 
12.5 Safeguards for vulnerable populations 
 
 
 
 
 
 
12.6 Qualifications of Investigators 
 
 
 
 
 
 
 
 
13.  Benefits 
 
 
 
 
 
 
 
 
 
32 
 
14.  Classification of Risk 
 
 
 
 
 
 
 
33 
 
15.  Consent documents and process  
 
 
 
 
 
33 
15.1 Who will obtain Consent 
 
 
 
 
 
 
 
 
 
16.  Data Safety and Monitoring Board 
 
 
 
 
 
33 
 
17.  Adverse Event Reporting  
 
 
 
 
 
 
33 
 
18. Alternatives to participation or alternative therapies 
 
 
 
34 
 
19. Confidentiality 
 
 
 
 
 
 
 
 
34 
19.1 Medical records 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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19.2 Research Data 
 
 
 
 
 
 
 
 
 
19.3 Sample Storage 
 
 
 
 
 
 
 
 
 
 
20.  Conflict of Interest/ Technology Transfer  
 
 
 
 
34 
 
21.  Compensation 
 
 
 
 
 
 
 
 
35 
 
 
References (Main Protocol) 
 
 
 
 
 
 
             52 
 
Appendices  (Main protocol)  
 
 
 
 
 
 
 
 
Appendix 1-   Study Flow Diagram 
 
 
 
 
 
 
58 
Appendix 2-  
Lands Equation  
 
 
 
 
 
 
60 
Appendix 3-  
Fatty acids and their metabolic derivatives 
 
 
 
61 
Appendix 4- 
Psychometric Assessment Scales  
 
 
 
 
64 
Appendix 5- 
Eligibility Checklist 
 
 
 
 
 
 
65 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 5 of 68 
 
 
3. Precis 
 
3.1 Objective 
To evaluate the effects of lowering the dietary nutrient linoleic acid (LA) as a controlled variable on: 1) 
tissue accretion of omega-6 and omega-3 highly unsaturated fatty acids (HUFA); 2) bioactive 
endocannabinoids derived from the omega-6 HUFA arachidonic acid (AA); 3) insulin sensitivity; 4) 
satiety and hunger; and 5) body composition. Modifications in dietary LA will be offset with reciprocal 
changes in the nutrient oleic acid (OA) to maintain equivalency of calories and nutrients.  
 
3.2 Study Population 
Up to 180 overweight, but otherwise healthy pre-menopausal women volunteers (aged 18 through 50) 
with body mass index (BMI) between 25 and 35 kg/m2, will be enrolled.   
With the anticipated dropout rates described in Section 7.4, we anticipate that 84 of these subjects will be 
randomized to study diets, and 64 subjects will complete the 12 week diet phase.  
 
3.3.1 Three Group Comparative Analysis 
A total of 84 subjects will be randomized to one of 3 healthy and nutritionally complete diets that differ 
only in the fatty acid composition of added visible oils. All foods will be provided through the NIH 
metabolic kitchen, with subjects visiting the kitchen daily Monday through Friday to obtain foods.  Each 
Friday, subjects will carry home foods packed for their weekend meals.  Baseline testing battery will 
include: 1) blood composition of omega-6 and omega-3 HUFA; 2) endocannabinoid, eicosanoid, and 
docosanoid derivatives of omega-6 and omega-3 HUFA; 3) insulin function assessment; 4) body 
composition assessment; 4) functional satiety assessment; and 5) self-reported measures of satiety, hunger 
and affective states. The full baseline testing battery will be repeated after 84 days of randomized 
treatment. An abbreviated testing battery will be performed after 28 and 56 days of randomized treatment.  
A subset of participants will undergo additional inpatient testing as described in Section 3.3.2. The 
remaining subjects will remain ambulatory throughout the study. Participants will be advised to continue 
their usual medical care and medications throughout the study. 
 
3.3.2 36-week Continuation Sub Study 
At completion of the 84 day (12 week) study, participants in the low LA group (Group B) will have the 
option of continuing their study diets for another 252 days (36 weeks).  Continuing subjects will consume 
the same low LA (1 en%) diet.  During the continuation phase, the abbreviated testing battery will be 
completed after 6, 12, 18, 24 and 30 weeks, and the full testing battery will be repeated after 36 weeks. 
 
3.4 Outcome Measures 
3.4A Primary Dependent Variables 
The primary outcomes will be the proportion of omega-6 HUFA in total HUFA (%omega-6 in HUFA) 
and two endocannabinoids derived from omega-6 AA, 2-Arachidonoylglycerol (2-AG) and N-
Arachidonoylethanolamine (Anandamide or AEA). 
 
3.4B Secondary Dependent Variables 
Secondary outcome measures will include blood concentrations of the omega-6 HUFA Arachidonic Acid 
(AA); the omega-3 HUFA Eicosapentaenoic Acid (EPA) and DHA; the rates of change in %omega-6 in 
HUFA, AA, EPA, DHA, 2-AG and AEA over time; adipose and muscle tissue fatty acid composition; 
endocrine hormones; gene expression profiles; psychometrics; functional satiety; 24-hr energy 
metabolism and substrate utilization; and body composition, as follows.  Omega-6 and omega-3 HUFA 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 6 of 68 
and total fat content will be measured in adipose and muscle tissue biopsies. Other endocannabinoid, 
eicosanoid and docosanoid derivatives of omega-6 and omega-3 HUFA will be measured in plasma via 
mass spectrometry.  
Psychometric outcomes will include self-reported measures of satiety and affective states. Functional satiety will 
be assessed via buffet-style food array testing. Body composition outcomes will include DEXA, 3T MRI, 
anthropometric measurements and bioelectrical impedance. Genotyping will also be performed to assess for 
interactions between selected gene variants and effects of the intervention. 
 
 
4. Introduction/ Scientific Rationale   
 
4.1 Role of endocannabinoids in craving and satiety 
Numerous crucial regulatory activities have been attributed to endocannabinoids, and their contribution to 
a host of seemingly unrelated chronic conditions is a matter of intense inquiry (1, 2). The 
endocannabinoid system includes the two endogenous ligands 2-arachidonoylglycerol (2-AG) and N-
arachidonoylethanolamine (Anadamide or AEA) and two cannabinoid receptors (CB-1 and CB-2). CB-1 
receptors are located primarily in the brain, but also in the GI tract, adrenal glands, liver, adipose tissue, 
and skeletal muscle (3). Excessive activity of the endocannabinoid system has recently emerged as having 
a central role in promoting obesity through at least three organ systems (4, 5). 1) Centrally, excessive 2-
AG stimulates the CB-1 receptor, which increases hunger-induced increases in food intake, and decrease 
in satiety. 2) In liver, CB-1 stimulation increases de novo hepatic lipogenesis through activation of the 
fatty acid biosynthetic pathway. 3) In adipose tissue, CB-1 expression has been implicated in the control 
of adiponectin secretion and lipoprotein lipase activity.  
 
Amid growing evidence that endocannabinoid signaling is up-regulated in obesity, pharmaceutical 
companies began developing CB-1 receptor antagonists. In 2006, the European Medicines Agency 
approved rimonabant, a CB-1 receptor inverse agonist, following impressive clinical trial results in both 
obese and diabetic patients. Specifically, placebo-controlled trials demonstrated considerable weight-loss 
(nearly 15 lbs), and significant improvements in: 1) waist circumference; 2) HDL-cholesterol; 3) 
triglycerides; 4) inflammatory markers; 5) blood pressure; 6) fasting glucose; and 7) fasting insulin (6, 7). 
In 2008, however, rimonabant was pulled from the market due to serious psychiatric side effects, 
including suicide (8). Because full pharmacological blockade of the CB-1 receptor is effective in treating 
obesity and related metabolic derangements, a safer alternative to pharmaceuticals for diminishing 
endocannabinoid hyperactivity may have widespread beneficial public health implications related to 
obesity and metabolic syndrome.  Given the fact that endocannabinoids have important physiological 
functions, partial CB-1 receptor blockade may be preferable to full inhibition. 
 
In a similar manner to food, endocannabinoid hyperactivity has been linked to impairment of satiety 
responses for alcohol (4, 9) and other substances of abuse (10). Impairment of satiety responses may be 
due to the shared downstream effects on signaling pathways (cAMP/PKA) regulated by CB-1 receptors 
and D2 dopamine receptors in the ventral tegmental area and striatum (11, 12). Thus, we posit that 
excessive endocannabinoid activity, and the ensuing neuroadaptations in the brain reward system, 
manifest phenotypically as deficits in satiety regulation for foods and substances of abuse, hepatic 
steatosis and excessive adipose accumulation. In this study we will not assess substances of abuse but will 
assess satiety for foods, and relevant metabolic changes. 
 
4.2 HUFA as precursors to endocannabinoids 
Endocannabinoids are not stored in cellular vesicles but rather are formed enzymatically from membrane 
phosopholipid-fatty acid precursors on demand (13). The two best characterized endocannabinoids, 2-AG 
and AEA, are both metabolic derivatives of a single fatty acid precursor, omega-6 arachidonic acid (AA). 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 7 of 68 
Because humans cannot synthesize AA de novo (14), tissue phospholipid concentrations of AA (PL-AA) 
are dependent upon dietary intake of: 1) omega-6 AA (15); 2) the precursor to AA–linoleic acid (LA) 
(16); and 3) competing fatty acids such as omega-3 EPA and DHA (14, 15, 17). Therefore, by restricting 
dietary omega-6 fatty acids and/or boosting intake of competing omega-3 fatty acids, humans may be able 
to reduce PL-AA content, subsequent endocannabinoid synthesis from PL-AA, and the metabolic 
consequences of high 2-AG and AEA levels.    
 
To test this hypothesis, we asked if selectively raising one omega-6 fatty acid in the rodent diet (while 
keeping total fat composition constant) would result in endocannabinoid hyperactivity and excess 
adiposity.  We first asked whether the selective increase of linoleic acid (LA), from 1 % of energy (en%) 
to 8 en%, could elevate PL-AA in membranes and in turn also elevate endocannabinoid levels in liver and 
brain.  To test if the proportion of PL-AA precursor pool was the critical variable, we also added 1 en% of 
energy as EPA and DHA to the 8 en% LA diet.  
 
Results  
Increasing dietary LA from 1 en% to 8 en% increased PL-AA (by 59%) and doubled liver 2-AG in the 
medium fat diet (see Table 1).  Conversely, the addition of 1 en% as EPA and DHA to the 8 en% LA diet 
decreased PL-AA and halved liver 2-AG, indicating that regulation of the PL-AA in the phospholipid 
pool is a critical determinant for endocannabinoid production. A similar pattern was seen in the high fat 
diet. A comparable pattern of adverse effects of 8 en% LA was observed for feeding efficiency (defined 
as weight gained per kcal of food consumption), leptin levels, adiponectin levels (inverse pattern) and 
increased adiposity (see Fig. 1, differing letters indicate statistical differences at p< 0.05) 
 
Table 1. Diet design, initiated in mothers, pups fed up to age 14 weeks, n=10 pups per group 
Medium fat (MF) 
High fat (HF) 
1 en% LA  8 en% LA  
8 en % LA + 
EPA/DHA  
1 en% LA  
8 en% LA  
8 en% LA + 
EPA/DHA  
Fat en%  
35 
35  
35  
60  
60  
60  
LA en%  
1  
8  
8  
1  
8  
8  
ALA en%  
1  
1  
1  
1  
1  
1  
EPA+DHA 
en%  
-  
-  
1  
-  
-  
1  
MUFA en%  
8  
8  
9  
7  
7  
7  
SFA en%  
25  
18  
16  
51  
44  
42  
PL-AA 
2.99+/- 1.26 
4.74+/- 0.86 
2.73+/-0.34 
1.94+/-0.28 
5.02+/-0.89 
2.92+/-0.36 
PL-EPA 
0.85+/-0.33 
0.13+/-0.02 
0.87+/-0.25 
1.07+/-0.19 
0.14+/-0.02 
1.17+-0.16 
PL-DHA 
4.73+/-2.02 
2.87+/-0.50 
3.49+/-0.65 
3.74+/-0.50 
3.07+/-0.59 
4.70+/-0.66 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 8 of 68 
 
 
Figure 1: Dietary linoleic acid increased the omega-6 endocannabinoid precursor pool and induced 
endocannabinoid hyperactivity and obesity in rats. 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 9 of 68 
 
In addition to raising PL-AA, the 8 en% LA diet reciprocally decreased both EPA (by 85%) and DHA (by 
39%) in liver phospholipids compared to their 1 en% LA counterparts.  Because PL-EPA and PL-DHA 
act as rival substrates with PL-AA for enzymatic conversion to omega-6 (or omega-3) endocannabinoid 
derivatives (18), these deficits in omega-3 EPA and DHA exacerbate omega-6 endocannabinoid 
hyperactivity. 
 
Our results agree in principle with landmark rodent studies conducted nearly 50 years ago, which clearly 
demonstrated that tissue PL-AA concentrations increase progressively as dietary omega-6 LA increases 
up to 5 en%. In their classic 1963 study, Mohrhauer and Holman showed that when dietary LA ranged 
from 0 to 4.87 en% for 100 days, the accumulation of PL-AA in liver lipids ranged from 1.8 to 14.7% by 
weight (19). Similar robust variability in tissue omega-6 PL-AA content was seen in brain tissue (20) and 
erythrocytes (21) when dietary LA ranged between 0 and 5 en%. (See Fig. 2). However, further increases 
in dietary LA above 5 en% had comparatively minor effects on tissue PL-AA content (22), presumably 
because AA accumulation has already ‘plateaued’.   
         
Figure 2: Dietary linoleic acid raises tissue arachidonic acid 
 
 
 
 
 
 
 
 
From Morhauer H 1963; 4;151 
4.3 Translation from rats to humans  
 
Do these principles apply to humans?  
Like rodents, humans lack the enzymatic machinery to synthesize AA de novo. Thus, human tissue 
concentrations of PL-AA and other omega-6 HUFA are dependent upon dietary consumption of AA; its 
precursor–LA; and omega-3 acids that compete with omega-6 acids for enzymatic elongation, 
desaturation, acylation, and incorporation into human tissue phospholipids (17).  Competition continues 
as omega-6 and omega-3 HUFA act as rival substrates for enzymatic cleavage and conversion into 
omega-6 and omega-3 endocannabinoids. Therefore, as in rodents, the reduction of dietary LA is a 
plausible strategy for decreasing tissue PL-AA and its endocannabinoid derivatives in human tissues.   
 
Pilot data from the combined analysis of the first 20 participants in a randomized dietary trial testing the 
effects of a 12-week reduction of dietary LA, found that AA declined significantly, and EPA+DHA 
increased significantly, with or without added EPA+DHA (Table 2).  These data indicate that 12 weeks is 
sufficient to detect significant changes in AA, EPA, and DHA.  However, dynamic equilibrium in 
erythrocyte fatty acids was not reached between weeks 8 and 12 weeks. 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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Table 2.  Means of erythrocyte fatty acids over the 12-week intervention,  
Chronic Daily Headache study, May 2010 
  
Baseline (n=20) 
Week 4 (n = 20) 
Week 8 (n=20) 
Week 12 (n=18) 
Variable 
Mean 
95% CI 
Mean 
95% CI 
Mean 
95% CI 
Mean 
95% CI 
Omega-3 index 
4.4 
3.9, 5.0 
6.2 
5.4, 7.1 
7.0 
5.9, 8.1 
7.8 
6.5, 9.0 
Omega-6 in HUFA 
76.3 
74.5, 78.0 
70.4 
67.5, 73.2 
67.4 
63.7, 71.0 
64.7 
60.3, 69.0 
DHA 
4.0 
3.5, 4.5 
5.2 
4.5, 5.8 
5.7 
4.9, 6.5 
6.3 
5.4, 7.3 
EPA 
0.46 
0.39, 0.54 
1.1 
0.8, 1.3 
1.3 
0.95, 1.6 
1.4 
1.0, 1.8 
AA 
14.8 
14.5, 15.2 
14.4 
13.7, 15.1 
13.8 
13.2, 14.5 
13.7 
12.9, 14.5 
LA 
11.5 
10.8, 12.1 
9.5 
8.8, 10.2 
9.3 
8.6, 10.0 
9.5 
8.8, 10.3 
  
The release of omega-6 and omega-3 fatty acids from adipose tissue stores is expected to prolong the time 
to necessary to reach dynamic equilibrium in plasma and erythrocyte phospholipids.  Because adipose 
tissue fatty acids have an estimated half-life of 300 to 600 days (23-25), interventions substantially longer 
than our 12-week (84 day) diet phase are expected to produce even more substantial metabolic and 
clinical effects. Therefore, a substantially longer dietary trial is likely to be necessary to attain maximal 
metabolic effects, and to provide new knowledge about the potential confounding role of omega-6 and 
omega-3 fatty acids in adipose tissue stores.  In conjunction with the preceding 84 day diet phase, subjects 
who complete the continuation phase will maintain study diets for 336 days, which will allow adequate 
time to assess the potential confounding role from the release of adipose tissue omega-6 and omega-3 
fatty acids.  The 36-week (252 day) duration of the continuation phase was therefore selected as a 
reasonable compromise between: 1) the desire to assess the confounding role of adipose stores of omega-
6 and omega-3 fatty acids, and 2) the practicality of keeping volunteers on study diets for prolonged 
periods. 
 
In conjunction with the plausible biological mechanisms described above, our rodent data and human 
pilot data provide proof of principle for our hypothesis that lowering dietary LA will raise tissue PL-EPA 
and PL-DHA, lower tissue PL-AA, attenuate excessive omega-6 endocannabinoid activity, enhance 
satiety and diminish excessive craving for pleasurable stimuli such as food or substances of abuse.  The 
critical knowledge gap, and the logical next step, is to test our hypothesis in randomized controlled trial in 
humans. 
 
 
 
 
5. Study objectives and hypotheses  
 
5.1 Study objective 
To evaluate the effects of modifying the dietary nutrient LA via controlled substitution for oleic acid 
(OA) on the blood omega-6 and omega-3 HUFA composition; endocannabinoids and other lipid 
mediators derived from omega-6 and omega-3 HUFA; and the rates of change in the omega-6 and omega-
3 HUFA and their metabolic derivatives over time.  We will also evaluate the effects of modifying dietary 
LA on insulin sensitivity; hunger and satiety; and body composition. 
 
5.2 Specific hypotheses   
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
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CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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5.2.1A Primary aims  
To evaluate the effects of modifying the dietary nutrient LA on the %omega-6 in HUFA; as well as two 
endocannabinoids derived from omega-6 PL-AA, 2-AG and AEA.  
 
