Vitamin D and Fish Oil for Autoimmune Disease, Inflammation and Knee Pain
Phase: NA
Conditions: Autoimmune Diseases, Systemic Inflammatory Process, Knee Pain Chronic, Osteoarthritis, Rheumatoid Arthritis
Sponsor: Brigham and Women's Hospital

PARTNERS HUMAN RESEARCH COMMITTEE 
PROTOCOL SUMMARY 
Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
1 
 
Answer all questions accurately and completely in order to provide the PHRC with the relevant 
information to assess the risk-benefit ratio for the study.  Do not leave sections blank. 
 
PRINCIPAL/OVERALL INVESTIGATOR 
Karen H. Costenbader 
 
PROTOCOL TITLE 
Vitamin D and Fish Oil for Autoimmune Disease, Inflammation and Joint Pain 
 
 
FUNDING 
 
National Institutes of Health R01 AR059086-01 
 
VERSION DATE 
 
November 9, 2009 
 
SPECIFIC AIMS 
Concisely state the objectives of the study and the hypothesis being tested. 
 
1. To test whether vitamin D3 and/or omega-3 fatty acid supplementation reduces the risk of 
incident autoimmune disease in five years in 20,000 elderly adults enrolled in an ongoing 
randomized controlled trial 
2. To assess the effects of vitamin D3 and/or omega-3 fatty acid supplementation upon levels 
of biomarkers of systemic inflammation over five years in a population of 2,000 older Americans 
in an ongoing randomized controlled trial 
3. To investigate whether vitamin D3 and/or omega-3 fatty acid supplementation decreases 
chronic knee pain among 2,000 older adults followed for five years in an ongoing randomized 
controlled trial 
 
 
BACKGROUND AND SIGNIFICANCE 
Provide a brief paragraph summarizing prior experience important for understanding the 
proposed study and procedures. 
 
Vitamin D and omega-3 fatty acids are two inexpensive and nontoxic nutritional supplements, 
widely held to have anti-inflammatory, immune- and pain-modulating effects. The purported 
health benefits of the relatively inexpensive and available dietary supplements vitamin D and 
omega-3 fatty acids have received enormous attention in both the medical literature and the 
popular press1-17. We propose to assess the roles of vitamin D and omega-3 fatty acids for three 
important outcomes in the elderly: prevention of autoimmune disease, effects on biomarkers of 
systemic inflammation, and modeulation of chronic knee pain. We will leverage the 
infrastructure of a newly NIH-funded randomized, double-blind, placebo-controlled clinical trial, 
the VITamin D and OmegA-3 TriaL (VITAL), for the primary prevention of cancer and 
cardiovascular disease. 
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
2 
Autoimmune diseases, including rheumatoid arthritis, autoimmune thyroid disease, inflammatory 
bowel disease, polymyalgia rheumatica and psoriasis, are increasingly prevalent with age and 
are associated with significant morbidity and medical expenditures. These diseases are 
associated with autoantibody production and systemic inflammation. No preventive therapy 
currently exists. Systemic inflammation associated with these diseases, as well as with 
increasing age, body mass index, and cigarette smoking, independently predicts cardiovascular 
disease, diabetes mellitus and mortality18-23. Inflammation and immune activation, as well as 
muscle weakening are involved in the development of chronic knee pain, one of the most 
common causes of pain and disability in elderly Americans. Chronic knee pain, largely due to 
osteoarthritis, imposes a huge economic burden, increasing with the aging of the “baby-boomer” 
generation.  
Understanding of the pluripotent immunomodulating and anti-inflammatory beneficial effects of 
vitamin D is rapidly advancing. In addition to its roles in regulating calcium homeostasis and 
bone turnover, vitamin D has effects on multiple cells of the immune system, inhibiting pro-
inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and 
lowering C-reactive protein (CRP)24. Data from laboratory studies and cross-sectional and 
observational epidemiologic investigations strongly suggest a protective effect for vitamin D in 
autoimmune disease susceptibility, but there have been no large randomized trials of high-dose 
vitamin D supplements for the primary prevention of autoimmune diseases or effects on 
systemic inflammation in a general population. Data on vitamin D and joint pain point to multiple 
potential therapeutic mechanisms, but the effects of nontoxic doses of vitamin D on chronic 
knee pain in the elderly have not been tested. 
Marine omega-3 fatty acids exert effects through the leukotriene and prostaglandin pathways, 
decreasing inflammatory mediators and cytokine production, and have multiple known anti-
inflammatory properties25. High dose omega-3 fatty acids have shown promise in the treatment 
of those with ongoing autoimmune diseases, reduction of biomarkers of systemic inflammation 
in large observational studies, and treatment of various types of pain and arthritis. However, no 
randomized controlled trials of these supplements have ever tested their effects in the primary 
prevention of autoimmune diseases, reduction of circulating biomarkers of systemic 
inflammation, or amelioration of chronic knee pain in a large elderly population.  
 
RESEARCH DESIGN AND METHODS 
Briefly describe study design and anticipated enrollment, i.e., number of subjects to be enrolled 
by researchers study-wide and by Partners researchers.  Provide a brief summary of the 
eligibility criteria (for example, age range, gender, medical condition).  Include any local site 
restrictions, for example, “Enrollment at Partners will be limited to adults although the sponsor’s 
protocol is open to both children and adults.”
 