5.2.1B Primary hypotheses 
Lowering dietary intake of LA as a controlled variable from 8% to 1% of daily food energy will 
significantly decrease the %omega-6 in HUFA and the endocannabinoid derivatives of omega-6 AA, 2-
AG and AEA.  
 
5.2.2A Secondary aims 
To assess the effects of modifying the dietary nutrient LA on the rates of change in omega-6 and omega-3 
HUFA, 2-AG, and AEA over time; adipose and muscle tissue fatty acid content, relevant metabolic 
mediators measured in plasma, body composition, functional satiety, and psychometrics, as follows.   
Metabolic outcomes will include other endocannabinoids, eicosanoids and docosanoids derived from omega-6 
and omega-3 HUFA, 24-hr energy metabolism, and endocrine hormones related to obesity.  
Adipose and muscle tissue outcomes will include tissue concentrations of omega-6 and omega-3 HUFA. Body 
composition outcomes will include DEXA, 3T MRI, anthropometric measurements and bioelectrical impedance.  
Functional satiety will be assessed via (buffet-style) food array testing.  
Psychometric outcomes will include self-reported measures of hunger, satiety, and affective states.  Genotyping 
will also be performed to assess for interactions between selected gene variants and effects of the intervention.  
 
5.2.2B Secondary hypotheses 
Lowering dietary intake of LA as a controlled variable from 8 en% to 1 en% will result in:  
• 
Increased omega-3 HUFA and reduced omega-6 HUFA in muscle and adipose tissue biopsies 
• 
Reduced total fat content of adipose and muscle tissue biopsies 
• 
Reciprocal increases in accretion of the omega-3 HUFA and their endocannabinoid, eicosanoid, 
and docosanoid derivatives.  
• 
Favorable effects on insulin sensitivity and endocrine hormone function. 
• 
Favorable effects on gene expression profiles. 
• 
Favorable effects on hunger, satiety, and affective states. 
• 
Increased functional satiety. 
• 
Reduced adiposity and increased lean body mass. 
 
6 Subjects 
 
6.1 Description of study population 
Subjects will be overweight, but otherwise healthy, pre-menopausal ambulatory volunteers aged 18 to 50. 
 
6.2 Inclusion criteria 
To be eligible for this research study, participants must: 
1. Be between 18 and 50 years of age. 
2. Have regular menstrual cycles. 
3. Be willing to use an effective method of birth control such as hormonal contraception, 
intrauterine device, barrier methods combined with spermicide, or surgical sterilization. 
4. Have a body mass index of 25 to 35 kg/m2. 
5. Be otherwise healthy as determined by history, medical examination and laboratory tests. 
6. Be able to come to the NIH Clinical Center every weekday for 3 months. 
7. Be able to understand the consent form, and provide informed written consent. 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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6.3 Exclusion criteria   
Participants are not eligible for this research study if they: 
1. Have been pregnant or have breast fed within the last 2 years. 
2. Work, or have an immediate family member who works, with a study investigator. Immediate 
family is defined as a spouse, parent, child or sibling, whether biological or legally adopted. 
3. Are HIV positive. 
 
4. Have any current, unstable medical conditions including respiratory insufficiency requiring 
oxygen therapy; cardiac ischemia; cardiac or hepatic failure; or acute neurological illness as 
assessed by history and physical exam. 
5. Have evidence of diseases that may influence metabolism (e.g. overt diabetes mellitus, thyroid 
disease, cancer) 
6. Have any current or past history of eating disorders such as binge eating or anorexia nervosa. 
7. Have had weight loss (bariatric) surgery. 
8. Have gained or lost more than 3% of your current weight in the past 3 months. 
9. Exercise strenuously, like running, swimming, or basketball, 4 or more times per week. 
10. Are planning to begin a strenuous exercise program in the next 4 months. 
11. Take supplements that contain omega-6 or omega-3 fatty acids (e.g. fish, cod liver, borage, 
evening primrose oils) 
12. Have significant dietary limitations (e.g. multiple food allergies/intolerances, vegan diet) or 
special dietary requirements that are difficult to accommodate with study diets 
13. Have significant claustrophobia that would preclude study tests. 
14. Have a history of alcohol or substance abuse or dependence in the past 5 years. 
15. Drink more than 2 alcoholic beverages per day, on average, or 6 drinks per sitting in the past 
year. Participants may drink up to one alcoholic beverage per day during the study. 
16. Used marijuana, amphetamines, cocaine, or heroin even once over the last year.  Participants 
may not use marijuana, amphetamines, cocaine, or heroin during the study. 
17. Have smoked 2 or more cigarettes per week, on average, over the past year. 
Participants may not use tobacco products during the study. 
18. Eat fish 3 or more times per week on average. 
19. Have a known bleeding disorder. 
20. Take medications or supplements that may interfere with this study by altering energy 
metabolism, nutrient absorption, or food intake. Regular use of the following compounds 
warrants exclusion: medications for the management of diabetes (Type 1 or 2), typical and 
atypical antipsychotics, thyroid medications, glucocorticoids, orlistat, decongestants, anti-
histamines, and medications for cardiac conditions (e.g beta blockers), attention disorders (e.g. 
amphetamine derivatives), blood lipid disorders (e.g. statins, fibrates), and hypertension (e.g. 
thiazide diuretics), as well as certain anti-depressants (e.g. tricyclics), and supplements that 
impact energy metabolism, such as ephedrine. 
21. Regularly take an anticoagulant medication, such as warfarin or aspirin.   
22. Have a pacemaker, brain stimulator, or other implanted electrical device, permanent eyeliner, 
metallic prosthesis (including metal pins and rods, heart valves, and cochlear implants) that 
would preclude MRI scans. 
23. Are currently participating in any other clinical research studies that include blood draws or 
other procedures.  
24. Have a serum FSH level greater than 20 units per liter. 
 
See Appendix 6: Eligibility Checklist 
 
7. Study Design and Methods: 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
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7.1 Study overview and design 
This randomized controlled, 3-arm dietary trial aims to assess the rate and extent of changes in the 
composition of omega-6 and omega-3 HUFA and their metabolic derivatives in response to marked 
dietary changes in the nutrient omega-6 LA, in ambulatory overweight premenopausal women. The main 
trial is divided into two phases: 1) a 4 week ‘baseline phase’, and 2) a 12 week ‘diet phase’. There is also 
an optional 36 week ‘continuation phase’ for participants receiving diet “B”, as discussed below.  During 
the baseline phase, prior to randomization, eligible subjects will complete 7-day food records. Those who 
are compliant with the food records and meet other eligibility criteria will undergo a baseline testing 
battery that includes: 1) blood parameters; 2) muscle and adipose tissue biopsies; 3) body composition 
assessment; 4) functional satiety assessment; and 5) psychometric testing. Participants will then be 
randomized to one of 3 healthy and nutritionally complete diets with equivalent macronutrient 
composition differing only in fatty acid content.  
After randomization, participants will receive all foods through the NIH Metabolic Kitchen for the 
duration of the 12-week diet phase (i.e. meal replacement). During the first 4 weeks, the iso-caloric 
‘weight maintenance’ phase, participants will be provided with the same amounts of daily food calories as 
they consumed during the 7-day record collection.  During the following 8 week ‘ad libitum’ phase (from 
4 to 12 weeks), participants will receive the same diets but will be allowed to eat as many servings of 
study foods as they desire.  After completion of the 12 week diet phase, the full testing battery will be 
repeated.   
 
At completion of the 12 week diet phase, a subset of participants (those receiving diet ‘B’) will have the 
option of continuing their study diets for an additional 36 weeks.  During this ‘continuation phase’, the 
diets will be identical to their preceding diets. After 6, 12, 18, 24, and 30 weeks of the continuation phase, 
subjects will participate in follow-up testing sessions.  At completion of the 36-week continuation phase, 
the complete baseline testing battery will be repeated. 
Participants will remain ambulatory throughout the study. Subjects will visit the NIH Metabolic Kitchen 
daily Monday through Friday to obtain foods, and will be advised to continue their usual medical care and 
medications throughout the study.  
 
7.2 Recruitment  
Subjects will be recruited through the NIH Normal Volunteer Office, by word of mouth and through local 
advertisement (IRB approved flyers, newsletters, websites).   
 
7.3 Screening Methods 
 
7.3.1 Pre-screening 
The NIH Patient Recruitment and Public Liaison Office will receive inquiries from interested study 
subjects.  Pre-screening by this office will exclude patients who cannot regularly attend the NIH 
Metabolic Unit to acquire study foods, and those with significant claustrophobia that will preclude the 
imaging tests that are required to participate in the full protocol. All others will be contacted by the 
protocol team to review exclusion criteria. Eligible subjects will be invited to Clinical Research Center for 
a screening visit. 
 
7.3.2 Screening visit (Baseline Phase Visit 1) 
Patients will be invited to come to the OP-9 (Outpatient 9) Multiple Medical Specialties Clinic to obtain 
informed consent by a member of the investigative team. A limited physical exam with current weight 
and evaluation of venous access will be performed.  A thorough medication history will be obtained with 
special attention given to drugs that may confound the interpretation of metabolic testing such as 
psychotropic agents, antiretroviral agents, sympathomimetic amines or beta blockers. An 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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electrocardiogram and baseline blood work (electrolytes, chronic liver disease work-up, renal function, 
vitamins, thyroid function, HgbA1C, HIV test), a serum pregnancy test and a urine test to screen for illicit 
drugs will be obtained. Instructions for completing detailed 7-day food records will be given to 
participants by a member of the Nutrition Dept staff. 
 
7.4 Study Design  
We anticipate that 84 subjects will need to complete the baseline phase and be randomized, with an 
anticipated dropout of about 20 subjects during the diet phase, in order for 64 subjects to complete the 12-
week diet phase. Based on previous NIH Clinical Center dietary protocols with a similar baseline phase, 
we anticipate that 30 to 55% of enrolled subjects will not complete the baseline phase due to lack of 
attendance, non-compliance with 7-day food records, and/or discovery of an underlying exclusionary 
condition during the baseline phase history, physical examination or laboratory testing. Therefore, up 
to180 subjects will be enrolled in the baseline phase so that a sufficient number of subjects (64) will 
complete the diet phase.  
  
After completing the baseline phase, 84 subjects will be randomized to 1 of 3 diets (Table 3), to be 
maintained for 12 consecutive weeks. We anticipate that about 20% of subjects will dropout during the 
diet phase. Therefore 64 subjects are expected to complete the diet phase. 
 
 
Table 3: Fatty Acids in 3 Study Diets 
Diet Group 
omega-6 
LA* 
omega-3 
ALA 
omega-6 
AA 
omega-3 
HUFA 
%omega-6 in 
HUFA** 
A. Orange 
8.00 
0.40 
0.045 
0.045 
80 
B. Blue 
1.00 
0.40 
0.045 
0.045 
50 
C. Green 
1.00 
0.40 
0.045 
0.81 
20 
*Fatty acid intake as percentages of daily food energy (en%).  
** Predicted percentage of omega-6 HUFA in total omega-6 and omega-3 HUFA, via the Lands empirical equation, 
as detailed in Appendix 2 
 
Please see Section 7.1: Study overview and design and Appendix 1: Study Design Flow Diagram. 
 
 
7.5 Study Procedures   
The main study lasts 16 weeks and is divided into 2 phases: 1) a 4 week ‘baseline phase’, and 2) a 12 
week ‘diet phase’.  There is also an optional 36 week ‘continuation phase’ for participants receiving diet 
“B”.   
 
During the 4 week baseline phase participants will visit the NIH outpatient clinics three times as shown in 
Table 3 below. Visit 1 is a screening visit lasting about 1 hour.  During Visit 2 laboratory tests will be 
reviewed, and participants will fill out questionnaires, which will take about 2 hours.  At Visit 3, 
participants will receive about 6 hours of testing and will be randomly assigned to 1 of 3 study diets.  
 
During the 12 week diet phase participants will eat only the study foods that are supplied by the NIH 
metabolic kitchen.  They will visit the NIH outpatient metabolic unit every morning for breakfast 
(Monday-Friday) for all 12 weeks of the diet phase.  Each of these 60 visits will last about 20 minutes, for 
a total of 20 hours.  They will fill out the Comprehensive Psychopathological Rating Scale-Self-Affective 
(CPRS) questionnaire (see Section 7.6.6) after breakfast once every 2 weeks, which takes about 10 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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minutes to complete.  Once every 4 weeks after breakfast, on Diet Day 28 and 56, they will have 
additional testing for about 1.5 hours; on Diet Day 84 they will have additional testing for about 6 hours 
as shown in Table 4 below. During the optional 9 month (36 week) continuation phase participants 
continue to eat only the study foods supplied by the metabolic kitchen.  They will continue to visit the 
NIH outpatient metabolic unit every morning for breakfast (Monday-Friday) for all 36 weeks of the 
continuation phase.  Each of these 180 visits will last about 20 minutes, for a total of 60 hours.  
Participants will fill out the CPRS questionnaire after breakfast once every 2 weeks, which requires about 
10 minutes to complete.  Once every 6 weeks after breakfast, participants will undergo additional testing 
for 3 to 6 hours as shown in Table 5 below.   
 
All study procedures are for research purposes only. 
 
 
Baseline Phase 
The sequence of procedures in the baseline phase is given in Table 4.  All time-points are targets, and 
may be adjusted based on practical considerations.  
 
Table 4:  Procedures during the Baseline Phase 
Baseline Phase Testing Visits (4 week phase, Days 1-28) 
 
 
 
Baseline phase: 1st Visit 
The first visit of the baseline phase is described in detail in Section 7.3.2 Screening Visit. 
 
Baseline phase: 2nd Visit  
Participants will return for an outpatient visit about 14 days after Baseline Visit 1. The 7-day food records 
and the results of the laboratory testing will be reviewed.  Questionnaires to evaluate eating, satiety and 
mood will be administered (see Section 7.6.6).  Those who remain eligible will be scheduled for a 3rd 
baseline visit.   
 
Baseline phase: 3rd Visit  
Procedure  
Visit 1 
(Day 1) 
Visit 2 
(Day 14) 
Visit 3 
(Day 21-28) 
Blood Draw   
▲ 
 
▲ 
Saliva DNA testing 
▲ 
 
 
Urine Drug Screen 
▲ 
 
 
Urine Laboratory Testing  
▲ 
 
▲ 
7-day Food Records  
▲ 
▲ 
 
Medical Exam and Interview 
▲ 
▲ 
 
Eating Questionnaires 
 
▲ 
 
Mood Questionnaires 
 
▲ 
 
Weight, Anthropometrics and Vital Signs 
▲ 
▲ 
▲ 
Electrocardiogram (EKG) 
▲ 
 
 
DEXA Scan 
 
 
▲ 
Muscle and Adipose Tissue Biopsies 
 
 
▲ 
Buffet Meal Test 
 
 
▲ 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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Participants will return for an outpatient visit about 7 to 14 days after the 2nd Visit. If their weight is 
stable, participants will have additional tests, which will require about 6 hours (Testing procedures are 
listed in Table 4).  
Participants will receive a schedule for the remaining study visits and tests. Participants will then be 
randomized to 1 of 3 diets as described in Section 7.6.2.  
 
Study Diets 
The 3 study diets differ only in the amounts of fatty acids that they contain, as described in Sections 8.2.  
The diets are designed by NIH Clinical center dietitians to be nutritionally complete.  All foods will be 
provided by the NIH metabolic kitchen. Participants are required to visit the NIH Clinical Center each 
morning, Monday through Friday, to eat breakfast and to pick up study foods, and to return uneaten food 
to the kitchen. Participants are also asked to keep a daily log of food and beverage intake.  
 
We will strive for 100% adherence to study diets and daily visit schedules, but recognize based on 
previous experience and reviews of literature that this may not be practical or achievable in an ambulatory 
setting for this length of time. Therefore, to maximize adherence participants are provided with a day of 
emergency meals to eat during missed days whenever possible. They will also receive group-specific 
food-based guidance from the nutrition team designed to mimic their diet group if they are unable to pick 
up study diets for more than one day. Subjects will record/report any non-study foods or beverages 
throughout the study and this will be analyzed as part of the study diet.  Finally, we will measure 
adherence to study diets using two proxies: (1) the LA content of circulating plasma measured at baseline 
and 4, 8, and 12 weeks of diet exposure; and (2) the number of days they did not pick up study meals. In a 
posthoc/exploratory manner, we will test for effect modification of the diet effects based on adherence. If 
effect modification is evident, we will present the results of the adherent subgroup as a posthoc analysis. 
Diet Phase 
The sequence of procedures in the Diet Phase is given in Table 5.  All time-points are targets, and may be 
adjusted based on practical considerations.  
 
Table 5: Procedures during the Diet Phase 
Diet Phase Testing Visits (12 week phase, Diet Days 1-84) 
 
 
During the 12 week diet phase participants will visit the NIH outpatient metabolic unit every morning for 
breakfast (Monday-Friday).  Each of these 60 meal visits will last about 20 minutes, for a total of 60 
hours.  Participants will complete the CPRS questionnaire after breakfast once every 2 weeks, which 
takes about 10 minutes to complete.  
 
Procedure 
Visit 1 
(Day 28) 
Visit 2 
(Day 56) 
Visit 3 
(Day 84) 
Blood draw 
▲ 
▲ 
▲ 
Urine Pregnancy Test  
▲ 
▲ 
▲ 
Mood Questionnaires 
▲ 
▲ 
▲ 
Weight, Anthropometrics and Vital Signs   
▲ 
▲ 
▲ 
DEXA Scan 
▲ 
▲ 
▲ 
MRI Scan 
 
 
▲ 
Muscle and Adipose Tissue Biopsies 
 
 
▲ 
Buffet Meal Test 
▲ 
 
▲ 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
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On 2 separate occasions after breakfast, once every 4 weeks on Days 28 and 56, participants will have a 
testing session lasting about 1.5 hours (Testing procedures are listed in Table 5).  Twelve weeks after 
beginning the diet phase (Day 84), participants will have testing that lasts about 6 hours.  They will have a 
shorter 2 hour testing session 4 and 8 weeks after beginning the diet phase.   
 
 
Optional Continuation Phase 
Those participants who were assigned to either diet “A” or diet “C” will have completed their 
participation after the 12-week diet phase of the study. Those who were assigned to diet “B” will be given 
the option of continuing a study diet for an additional 9 months (36 weeks).  
 
The sequence of procedures in the Continuation Phase is given in Table 6.  All time-points are targets, 
and may be adjusted based on practical considerations.  
 