The VITAL trial (Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov number 
01169259) will involve 20,000 men and women from across the U.S., selected on age only (men 
aged ≥60 and women aged ≥65), with an oversampling of Blacks. In a 2x2 factorial design, 
participants will be randomized to moderate-to-high dose vitamin D3 (cholecalciferol; 2000 IU 
[50 µg]/d) and marine omega-3 fatty acids (EPA [500 mg/d] + DHA [500 mg/d]) supplements (or 
placebos). 
 
The proposed investigations will take advantage of an NIH-funded large RCT of vitamin D and 
omega-3 fatty acid supplementation for the prevention of cancer and cardiovascular disease. 
The VITamin D and OmegA-3 TriaL (VITAL ,JoAnn Manson, MD, DrPH, PI) is a randomized, 
double-blind, placebo-controlled trial of the benefits and risks of vitamin D (vitamin D3 
[cholecalciferol], 2000 IU/d) and omega-3 fatty acids (800 mg/d; EPA to DHA ratio, 1:1) among 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
3 
20,000 men and women, aged ≥60 and ≥65, respectively. VITAL cohort assembly has begun 
and baseline mailings will begin in January 2010. The three outcomes that we will test in this 
ancillary grant include: prevention of autoimmune diseases, and effects on systemic 
inflammation and chronic knee pain. At the end of a 3 month run-in period, participants will be 
randomly assigned to one of four treatment groups for 5 years: vitamin D3 (2000 IU/d) and fish 
oil (EPA+DHA, 800 mg/d); vitamin D3 and fish oil placebo; placebo vitamin D3 and fish oil; and 
placebo vitamin D3 and placebo fish oil. At 1-year intervals, participants will receive a new 
supply of pills, and assessments of incident autoimmune diseases, compliance and potential 
side effects. All autoimmune disease endpoints will be confirmed by medical record review by 
the physician endpoints committee. We anticipate validation of 600 incident cases of 
autoimmune disease, giving us sufficient statistical power to assess supplement effects on 
disease incidence. Blood samples at baseline and after 1-2 years follow-up will be collected in a 
subcohort of 2000 individuals and analyzed for changes in biomarkers of systemic inflammation: 
C-reactive protein, interleukin-6, and tumor necrosis factor-. Approximately 2000 individuals 
with chronic, daily knee pain on baseline screening will be followed with annual questionnaires, 
providing ample statistical power to evaluate the effects of vitamin D and fish oil upon changes 
in chronic knee pain. This trial will be very cost-effective as it will be conducted by mail. Each 
participant will be randomly assigned in a 2x2 factorial design to vitamin D, fish oil, both agents, 
or both placebos (anticipated mean treatment duration of 5 years). 
 
Study population. The study population will be that of the parent VITAL trial, described in 
Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov NUMBER01169259. It will 
include approximately 20,000 apparently healthy participants—10,000 men aged ≥60 and 
10,000 women aged ≥65, ages at which rates of chronic diseases, including autoimmune 
diseases and knee OA pain, increase substantially.  
Cohort assembly, eligibility, run-in and randomization are described in Partners’ IRB 
approved protocol 2009-P-00127; clinicaltrials.gov number 01169259.  
Baseline autoimmune disease prevalence assessment. The enrollment questionnaire 
accompanied by a cover letter will be sent to all participants 
and will include a question regarding personal and family 
history of five specific autoimmune diseases (AITD, IBD, 
psoriasis, RA and PMR) as well as space to write in others. 
Individuals will also be asked about their family history of any 
autoimmune disease.  
Screening for chronic daily knee pain during run-in. As 
VITAL participants will be involved in the parent trial of cancer 
and cardiovascular outcomes and potentially other ancillary 
studies, to limit participant burden we will screen the entire 
population and select 2000 participants with chronic daily and 
severe knee pain who are willing and eligible to be in this 
subtrial. This number will provide ample statistical power for 
chronic knee pain outcomes. 
For our purposes, the outcomes of interest will be chronic 
frequent knee pain (not radiographic knee OA). We will thus 
screen our trial population and classify those with: knee pain 
with walking 2-3 blocks, a doctor’s diagnosis of knee OA, pain 
> 5 days per week and for > one year and classify those with 
all four of these as having chronic frequent knee pain. (Fig. 
2). Individuals with total knee replacements at baseline will be 
excluded. Our goal is to include only those with the most 
severe, chronic knee pain at basline in this longitudinal RCT 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
4 
of the two investigational agents. Based on estimates of the prevalence of knee pain in a 
population of adults over age 60123, 404 and the added specificity of our screening questions, we 
anticipate that the prevalence of chronic severe knee pain by this method will be approximately 
12.5%, or 5000 individuals of the 40,000 individuals in the run-in phase. Although the sensitivity 
and specificity of our screening questions for chronic frequent knee pain (highly likely to be OA) 
in this population is not known, it is likely higher that that of Lavalley and ultimately will ensure 
that we include individuals with chronic, frequent knee pain, regardless of diagnosis.  
Baseline knee pain severity assessment. We will include supplementary longitudinal knee 
pain assessment questions (Appendix B) and a cover letter with run-in questionnaire to 5000 
individuals during the run-in phase. After the run-in phase, 50% of the individuals who were 
compliant with > 66% of study medications and supplementary requests, and continue to be 
willing and eligible, will be enrolled in VITAL. Thus, we anticipate that 2500 of the 5000 will be 
eligible for this sub-trial, and of these 2000 individuals with chronic, severe knee pain will 
complete the baseline assessment and will be followed annually. In a subset of 100 men and 
100 women participants, we will request access to medical records and radiograph reports and 
have them forwarded to us for review to validate the sensitivity, specificity, positive and negative 
predictive value of our screening strategy for the diagnosis of knee OA. A standard definition of 
knee OA for epidemiologic studies will employed: physician diagnosis of OA and the presence 
of one or more osteophytes on AP knee radiographs405.  
Baseline Blood Collection and Randomization are those of the parent VITAL trial, described 
in Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov number 01169259.  
 