Table 6: Procedures during the Continuation Phase 
Diet Continuation Testing Phase Visits (36 week phase, Diet Days 85-336)  
 
 
During the 36-week continuation phase, participants will continue to receive the study diet “B” that they 
received in the 12 week diet phase as shown in Appendix 1: Study Design Flow Diagram. 
As in the main diet phase, participants will continue to visit the NIH outpatient metabolic unit every 
morning for about 20 minutes for breakfast (Monday-Friday). Each of these 180 visits will last about 20 
minutes, for a total of 60 hours. Participants will fill out the CPRS questionnaire for about 10 minutes 
after breakfast once every 2 weeks.  
 
On 5 separate occasions after breakfast, at 6, 12, 18, 24 and 30 weeks into the continuation phase, 
participants will have a testing session lasting about 1.5 hours.  At 36 weeks into the continuation phase 
participants will have a testing session lasting about 6 hours (Testing procedures are listed in Table 6).  
 
 
7.6 Study Procedure Specifics  
 
7.6.1 Food Records 
Food records will be used to assess recent and usual intake of kilocalories, macro- and micronutrients as well as 
assess usual eating patterns.  The information will be used to plan the caloric content of the diet to be served from 
Procedure 
Visit 1 
Day 
126 
Visit 2 
Day 
168 
Visit 3 
Day 
210 
Visit 4 
Day 
252 
Visit 5 
Day 
294 
Visit 6 
Day 
336 
Blood draw 
▲ 
▲ 
▲ 
▲ 
▲ 
▲ 
Urine Pregnancy Test 
 
▲ 
 
▲ 
 
▲ 
Mood Questionnaires  
▲ 
▲ 
▲ 
▲ 
▲ 
▲ 
Weight, Anthropometrics and Vital 
Signs 
▲ 
▲ 
▲ 
▲ 
▲ 
▲ 
DEXA Scan 
 
▲ 
 
▲ 
 
▲ 
MRI Scan 
 
 
 
 
 
▲ 
Muscle and Adipose Tissue 
Biopsies 
 
 
 
 
 
▲ 
Buffet Meal Test 
 
 
 
 
 
▲ 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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metabolic kitchen.  The food record will be analyzed using the Nutrition Data System for Research (NDS-R), 
developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN.  
 
7.6.2 Randomization and Masking  
At completion of the pre-intervention phase, participants will be randomized to 1 of the 3 treatment groups by 
computer.  This process eliminates experimenter bias in group assignment. The computer program uses a random 
numbers generator and is programmed to ensure appropriate numbers of subjects in each arm of the study within a 
permuted variable block size of 3-4 patients.  The computer program generates a treatment assignment associated 
with each eligible subject’s name and study ID number and documents these in an uneditable form with a date 
stamp. The research dietitian will have password-protected access to the computer-generated treatment 
assignments.  The dietitian will use this information to assign each participant to one of the 3 treatment arms, 
which will be designated by a letter and color (e.g. Diet A: Orange).  Because the 3 diets differ only in the fatty 
acid content of added visible oils, which are clear and can be distinguished only by nutritional analysis, the 
participants will remain blinded to their treatment.  The research dietitian, by necessity, needs to know which 
group the patient is assigned in order to provide appropriate foods. No other research or clinical investigators will 
be told which arm the participants have been assigned.   
 
7.6.3 Specimen collection for biomarker testing 
 
Blood Collection  
To assess the blood compositions of omega-6 and omega-3 fatty acids and their metabolic derivatives, 
fasting venous blood will be collected at 4 timepoints in the main study (Baseline Visit 3; Diet Phase 
Visits 1, 2 and 3).  Blood will be collected from participants in the optional continuation phase at 6 
additional time points (see Table 5: Continuation Phase Visits).  Blood will be immediately processed and 
stored at -80 degrees C. See Table in Appendix 2 for a list of fatty acids and their metabolic mediators 
that will be assessed.   
 
Genotyping 
This project will collect DNA samples from the saliva of well phenotyped overweight subjects for genotyping.  
Genotyping will be performed on all study participants. Single gene mutations/polymorphisms of interest include: 
1) those involved in fatty acid metabolism; 2) those implicated in the pathogenesis of obesity; 3) those implicated 
in the pathogenesis of medical and psychiatric conditions associated with obesity; 4) those linked to certain 
behavioral characteristics relevant to obesity and obesity-related comorbidities. On the consent form, participants 
can designate whether or not the DNA samples can also be stored for future research projects. 
 
Muscle and adipose tissue biopsies and tissue procurement and post-biopsy pain assessment 
Fat and muscle biopsies will be obtained before and after the dietary interventions, by the study 
physicians and/or nurse practitioner. In accordance with established NIDDK practices, biopsies will be 
performed on the Metabolic Unit or the adjacent 5SW day hospital, as described below.  
Subcutaneous adipose tissue (3-5 g) will be removed from the abdominal region by aspiration with a 16-
gauge needle under local anesthesia (e.g. 2% xylocaine).  
Muscle biopsies (100-250 mg) will be obtained under local anesthesia from the vastus lateralis, as 
follows. The patient will be prepped and draped in sterile fashion. The muscle biopsy procedure involves 
numbing a nickel-sized portion of the skin of the thigh with a local anesthetic and making a small 
incision, approximately 1/4 inch. The biopsy needle is then inserted via the incision through adipose 
tissue and muscle fascia into the muscle.  A small sample of muscle tissue is removed.  The incision will 
then be closed with steri-strips and covered with a dressing.  If the muscle is too deep to be reach, the 
muscle biopsy will not be attempted.  

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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The biopsy tissue specimens will be separated into two aliquots for muscle and three aliquots for adipose tissue 
for assessment of fatty acid composition and other biochemical assays, frozen in dry ice and stored at -80ºC for 
future studies.   
Post-biopsy pain will be evaluated using the standard Clinical Center Numeric Rating Scale (Score range 0-10) 
before and after the biopsy, and twice daily (upon awakening and at lunch time) for 3 additional days. 
 
 
7.6.4 Insulin Sensitivity 
Homeostasis Model of Assessment – Insulin Resistance (HOMA-IR)  
To assess insulin sensitivity, fasting blood glucose and insulin will be collected at each testing visit, for 
calculation of the HOMA-IR, a validated biomarker for estimating insulin sensitivity.  
 
 
 
7.6.5 Functional Satiety assessment 
Ad-libitum Buffet Meal 
Participants will be fed a standard breakfast meal containing 15% of energy needs (calculated using the 
Mifflin St. Joer equation with an activity factor of 1.5).  Between the breakfast and buffet meal 
participants will be asked to fill out Visual Analogue Scales asking questions about hunger and satiety at 
30-60 minute intervals.  Six hours following the standard breakfast the patient will be placed in a food 
laboratory and presented with a buffet meal.  They will be instructed to “eat until they are full.”  The 
buffet meal will contain foods that will be weighed pre and post consumption in a metabolic kitchen so 
that the precise amounts of foods consumed can be calculated. 
 
Buffet Meal Screening 
Food Preference Questionnaire:   
At screening patients will fill out a 50 item food preference questionnaire with buffet items embedded 
within it.  If the patient reports dislike of > 50% of the food items on the buffet, they will be excluded 
from participation. 
 
7.6.6 Psychometric assessments 
Questionnaires to Evaluate Eating Behavior and Satiety   
WALI- Weight and Lifestyle Inventory:  This is a comprehensive questionnaire encompassing the subject’s weight 
and health history.   It includes sections on self-reported eating and physical activity patterns.  Eating disorders 
such as binge eating, night eating syndrome and other abnormal eating patterns can be evaluated with this 
assessment.  Estimated completion time is 60-90 minutes. 
 
Three Factor Eating Questionnaire:  An instrument developed by Stunkard and Messick to measure dietary 
restraint, disinhibition and hunger.  Consists of 51 questions related to these 3 factors of human eating behavior.  
Estimated completion time is 20-30 minutes. 
 
Questionnaires to Evaluate Mood  
POMS- Profile of Mood States:  The POMS assessment provides a rapid, economical method of assessing 
transient, fluctuating active mood states.  The POMS can be readministered as frequently as every week.  The 
scale contains 65 items, which subjects rate on the Likert scale (0 to 4).  A summation of scores for tension, 
depression, anxiety, fatigue, and confusion are subtracted from the vigor score to yield an overall description of 
subject’s mood state.  Estimated completion time is 10-15 minutes. 
 
FS- Flourishing Scale:  This scale consists of 8 questions related to subject’s feelings about their personal 
accomplishments in life.  Subjects rate each item on a numerical scale.  High scores represent people who feel 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
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they are leading thriving, resourceful and capable lives, and thus is a measure of positive affect.  Estimated 
completion time is 5-15 minutes. 
 
PANAS- Positive and Negative Affect Schedule:  This assessment is a self-rated affect scale which distills 
emotionally descriptive words into either positive or negative mood valences. The subject gives a numerical 
rating to each emotionally descriptive word given in the list of words.   Positive and Negative affectivity are 
constructed by summing the ratings for these words.  The test can be repeatedly administered with valid results; 
even within a single day.  Estimated completion time is 10-15 minutes. 
 
CPRS-S-A- Comprehensive Pyschopathological Rating Scale-Self-Affective:  Contains 19 questions condensed 
and rephrased from the full CPRS for self-implementation to quickly assess the subject’s mood and anxiety level.  
It can be repeatedly administered, up to every three days.  It will be administered weekly in this study.  Estimated 
completion time 5-15 minutes. 
 
 
7.6.7 Body Composition 
Anthropometric measurements and bioelectrical impedance 
Height, weight, and circumferences will be obtained using standardized procedures. Bioelectric impedance 
spectroscopy (BIS, Xitron 4200, Xitron Technologies, San Diego, CA) testing will be used to assess fluid status 
(ECW, ICW, TBW).  Subjects are measured while lying supine on a nonconductive surface.  Bioelectric 
resistance and reactance values are measured after induction of electrical signals with a maximum current of 800 
µA at multiple frequencies.   
 
Dual Energy X-ray Absorptiometry (DEXA) scan  
DEXA (iDXA, GE Medicalsystems, Madison WI) will be used to measure body composition. This technique has 
found extensive clinical and research applications in addition to the assessment of bone mineralization status. 
With this technique, one can determine total and regional body fat and fat-free masses and can estimate 
appendicular muscle. DEXA produces photons at two different energy levels, 40 and 70 KeV. The photons pass 
through tissues and attenuate at rates related to elemental composition. Bone mineral, with highly attenuating 
calcium and phosphorous, is readily distinguished from soft tissues. The different elemental profiles of fat and 
bone-mineral free lean allows for the analysis of soft tissue fat content, so that bone mineral, fat, and bone mineral 
fat-free lean components may be resolved.  The DEXA has a slight radiation associated (an effective dose of 
about 1 mrem total exposure to the tissue/scan, approximately equal to one day of background exposure).   Half-
body scans may need to be performed for subjects that extend beyond the scanning area (26, 27). 
 
Assessment of visceral, liver and cardiac adipose (3T MRI) 
Overweight and obesity are associated with fat accumulation in the abdominal cavity (visceral fat) and 
other important sites such as the liver (28, 29) and muscle, referred to collectively as “ectopic fat” (30). 
Magnetic resonance imaging (MRI) is used to quantify triglyceride content in the abdominal cavity, liver 
and heart.  Two techniques will be employed to measure ectopic fat: proton MR spectroscopy (MRS) (29) 
and multi-echo imaging using the 3.0 T magnet (31).   
 
7.6.8 Assessment of Dietary Adherence 
Adherence to study diets will be assessed via multiple methods.  The fatty acid compositions of all study 
foods will be determined and all study foods that are dispensed to study subjects will be recorded.  
Participants will be asked to return any foods they do not eat. They will be instructed to record any 
consumption of outside foods in a food log, including the specific food type (name brand if possible) and 
serving size.  Missed breakfast and food pick-up visits will be recorded.  In addition, blood concentration 
of LA will be measured every 4 weeks during the diet phase, and every 6 weeks during the continuation 
phase.  

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
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7.6.9 Follow-up/ termination procedures 
There is no required follow-up for this study after completion of the experimental protocol. Participants will be 
advised to continue receiving their usual medical care throughout the duration of the study.  With patient consent, 
clinically-relevant information obtained through participation in the protocol may be shared with participants and 
appropriate health care providers.   
 
7.6.10 Radiation exposure 
DEXA scans will be performed solely for research purposes.  Please see sections 8.7 and 8.9 for risks. 
 
7.6.11 Medications and devices requiring IND/IDE 
No medications or devices requiring IND/IDE will be used in this study. 
 
7.6.12 Storage of research samples and data 
Since many potentially important markers and genes relevant to obesity and related comorbidities are unknown at 
the present time, all research samples including DNA will be coded for storage in refrigerators and freezers in a 
locked laboratory. Retention of codes to identify the sources of research samples is necessary to perform 
correlations between genotype and phenotype, and between dietary intervention and outcomes.  The plan is to 
store the samples until they are analyzed.  These samples will be stored indefinitely.  The IRB will be notified in 
the event these samples are accidentally destroyed, lost or are anonymized.  Tissue banking of DNA and tissue 
specimens will only include participants who specifically provide consent for future studies.  
Some clinically relevant research data will be stored indefinitely in the medical record and will be accessible to 
the patient for review by others of their choosing (doctors, insurance companies etc.) after executing a release of 
information.  The validated psychometric instruments used in this protocol have been converted to an electronic 
web-based securedata entry platform administered by the NIAAA.  .  The security of the data management system 
f is curated by the NIAAA in agreement with NIH data security policies.  Most data generated in this study will 
be stored and maintained in the NIAAA clinical database, which is password protected and secure.  
 
 
Use of research samples and data 
Currently, the research use of samples is outlined in the protocol. Stored materials will be used for future 
evaluation of metabolic mediators derived from fatty acids, as well as metabolic mediators implicated in obesity 
and related medical and psychiatric comorbidities as the field evolves.   
 
Collaborations on stored tissue and blood samples  
Any collaborations requiring transfer of patient material will be done in a coded fashion after informing the IRB 
and obtaining necessary assurances from the outside institution.  The protocol will be amended and IRB approval 
will be sought when such collaborations are established.     
 
 
 
8 Risks and Discomforts  
8.1 Procedures with No Medical Risk and Minimal Discomfort 
The history and physical exam, body measurements, bioelectrical impedance, vital signs, 
electrocardiogram, buffet meal test, and questionnaires to evaluate eating history and quality of life have 
no medical risk and minimal discomfort. 
 
8.2 Study Diets 

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We will provide healthy and nutritionally complete diets to all intervention groups. The diets will differ in 
their fatty acid composition. In general, there is minimal risk associated with adverse events as fatty acids 
in edible form are GRAS (generally recognized as safe) by federal regulatory agencies.   
 
The controlled dietary variable to be changed in this study is linoleic acid (LA), a common nutrient in 
foods. The minimal dietary requirement for LA to prevent signs and symptoms of deficiency, as reviewed 
and developed by William EM Lands, PhD is between 0.05% and 0.50% of energy (see Figure below, 
available at http://efaeducation.nih.gov/sig/dri.html). Furthermore, several modern populations consume 
less than 1 % of energy from LA with no apparent ill-effects (32). The American Heart Association 
recently advised that it is safe to consume “at least 5 to 10% of energy” from LA (33). In our study diets, 
LA intakes will range from 1.0 % to 8.0 % of energy. At these ranges, there is little concern for risk as 
they are well above the minimal dietary requirement and in ranges that are commonly consumed by 
modern humans.   
 Dietary Reference Intake (DRI) for LA 
 
Some published articles with information related to these four categories are listed below.  
EAR-Unwanted dermal signs were present in 100% infants receiving 0.04 % energy as linoleate, but in 
only 40% infants receiving 0.07 % energy as linoleate and not in any receiving 1.3 %. The EAR may be 
near 0.06 % of energy (34); RDA-Thousands of healthy infants were raised with diets containing omega-
6 as 0.4 to 0.9 % energy, and calculation showed that probably less than 0.5 % energy was needed to 
prevent deficiency symptoms in infants. This level may meet requirements for 97% to 98% of infants (35, 
36); AI- Although not rigorously determined, intakes from 1 % to 3 % of daily energy as omega-6 seem 
adequate (37); UL-Excessive omega-6 eicosanoid actions and thrombotic deaths seem less frequent with 
balanced omega-3/omega-6 intakes (38). Quintiles of dietary intake of LA-in the USA have the 
following means: 2.9; 4.4; 5.5; 6.9; 10.3 en% (39).  
 
A subset of participants will also receive 1 to 2 grams of omega-3 EPA and DHA from fatty fish. There is 
a theoretical increase in bleeding times with omega-3 fatty acid intake.  However, human supplement 
trials, including trials with considerably higher doses of omega-3 fatty acids, have not found increased 
bleeding risk (40, 41), with or without concurrent use of antithrombotic medications. We will use doses 
from foods that well within the usual daily intakes of populations with regular fish intake, such as Japan 

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and Iceland (42, 43). Therefore, there is little concern for increased risk due to increased omega-3 fatty 
acid intake.   
 
It is possible that participants could have an allergic response to study foods.  During the screening visit, 
participants will provide information on known dietary allergens and the diets will be adapted 
accordingly, or if necessary the participants will be excluded.   Participants will also be advised to alert 
the research staff as soon as possible if they experience symptoms such as rash, hives, wheezing, or other 
signs of food allergy.  
With any change in dietary intake there is potential for gastrointestinal distress in human subjects. 
Subjects will be asked to report signs of distress. Participants will be advised to use appropriate 
medications and/or digestive aids (H2 blockers, bean-o, etc) as necessary.  
This study also requires daily workday visits to NIH for 12 weeks.  It requires that participants only eat 
the food we provide. They may find these requirements inconvenient or burdensome.  They may not like 
the study foods.  Each of the study diets is nutritionally complete so it is unlikely that eating the diet will 
compromise health. 
8.3 Specimen Collection for biomarker testing 
Blood Collection 
Participants may have some discomfort and bruising at the site of needle entry.  There is a very small risk 
of fainting.  Infection in the area of the needle insertion is rare. 
The amount of blood drawn from participants shall not exceed the specified limit of 550 ml (or 10.5 
mL/kg) for any 8-week period for adult volunteers, provided in “Guidelines for Limits of Blood Drawn for 
Research Purposes in the Clinical Center” (M95-9 (rev.) 5 June 2009). 
Since participants will have BMI between 25-35, the limit of 550 mL per 8-week period will apply. 
During the main study, which includes the baseline phase and 12-week diet phase, we will draw no more 
than 1 cup of blood (1 cup = 8 fl oz = 235 mL) for research purposes at any one time and no more than 3 
cups during the entire main study.  We will draw no more than 2 cups (470 mL) of blood during any 8-
week period.  
For participants in the optional 36-week continuation phase, we will draw no more than 3 additional cups 
of blood for research purposes, and no more than 1.5 cups (353 mL) during any 8-week period.   
 
Genetic Testing 
The genetic testing that will be done as part of this study is done for research purposes only.  It will not 
provide any information about participants’ health or ancestry. It is our policy to not provide the results of 
such genetic testing.  
 
 
 
8.4 Muscle and adipose tissue biopsies and tissue procurement 
The major risks/discomforts include pain, bruising, infection, and scarring. The procedures will be 
performed under sterile technique to minimize the chances of infection. Local anesthetic will be used to 
minimize pain.  Ice will be applied to the site immediately after the procedure to limit bruising, swelling 
and tenderness. After subcutaneous tissue biopsy, patients will be monitored by a nurse. Post-biopsy the 
incision site will be cleaned and closed with adhesive wound closures and covered with gauze and 
translucent dressing tape. Study participants will be instructed to report to the clinical staff any changes at 

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the biopsy site including bleeding, secretion, erythema, pain, and signs and symptoms of infection. Study 
participants will be instructed to self-monitor the incision site after discharge from the Clinical Center.  
 
8.5 Questionnaires to Assess Mood  
Some subjects may find the questions or tests boring or frustrating.  They may feel uncomfortable discussing their 
mood and personality with the study staff.  They will be instructed to answer the questions as well as and as 
honestly as they can.  There are no direct physical risks associated with these tests.  It is possible that important 
information may be revealed about the subject that in some cases may benefit from medical intervention.  In this 
case, the patient will be advised to share this information with their doctor for follow-up.  Furthermore, with the 
patient’s consent, results from these tests will be forwarded to their physician of choice for follow-up.   
 
8.6 Body Composition 
Dual Energy X-ray Absorptiometry (DEXA) SCAN 
DEXA will be used to measure body composition using x-rays to non-invasively assess skeletal and soft 
tissue density.  The procedure involves low-level radiation exposure.  The expected exposure for a scan is 
(0.001 rem) or the equivalent of less than one day’s exposure to natural background radiation.  For more 
information see Radiation in Section 8.9. 
 
Magnetic Resonance Imaging (MRI) assessment of visceral, liver and cardiac fat  
People are at risk for injury from the MRI magnet if they have pacemakers or other electrical devices, 
brain stimulators, some types of dental implants, aneurysm clips (metal clips on the wall of a large artery), 
metallic prostheses (including metal pins and rods, heart valves, and cochlear implants), permanent 
eyeliner, implanted delivery pump, or shrapnel fragments.  Welders and metalworkers are also at risk for 
injury because of possible small metal fragments in the eye of which they may be unaware.  Subjects will 
be screened for these conditions before having any scan, and if any are found, the subject will not receive 
an MRI scan.    It is not known if MRI is completely safe for a developing fetus. Therefore, all women of 
childbearing potential will have a pregnancy test performed no more than 24 hours before each MRI scan. 
The scan will not be done if the pregnancy test is positive.  
People with fear of confined spaces may become anxious during an MRI. Those with back problems may 
have back pain or discomfort from lying in the scanner.  The noise from the scanner is loud enough to 
damage hearing, especially in people who already have hearing loss. Everyone having a research MRI 
scan will be fitted with hearing protection. Subjects will be asked to complete an MRI screening form for 
each MRI scan.   
 
8.7 Radiation from DEXA Scans 
This research study involves exposure to radiation from the 4 DEXA scans in the main study and 3 
additional DEXA scans in the optional continuation phase. This radiation exposure is not required for 
medical care and is for research purposes only.  The amount of radiation the subject will receive in the 
main study is 0.004 rem, and in continuation phase is an additional 0.003 rem, which are below the 
guideline of 5 rem per year allowed for research subjects by the NIH Radiation Safety Committee. The 
average person in the United States receives a radiation exposure of 0.3 rem per year from natural 
sources, such as the sun, outer space, and the earth’s air and soil.  If a subject would like more 
information about radiation a copy of the pamphlet An Introduction to Radiation for NIH Research 
Subjects will be provided.  
 
While there is no direct evidence that the amount of exposure from participating in this study is harmful, 
there is indirect evidence it may not be completely safe.  There may be a very slight increase in the risk of 
cancer. 
 

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Participants will be instructed to tell the doctor if they have had any radiation exposure in the past year, 
either from other research studies or from medical tests or care, so the research staff can make sure that 
the subject will not receive too much radiation.  Radiation exposure includes x-rays taken in radiology 
departments, cardiac catheterization, and fluoroscopy as well as nuclear medicine scans in which 
radioactive materials were injected into the body.   
 
If a subject is pregnant or breast feeding, she may not participate in this research study. It is best to avoid 
radiation exposure to unborn or nursing children since they are more sensitive to radiation than adults. 
 
 
 
9 Subject Monitoring, adverse event grading and withdrawal 
 
9.1 Parameters to be monitored 
Participants will be closely monitored for adverse reactions by medically qualified staff during the 
invasive procedures; including the tissue biopsy procedures, the blood sample collection. Appropriate 
medical care and treatment will be provided if necessary. Study participants will have daily contact with 
the metabolic kitchen staff during weekdays and will be encouraged to report any potential adverse 
reactions to study foods. 
 
During the diet phase and continuation phase, the CPRS will be administered to monitor participants’ mood.  It is 
possible that important information may be revealed about the subject that in some cases may benefit from 
medical intervention.  In this case, the patient will be advised to share this information with their doctor for 
follow-up.  Furthermore, with the patient’s consent, results from these tests will be forwarded to their physician of 
choice for follow-up.   
 
Participants will be withdrawn from the study if they do not follow the instructions outlined in the 
protocol and informed consent form.  Non-compliance will be set as a threshold of deviation from the 
provided diet and will be determined by medically qualified staff.  Participants may also, at any time, 
choose to discontinue their participation from the study. 
 
 
10. Outcome Measures  
 
10.1 Primary Outcome Measures  
• 
Plasma phospholipid fatty acid composition 
• 
Plasma endocannabinoids derived from PL-AA 
 
10.2 Secondary Outcome Measures  
• 
Muscle and adipose tissue fatty acid composition 
• 
Metabolic derivatives of omega-3 HUFA and omega-6 HUFA in plasma 
• 
Pre- and postprandial endocrine hormones 
• 
24-hr energy metabolism and substrate utilization 
• 
Self-reported eating behavior, satiety, and affective states  
• 
Functional satiety assessment with buffet-style food array  
• 
Anthropomorphic measures and DEXA scan  
• 
3T MRI for body composition 
 
 

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11. Statistical Analyses  
 
11.1 Overview  
This study is a three-arm dietary intervention trial with a baseline measurement and with repeated 
measures of the outcome variables over time. The primary aim is to measure the pre- post- changes in our 
primary outcome variables: %omega-6 in HUFA, 2-AG and AEA.  In secondary Aim 1, we will measure 
the rates of change in our primary outcome variables.  Exploratory aims will be directed towards the 
extent and rates of change in our secondary outcome variables (see Section 10.2) in response to dietary 
interventions.  
 
11.2 Analysis of Data 
Fatty acid concentration data will be examined for homogeneity of variance and normality of distribution, 
and if necessary transformed to meet this criterion.  A probability of 0.05 or lower will be considered 
significant. 
 
Primary Aim: The sample size was selected to provide adequate power (0.8) to test our Primary Aim, as 
described in section 11.3.  In this Aim we plan to assess the extent of changes in %omega-6 in HUFA; 2-
AG and AEA; after 84 days of targeted dietary interventions. To achieve this aim we will treat the data 
coming from a four-group (control and two treatment groups), pre-post study design. There will be a 
measurement for all the individuals in the study at randomization and at 84 days. The outcome will be the 
measurement at 84 days.  
 
Secondary Aim 1: To assess the rate of changes in %omega-6 in HUFA; PL-AA; 2-AG and AEA; PL-
EPA; and PL-DHA, we will make use of the repeated measures of the outcome variables measured at 
baseline, and 28, 56 and 84 days into the diet phase. The goal is to compare the rates of change in these 
outcome variables over time in the three groups. It is expected that the rates of change will be fastest in 
Group C (the low LA plus omega-3 HUFA diet group). 
 
Exploratory Aims: Additional exploratory analyses will be performed to further examine the metabolic 
and clinical effects the of dietary interventions by: 1) assessing the extent and rates of change of the other 
metabolic mediators and clinical outcome measures, as detailed in Section 10; 2) examining the association 
between the changes in blood concentrations of fatty acid precursors (e.g. PL-AA) and their metabolic 
derivatives (e.g. 2-AG); 3) examining the association between changes in precursor fatty acids and clinical 
outcome measures; and 4) examining the association between changes in the metabolic derivatives of 
precursor fatty acids and clinical outcome measures.   
 
 
11.3 Estimation of Power and Sample Size 
We will use one-way analysis of variance to compare the proportion of %omega-6 in HUFA measured at 
12 weeks among three groups. Assuming that the %omega-6 in HUFA will be, on average, 0.8, 0.5 and 
0.2 in the three groups at the end of the 12 weeks (as predicted by Lands’ empirical equation and the 
nutrient intake targets), we calculated required sample size at three values of standard deviation:  the 
maximum value of 0.5, at 0.4 and 0.3. These values translate to root mean square standardized effect is 
calculated as 0.6, 0.75, and 1.0. To detect these effect sizes, at a power of 80% and an alpha of 0.05, we 
will need either 15 individuals per group (s = 0.5), 10 (s = 0.4), or 6 (s = 0.3). 
 
Fig. 6. Sample Size and Power Curve  

PI name: Christopher E. Ramsden, MD 
 
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12 Human Subjects protection 
 
12.1 Regulatory and Ethical Considerations 
The study will be conducted in accordance with Good Clinical Practice (GCP), 21 CFR Parts 50 and 56 
and all applicable regulatory requirements. 
 
Pre-menopausal women, post-menopausal women, and men are known to have major differences in their 
metabolism of omega-6 and omega-3 fatty acids (44-48).  Specifically, pre-menopausal women efficiently 
convert dietary omega-6 and omega-3 18-carbon precursors (omega-6 LA and omega-3 alpha-linoleic 
acid) into omega-6 AA and omega-3 DHA, which compete for incorporation into tissue membranes (44, 
46, 49, 50).  Critically omega-6 AA is the immediate metabolic precursor to endocannabinoids (13), the 
primary outcome of the study.  This efficient conversion is due to the effects of estrogen on liver enzymes 
(46, 49).  Because endocannabinoids regulate hunger, satiety and metabolism, we expect pre-menopausal 
women to uniquely benefit from lowering dietary LA.  By contrast, men and post-menopausal women are 
inefficient converters of omega-6 and omega-3 18-carbon precursors to AA and DHA (14, 44, 46).  
Because low LA diets are not expected to efficiently reduce tissue concentrations of AA, or its 
endocannabinoid derivatives in post-menopausal women and men, they are considerably less likely to 
benefit from lowering dietary LA. 
 
12.2 Equitable Inclusion 
The study population will consist of non-pregnant, non-lactating female subjects.  Subjects that fulfill the 
study criteria will be included regardless of race, ethnicity, socioeconomic, religious, political or similar 
factors.  Based on open recruitment from the NIH federal employee population, the expected racial 
composition of the study population will be 20.3% African-American, 2.8% Hispanic, 15.9% Asian or 
Pacific Islander, 0.6% Native American and 60.4% White (based on Office of Personnel Management 
Demographic Statistics available at: https://www.opm.gov/policy-data-oversight/data-analysis-
documentation/federal-employment-reports/demographics/2010/table1mw.pdf). 
Only English speaking volunteers will be included in this study because several of the psychometric 
instruments (e.g. POMS, PANAS, FS, CPRS) have not been validated in other languages.  
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12.3 Inclusion of children or other vulnerable subjects 
No children will participate in this protocol because children metabolize fatty acids differently than 
adults, and weight gain is not necessarily undesirable in this growing population.   
  
12.4 Justification of sensitive procedures 
The controlled dietary variables to be modified in this study are linoleic acid and oleic acid, which are 
common nutrients in foods.  There is minimal risk associated with adverse events as fatty acids in edible 
form are GRAS (generally recognized as safe) by federal regulatory agencies.   
 
Procedures that may cause some discomfort to subjects: 
1) Blood sampling: Trained medical personnel will carry out the blood draws which may cause 
some discomfort to the subject. 
2) DEXA scan:  Trained medical personnel will perform the scan to reduce discomfort and risk to 
the subject.  The expected exposure for a scan is (0.001 rem) or less than one day’s exposure to 
natural background radiation. 
3) Psychiatric and Neuropyschological Assessment measures:  Research team personnel trained in 
the administration of psychometric measures will assist the subject in answering survey questions 
in the vicinity of trained medical personnel. Any confusion or unexpected response will be taken 
into consideration for follow-up by the trained medical personnel. 
4) Tissue Biopsy:   Muscle and adipose tissue biopsies will be performed by trained medical 
personnel.    Subject will be monitored for signs of undue discomfort .  Medication and 
appropriate palliative care will be provided if necessary. 
5) 3T MRI Scan:   Trained personnel will perform the scan to reduce discomfort and risk to the 
subject.  In the 3T scanner, it is possible that the patient may experience peripheral nerve 
stimulation resulting in a sensation of mild twitching, warmth or vibration.  Medical staff will 
monitor the subjects and administer appropriate care if necessary. 
6) Storage of Private Health Information in a Research Database:  Clinical information from each 
subject will be stored in a retrievable database at NIH.  This information will remain confidential 
and protected like the medical record of the subject.  Information with identifiers will not be 
released to any investigators or agencies/companies without the explicit consent of the subject. 
7) Storage of Patient Samples:  Coded blood and tissue samples without patient names or personal 
identifiers will be stored in secured freezers at 5625 Fishers Lane, Room 3N-01 or in the NIAAA 
Clinical Core lab freezers in the 1-SE area of the clinical center.  Samples will be processed upon 
completion of the study and destroyed when analysis is complete. 
 
 
Reasons for Sensitive Procedures: 
1) Blood sampling:  Procedure is necessary to find the tissue fatty acids concentrations and [eCBs].  
Previous work in this area has been performed successfully in rodents, but has not been translated 
to humans in a clinical trial. 
2) DEXA Scan:  Accurate determination of body composition is essential for determining the 
contribution of dietary fatty acids on body composition. 
3) Psychiatric and Neuropsychological Assessment measures:  These assessments are critical for 
determining each subject’s eating habits, mood, and propensity for addictive behavior at each 
stage in the dietary intervention. 
4) Tissue Biopsy: Muscle and adipose tissue biopsies are needed to determine local and specific 
tissue compositional changes due to the changed LA composition of the diets. 

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5) 3T MRI Scan: Non-invasive imaging for body composition is necessary to avoid the risks of deep 
tissue sampling of subjects for visceral fat and liver composition information.   
6) Storage of Private Health Information in a Research Database: The storage of clinical 
information in retrievable databases is necessary to facilitate collaborative input and analysis.  
This information will remain confidential and protected like the medical record of the subject. 
7) Storage of Patient Samples: The storage of clinical samples is necessary for collaboration and 
assurance of similar analysis conditions across all of the study samples.   
 
 
Notes on Secure Data and Sample Storage: 
Data will be collected and entered on form sets labeled with a unique coded identifier that is not related to 
the patient’s name or any other personal identifier (Soc Security number, NIH employee number, etc).  
Data will be kept in a locked cabinet or in secure computer files with access only for the Principal 
Investigator (PI) and Associate Investigators (AIs) involved with the study. When entered into the 
electronic databases, information will be under the CRC and NIH network regular access control.  In 
addition, any information allowing the identification of the participants will be removed.  The only 
possibility to reconstruct the link between database records and information will be through the unique 
identifier.  The translation key will be stored in a locked cabinet or secure computer file with access 
available only to the PI and AIs named in this protocol document.  
Blood and tissue samples will be destroyed after all of the analyses are completed.  Research data will be 
stored for seven years following the study and destroyed according to NIH guidelines. 
 
 
 
12.5 Safeguards for vulnerable populations 
No vulnerable populations are recruited into this study.  Although NIH employees may participate, study 
investigators and their superiors, subordinates and immediate family members (adult children, spouses, 
parents, siblings) will be excluded. Non-pregnant, non-lactating females will be recruited; but pregnancy 
testing will be provided for these females before sensitive procedures if deemed reasonable and/or 
necessary.   
 
12.6 Qualifications of Investigators 
The Laboratory of Membrane Biochemistry and Biophysics (LMBB), Section of Nutritional 
Neuroscience (SNN) program at NIAAA is an internationally respected laboratory in the area of fatty acid 
metabolism and nutritional neuroscience. The central focus of the SNN is to examine the impact of 
deficiencies in long chain omega-3 fatty acids on adverse neurodevelopmental and psychiatric outcomes. 
This section translates basic biochemical and biophysical fatty acid and lipid results into clinical 
applications.  
The first in vivo studies of the essential fatty acid (EFA) metabolism in alcoholics and smokers have been 
performed within the SNN. Highly sensitive and selective methodology has been developed within our 
research group allowing for the safe and non-invasive assessment of EFA metabolism in vivo. Thus, 
arguably the worldwide lead in the metabolomics of EFA has been gained at the SNN. 
 
Christopher Ramsden, MD is a Lieutenant Commander, Commissioned Corps of the USPHS for 
LMBB/SNN at NIAAA. Dr. Ramsden is a licensed and board certified physician with current credentials 
at the NIH Clinical Center.  He completed a transitional medicine internship and a clinical medical 
residency training in Physical Medicine and Rehabilitation (PM&R). He has research experience at the 
Rehabilitation Institute of Chicago-Northwestern Feinberg School of Medicine, and as a postdoctoral 
research fellow at UNC-Chapel Hill. He received a grant from the Mayday Fund to develop and 
implement dietary interventions for 70 patients with treatment-resistant Chronic Daily Headache at UNC-

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Chapel Hill. Dr. Ramsden is Adjunct Research Faculty in the Department of PM&R at UNC, where the 
translational project is ongoing. The low omega-6 diets and low omega-6 plus high omega-3 diets 
employed in the UNC study serve as a basis for our study interventions.  Dr. Ramsden may obtain 
consent. 
 