Briefly describe study procedures.  Include any local site restrictions, for example, “Subjects 
enrolled at Partners will not participate in the pharmacokinetic portion of the study.”  Describe 
study endpoints.
 
Trial Procedures and Data Collection   
Treatment with active vitamin D3 and/or fish oil or placebo is described in Partners’ IRB 
approved protocol 2009-P-00127; clinicaltrials.gov number01169259. 
Follow-up procedures, dietary and supplemental intake assessments, are those of the  
parent VITAL trial, described in Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov 
number 01169259. 
Incident autoimmune disease ascertainment and validation. Incident autoimmune disease 
case identification during the trial will be a 2-stage procedure: 
1. Questions inquiring about new onset autoimmune diseases will be sent with the annual 
questionn
aires. 
Participan
ts who 
self-report 
an 
incident 
autoimmu
ne 
disease 
endpoints 
on any of 
these 
questionn

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
5 
aires will be mailed a letter asking them to sign a medical release form authorizing VITAL staff 
to obtain hospital/physician records. The request will be accompanied by a cover letter 
expressing sympathy for the diagnosis and explaining guidelines and the scientific importance 
of record validation. Non-responders will be sent two additional requests for the medical release 
form. During the 10 year Women’s Health Study trial, only 5% of cases refused to sign a 
medical release. After the release is obtained, a copy will be sent to the treating hospital or 
physician. If there is no response within 1 month, a second request will be mailed, followed by a 
phone call. In the Women’s Health Study, the staff was able to obtain hospital/physician records 
99% of the time. 
2. Medical records will be reviewed independently by two trained physicians (including board 
certified rheumatologist, endocrinologist, and gastroenterologists) blinded to the randomized 
treatment assignment, will confirm or disconfirm the autoimmune disease according to 
classification criteria (Table 3). The specificity of autoimmune connective tissue disease 
detection using a staged series design is very high, reducing misclassification of healthy 
subjects406. Similar procedures have been used in the Nurses’ Health Study for the validation of 
RA and SLE by self-report, with case confirmation rates of 69-70% of medical record 
reviewed386, 387. All participants who report autoimmune diseases that cannot be validated will be 
censored from analyses at the time of the ambiguous self-report. The primary autoimmune 
disease endpoint of VITAL will be total autoimmune disease incidence. We estimate that 1000 
or more new cases of autoimmune disease should occur during the five years of this trial in this 
elderly population, although we will not be able to capture them all. We anticipate some over-
reporting of incident autoimmune diseases, and may need to mail up to three separate medical 
record requests in some cases. We estimate a final confirmation of 60% of the true incident 
cases, or 600 cases. Individuals who report an autoimmune disease at baseline or in follow-up 
will not be excluded from analyses of other incident autoimmune diseases (e.g., an individual 
who reported pre-existing AITD will not be excluded from analyses of new autoimmune 
diseases).  
Follow-up blood collection, biochemical assays for circulating 25(OH)D, EPA and DHA 
are those of the parent VITAL trial, described in Partners’ IRB approved protocol 2009-P-00127; 
clinicaltrials.gov number 01169259.   
Biochemical assays for Inflammatory biomarkers: IL-6, TNFreceptor2 (TNFR2) and 
hsCRP assays. (Children’s Hospital, Boston, MA, Nader Rifai, PhD) IL-6 will be measured by 
an ultra-sensitive quantitative sandwich enzyme immunoassay from R&D Systems. IL-6 is 
quantified in pg/ml and assay sensitivity is 0.94 pg/mL. IL-6 is a strong predictor of 
cardiovascular disease and type 2 diabetes23, 412, 413, suggesting that a single sample represents 
long-term levels. TNF cannot be reliably measured in stored plasma as it degrades rapidly.  
TNFR2 expression parallels TNF levels and is a surrogate marker for inflammation414, 415. 
TNFR2 will be measured by a quantitative sandwich ELISA assay from R & D Systems. Day-to-
day assay variabilities at concentrations of 54.8, 252 and 356 pg/mL are 8.8, 3.7 and 5.8%, 
respectively.  TNFR2 stability was assessed in 17 fresh blood samples, at baseline, and after a 
delay of 24 hours and 36 hours, and the CV was 0.8 for the comparison of 0 to 36 hours 
(personal communication, Nader Rifai). High sensitivity CRP will be measured by high 
sensitivity latex-enhanced immunonephelometric assay on a BNII analyzer (Dade Behring, 
Newark, Delaware), with a coefficient of variation of < 5%. CRP levels by this technique, in 
mg/L, are predictive of cardiovascular disease and type 2 diabetes in cohort studies23, 416. 
Knee Pain Assessments Among those who screen positive for chronic knee pain (knee pain 
with walking 2-3 blocks, pain > 5 days per week for > 1 year, and a doctor’s diagnosis of knee 
OA and no history of total knee replacement) during the run-in phase, baseline knee pain 
symptoms will be assessed prior to randomization and receipt of any study medications. 
Thereafter, knee pain symptoms will be assessed annually during the same season (month) in 
this chronic knee pain subcohort of 2000 participants. We will use the following validated, short 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
6 
instruments to assess knee pain, analgesic use and depression:  
a. Western Ontario McMaster Universities (WOMAC) Knee Osteoarthritis Index Developed 
in 1988, the WOMAC knee questionnaire is a multidimensional measure of pain, stiffness, and 
physical functional disability consisting of 24 items graded in a numerical rating scale ranging 
from 0 (“no symptoms”) to 10 (“extreme symptoms”)417, 418. It is the most widely used and 
accepted measure for monitoring419 the course of the disease or to determine the effectiveness 
of anti-rheumatic medications in many knee OA trials. As several studies pointed to redundancy 
in the WOMAC function scale68, 420, Whitehouse and colleagues have developed and tested a 
reduced WOMAC function scale that contains seven functional items421. This scale, when tested 
in 1578 total joint replacement patients, had a Cronbach’s alpha of 0.85 with the WOMAC, 
indicating reliability and responsiveness to change was even greater than that of the extended 
scale. We will utilize the reduced WOMAC function scale, along with the pain, stiffness, and 
overall WOMAC scales. 
b. Analgesic medications Participants will be asked to report which medications they are 
taking from a short list of typical medications taken for knee pain, how frequently and for how 
long they have taken them. Medication use will be categorized as follows: (0) none, (1) 
acetominophen/non-steroidal anti-inflammatory drugs (NSAIDs), (2) narcotics/adjunctive pain 
medications (such as anti-depressants and antiepileptics). A decrease in analgesic medication 
use (from higher to lower category) will be considered as a secondary outcome, and analgesic 
use will also be included as a covariate in other knee pain analyses.    
c. Mental Health Inventory-5 (MHI-5) from the Short- form (SF-36) Health Survey The SF-
36 is a widely-used and well-validated questionnaire that assesses eight domains of general 
health422-425. The SF-36 includes a subscale of five questions about mental health (the Mental 
Health Index-5,MHI-5)426. The MHI-5 is comprised of 5 questions and there are six possible 
responses to each question, scored between 1 and 6. Individuals’ scores thus range from 5 to 
30. This is then transformed into a variable from 0-100 using a standard linear transformation. 
This subscale will provide useful a covariate as mental health could potentially explain some of 
the outcome variance. The responses range from ‘‘never’’ to ‘‘most of the time’’ and will be 
scored following a standard protocol. The recommended cut-off for a mood disorder/depression 
is a score of 65 on the mental health questions (MHI-5). The SF-36v2, its most recent version, is 
copyrighted, and we have obtained permission to use it from the Medical Outcomes Trust and 
QualityMetric Incorporated.   
Procedures for the Validation of Deaths, Compliance and Data Management will be those 
of the parent VITAL trial, described in Partners’ IRB approved protocol 2009-P-00127; 
clinicaltrials.gov number 01169259.  
For studies involving treatment or diagnosis, provide information about standard of care at 
Partners (e.g., BWH, MGH) and indicate how the study procedures differ from standard care.  
Provide information on available alternative treatments, procedures, or methods of diagnosis.
 
This trial will not affect standard of care for any disease state. The medications tested will not be 
for treatment or diagnosis.  
 
Describe how risks to subjects are minimized, for example, by using procedures which are 
consistent with sound research design and which do not unnecessarily expose subjects to risk or 
by using procedures already being performed on the subject for diagnostic or treatment purposes.
 
Procedures to minimize risks including recruitment and informed consent, protection of medical 
records and personal health information, minimization of risks of the trial medications, DSMB 
structure and oversight, will be those of the parent VITAL trial, described in Partners’ IRB 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
7 
approved protocol 2009-P-00127; clinicaltrials.gov number 01169259. 
For this ancilllary study, investigations of autoimmune disease incidence (Aim 1) and 
biomarkers of systemic inflammation (Aim 2) will involve subjects already consented for 
involvement in the parent VITAL trial and the ancillary studies utilizing mailings a separate blood 
draw consent form (already approved for parent trial) to obtain follow-up blood samples. For 
self-reports of autoimmune disease, a letter will be mailed describing the study and ask for 
consent for review of medical records. Medical record will be requested from physicians and 
hospitals and reviewed for published criteria autoimmune diseases.  For the chronic knee pain 
investigations (Aim 3), a letter will be mailed to potential participants with the supplementary 
knee pain assessments, stating that they have reported chronic knee pain on the past 
questionnaire and requesting that they complete another annual questionnaire regarding their 
chronic knee pain symptoms. Return of these supplementary questionnaires will imply informed 
consent and subjects will also be given the option of stating "I am not interested", so that they 
are not followed further.  
 
Describe explicitly the methods for ensuring the safety of subjects.  Provide objective criteria for 
removing a subject from the study, for example, objective criteria for worsening disease/lack of 
improvement and/or unacceptable adverse events.  The inclusion of objective drop criteria is 
especially important in studies designed with placebo control groups.
Procedures for protection against risks will be those of the parent VITAL trial, described in 
Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov number 01169259. This 
ancillary study poses only the minimal risk of inadvertent disclosure of private health 
information. 
The potential risk of disclosure of confidential information is guarded against by maintaining 
completed questionnaires, including ancillary questionnaires and blood test results in locked 
files accessible by authorized personnel only. In these files, participants are identified only by 
study ID. Subject identifiers are stored in separate computer files with password protection, and 
results will be presented only in the aggregate. In addition, employees involved with the 
proposed study will be asked to sign a form agreeing not to disclose any information to which 
they might have access, regardless of their personal perception about its confidentiality. Each 
employee of the study with human subject contact participates in an institutional (Brigham and 
Women’s Hospital) human subjects’ educational program, which consists of reviewing 
regulatory and informational documents pertaining to human subjects’ research; passing a test 
on ethical principles and regulations governing human subjects research; and signing a 
statement of commitment to the protection of human subjects.  Finally, all such employees are 
required to participate in HIPAA training. Medical records will be reviewed by Brigham and 
Women’s physicians who have completed Human Subjects Certification. 
 