CAPT Joseph Hibbeln, MD, USPHS is the Acting Chief of the LMBB/SNN at NIAAA.  Dr. Hibbeln has 
participated in extensive international collaborative clinical trials of omega-3 fatty acids for the 
prevention of suicide, postpartum depression, and violence. He is a primary collaborator in the Avon 
Longitudinal Study of Parents and Children, Bristol, UK, examining the residual effect of nutritional 
insufficiencies in pregnancy in childhood neurodevelopmental outcomes and relevant gene-nutrient 
interactions. Dr. Hibbeln was one of the first investigators to draw attention to the importance of omega-3 
fatty acids in psychiatric disorders. Dr. Hibbeln conceived and implemented the rodent study that 
provided pilot data for this project.  
Dr. Hibbeln may obtain consent. 
 
Amber Courville, PhD, RD is a Registered Dietitian with a doctorate in nutrition. She is a dietitian for the 
NIH clinical center nutrition dept and researcher in the metabolic unit of the NIDDK at the NIH clinical 
center.  Dr. Courville has clinical research experience in measuring satiety and hunger in the context of 
the endocannabinoid metabolome, and in conducting clinical trials with variable intakes of fatty acids. 
Her expertise includes the development of relevant research paradigms for study of many facets of human 
appetite. Dr. Courville may obtain consent. 
 
Kong Y. Chen, PhD is an experienced metabolic researcher at the Clinical Endocrine Metabolism unit of 
the NIDDK.  He joined the NIDDK in 2006 after serving as Director of the Energy Balance Core Lab and 
Human Metabolism and Exercise Physiology Lab at Vanderbilt University. Dr. Chen has focused in the 
measurements of energy metabolism using whole-room indirect calorimeters (metabolic chambers). Dr. 
Chen and his lab are also investigating the physiological regulations of energy metabolism in lean and 
obese individuals, as well as improving the technologies for measuring body composition and physical 
activity. Dr. Chen may obtain consent. 
 
Nancy Diazgranados, M.D., M.Sc., is a Staff Clinician and Deputy Clinical Director, Office 
of the Clinical Director (OCD), NIAAA.  She received her Doctoral Degree in Medicine and 
Surgery from the Pontificia Universidad Javeriana in Bogota, Colombia in 2001. She 
completed her Psychiatry residency at Albert Einstein Medical Center and a Master in Science 
Degree in Pharmacology at Thomas Jefferson University in 2007.  In 2008 she became a 
diplomat of the American Board of Psychiatry and Neurology. She continued her training as 
a Post-Doctoral Clinical Research Fellow in the Intramural Program at NIMH.  In 2013 she 
joined NIAAA. Dr. Diazgranados will serve as an Associate Investigator. She will evaluate 
participants and provide clinical care as required.  She is experienced with conducting the 
Informed Consent process and has completed the NIMH, HSPU Informed Consent 
training.  Dr. Diazgranados may obtain consent for this study. 
Tonette Vinson, C.R.N.P., OCN,  is an Adult Nurse Practitioner in NIAAA.  She obtained her 
Masters of Science in Nursing degree from The University of Pennsylvania in 2003. Tonette 
will serve as an Associate Investigator. She will evaluate participants and provide clinical care 
as required. She will aid in data collection for the study.  She is experienced with conducting 

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the Informed Consent process and has completed the NIMH, HSPU Informed Consent 
training.  Ms. Vinson may obtain consent for the study. 
 
 
Yvonne Horneffer, M.S.N., CRNP, is a board certified Adult Nurse Practitioner. She obtained her Master 
of Science in Nursing in 2001 from George Mason University in collaboration with the George 
Washington University School of Medicine. Prior to joining NIAAA Yvonne was an Associate 
Investigator at the National Cancer Institute’s early drug development program from 2006 to 2016 
conducting Phase 0 and Phase 1 clinical trials. Yvonne is also a Certified Nephrology Nurse who served 
as an Associate Investigator at NIDDK’s kidney and pancreas transplant program from 2004 to early 
2006. With prior background in nursing management and surgical nursing, in her spare time, Yvonne 
organizes, plans, and leads a team of volunteer surgeons and other health care providers on an annual 
surgical mission to the Philippines. She will evaluate participants and provide clinical care as required. 
She will aid in data collection for the study.   She is experienced with conducting the Informed 
Consent process and has completed the NIMH, HSPU Informed Consent training.  Ms. Horneffer 
may obtain consent for the study 
 
Yuhong Lin, PhD is a staff scientist in the LMBB/SNN at NIAAA.  Dr. Lin has a doctorate in Medical 
Science from Shanghai Jiao Tong University School of Medicine was also a former Associate Professor 
in the Department of Experimental Nuclear Medicine there.  Dr. Lin has 20+ years of experience in 
analytical biochemistry and 10 years of specialized experience in the gas chromatography-mass 
spectrometry of fatty acids and their metabolites; including multiple stable isotope techniques.   Dr. Lin 
will not obtain consent. 
 
Laura Musse, CRNP, MS is a Special Volunteer Researcher  in the Clinical Endocrine Metabolism unit of 
the NIDDK at the NIH Clinical Center.  She is a Certified Nurse Practioner with procedural experience 
performing bone marrow biopsies, and has been trained in our fat and in muscle tissue biopsy procedures 
by Drs. Louis Simchowitz and Asem Ali.  Ms. Musse may obtain consent. 
 
Sharon Majchrzak-Hong, MS is a chemist in the LMBB/SNN at NIAAA.  She has 12 years experience in 
the field of lipid research; including chemical and data analysis from both animal laboratory experiments 
and human clinical studies.  Her previous non-research experience in education includes handling 
confidential legal documents.  Ms. Majchrzak-Hong will not obtain consent. 
 
Beth MacIntosh, MPH, RD, is a Research Nutritionist at the UNC-Chapel Hill Clinical Translational 
Research Center. She has experience in developing palatable dietary menus, meal plans and recipes with 
varying levels of essential fatty acids.  Along with Dr. Ramsden, she designed and implemented low 
omega-6 and low omega-6 plus high omega-3 diets for an ongoing study in patients with Chronic Daily 
Headache. Ms. MacIntosh will not obtain consent. 
 
 
 
 
D5-LNA Infusion Amendment Collaborators 
 
 
Stanley I. Rapoport, M.D., is Chief of the BPMS NIA, who has published extensively in the areas of lipid 
metabolism and brain function in health and disease.  He received his MD degree from Harvard Medical School, 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
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and is clinically credentialed at the NIH Clinical Center. The laboratory that he directs has developed and 
validated the infusion method and analysis in rodents.  These methods form a major basis for the Infusion 
Amendment employed in this human protocol. He is involved in the design of the protocol, and its conduct, and in 
data analysis and writing up results. 
Dr. Rapoport will not obtain consent. 
 
 
 
13.  Benefits 
This study will provide generalizable knowledge on the impacts of dietary fatty acids on endocannabinoid 
synthesis, satiety, eating behavior, and obesity. This is an experimental, unproven approach that may not prove 
beneficial to study participants.   
 
14.  Classification of Risk 
▪ 
No direct benefit, but likely to yield generalizable knowledge for preventative nutrition 
▪ 
More than minimal risk 
▪ 
The risks are reasonable in relation to anticipated benefits.  We will discover critical new 
information on how modifying LA in the diet affects metabolism, satiety and body 
composition.  We will also gain valuable insights the plausibility of future safe dietary 
interventions for the treatment and prevention of obesity as alternatives to risky surgical 
procedures and pharmaceutical products.  
 
15.  Consent documents and process 
 
15.1 Who will obtain Consent  
A copy of the consent document may be mailed or emailed to potential participants for their review prior 
to the screening visit.  The informed consent process will include a face to face meeting with the study 
investigators that are designated to obtain consent in Section 12 of this protocol.  The training and 
qualifications of non- MD and RN investigators who will obtain consent are as follows:  Amber 
Courville, RD, PhD is a registered dietitian with a doctorate degree in nutrition. Amber has expertise 
specifically related to medical procedures done under our protocol, and currently obtains consent on 
several protocols that have the same outcome measures and medical procedures.  Kong Chen, PhD has 
expertise specifically related to medical procedures done under our protocol, and currently obtains 
consent on several protocols that have the same outcome measures and medical procedures.  
 
 
16.  Data Safety and Monitoring & 17.   Adverse Event Reporting 
 
16.1  Data Safety and Monitoring  Board 
The main dietary intervention protocol uses standard clinical and laboratory procedures to study the 
effects of modifying dietary LA on body composition and response.  Since the research procedures are to 
be performed in adult subjects and such procedures involve minor increase over minimal risk, the 
collection, monitoring and analysis of adverse events will be the responsibility of the Principal 
Investigator and the investigative team.  No DSMB will be convened. 
 
16.2 Data Safety and Monitoring Plan 
Elements of the Data Safety and Monitoring Plan 
(1) Monitoring mechanism:   

PI name: Christopher E. Ramsden, MD 
 
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The Principal Investigator and the investigative team will monitor the protocol 
participant daily meal visit data for signs of participant distress and signs of dietary 
non-compliance. 
(2) Frequency:   
The Principal Investigator will meet with study staff weekly to discuss participant 
well-being and study progress. 
(3) Stop or change rules:   
The study will be stopped when 64 participants finish the diet phase.  In the unlikely 
situation that multiple participants experience adverse events resulting from the 
consumption of the study foods, these events will be reported to the IRB and the study 
will be evaluated for continuation if these events are determined to be serious and life-
threatening.  Participants will be removed from the diet at the recommendation of the 
nutrition team or if they display signs of a serious adverse event or non-compliance as 
evaluated by the Principal Investigator, Lead Associate Investigator(s) or nutrition or 
medical staff.   
 
(4) Advanced Plans:   
No interim or futility analyses  are planned.  The study requires more cohort to reach the 
estimated sample size of 64 participants.   
(5) Information to be monitored:   
a. Attendance at daily meal visits as recorded in participant medical record; 
b. Daily vital signs as recorded in participant medical record; 
c. CPRS to monitor participant mood as stated on page 26. 
(6) Communication:   
A record of participants removed from the diet phase will be reported to the Addictions IRB at 
the time of annual review.  Serious adverse events will be reported according to the required NIH 
Clinical Center guidelines as stated in section 17 on the next page. 
 
16.3 Data Monitoring for Quality Assurance 
Data monitoring for quality assurance will be the responsibility of the Principal Investigator and 
the investigative team.   
(1) Monitoring mechanism:   
The Principal Investigator and the investigative team will monitor the protocol 
participant questionnaire data and participant binder data. 
(2) Frequency:   
As stated above, paper questionnaire data and other information in the participant 
binders will be reviewed bimonthly.   
(3) Information to be monitored: 
PANAS, POMS and FS psychometric data for each participant will be evaluated for 
self-report form completeness and status of data entry.  Location and status of 
participant biospecimens will also be tracked and recorded in study sample 
spreadsheet logs.  Status of imaging data will also be noted.  
(4) Communication: 

PI name: Christopher E. Ramsden, MD 
 
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Missing psychometric data will be reported to the Principal Investigator and summarized in a 
report to the IRB at the time of annual review.  A summary record of participants who refuse 
participation in the biopsies and MRI scan will also be reported to the IRB at the time of annual 
review. 
 
 
 
17.  Adverse Event Reporting 
 
Adverse events 
In the course of a dietary study, adverse events may occur. Unanticipated adverse events that occur as a direct 
result of research studies initiated at the NIH, but not related to the natural history of the underlying disease will 
be summarized at the time of the annual review.    
Serious Adverse events  
A serious adverse event is any adverse experience that: 
1. Results in death; 
2. Is life threatening; 
3. Results in hospitalization or prolongs hospitalization; 
4. Results in persistent or significant disability/incapacity;  
5. Results in congenital anomaly/birth defect; or 
6. Results in a condition, which in the judgment of the investigator represents a significant hazard.   
 
Serious adverse events that are possibly, probably or definitely related to participation in research studies 
performed at the NIH Clinical Center will be reported.  However, serious adverse events (other than death) that 
are clearly and unambiguously due to extraneous causes (accidental, environmental or coincidental events) will 
not be reported.  
All serious adverse events will be reported using the NIH Serious Adverse Event Report Form to the NIAAA 
CNS IRB, and NIAAA Clinical Director as soon as possible, but no later than seven days of death or life 
threatening serious adverse event or within fifteen days after the occurrence of all other forms of serious adverse 
events.    
 
 
18.  Alternatives to participation or alternative therapies 
 
Subjects are healthy volunteers and are not recruited for the treatment of a medical condition.  
Participants are counseled to continue their usual medical care for the duration of the study. 
 
19.  Confidentiality 
 
19.1 Medical records 
This protocol is covered by a Certificate of Confidentiality (CoC) issued by the US Department of Health 
and Human Services (US:DHHS), National Institutes of Health (NIH). This information will remain 
confidential and protected according to NIH guidelines.  Some clinically relevant research data will be 
stored indefinitely in the medical record and will be accessible to the patient for review by others of their 
choosing (doctors, insurance companies etc.) after executing a release of information. 
 
19.2 Research Data 
 

PI name: Christopher E. Ramsden, MD 
 
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Data will be collected and entered on form sets labeled with a unique coded identifier that is not related to 
the patient’s name or any other personal identifier (Soc Security number, NIH employee number, etc).  
Data will be kept in a locked cabinet or in secure computer files with access only for the Principal 
Investigator (PI) and Associate Investigators (AIs) involved with the study. When entered into the 
electronic databases, information will be under the CRC and NIH network regular access control.  In 
addition, any information allowing the identification of the participants will be removed.  The only 
possibility to reconstruct the link between database records and information will be through the unique 
identifier.  The translation key will be stored in a locked cabinet or secure computer file with access 
available only to the PI and AIs named in this protocol document. 
 
The validated psychometric instruments used in this protocol have been converted to an electronic web-based 
secure data entry platform administered by the NIAAA.  The security of the data management system  is curated 
by the NIAAA in agreement with NIH data security policies. 
Most data generated in this study will be stored and maintained in the NIAAA clinical database which is 
password protected and secure. 
 
 
19.3 Sample Storage 
Coded blood and tissue samples without patient names or personal identifiers will be stored in secured 
freezers at 5625 Fishers Lane, Room 3N-01 or in the NIAAA Clinical Core lab freezers in the 1-SE area 
of the clinical center.  Samples will be processed upon completion of the study and destroyed when 
analysis is complete.   Any additional collaborations requiring transfer of patient material will be done in 
a coded fashion after informing the IRB and obtaining necessary assurances from the outside institution.  
The protocol will be amended and IRB approval will be sought when such collaborations are established.     
 
 
20.  Conflict of Interest/Technology Transfer 
NIH guidelines on conflict of interest have been distributed to all investigators. There are no conflicts of 
interest to report.  Technology transfer is not applicable to this protocol. 
 
 
21.  Compensation  
Remuneration 
Remuneration for participation in this study is based on existing NIH guidelines based on inconvenience, to offset 
potential loss of earnings and travel expenses. The amount paid will be pro-rated based upon participation, and 
does not include the free foods that will be provided.   
 
For the baseline phase, participants will be compensated a maximum of 250 dollars. 
Baseline Phase Visits 
Compensation 
Visit 1 
50 
Visit 2 
50 
Visit 3 
150 
Maximum Baseline Phase Compensation  
250.00 
 
 
 
For the diet phase, participants will be compensated a maximum of 450 dollars. 
Diet Phase 
Compensation 
Attendance at Daily Meals 
150 

PI name: Christopher E. Ramsden, MD 
 
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Visit 1 
50 
Visit 2 
50 
Visit 3 
200 
Maximum Diet Phase Compensation 
450.00 
 
For the continuation phase, participants will be compensated a maximum of 960 dollars. 
 
Continuation Phase 
Compensation 
Attendance at Daily Meals  
360 
Visit 1 
50 
Visit 2 
50 
Visit 3 
50 
Visit 4 
50 
Visit 5 
50 
Visit 6 
350 
Total Compensation for Continuation Phase 
960.00 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
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S1.  Table of Contents (d5-LNA Infusion Substudy)                             35-51 
 
 
 
 
 
 
 
 
S2. Precis 
 
 
 
 
 
 
                          38 
 
 
 
Objective 
 
 
 
 
 
 
 
 
 
 
 
Study Population 
 
 
 
 
 
 
 
 
 
 
Three Group Comparative Analysis 
 
 
 
 
 
 
 
 
Continuation Phase Sub Study  
 
 
 
 
 
 
Outcome Measures 
 
 
 
 
 
 
 
 
 
Primary Dependent Variables 
 
 
 
 
 
 
 
Secondary Dependent Variables 
d5-LNA Infusion Outcomes 
 
 
 
 
 
 
 
 
S3. Introduction/ Scientific Rationale  (Infusion Substudy) 
 
 
38 
 
           
 
 
 
 
 
 
 
 
 
 
 
 
S4. Study objectives and hypotheses  (Infusion Substudy) 
 
 
40 
 
 
Study objectives 
 
 
 
 
 
 
 
 
 
 
Specific hypotheses   
 
 
 
 
 
 
 
 
Primary aims   
 
 
 
 
 
 
 
 
Primary hypotheses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
S5. Subjects  (Infusion Substudy) 
 
 
 
 
 
40 
 
 
 
Description of study population  
 
 
 
 
 
 
Inclusion criteria (Eligibility Checklist as attachment) 
 
 
 
 
Exclusion criteria   
 
 
 
 
 
 
 
 
 
 
S6. Study Design and Methods  (Infusion Substudy)  
 
 
41 
 
Recruitment 
Overview of Infusion Substudy   
 
 
 
 
 
 
 
 
Screening Methods 
 
 
 
 
 
 
 
 
 
Characteristics of Stable Isotope d5-LNA Infusion 
 
 
 
 
 
 
 
Estimation of Plasma Volume 
Timepoints for Blood Collection for Fatty Acid Analyses 
Genotyping 
Infusion Substudy Procedure Specifics  
Inpatient Admission with standard meal and overnight fast 
 
 
 
 
 
Follow-up/ termination procedures 
 
 
 
 
 
 
Radiation exposure 
 
 
 
 
 
 
 
 
Medications and devices requiring IND/IDE 
 
 
 
 
 
Storage of research samples and data 
 
 
 
 
 
 
 
S7. Risks and Discomforts (Infusion Substudy) 
 
 
 
44 
 
 
 
Procedures with No Medical Risk and Minimal Discomfort  
 
 
 
 
Specimen Collection for Biomarker Testing 
 
 
 
 
 
  
Risks and Discomforts - Infusion Amendment 
 
 
 
 
 
 
 
 
S8. Subject Monitoring, adverse event grading and withdrawal (Infusion Substudy) 45 
 
 

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Parameters to be monitored 
 
 
 
 
 
 
 
 
 
S9. Outcome Measures (Infusion Substudy) 
 
 
 
 
45 
 
 
S10. Statistical Analyses  (Infusion Substudy)  
 
 
 
47 
 
 
 
 
 
 
 
S11. Human Subjects protection (Infusion Substudy)  
 
 
49 
 
 
 
Issues applying to both the main protocol and Infusion Substudy  
 
 
 
 
Justification of sensitive procedures 
 
 
 
 
 
 
 
Qualifications of Investigators  
 
 
 
 
 
 
 
S12. Benefits (Infusion Substudy) 
 
 
 
 
 
49 
 
 
 
 
 
 
 
 
 
 
S13. Classification of Risk (Infusion Substudy) 
 
 
 
49 
 
 
 
S14. Consent documents and process (Infusion Substudy) 
 
 
50 
 
 
 
Who will obtain Consent 
 
 
 
 
 
 
 
 
 
S15. Data Safety and Monitoring Board (Infusion Substudy)  
 
51 
 
 
 
 
S16. Adverse Event Reporting (Infusion Substudy) 
 
 
 
51 
 
 
 
 
S17. Alternatives to participation or alternative therapies (Infusion Substudy)  51 
 
 
 
 
S18. Confidentiality (Infusion Substudy) 
 
 
 
 
51 
 
Medical records 
 
 
 
 
 
 
 
 
Research Data  
 
 
 
 
 
 
 
 
Sample Storage  
 
 
 
 
 
 
 
 
 
S19. Conflict of Interest/ Technology Transfer (Infusion Substudy)                 52 
 
 
 
 
 
 
S20. Compensation (Infusion Substudy) 
 
 
                          52 
 
S21  References  (References 51-71 apply to Infusion Substudy)                       52 
 
 
 
 
 
 
 
 
 
 
 
S22. Appendix 7: Eligibility Criteria for d5-LNA Infusion Amendment (Infusion Substudy) 65 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
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S1. Deuterated alpha linolenic acid (d5-LNA) Infusion Substudy 
 
 
 
 
 
 
 
 
 
S2. Precis 
 
 
 
 
 
 
 
 
 
 
Objective 
To quantify hepatic synthesis-secretion rates of long-chain omega-3 polyunsaturated fatty acids (omega-3 
LCPUFAs) from circulating unesterified alpha-linolenic acid (LNA) in humans consuming a typical US 
diet.  To evaluate the effects of lowering the dietary nutrient linoleic acid (LA) as a controlled variable on 
omega-3 LCPUFA synthesis-secretion rates.  Modifications in dietary LA will be offset with reciprocal 
changes in the nutrient oleic acid (OA) to maintain equivalency of calories and nutrients.  
 
Study Population 
A subset of up to 24 participants who are enrolled in the main protocol NIAAA-11-AA-0028 ‘Dietary 
linoleic acid regulation of omega-3 PUFA metabolism; satiety and body composition among overweight 
female subjects’, will be invited to participate in this Infusion Substudy.  Participants must be in the final 
two weeks of the main protocol (11-AA-0028) intervention phase in order to participate in the Infusion 
Substudy.  
 
Infusion Substudy Overview  
Participation in the Infusion Substudy requires one approximately 22-hour hospital admission visit, 
including an 8-hour infusion of d5-LNA with serial blood draws, in addition to the visits of the primary 
protocol.  This additional visit will occur during the final two weeks of the 12-week diet phase, i.e., after 
10-12 weeks of exposure to the study diets.  As in the main protocol, participants will be advised to 
continue their usual medical care and medications throughout the study. 
 
Outcome Measures 
Primary Dependent Variables 
The primary outcome will be the hepatic synthesis-secretion rate of docosahexaenoic acid (DHA) from 
infused d5-LNA. 
 
 
S3. Scientific Rationale for Infusion Substudy 
Potential importance of DHA and its metabolites in addictive disorders  
DHA (22:6n-3) is the most abundant polyunsaturated fatty acid in the human brain and is particularly 
abundant in synaptic phospholipids. DHA has unique chemical and biophysical properties (51), and its 
docosanoid and endocannabinoid metabolites favorably modulate an array of biochemical processes 
including neurogenesis (52, 53) and neuro-inflammation (54). Deficits in DHA and its bioactive 
metabolites are proposed to contribute to suboptimal neurological function, increased relapse 
vulnerability and aggression in cocaine addicts and other substance abusers (55-58).  
 
Can dietary linoleic acid lowering increase hepatic synthesis-secretion rates and tissue 
accumulation of n-3 DHA? 
It is generally believed that synthesis of omega-3 DHA from dietary LNA is not sufficient to maintain 
adequate DHA in humans (59). However the long-term synthesis of DHA from LNA has not yet been 
evaluated at ‘low’ LA intakes that are consistent with evolutionary and historical US intakes, which are 
expected to result in increased DHA synthesis and esterification into tissue phospholipids. High omega-6 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
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LA (18:2 n-6) consumption may reduce the omega-3 EPA and DHA content of human tissues by: (1) 
impairing enzymatic conversion of omega-3 LNA (18:3n-3) to 18:4n-3 (the precursor to EPA) by 
competing for the active site of D6-desaturase (60); (2) impairing enzymatic conversion of 24:5n-3 to 
24:6n-3 (61), the precursor to DHA (22:6n-3) by competing for the active site of D6-desaturase in the 
endoplasmic reticulum; and (3) competing with omega-3 EPA and DHA for esterification into the sn-2 
position of phospholipids (62)(Fig. 7: Omega-3 HUFA biosynthetic pathway).  
 
Fig. 7: Omega-3 HUFA Biosynthetic Pathway 
D-6 desaturase is rate limiting for enzymatic conversion of the omega-3 precursor LNA (18:3n-3) to the omega-3 LCPUFA 
products EPA (20:5n-3) and DHA (22:6n-3).  Red lines indicate that omega-6 LA (18:2n-6) competes with 18:3n-3 and 24:5n-3 
for the active site of D-6 desaturase, impairing conversion of LNA to EPA and DHA. LA also competes for incorporation into 
tissue phospholipids (not shown). Variants in theFADS2 gene, which codes for the D-6 desaturase enzyme, also modify the 
efficiency of conversion from LNA to EPA and DHA.  
 
A randomized trial that compared the effects of an infant formula with high LA (6.7 en%) with a low LA 
infant formula (1.7 en%) on infant tissue omega-3 EPA and DHA supports the proposition that high-LA 
intake decreases tissue omega-3 long-chain PUFA status.  Infants in the low-LA group, compared with 
infants who received the high-LA formula, had 228% higher EPA (1.28 weight% compared with 0.39 
weight%, respectively) and 29% higher DHA (4.48 weight% compared with 3.47 weight%, respectively) 
in erythrocyte membrane phospholipids. Importantly, however, these infant formulas did not contain the 
preformed omega-6 and omega-3 LCPUFAs present in adult omnivorous diets. Because dietary HUFA 
alter enzymatic conversion of LNA to EPA and DHA (via feedback inhibition) (63), it is not yet clear 
what extent dietary LA lowering increases the accumulation of EPA and DHA in normative human 
omnivorous diets.    
 
Advantages of hepatic synthesis secretion rates of EPA and DHA versus fatty acid composition data 
Assessment of the EPA and DHA composition of blood and other tissues provides a useful snapshot for 
assessing present omega-3 status.  However, fatty acid compositional data do not allow for 
characterization of rates of synthesis-secretion of individual fatty acids from their precursors.  
Compositional data are therefore of limited use in for quantifying the dose of dietary LNA necessary to 
deliver a given dose EPA and DHA to human tissues. Stanley Rapoport et al. in the National Institute of 
Aging developed and validated a stable isotope LNA infusion methodology in a rodent model to ascertain 
18:3n-3 (! -LNA)
18:4n-3
20:4n-3
20:5n-3 (EPA)
22:5n-3 (DPAn-3)
24:5n-3
24:6n-3
22:6n-3 (DHA)
Elongation
(² 6 Desaturase)
#-Oxidation
n-3 PUFA synthesis pathway
Desaturation
(² 5 Desaturase)
Desaturation
Elongation
Elongation
(² 6 Desaturase)
Desaturation
(Acyl-CoA oxidase)
(Elongase 5)
(Elongase 2 and 5)
(Elongase 2)
18:3n-3 (! -LNA)
18:4n-3
20:4n-3
20:5n-3 (EPA)
22:5n-3 (DPAn-3)
24:5n-3
24:6n-3
22:6n-3 (DHA)
Elongation
(² 6 Desaturase)
#-Oxidation
n-3 PUFA synthesis pathway
Desaturation
(² 5 Desaturase)
Desaturation
Elongation
Elongation
(² 6 Desaturase)
Desaturation
(Acyl-CoA oxidase)
(Elongase 5)
(Elongase 2 and 5)
(Elongase 2)
18:2n-6 (LA) 
18:2n-6 (LA) 
FADS2 gene 
FADS2 gene 

PI name: Christopher E. Ramsden, MD 
 
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the synthesis-secretion rates of EPA and DHA  (64, 65).  In this collaborative amendment we intend to 
translate their methodology to our human trial to assess whether the omega-6 LA content of human diet is 
a major determinant of whole-body (mainly liver) synthesis-secretion rates of EPA and DHA from 
labeled LNA.  Ascertainment of synthesis-secretion rates will allow for more accurate quantitative 
estimation of amounts of dietary LNA necessary to maintain optimal EPA and DHA, and determination 
of whether dietary LA lowering affects these estimates.  These data may ultimately prove useful in the 
formulation of dietary guidelines, and the human infusion methods piloted here can be adapted to evaluate 
the effects of other variables (i.e., nutrients, drugs, alcohol abuse) on LNA conversion to EPA and DHA. 
 
Is the relationship between dietary linoleic acid lowering and liver synthesis-secretion of omega-3 
EPA and DHA modified by variants in the FADS1 and FADS2 genes? 
Increases in synthesis-secretion rates of omega-3 EPA and DHA in response to controlled dietary LA 
lowering maybe altered by allele variants in the FADS gene locus (which code for rate limiting enzymatic 
conversion of LNA into EPA and DHA) (Figure 7). The ‘fast converter’ FADS allele variant indicates 
substantial conversion of the omega-3 precursor LNA to EPA and DHA.  By contrast, the ‘slow 
converter’ allele variant indicates minimal conversion of the omega-3 precursor LNA to EPA and DHA.  
Homozygosity for the ‘slow converter’ allele may confer neuro-developmental and other risks when 
accompanied by suboptimal dietary intakes of seafood sources of preformed EPA and DHA(66).  
However, associations between alleles and adverse neuro-developmental outcomes have thus far been 
evaluated only in populations with uniformly high LA intakes (considerably higher than historical and 
evolutionary diets without added seed oils).  Comparison of the 8 en% and 1 en% LA dietary 
interventions in this amendment will allow for evaluation of whether increases in whole-body synthesis-
secretion rates of EPA and DHA after dietary LA lowering are dependent upon FADS gene variants. 
 
S4. Study Objectives and Hypotheses (d5-LNA Infusion Amendment)  
 
Study objectives 
To demonstrate feasibility for characterizing the hepatic synthesis-secretion rates of DHA and other 
omega-3 HUFA from infused d5-LNA in humans. To evaluate the effects of lowering the dietary nutrient 
LA via controlled replacement with OA on DHA synthesis-secretion rates.  
 
Specific hypotheses   
 
Primary aims 
To demonstrate feasibility for characterizing the human hepatic synthesis-secretion rates of DHA using a 
d5-LNA infusion methodology adapted from an NIH rodent model. 
To assess increases in hepatic synthesis-rates for DHA from d5-LNA in response to controlled lowering 
of dietary omega-6 LA from 8 en% to 1 en%. 
 
Primary hypotheses 
Hepatic synthesis-secretion rates for DHA from infused d5-LNA can be successfully quantified in 
humans.  
Lowering dietary intake of LA as a controlled variable from 8 en% to 1 en% will significantly increase 
synthesis-secretion rates for DHA and other omega-3 LCPUFAs.  
 
S5. Subjects 
 
Description of study population 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
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Only participants that are currently enrolled in the main protocol 11-AA-0028, and nearing completion of 
12-week intervention phase will be invited to participate in the Infusion Substudy. In order to enroll in the 
Infusion Substudy, subjects must continue to meet all eligibility criteria for the main protocol in order to 
enroll (see inclusion and exclusion criteria in Section 6 (pages 12-13) and Appendix 7).  
 
 
 
 
S6. Study Design and Methods – Infusion Substudy 
 
 
 
 
 
Recruitment 
Participants who were randomized to either diet ‘A’ or diet ‘B’ and are in their final two weeks of the 12-
week diet phase will be invited to participate in the Infusion Substudy. The study dietitian, the only 
investigator who is unblinded to treatment allocation, will do an initial screen of participant in diet ‘A’ or 
diet ‘B’ who may be eligible to participate. However, enrollment in the Infusion Substudy will require a 
new informed consent administered by the physicians and/or nurse practitioner specified in Section S11 
(page 49).  
  
 
 
 
 
 
 
 
 
 
Overview of Infusion Substudy Design and Methods 
After at least 10 consecutive weeks of exposure to study diets, participants randomized to diet ‘A’ or diet 
‘B’ (Table 7, Figure 8) will be invited to participate in the d5-LNA Infusion Substudy. No matching 
procedure will be employed. A maximum of 12 subjects in each group will participate.  
 
Table 7: Fatty Acids of 2 Study Diets in the Infusion Amendment 
Diet Group omega-6 LA* omega-3 LNA omega-6 AA omega-3 EPA+DHA 
A. Orange 
8.00 
0.40 
0.045 
0.045 
B. Blue 
1.00 
0.40 
0.045 
0.045 
Fatty acid intakes as percentages of daily food energy (en%). LA indicates linoleic acid; LNA indicates alpha-linolenic acid; AA 
indicates arachidonic acid; EPA indicates eicosapentaenoic acid; DHA indicates docosahexaenoic acid. *Indicates that LA will 
be modified as a controlled variable via isocaloric substitution for oleic acid. 
 
Participation requires a new consent specific to the Infusion Protocol and one additional visit lasting 
approximately 22-hours, including: 
(1) Admission to the 5SW Metabolic Unit; 
(2) Insertion of two intravenous catheters; 
(3) Overnight fast; 
(4) 8-hour infusion of d5-LNA with serial blood sampling. 
(5) Completion of craving/hunger questionnaires. 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 43 of 68 
 
 
Fig. 8. Infusion Substudy Flow Diagram: Participants in Diet Group A (8 en% as LA) and Diet group B (1 en% as LA) are 
invited to participate in the Infusion Substudy. Participation requires one additional 22-hour study visit after 10-12 consecutive 
weeks of consuming study diets. Saliva is collected for FADS genotyping in the baseline phase of the main protocol. LA 
indicates omega-6 linoleic acid; d5-LNA indicates deuterated omega-3 alpha-linolenic acid. 
 
Characteristics of Stable Isotope d5-LNA Infusion  
Non-radioactive stable isotope-labeled alpha-linolenic acid (17,17,18,18,18-D5, 98%) (d5-LNA) in 
potassium salt will be prepared by Cambridge Isotope Laboratory; the product will be tested and verified 
as sterile and non-pyrogenic by Cambridge before delivery to NIH. The NIH Pharmacy will then mix the 
d5-LNA potassium salt into sterile water and then reconstitute with 25% human albumin, and then verify 
the identity, purity, and dose, sterility and non-pyrogenicity of these infusion materials. These procedures 
are performed in order to ensure that the infusion materials meet NIH Pharmacy standards for human 
infusion. George Grimes (Chief of the Pharmaceutical Development Section) is our Clinical Center 
Pharmacy contact (GGrimes@cc.nih.gov 301.496.1031). 
 
A total dosage of d5-LNA of approximately 19.2 μmol per kg of body weight will be infused 
intravenously through a 0.2 micron filter at a constant rate for up to 8 hours during the feasibility stage. 
This initial dose estimate was calculated based on published human infusion trials (68) using deuterated 
palmitate and the endogenous steady state concentration of LNA in plasma (23) for an obese subject from 
the main protocol with body weight of 95kg. The half-life of unesterified infused fatty acids in human 
plasma is about 2 minutes, so that a steady state plasma concentration should be reached in about 10 
minutes (5 half-lives). After the feasibility stage, we will adjust infusion rate, dose and duration.  
 
Estimation of Plasma Volume 
DEXA scans performed as part of the main protocol (11-AA-0028) will be used to estimate lean body 
mass for normalization of body fluid volumes as described by Boer.  These estimates for plasma volume 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 44 of 68 
will be used in calculating the infusion amount and rate, and in interpreting results as described in 
Section S.9. 
 
Time points for Blood Collection and Fatty Acid Analysis 
Timed venous blood samples will be collected from the start of d5-LNA infusion until 480 min (8 hours) 
to determine plasma levels of esterified and unesterified labeled fatty acids.  Sampling will be at -10, -5, 
0, 5, 10, 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, 300, 360, 420, and 480 minutes (19 time points). A  
total blood volume of 80 mL will be collected. Blood samples will be used to determine the plasma 
concentrations over time of endogenous unesterified omega-6 and omega-3 fatty acids (nmol/ml) and d5-
labeled unesterified and esterified omega-3 fatty acids (nmol/ml) and endocannabinoids with GC and 
GC-MS. Collected blood will be centrifuged at 2000g for 10 min, and plasma will be transferred to 
another tube, and kept at -80°C until assay.  After lipid analysis of esterified and unesterified labeled and 
unlabeled fatty acids, hepatic synthesis-secretion rates will be calculated for EPA, DHA and other 
omega-3 fatty acids as described in Section S.9. 
 
Genotyping 
DNA is collected from sputum for analysis of FADS gene variants as part of the main protocol.  
 
Infusion Substudy Procedure Specifics 
 
Infusion Substudy Timeline 
All study procedures are for research purposes only. The sequence of procedures is given in Table 8.  All 
time-points are targets, and may be adjusted based on practical considerations.  
 