FORESEEABLE RISKS AND DISCOMFORTS 
Provide a brief description of any foreseeable risks and discomforts to subjects.  Include those 
related to drugs/devices/procedures being studied and/or administered/performed solely for 
research purposes.  In addition, include psychosocial risks, and risks related to privacy and 
confidentiality.  When applicable, describe risks to a developing fetus or nursing infant.
As above, there is the risk of inadvertent disclosure of private health information. The strict 
procedures we will follow to safeguard against this are outlined above.  
 
The foreseeable risks and discomforts of the parent VITAL trial are described in Partners’ IRB 
approved protocol 2009-P-00127; clinicaltrials.gov number 01169259. 
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
8 
EXPECTED BENEFITS 
Describe both the expected benefits to individual subjects participating in the research and the 
importance of the knowledge that may reasonably be expected to result from the study.  Provide 
a brief, realistic summary of potential benefits to subjects, for example, “It is hoped that the 
treatment will result in a partial reduction in tumor size in at least 25% of the enrolled subjects.”  
Indicate how the results of the study will benefit future patients with the disease/condition being 
studied and/or society, e.g., through increased knowledge of human physiology or behavior, 
improved safety, or technological advances. 
 
For the majority of participants, there will be few direct benefits from participating in this primary 
prevention study other than the awareness of being involved in a large endeavor to answer 
relevant and timely questions regarding the possible benefit of vitamin D and fish oil. During the 
trial itself, we will receive inquiries from the participants regarding both specific and general 
health concerns. We will respond to each of those questions, primarily directing participants to 
published sources or recommending that they see their local health provider who is familiar with 
their medical history.  
The potential benefits to society relate to the increasing use of both vitamin D and fish oil, with 
data not yet clearly indicating efficacy in the general population with respect to prevention of 
autoimmune disease, inflammation and knee pain. Such data will help guide individual 
decisions, clinical recommendations, and public health guidelines. 
 
EQUITABLE SELECTION OF SUBJECTS 
The risks and benefits of the research must be fairly distributed among the populations that stand 
to benefit from it.  No group of persons, for example, men, women, pregnant women, children, 
and minorities, should be categorically excluded from the research without a good scientific or 
ethical reason to do so.  Please provide the basis for concluding that the study population is 
representative of the population that stands to potentially benefit from this research.
 
The gender distribution of the 20,000 participants in VITAL will be 50% male and 50% female—
specifically, 10,000 men aged ≥60 and 10,000 women aged ≥65. Estimates of the race/ethnicity 
of the participants come from the pilot study, in which the source list of individuals to whom we 
were mailing was enhanced with names identified as possible minorities. Based on the pilot 
results, we will be able to identify approximately 40,000 willing and eligible participants to enter 
the run-in, with at least 25% underrepresented minorities; 20,000 of these will be randomized 
into the trial, with the same proportion minority. Specifically, of the 20,000 randomized 
participants, we anticipate the ethnic distribution to be 1400 (7.5%) Hispanic and 18,600 (93%) 
non-Hispanic; with regard to race, we anticipate 5000 (25%) African-American, 500 (2.5%) 
Asian, 400 (2%) American Indian, 80 (0.4%) Pacific Islander, and 14,020 (70.1%) white 
individuals. Note that ethnic and racial categories can overlap—e.g., participants can be 
Hispanic-white or Hispanic-black.  
 
 
When people who do not speak English are excluded from participation in the research, provide 
the scientific rationale for doing so.  Individuals who do not speak English should not be denied 
participation in research simply because it is inconvenient to translate the consent form in 
different languages and to have an interpreter present.
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
9 
Individuals who do not speak English will not be eligible for this trial as it will not be possible to 
translate informed consent forms and all study questionnaires and documents into other 
languages.  
 
For guidance, refer to the following Partners policy: 
          Obtaining and Documenting Informed Consent of Subjects who do not Speak English
          http://healthcare.partners.org/phsirb/nonengco.htm 
 
 
 
RECRUITMENT PROCEDURES 
Explain in detail the specific methodology that will be used to recruit subjects.  Specifically 
address how, when, where and by whom subjects will be identified and approached about 
participation.  Include any specific recruitment methods used to enhance recruitment of women 
and minorities.
 
Recruitment procedures will be those of the parent VITAL trial, described in Partners’ IRB 
approved protocol 2009-P-00127; clinicaltrials.gov number 01169259.  
 
Provide details of remuneration, when applicable.  Even when subjects may derive medical 
benefit from participation, it is often the case that extra hospital visits, meals at the hospital, 
parking fees or other inconveniences will result in additional out-of-pocket expenses related to 
study participation.  Investigators may wish to consider providing reimbursement for such 
expenses when funding is available
 
VITAL subjects will not receive remuneration for their participation in the trial. 
 