Table 8:  Timeline for Infusion Visit 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inpatient Admission with standard meal and overnight fast 
Participants will be admitted to the 5-SW Metabolic Inpatient Unit the evening before the 8-hour d5-LNA 
infusion, and consume dinner. Their vital signs, weight, and height will be measured.  Participants will consume a 
standardized ‘meal’ at 2300, and then begin an overnight fast at 2400.  At 0630 the following morning, one 
catheter will be inserted into a forearm vein to administer stable isotopically-labeled tracers and a second catheter 
Procedure 
Time 
Admission to 5/7SW Metabolic Unit  
1800 
Standardized research meal 
2300 
Craving/Hunger Visual Analog Scale 
(VAS) 
2400 (at completion of 
research meal) 
Begin 8-hour overnight fast 
2400 
Insertion of two IV lines:  
Antecubital (infusion) 
Heated hand (sample collection) 
0630 
Sample collection begins  
0745 
Repeat  administration of craving/hunger 
VAS  (0805, 1000, 1200, 1600) 
0805 
d5-LNA Infusion begins 
0800 
End Infusion/post-infusion meal 
1600 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 45 of 68 
will be inserted retrograde into the contralateral hand, which will be warmed with a thermostatically controlled 
box to obtain arterialized blood samples. The study will begin at 0745. At 0800, constant infusion of d5-LNA 
bound to 25% albumin will be started and maintained for up to 8 hours. To assess whole-body synthesis-secretion 
rates of EPA and DHA, fasting blood will be collected before (at -10, -5, 0 minute time points) and during (at 5, 
10, 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, 300, 360, 420, 480 minute time points) the d5-LNA Infusion. 
Participants will be instructed to rest 30 minutes prior to, and during the full 8-hour Infusion period. Participants 
can ambulate to the restroom as needed.  At the conclusion of the Infusion, participants will be provided with a 
post-fasting meal consistent with their study diet.   
 
Visual Analog Scale to assess craving and hunger 
Upon completion of the standardized research meal (between 2300 and 2400), participants will complete a brief 
VAS scale to assess their craving and hunger for food.  The same VAS will be administered after 8, 10, 12 and 16 
hours of fasting (Infusion day times of 0805, 1000, 1200, and 1600). 
 
Follow-up/ termination procedures 
There is no required follow-up for this study after completion of the experimental protocol. Participants will be 
advised to continue receiving their usual medical care throughout the duration of the study.  With patient consent, 
clinically-relevant information obtained through participation in the protocol may be shared with participants and 
appropriate health care providers.   
 
Radiation exposure 
There is no radiation exposure from participation in the Infusion Substudy.   
 
Medications and devices requiring IND/IDE 
No medications or devices requiring IND/IDE will be used in this study. 
It is important to note that only a trace amount of d5-LNA is provided in the infusion materials. This 
labeled d5-LNA is a tiny fraction of the non-deuterated LNA pool present in human plasma and tissues.  
There is a precedent for using intravenous infusion of (non-radioactive) deuterated stable isotopes of fatty 
acids in human clinical trials. Bernard V. Miller III, MD (NIDDK, Co-I on this Infusion Amendment), 
has performed over 100 stable isotope tracer infusion procedures in human clinical trials in Washington 
University School of Medicine-St. Louis, Missouri. A recently published manuscript (68) describing these 
methods is attached to our amendment submission memorandum.   
 
In 2008, The FDA Division of Metabolism and Endocrinology Products expressed the following policy 
regarding the use of stable isotopes in investigational metabolic studies: If a substance would not 
otherwise require submission of an IND, then that substance enriched with a stable isotope will not 
require an IND. If the isotope enrichment is for metabolic tracer studies employing drugs or non-
pharmacological substances, such as glucose and individual amino acids, for which there are adequate 
data demonstrating their safety and if the substances are used within the limits for which the data are 
adequate, no IND will be required. However, oversight by an IRB and obtaining informed patient consent 
are recommended.  
 
Storage of research samples and data 
Please see section 7.6.12 for the main protocol procedures for storage and use of research samples and data, and 
collaborations on stored research samples and data. The same procedures apply to this Substudy. 
 
S7. Risks and Discomforts – Infusion Substudy 
 
Allergic Responses 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 46 of 68 
It is possible that participants could have an allergic response to infusion materials.  During the Infusion 
Substudy informed consent visit, and again during the infusion visit (prior to infusion), participants will 
provide information on known allergies and if necessary the participants will be excluded.  Participants 
will also be advised to alert the research staff as soon as possible if they experience symptoms such as 
rash, hives, wheezing, or other signs of allergy or distress. A physician or nurse practitioner study 
investigator will be present at the bedside for the first two hours, and clinically available throughout the 
remainder of the infusion. 
 
Infection 
It is possible, but highly unlikely, that participants could be exposed to an infectious agent present in the 
human albumin or another component of the infusion materials.  In order to minimize the risk of 
infection, the NIH pharmacy will perform pyrogenicity and sterility testing to certify that infusion 
materials meet NIH standards for human infusion. In addition, a 0.2 micron filter will be used to 
administer the infusion materials at the bedside. 
 
Heat-related Injury 
In order to collect arterialized blood from a hand vein, participants’ hands will be heated to between 45 
and 55°C. Participants will be advised to alert research staff if they experience a pain or a burning 
sensation. It is possible, but unlikely, that participants could experience a burn. 
  
IV Insertion for Infusion and Specimen Collection  
Participants may have some discomfort and bruising at the site of IV insertion for the infusion and serial 
blood collection. Infection in the area of the needle insertion is rare. There is a very small risk of fainting.   
 
Fasting for the Infusion 
Participants will undergo a 16-hour fast beginning at midnight (2400) the day before the infusion and ending at 
the conclusion of the infusion (1600).  Participants will receive IV fluids with electrolytes throughout the infusion 
and a meal at the conclusion of the infusion. 
 
S8. Subject Monitoring, adverse event grading and withdrawal – Infusion Substudy 
 
Parameters to be monitored 
Participants will be closely monitored for adverse reactions by medically qualified staff during the 
invasive procedures.  The participants’ vital signs will be closely monitored during the Infusion Procedure 
testing for any signs of subject distress.  Appropriate medical care and treatment will be provided if 
necessary. 
Participants will be withdrawn from the Infusion Substudy if they do not follow the instructions outlined 
in the protocol and informed consent form. Participants may also, at any time, choose to discontinue their 
participation from the Substudy. 
 
S9. Outcome Measures (Infusion Substudy) 
 
Primary Outcome Measure 
Synthesis-secretion rates for omega-3 DHA with d5-LNA infusion, after 12-weeks of exposure to diets 
containing either 8 en% or 1 en% as omega-6 LA. 
 
Human d5-LNA Infusion Model (adapted from Rapoport rodent model (64,65)).  

PI na me : C hrist o p her E. Ra ms de n, M D 
 
Date:  0 1 /0 3 / 2 0 18  
 
 
 
Pr ot oc ol n u m ber : 1 1-A A -0 0 2 8  
Versi o n :  9. 1 
 
 
C N S I R B Pr ot o c ol Te m plate (re v. 1 -1 7 -0 8)  
 
 
 
 
 
 
pa ge 4 7  of 6 8  
T he o bjecti ve of t his st u d y is t o q ua ntif y  c o n versi o n rates of circ ulati n g u nesterifie d plas ma L N A i nt o 
l o n ger c hai n o me ga-3 fatt y aci ds.  It is ass u me d t hat m ost of t his c o n versi o n will ta ke place i n t he li ver.  
 
T he i dea is t o i nf use la bele d d 5 -L N A, he ncef ort h desi g nate d as L N A *, i ntra ve n o usl y at  a c o nsta nt rate f or 
u p 8 h o urs, a n d t o sa m ple bl o o d plas ma (t hr o u g h a sec o n d i ntra ve n o us cat heter) peri o dicall y f or 
meas uri n g esterifie d a n d u nesterifie d plas ma c o nce ntrati o ns of L N A * a n d o me ga -3 L C P U F As ( 1 8: 4 n -3 *, 
2 0: 4 n -3 *, 2 0: 5 n -3 * ( E P A), 2 2: 6 n -3 * ( D P A) , 2 2: 5 n-3 *, 2 4: 6 n -3 *, a n d 2 2: 6 n -3 * ( D H A). A d diti o nall y, 
D E X A sca n data fr o m t he pri mar y pr ot oc ol ( 1 1 -A A -0 0 2 8) will be use d t o esti mate plas ma v ol u me V pl as m a. 
Meas ure me nts als o will be ma de of u nesterifie d u nla bele d L N A, D P A, E P A a n d D H A c o nce ntrati o ns i n 
p las ma, t o calc ulate act ual c o n versi o n rates, as f ut ure st u dies ma y l o o k at rates t hat ma y de pe n d o n t hese 
c o nce ntrati o ns altere d i n disease or dietar y states.  
 
I nitiall y, a c o nsta nt i nf usi o n rate of a p pr o xi matel y 2. 2 u m ol/ mi n will be esta blis he d a n d i nf usio n ti me will 
be 8 h o urs, f or a t otal la bele d i nf usi o n a m o u nt of 3 5 0 m g.  Si nce t he half -life of i nj ecte d u nesterifie d fatt y 
aci d is a b o ut 2 mi n utes i n h u ma n plas ma, a stea d y state c o nce ntrati o n L N A * will be esta blis he d wit hi n 
a p pr o xi matel y 1 0 mi n utes after  i nf usi o n is i n i nitiate d. D urati o n a n d rate ca n be a dj uste d as necessar y t o 
pr o perl y deter mi ne t he e nric h me nt of t he tracer a n d fatt y aci d ki netics.  
 
E q u ati o ns  
Ass u mi n g li nearit y, t he net rate of s y nt hesis of a la bele d o me ga -3 L C P U F A (e. g. D H A *) wit hi n s ecrete d 
V L D Ls or ot her li p o pr otei ns fr o m la bele d L N A * is gi ve n as:  
  
 
T he rate of disa p peara nce of D H A * fr o m plas ma is gi ve n as:   
 
w here c o nce ntrati o ns C are n m ol ( or ot her u nits)/ ml plas ma, t is ti me after i nf usi o n has be g u n, k-
a p peara nce a n d k -disa p peara nce are s y nt hesis c oefficie nts i n u nits of ti me -1, a n d V pl as m a is plas ma v ol u me 
(esti mate d via D E X A sca n). Beca use t here is n o is ot o pe effect o n e nz y me ki netics, rate c oefficie nts k-
a p peara nce a n d k -disa p peara nce are vali d f or t he u nla bele d P U F As as well.  
 
T he c ha n ge i n q ua ntit y of D H A * i n plas ma at a n y ti me after i n f usi o n be gi ns e q uals: 
 
 
D H A ca n e xist i n t w o f or ms i n plas ma, esterifie d i n li p o pr otei ns a n d u nesterifie d (after release fr o m 
li p o pr otei ns) a n d release of la bele d u nesterifie d fatt y aci d ta kes a b o ut 3 0 mi n t o bec o me e vi de nt i n rat 
plas ma after ga va ge ( 7 1). Beca use o xi dati ve meta b olis m is 2. 5 ti mes faster i n rats t ha n i n h u ma ns ( 6 9), it 
is e x pecte d t hat t he dela y ma y be u p t o 6 0-9 0 mi n. A d diti o nall y, d uri n g i ntra ve n o us i nf usi o n of * L N A, 
esterifie d la bele d n -3 P U F As a p pear i n plas ma o nl y after 3 0 mi n, a n d it  is e x pecte d t hat i n t he prese nt 
pr ot oc ol t he y will als o a p pear wit h a pr ol o n ge d dela y, bet wee n 6 0 a n d 9 0 mi n. I n rat st u dies, t he d urati o n 
of i ntra ve n o us i nf usi o n f or 2 h has pr o ve n satisfact or y ( 6 4, 6 5). F or t hese reas o ns, we will start i nitiall y i n 
h u ma ns wit h a n i nf usi o n ti me of 8 h o urs, b ut ma y alter t he d urati o n de pe n d o n preli mi nar y res ults.  
 
H o we ver, t he half -li ves of L N A a n d D H A esterifie d i n plas ma li p o pr otei n ( V L D L, L D L, H D L) i n 
h u ma ns are 1 h o ur a n d 2 0 h o urs, res pecti vel y ( 7 0, 7 1).  T his mea ns that after a dela y i n reac hi n g stea d y -
state rates of c o n versi o n of -L N A t o D H A a n d s h orter c hai n n -3 fatt y aci d prec urs ors i nt o plas ma 
li p o pr otei ns, t he i nitial sl o pe of i ncrease d acti vit y will esse ntiall y gi ve t he c o n versi o n c oefficie nt k-
*
*
L N A
D H A
pl as m a
C
k
dt
d C
V
-
=
a
  
  
 
k C D H A *
*
*
*
D H A
L N A
D H A
pl as m a
C
k
C
k
dt
d C
V
-
=
-
a

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 48 of 68 
appearance. Any unesterified DHA* or other labeled unesterified omega-3 fatty acids that is released over 
time will have a half-life in plasma of about 2 minutes, and this will be taken into account as well. 
 
In this study, we will use an infusion schedule to raise the concentration of d5-LNA as a step function, 
and will plot 
 against time.  The initial slope relating the 
 against time after a 
delay will approximate the synthesis coefficient k* for DHA*; similar coefficients can be obtained for the 
shorter chain labeled n-3 fatty acids. 
 
For more explicit analysis, as time proceeds, we will fit equation 4 to the data after inserting a time delay 
using a curve fitting procedure, to obtain 
for the different n-3 products. 
is the coefficient of 
conversion by whole body per given concentration of plasma LNA, in units of nmol/time, e.g. (nmol/ml, 
concentration) per unit time multiplied by ml (volume).  This rate can be compared to our estimated rates 
of liver synthesis at the steady state, 
, in rats following the intravenous infusion of LNA*
. is the 
plasma unesterified concentration of LNA.  
 
 
The concentration curves representing LNA* and DHA* should look like Figure 9: 
 
Fig. 9. Graphical depiction of concentration curves for d5-LNA and DHA. 
 
S10. Statistical Analyses (Infusion Substudy) 
Overview 
This Infusion Substudy first demonstrates feasibility for characterizing the hepatic synthesis-secretion rates 
of DHA from infused d5-LNA in humans, and then compares synthesis-secretion rates of omega-3 DHA in 
two diet groups that differ only in dietary omega-6 LA content.   
 
Analysis of Data 
Primary Aim 1:In the feasibility portion of this study we will define the dose, rate and duration of the d5-
LNA infusion necessary to characterize synthesis-secretion rates of DHA from infused d5-LNA.  Based on 
our previous experiences using this methodology in a rodent model, we anticipate that these data can be 
obtained from the first 4 infusion participants. 
 
Primary Aim 2: 
In the diet group comparison, we will compare synthesis-secretion rates for omega-3 DHA from d5-LNA 
in response to controlled dietary omega-6 LA lowering from 8 en% to 1 en%.  The synthesis-secretion 
VplasmaCDHA*
VplasmaCDHA*
 
k 
 
k 
 
k 
 
k 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 49 of 68 
rate of labeled serial concentration data for labeled DHA will be compared in the two diet groups using a 
t-test. A probability of 0.05 or lower will be considered significant.  
The FADS genotype will be measured as part of the main protocol.  This will allow us to assess whether 
the “low converter” FADS genotype is a modifying variable, or to rule it out as a confounder. 
 
 
Estimation of Sample Size  
We estimate that 4 to 12 individuals in each group are needed to provide adequate power (0.8) to test 
Primary Aim 2, as shown in Figure 10.   
 
Estimation of Power and Sample Size 
We will use a t-test to compare the synthesis-secretion rates and coefficients measured at 4 weeks in the 
two groups (A and B). Assuming a 20-30% standard deviation in the synthesis-secretion rates based on 
our prior kinetic work in humans (74) and rodents (64) and a 67% difference in expected change in 
kinetics consistent with Pawlosky et al (74) and prior measured changes in DHA concentrations, we 
calculated required sample size(s) (Fig. 10). To detect these effect sizes, at a power of 80% and an alpha 
of 0.05, we will need 4-12 individuals per group. 
 
 
Fig. 10. Sample Sizes for Endogenous DHA Synthesis Amendment 
Sample Size Calculation (Spreadsheet1)
Two Means, t-Test, Ind. Samples
H0: Mu1 = Mu2
Value
Population Mean Mu1
Population Mean Mu2
Population S.D. (Sigma)
Standardized Effect (Es)
Type I Error Rate (Alpha)
Critical Value of t
Power Goal
Actual Power for Required N
Required N (per group)
14.1000
23.5470
7.7620
-1.2171
0.0500
2.0739
0.8000
0.8130
12.0000
 
Sample Size Calculation (Spread
Two Means, t-Test, Ind. Samples
H0: Mu1 = Mu2
Value
Population Mean Mu1
Population Mean Mu2
Population S.D. (Sigma)
Standardized Effect (Es)
Type I Error Rate (Alpha)
Critical Value of t
Power Goal
Actual Power for Required N
Required N (per group)
14.1000
23.5470
6.4684
-1.4605
0.0500
2.1199
0.8000
0.8284
9.0000
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 50 of 68 
Sample Size Calculation (Spreadsheet1)
Two Means, t-Test, Ind. Samples
H0: Mu1 = Mu2
Value
Population Mean Mu1
Population Mean Mu2
Population S.D. (Sigma)
Standardized Effect (Es)
Type I Error Rate (Alpha)
Critical Value of t
Power Goal
Actual Power for Required N
Required N (per group)
14.1000
23.5470
3.7647
-2.5094
0.0500
2.4469
0.8000
0.8386
4.0000
 
 
 
 
 
 
 
S11. Human Subjects protection – Infusion Substudy 
 
For human subjects protection issues that apply to both the main protocol and the Infusion Substudy, 
please see Section 12 (Page 38) of the main protocol. Additional issues that apply to the Infusion 
Substudy are addressed in the following sections. 
 
Justification of sensitive procedures 
Procedures that may cause some discomfort to subjects: 
Prolonged fast for d5-LNA Infusion:  Participants are required to fast for up to 16 continuous hours.  
Participants will be provided with IV fluids but are likely to experience some hunger during the 8-hour 
infusion. They will be provided with a meal immediately after infusion. 
IV insertion for d5-LNA Infusion: Trained medical personnel will carry out the insertion of two IV 
catheters, which may cause some discomfort to the subject. 
Heated hand: Each participant’s non-dominant hand will be placed in a heated box for approximately 4 
hours during the infusion procedure to obtain “arterialized” blood samples for the proper determination of 
stable isotope tracer enrichment. The temperature inside the box will be set to between 44 to 54˚C 
(between 110 and 129˚F). A controller will maintain the temperature within ±2 degrees of the selected 
temperature. No significant adverse effects (including burning) have been reported using this equipment 
for extended time periods during stable isotope tracer infusions studies. 
Blood sampling: Trained medical personnel will carry out the blood draws, which may cause some 
discomfort to the subject. 
Storage of Patient Samples:  Coded blood and tissue samples without patient names or personal 
identifiers will be stored in secured freezers at 5625 Fishers Lane, Room 3N-01 or in the NIAAA Clinical 
Core lab freezers in the 1-SE area of the clinical center.  Samples will be processed upon completion of 
the study and destroyed when analysis is complete. 
 
Reasons for Sensitive Procedures: 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 51 of 68 
Prolonged fast for Infusion: Continuation of the typical 8-hour overnight fast for an additional 8 hours 
(the maximum duration of the infusion) is necessary in order to minimize the introduction of uncontrolled 
dietary variables that may alter the measured synthesis-secretion of omega-3 LCPUFAs (i.e. consumption 
of unlabeled LNA, LA and other competing fatty acids through diet).  
IV insertion and blood sampling for Infusion: These IV catheters will be used for the Infusion and serial 
blood sampling, allowing for collection of blood on 19 occasions without serial blood draws. Blood 
sampling is necessary to determine the concentrations and labeled and unlabeled fatty acids for 
assessment of synthesis-secretion rates of omega-3 LCPUFAs.  Previous work in this area has been 
performed successfully in rodents, but has not been translated to humans in a clinical trial. 
Heated hand:  The heated hand vein technique is used to obtain blood samples that are “arterialized”, 
meaning they are more like arterial blood than venous blood. Using the heated hand vein technique is 
standard procedure used during stable isotope tracer infusion studies to ensure proper assessment of 
substrate kinetics. The heated hand vein technique avoids the use of more invasive procedures such as an 
arterial line.  
Blood sampling:  Procedure is necessary to assess the concentrations of labeled and unlabeled fatty acids 
in order to calculate hepatic synthesis-secretion rates for omega-3 DHA.  Previous work in this area has 
been performed successfully in rodents, but has not been translated to humans in a clinical trial. 
Storage of Patient Samples: The storage of clinical samples is necessary for collaboration and assurance 
of similar analysis conditions across all of the study samples.   
 
 
 
 
 
Qualifications of Investigators – Infusion Substudy 
 
Introduction 
Enrollment in the Infusion Substudy will require a new informed consent, obtained via a face-to-face 
meeting with Dr. Ramsden and/or Laura Musse, CRNP, MS. 
The Brain Physiology and Metabolism Section (BPMS) of the National Institute on Aging (NIA), of 
which Stanley I. Rapoport is Chief, is collaborating on the d5-LNA Infusion Amendment. The BPMS 
conducts basic and clinical research related to factors that regulation brain function and structure in health 
and disease. One focus is to identify the influence of dietary composition and liver metabolism of 
polyunsaturated fatty acids (PUFAs). Methods are developed to image regional brain metabolism of 
omega-6 AA and omega-3 DHA in unanesthetized animals and in humans (with positron emission 
tomography), and to quantify the ability of the liver to synthesize these PUFAs from their precursors LA 
and LNA, respectively, in vivo. The BPMS has developed the infusion methodology and analysis in 
rodents, which is a basis of this clinical amendment.  
 
D5-LNA Infusion Amendment Collaborators 
 
Stanley I. Rapoport, M.D., is Chief of the BPMS NIA, who has published extensively in the areas of lipid 
metabolism and brain function in health and disease.  He received his MD degree from Harvard Medical School, 
and is clinically credentialed at the NIH Clinical Center. The laboratory that he directs has developed and 
validated the infusion method and analysis in rodents.  These methods form a major basis for the Infusion 
Amendment employed in this human protocol. He is involved in the design of the protocol, and its conduct, and in 
data analysis and writing up results. 
Dr. Rapoport will not obtain consent. 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 52 of 68 
 
 
 
S12. Benefits 
This Infusion Substudy will provide generalizable knowledge on the normal DHA synthesis-secretion rates in 
humans, and the impact of dietary fatty acids on hepatic synthesis-secretion of DHA. This experimental procedure 
has no anticipated benefits for study participants.   
 
S13.  Classification of Risk 
▪ 
No direct benefit, but likely to yield generalizable scientific knowledge  
▪ 
More than minimal risk 
▪ 
The risks are reasonable in relation to anticipated benefits. We will obtain important 
insights into normal DHA synthesis-secretion rates in humans, and learn whether 
modifying dietary LA alters synthesis-secretion rates of DHA.  Both findings could 
ultimately have important implications for the prevention and/or adjunctive management 
of conditions including addictive disorders. 
 
S14. Consent documents and process 
 
Who will obtain Consent  
The informed consent process for the Infusion Substudy will include a face-to-face meeting with Dr. 
Ramsden, Dr. Miller and/or Laura Musse, CRNP, MS. 
 
S15. Data Safety and Monitoring & S16. Adverse Event Reporting 
 
S15.1 Data Safety and Monitoring Board 
The Infusion Substudy is two-arm study with an 8-hour intravenous infusion of a non-radioactive nutrient 
in a buffered solution with human albumin.  Since the research procedures are to be performed in adult 
subjects and such procedures involve minor increase over minimal risk, the collection, monitoring and 
analysis of adverse events will be the responsibility of the Principal Investigator and the investigative 
team. No DSMB will be convened. 
 
S15.2 Data Safety and Monitoring Plan 
Elements of the Data Safety and Monitoring Plan 
(1) Monitoring mechanism:   
The Principal Investigator and/or medically qualified staff will monitor the infusion 
protocol participant vital signs data for signs of participant distress as stated on page 
44. 
(2) Frequency:   
The Principal Investigator and/or medically qualified staff will be present at the 
beginning of the infusion and will stay on the unit for the first 2 hours of the 
procedure.  Medically qualified staff will check the participant vital sign status at 
least hourly after these first 2 hours.   
(3)  Stop or change rules:   
Participants may choose to stop the infusion procedure at any time.  Participants will 
be reminded to report any signs of distress or discomfort to medically qualified staff 

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members.  Medically qualified staff will determine if any signs of participant distress 
are present and are severe enough to abort the infusion procedure.   
(4) Advanced Plans:   
As stated on page 46, this study is designed with the first primary aim as a feasibility 
study and the data will be reviewed after the first 4 participants.   
(5) Information to be monitored for participant safety:   
Participant vital signs (as stated above) 
(6) Communication:   
A record of participants removed from the infusion substudy will be reported to the Addictions 
IRB at the time of annual review.  Serious adverse events will be reported according to the 
required NIH guidelines as stated in section S16 on the next page. 
 
S15.3 Data Monitoring for Quality Assurance 
Data monitoring for quality assurance will be the responsibility of the Principal Investigator and 
the investigative team.   
(1) Monitoring mechanism:   
The Principal Investigator and the investigative team will monitor the protocol 
participant free and esterified fatty acid data. 
(2) Frequency:   
After each infusion, the blood samples will be analyzed for deuterated fatty acid 
concentration and the results will be reviewed by the Principal Investigator and the 
investigative team. 
(3) Information to be monitored: 
Deuterated fatty acid concentration will be monitored for appropriateness of infusion 
dose, rate and sampling schedule  
(4) Communication: 
Infusion substudy progress will be reported to the Addictions IRB at the time of annual review in 
the form of a summary report.  This summary report will include the numbers of recruited, and 
enrolled participants in this substudy.  It will also include a brief data report noting any need for 
adjustments in the infusion procedure or sampling schedule. 
 
 
S16. Adverse Event Reporting 
 
Adverse events 
In the course of a dietary study and infusion amendment, adverse events may occur. Unanticipated adverse events 
that occur as a direct result of research studies initiated at the NIH, but not related to the natural history of the 
underlying disease will be summarized at the time of the annual review.    
Serious Adverse events  
A serious adverse event is any adverse experience that: 
1. Results in death; 
2. Is life threatening; 
3. Results in hospitalization or prolongs hospitalization; 
4. Results in persistent or significant disability/incapacity;  
5. Results in congenital anomaly/birth defect; or 

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6. Results in a condition, which in the judgment of the investigator represents a significant hazard.   
 
Serious adverse events that are possibly, probably or definitely related to participation in research studies 
performed at the NIH Clinical Center will be reported.  However, serious adverse events (other than death) that 
are clearly and unambiguously due to extraneous causes (accidental, environmental or coincidental events) will 
not be reported.  
All serious adverse events will be reported using the NIH Serious Adverse Event Report Form to the NIAAA 
CNS IRB, and NIAAA Clinical Director as soon as possible, but no later than seven days of death or life 
threatening serious adverse event or within fifteen days after the occurrence of all other forms of serious adverse 
events.    
 
 
S17. Alternatives to participation or alternative therapies 
 
Subjects are healthy volunteers and are not recruited for the treatment of a medical condition.  
Participants are counseled to continue their usual medical care for the duration of the study. 
 
S18.  Confidentiality 
 
Medical records 
This information will remain confidential and protected according to NIH guidelines.  Some clinically 
relevant research data will be stored indefinitely in the medical record and will be accessible to the patient 
for review by others of their choosing (doctors, insurance companies etc.) after executing a release of 
information. 
 
Research Data 
 
Data will be collected and entered on form sets labeled with a unique coded identifier that is not related to 
the patient’s name or any other personal identifier (Soc Security number, NIH employee number, etc).  
Data will be kept in a locked cabinet or in secure computer files with access only for the Principal 
Investigator (PI) and Associate Investigators (AIs) involved with the study. When entered into the 
electronic databases, information will be under the CRC and NIH network regular access control.  In 
addition, any information allowing the identification of the participants will be removed.  The only 
possibility to reconstruct the link between database records and information will be through the unique 
identifier.  The translation key will be stored in a locked cabinet or secure computer file with access 
available only to the PI and AIs named in this protocol document. 
 
The validated psychometric instruments used in this protocol have been converted to an electronic web-based 
secure data entry platform) administered by the NIAAA.  The security of the data management system  is curated 
by the NIAAA IT department in agreement with NIH data security policies. 
Most data generated in this study will be stored and maintained in the NIAAA clinical database which is 
password protected and secure. 
 
Sample Storage 
Coded blood and tissue samples without patient names or personal identifiers will be stored in secured 
freezers at 5625 Fishers Lane, Room 3N-01 or in the NIAAA Clinical Core lab freezers in the 1-SE area 
of the clinical center.  Samples will be processed upon completion of the study and destroyed when 
analysis is complete.   Any additional collaborations requiring transfer of patient material will be done in 
a coded fashion after informing the IRB and obtaining necessary assurances from the outside institution.  
The protocol will be amended and IRB approval will be sought when such collaborations are established.     

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S19.  Conflict of Interest/Technology Transfer 
NIH guidelines on conflict of interest have been distributed to all investigators. There are no conflicts of 
interest to report.  Technology transfer is not applicable to this Infusion Substudy. 
 
S20.  Compensation  
Remuneration for Infusion Substudy 
Remuneration for participation in the Infusion Amendment is based on existing NIH guidelines based on 
inconvenience, to offset potential loss of earnings and travel expenses. Since participation involves one 
visit, the amount paid will not be pro-rated based upon participation.  For the completion of all aspects 
Infusion Amendment Visit, participants will be compensated a maximum of 250 dollars. 
 
Note: References for the Infusion Substudy are in blue text in the Bibliography Section. 
 
 
 
 
 
 
 
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PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 61 of 68 
23. Appendices 
Appendix 1: Study Flow Diagram 
 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 62 of 68 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 63 of 68 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 64 of 68 
Appendix 2: Lands Equation for Predicting Percentage of omega-6 in HUFA (%omega-6 in HUFA) 
 
In a series of human experiments, Lands et al. assessed the relationships between 18, 20, and 22 carbon 
omega-6 and omega-3 fatty acid composition of diets, within typical U.S. dietary ranges, and percentages 
of omega-6 and omega-3 HUFA in total HUFA (17). Within these ranges, tissue accumulation of omega-
6 and omega-3 HUFA were surprisingly consistent with the principles that Mohrhauer and Holman 
observed in rats. By combining human and animal data, Lands developed empirical equations relating 
fatty acid composition of diet to tissue HUFA content.  
 
 
 
The Lands equation accurately predicts mean population omega-6 and omega-3 HUFA proportions in 
populations with radically different dietary omega-6 and omega-3, supporting validity of the equations. 
Mean tissue percentages of omega-6 in total HUFA (%omega-6 in HUFA) varied widely, from less than 
30% to more than 80% of total HUFA, in populations consuming dissimilar diets. 
 
 
In a recent study, the %omega-6 in HUFA measured in whole blood correlated closely with liver, heart, 
and brain HUFA status in autopsied rats and pigs (75). In this respect, %omega-6 in HUFA outperformed 
other HUFA biomarkers including erythrocyte EPA+DHA, which did not correlate with EPA+DHA 
content of any of these tissues (75). Thus, the demonstration that the empirical equation of Lands 
accurately predicts changes in %omega-6 in HUFA in blood following major reductions in dietary intakes 
of LA would have important implications for any condition affected by membrane AA content, or any 
disease in which AA-derived mediators have been implicated, no matter which tissues are involved in the 
disease process. 
 
 
 
 
 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 65 of 68 
 
Appendix 3: Omega-6 (omega-6) and omega-3 Fatty Acids and their Metabolic Derivatives 
 
omega-6 Fatty Acids 
Formula 
LA  
18:2 omega-6 
AA 
20:4 omega-6 
DTA 
22:4 omega-6 
DPA (omega-6) 
22:5 omega-6 
%n6 in HUFA (Lands equation) 
100* [(Total omega-6 HUFA)/ (Total omega-6 and omega-3 
HUFA)] 
 
 
omega-3 Fatty Acids 
 
ALA 
18:3 omega-3 
EPA 
20:5 omega-3 
DPA (omega-3) 
22:5 omega-3 
DHA 
22:6 omega-3 
omega-3 Index 
EPA + DHA 
 
 
 
Precursor 
omega-6 AA derived Endocannabinoids 
 
2-AG 
PL-AA 
AEA 
PL-AA 
 
 
omega-3 EPA/DHA derived Endocannabinoids 
 
2-DHG 
PL-DHA 
2-EPG 
PL-EPA 
DHEA 
PL-DHA 
EPEA 
PL-EPA 
 
 
Other Endocannabinoids 
 
OEA 
PL-Oleic acid (18:1 n-9) 
PEA 
PL-Palmitic acid (16:0) 
 
 
omega-3 Docosanoids and Resolvins 
 
10s, 17s – diDHA 
DHA 
NPD1 
DHA 
RvD1 
DHA 
RvD2 
DHA 
RvE1 
EPA 
 
 
omega-6 AA derived Eicosanoids 
 
PGE2 
AA 
LTB4 
AA 
TXB2 
AA 
Key: LA linoleic acid; AA arachidonic acid; DTA docosatetraenoic acid; DPA docosapentaenoic acid; ALA alpha-linolenic acid; 
EPA eicosapentaenoic acid; DHA docosahexaenoic acid; 2-AG 2-arachidonoyl-glycerol; AEA anandamide; 2-DHG 2-
docosahexaenoyl-glycerol; 2-EPG 2-eicosahexaenoyl-glycerol; DHEA docosahexaenoyl-ethanolamide; EPEA eicosapentaenoyl-
ethanolamide; OEA oleoyl-ethanolamide; PEA palmitoyl-ethanolamide; NP neuroprotectin; Rv resolvin; PG prostaglandin; LT 
leukotriene; TX thromboxane 
 
 
 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 66 of 68 
 
  
Appendix 4: Psychometric Questionnaires  (pdfs) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix 5: Eligibility Checklist – Main Protocol 
INCLUSION CRITERIA 
Yes         No 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 67 of 68 
 
 
Female gender 
 
 
18 to 50 years of age 
 
 
BMI between 25 and 35 kg/m2 
 
 
Use of effective contraception with regular menstrual cycles  
         
 
FSH less than 20  
 
 
ECG that is clinically unremarkable   
 
 
Lab tests that are within normal limits or clinically unremarkable  
 
 
Able to come to NIH Clinical Center every weekday for 3 months 
 
 
Negative urine drug screen 
 
 
 
 
Menstruating 
Negative serum pregnancy test (b-HCG)  
EXCLUSION CRITERIA 
Yes          No 
 
 
 
Major medical problems (CNS, cardiovascular, respiratory, GI, hepatic, renal):  
 
Major organ or body system dysfunction (Specify: ___________) 
 
Endocrine disorders (Specify: ____________________________) 
 
Other (Specify: _______________________________________) 
 
 
 
Study investigator or their superior, subordinate, or immediate family member  
 
 
Positive HIV test 
 
 
Significant Dietary Limitations (Multiple Food Allergies/intolerances, vegan 
diet) or special dietary requirements that are difficult to accommodate 
 
 
Past or present history of Eating Disorder (including binge eating) or psychiatric 
disease (Specify: __________________) 
 
 
Pregnancy and/or Lactation within the last 2 years 
 
 
Have had bariatric surgery  
 
 
Have gained or lost more than 3% of your current weight in the past 3 months 
 
 
Seafood consumption more than 3 x per week 
 
 
Regular use of supplements that contain omega-6 or omega-3 fatty acids (e.g. 
fish, cod liver oil, borage or evening primrose oil) 
 
 
History of alcohol dependence or abuse in the past 5 years, or current regular use 
of alcohol, defined as more than 2 drinks per day or 6 drinks per sitting  
 
 
Smoker (more than 2 cigarettes/week in the past year) 
 
 
History of substance dependence or abuse in the past 5 years 
 
 
Have a known bleeding disorder, or regularly use anti-coagulant medications 
 
 
Regular use of prescription or OTC medications or supplements known to alter 
energy expenditure, affect nutrient absorption or alter food intake. 
[Examples include medications for diabetes (type I or II), antipsychotics, 
tricyclic anti-depressants, thyroid medications, cholesterol medications, anti-
hypertensives, beta-blockers, orlistat, glucocorticoids, decongestants, 
antihistamines, ephedrine, methylphenidate, L-dopa] 
Others (Specify: ______________________________) 
 
 
 
 
Appendix 7: Eligibility Checklist – d5-LNA Infusion Amendment 
 

PI name: Christopher E. Ramsden, MD 
 
Date: 01/03/2018 
 
 
 
Protocol number: 11-AA-0028 
Version:  9.1 
 
 
CNS IRB Protocol Template (rev.1-17-08) 
 
 
 
 
 
 
page 68 of 68 
 
INCLUSION CRITERIA 
Yes          No 
 
 
 
Met all eligibility criteria for enrollment in protocol 11-AA-0028 
 
 
Presently enrolled and nearing completion of 12-week diet phase of  
11-AA-0028 
 
 
Negative pregnancy test  
EXCLUSION CRITERIA 
Yes          No 
 
 
 
History of allergic or other adverse reaction to infusion components