For guidance, refer to the following Partners policies: 
          Recruitment of Research Subjects 
          http://healthcare.partners.org/phsirb/recruit.htm
 
          Guidelines for Advertisements for Recruiting Subjects
          http://healthcare.partners.org/phsirb/advert.htm
 
          Remuneration for Research Subjects
          http://healthcare.partners.org/phsirb/remun.htm
 
 
CONSENT PROCEDURES 
Explain in detail how, when, where, and by whom consent is obtained, and the timing of consent 
(i.e., how long subjects will be given to consider participation).  For most studies involving more 
than minimal risk and all studies involving investigational drugs/devices, a licensed physician 
investigator must obtain informed consent.  When subjects are to be enrolled from among the 
investigators’ own patients, describe how the potential for coercion will be avoided.
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
10 
Subjects will be mailed an informed consent document along with a letter explaining the parent 
VITAL trial and a screening questionnaire. Return of the signed informed consent document by 
mail will constitute informed consent.   
 
NOTE: When subjects are unable to give consent due to age (minors) or impaired decision-
making capacity, complete the forms for Research Involving Children as Subjects of Research 
and/or Research Involving Individuals with Impaired Decision-making Capacity, available on 
the New Submissions page on the PHRC website: 
http://healthcare.partners.org/phsirb/newapp.htm#Newapp
 
For guidance, refer to the following Partners policy: 
          Informed Consent of Research Subjects
          http://healthcare.partners.org/phsirb/infcons.htm
 
 
 
DATA AND SAFETY MONITORING 
Describe the plan for monitoring the data to ensure the safety of subjects.  The plan should 
include a brief description of (1) the safety and/or efficacy data that will be reviewed; (2) the 
planned frequency of review; and (3) who will be responsible for this review and for determining 
whether the research should be altered or stopped.  Include a brief description of any stopping 
rules for the study, when appropriate.  Depending upon the risk, size and complexity of the 
study, the investigator, an expert group, an independent Data and Safety Monitoring Board 
(DSMB) or others might be assigned primary responsibility for this monitoring activity.        
 
NOTE: Regardless of data and safety monitoring plans by the sponsor or others, the principal 
investigator is ultimately responsible for protecting the rights, safety, and welfare of subjects 
under his/her care. 
 
 
 
Describe the plan to be followed by the Principal Investigator/study staff for review of adverse 
events experienced by subjects under his/her care, and when applicable, for review of sponsor 
safety reports and DSMB reports.  Describe the plan for reporting adverse events to the sponsor 
and the Partners’ IRB and, when applicable, for submitting sponsor safety reports and DSMB 
reports to the Partners’ IRBs.  When the investigator is also the sponsor of the IND/IDE, include 
the plan for reporting of adverse events to the FDA and, when applicable, to investigators at 
other sites.   
 
NOTE: In addition to the adverse event reporting requirements of the sponsor, the principal 
investigator must follow the Partners Human Research Committee guidelines for Adverse Event 
Reporting
 
The parent VITAL trial has established a DSMB for the parent trial and all ancillary studies. This 
is described in Partners’ IRB approved protocol 2009-P-00127; clinicaltrials.gov number 
01169259. 
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
11 
MONITORING AND QUALITY ASSURANCE 
Describe the plan to be followed by the principal investigator/study staff to monitor and assure 
the validity and integrity of the data and adherence to the IRB-approved protocol.  Specify who 
will be responsible for monitoring, and the planned frequency of monitoring.  For example, 
specify who will review the accuracy and completeness of case report form entries, source 
documents, and informed consent.   
 
NOTE: Regardless of monitoring plans by the sponsor or others, the principal investigator is 
ultimately responsible for ensuring that the study is conducted at his/her investigative site in 
accordance with the IRB-approved protocol, and applicable regulations and requirements of the 
IRB.
 
Dr. Costenbader, PI of this ancillary VITAL study, will be responsible for collection and 
monitoring of data, including those concerning any adverse events, for the outcomes of 
autoimmune disease, inflammatory biomarkers, and knee pain. She will work closely with the 
parent trial PIs, steering committee and DSMB. A panel of BWH physicians, including Dr. Erik 
Alexander, Dr Abrar Qureshi, Dr. Sonia Freidman, Dr. Elizabeth Karlson, Dr. Jeffrey Katz and 
Maura Iversen, PT, DrPH will be involved in validating outcomes. They will follow hospital and 
research privacy procedures strictly and will be required to pass required Partners research 
privacy courses ad certifications.    
The trial DSMB is also described in Partners’ IRB approved protocol 2009-P-00127; 
clinicaltrials.gov NUMBER01169259.  
 
 
For guidance, refer to the following Partners policies: 
          Data and Safety Monitoring Plans and Quality Assurance
          http://healthcare.partners.org/phsirb/datasafe.htm
 
          Adverse Event Reporting Guidelines
          http://healthcare.partners.org/phsirb/adverse_events.htm
 
 
 
PRIVACY AND CONFIDENTIALITY 
Describe methods used to protect the privacy of subjects and maintain confidentiality of data 
collected.  This typically includes such practices as substituting codes for names and/or medical 
record numbers; removing face sheets or other identifiers from completed 
surveys/questionnaires; proper disposal of printed computer data; limited access to study data; 
use of password-protected computer databases; training for research staff on the importance of 
confidentiality of data, and storing research records in a secure location.   
 
NOTE: Additional measures, such as obtaining a Certificate of Confidentiality, should be 
considered and are strongly encouraged when the research involves the collection of sensitive 
data, such as sexual, criminal or illegal behaviors.
 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
12 
The potential risk of disclosure of confidential information is guarded against by maintaining 
completed questionnaires, including ancillary questionnaires and blood test results in locked 
files accessible by authorized personnel only. In these files, participants are identified only by 
study ID. Subject identifiers are stored in separate computer files with password protection, and 
results will be presented only in the aggregate. In addition, employees involved with the 
proposed study will be asked to sign a form agreeing not to disclose any information to which 
they might have access, regardless of their personal perception about its confidentiality. Each 
employee of the study with human subject contact participates in an institutional (Brigham and 
Women’s Hospital) human subjects educational program, which consists of reviewing regulatory 
and informational documents pertaining to human subjects research; passing a test on ethical 
principles and regulations governing human subjects research; and signing a statement of 
commitment to the protection of human subjects.  Finally, all such employees are required to 
participate in HIPAA training. Medical records will be reviewed by Brigham and Women’s 
physicians who have completed Human Subjects Certification. 
 
SENDING SPECIMENS/DATA TO RESEARCH COLLABORATORS OUTSIDE 
PARTNERS 
Specimens or data collected by Partners investigators will be sent to research collaborators 
outside Partners, indicate to whom specimens/data will be sent, what information will be sent, 
and whether the specimens/data will contain identifiers that could be used by the outside 
collaborators to link the specimens/data to individual subjects.
 
Specimens and data from this study will not be shared with collaborators outside of Partners. 
 
Specifically address whether specimens/data will be stored at collaborating sites outside 
Partners for future use not described in the protocol.  Include whether subjects can withdraw 
their specimens/data, and how they would do so.  When appropriate, submit documentation of 
IRB approval from the recipient institution.
 
Not applicable.  
 
RECEIVING SPECIMENS/DATA FROM RESEARCH COLLABORATORS OUTSIDE 
PARTNERS 
When specimens or data collected by research collaborators outside Partners will be sent to 
Partners investigators, indicate from where the specimens/data will be obtained and whether the 
specimens/data will contain identifiers that could be used by Partners investigators to link the 
specimens/data to individual subjects.  When appropriate, submit documentation of IRB 
approval and a copy of the IRB-approved consent form from the institution where the 
specimens/data were collected.
 
 
Not applicable.  
 
----------------------------------------- 
Protocol Biostatistical Analyses  
Separate analyses will be conducted for the 3 endpoints and for the 2 nutritional supplements, 
vitamin D and omega-3 fatty acids. Analyses of treatment effects will be based on the intent-to-
treat principle. The first analysis will compare baseline characteristics by randomized treatment 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
13 
assignment to ensure that balance was achieved by the randomization. Characteristics to be 
examined include known risk factors for autoimmune diseases, systemic inflammation, and 
chronic knee pain including: age; gender; race/ethnicity; family history of autoimmune disease; 
BMI; smoking; alcohol use; physical activity; co-morbid medical conditions such as 
hypertension, hyperlipidemia, diabetes, and cardiovascular disease; and baseline vitamin D and 
omega-3 fatty acid levels as assessed by dietary questionnaire in all participants and blood 
assays in the case-cohort subsample. The large sample size, as well as successful balance of 
known potential confounders, will provide assurance that unmeasured or unknown potential 
confounders will also be equally distributed across randomized treatment groups. For 
autoimmune disease, we will estimate the intervention effects on the primary endpoint of total 
incident autoimmune disease. For inflammatory biomarkers, we will assess changes in linear 
biomarkers (log transformed if necessary). For chronic knee pain, the primary endpoint will be 
assessed by the WOMAC pain scale; secondary analyses will test for decreases in WOMAC 
stiffness, function and global scales, as well as analgesic use separately. Data analyses will be 
performed using SAS 9.2 (SAS Institute Inc., Cary, North Carolina).  
Autoimmune Disease Incidence. In this 2x2 factorial design, the primary aim is to compare 
the main effects of intention-to-treat with vitamin D and with fish oil upon autoimmune disease 
incidence. Initially, we will compare Kaplan-Meier curves to graphically display the survival 
functions across different treatment groups and a log-rank test to test the difference. We will use 
Cox proportional hazards models to assess the main and interaction effects of the treatment 
groups. The unadjusted risk ratios and their 95% CIs will be used to measure the strength of 
risk across different treatment groups. The proportional hazard assumption will be tested based 
on the smoothed plots of the scaled Schoenfeld residuals 386. To estimate the cumulative 
incidence function, a nonparametric estimate will be obtained using the Kaplan–Meier method. 
Given randomization and blinding of 20,000 individuals in this RCT, most important covariates 
will be equally distributed. But, in secondary analyses, we will assess the effects of controlling 
for age, sex and other possible strong predictors, such as family history of autoimmune disease; 
baseline levels of inflammatory biomarkers, vitamin D, EPA and DHA; dietary vitamin D, EPA 
and DHA intake; BMI and physical activity.  
Inflammatory Biomarkers. In a randomly selected subcohort of 2000 participants, blood will be 
collected at both baseline and follow-up and changes in levels of plasma biomarkers of 
inflammation will be investigated. As inflammatory biomarkers are associated with age, BMI, 
medication use and cigarette smoking, we will again ensure that baseline characteristics are 
balanced in the four treatment groups in this subcohort and will additionally adjust for them in 
our analyses. Inflammatory Biomarkers (hsCRP, IL-6, TNFR2) will be analyzed as continuous 
outcomes. Descriptive statistics such as the minimum, maximum, range, median, and mean are 
used to summarize the variable as well as detect outliers and missing values. Exploratory 
graphical techniques such as Boxplots, Histograms, Quantile-Quantile plots, and Stem and Leaf 
plots will be used to further examine the distribution of each biomarker. Natural log 
transformation may be necessary based on our past analyses105. Unadjusted treatment effects 
will be evaluated using two-way Analysis of Variance (ANOVA) or Kruskal–Wallis nonparametric 
test. Multiple linear regression will be used to test the association between the biomarkers (or 
transformed scale) and treatment groups, while adjusting for age and gender. Model 
assumptions and goodness of fit will be evaluated using applicable techniques387. In a 
secondary analysis, additional adjustment for the other potential predictors including baseline 
biomarkers will be performed.  
Chronic Knee Pain. In a separate subcohort of approximately 2000 individuals with baseline 
chronic daily knee pain and no history of total knee replacement surgery, we will evaluate 
changes in level of knee pain over the course of the RCT. The annual measure of WOMAC 
knee pain subscale will be the primary outcome. Angst and colleagues examined data from 122 
prospectively-followed individuals with knee OA in a comprehensive rehabilitation program to 
define the minimally clinically important difference (MCID) in WOMAC scores (range 0-10), for 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
14 
pain, stiffness and function388. Analgesic use will be secondary outcome, defined on a scale of 
0-2: 0 (none), 1 (NSAIDS or acetaminophen only) and 2 (narcotics). As chronic knee pain is 
associated with age, BMI, medication use, comorbidities and depression (MHI-5), we will again 
ensure that baseline characteristics are balanced in the four treatment groups in this knee pain 
subcohort and will additionally adjust for these potential confounders in our analyses. Similarly, 
we will analyze repeated measures of WOMAC knee pain score, stiffness score, function score 
and globe score as continuous outcomes. The descriptive statistics and the distribution of each 
outcome variable will firstly be examined. Transformation will be considered if applicable. We 
will use two-way analysis of variance (ANOVA) or Kruskal–Wallis test for bivariable analysis and 
general linear mixed model for multivariable repeated measure analysis. For another secondary 
outcome, we will assess the effects of the supplements upon analgesic use, classified as: no 
medications, acetaminophen or non-steroidal use; and narcotic medication). Generalized linear 
mixed models for repeated binary outcomes will be used to examine the adjusted associations.  
Statistical issues in resampling from a RCT. Our investigations of the effects of these agents 
on biomarkers of systemic inflammation and knee pain will not utilize the entire 20,000 
individuals enrolled in the funded VITAL trial. Rather, each will follow a selected subcohort of 
approximately 2000 individuals. It is possible that these individuals will not be drawn completely 
equally or randomly from the 4 treatment groups. Possible unbalance of sample characteristics 
will be evaluated and be controlled for in multivariable analyses.  
Secondary Analyses. Beyond the primary analyses, we will examine several pre-specified 
secondary outcomes for autoimmune diseases, inflammatory biomarkers, and chronic knee 
pain. We will examine effect modification by the other randomized intervention, by baseline risk 
factors, and by time. Given past studies showing interactions between intake of n-6 fatty acids 
and n-3 in reductions in biomarkers of systemic inflammation285, we have a particular interest in 
exploring interactions between the interventions, as well as with the baseline plasma biomarkers 
of 25(OH)D for vitamin D and of EPA+DHA for fish oil, and dietary intake of both. We 
hypothesize that the intervention effects may be larger among those with below-median 
baseline levels and will examine treatment effects by quartiles of these biomarkers. When 
stratifying by 25(OH)D, season (assessed by date of return) and geographic location (assessed 
by zip code) will be considered. We also have a prior interest in the effects of the vitamin D 
intervention within groups defined by race/ethnicity and skin pigmentation, and by BMI. In 
addition, we will evaluate effect modification by age and gender, as well as by sunlight 
exposure, calcium and phosphorus intakes from the FFQ (as these nutrients affect vitamin D 
bioavailability1), and baseline risk factors for autoimmune disease, systemic inflammation and 
chronic knee pain. Interaction effects will be interpreted cautiously, as hypothesis-generating. 
Finally, we will examine whether treatment effects vary over time and duration of treatment by 
examining survival plots and interactions with time. There may be latent effects on outcomes, 
depending on the stage at which these agents act. 
Analysis of potential adverse effects. We will also compare the incidence of potential side 
effects in the active vs. placebo groups for each agent, including the incidence of kidney stones 
with vitamin D assignment389 and the incidence of GI symptoms, skin abnormalities, and 
bleeding with fish oil assignment.329 
Power calculations. This section provides a detailed power analysis for each of the 3 aims. To 
calculate 
power, the following assumptions were made: (1) a 2x2 factorial trial in 10,000 men aged >60 
and 10,000 women aged >65; (2) independent and equal allocation of participants to each 
treatment in the trial (for Aim 1); (3) an age distribution based on that observed at baseline in 
our past RCTs for men aged ≥60 and women aged ≥65; (4) trial follow-up of 5 years, with little 
loss to follow-up as achieved in our past RCTs; and (5) compliance of 80% similar to that in 
published trials upon which our estimated rate ratio (RR) reductions are based. Power 
calculations were performed using the observed RR, given past observed effects and assuming 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
15 
a compliance of 80%. The corresponding true relative risk (RR) with perfect compliance is also 
calculated.  
a. Incident autoimmune diseases. As in table 1, we estimate the annual incidence of total 
autoimmune diseases in a U.S. population over age 60 to be approximately 1000 cases per 
100,000 individuals per year: thus, in this trial of 20,000 individuals for 5 years, 1000 incident 
cases.(Table 2) Conservatively, we will validate 60% of these 1000 cases, or 600 cases, with 
150 occurring in the placebo group and fewer in the intervention groups if they are effective. 
Given the clustering of autoimmune diseases within individuals, some incident cases will occur 
within the same individuals, and these will be analyzed as discrete endpoints. Power is given for 
a a logrank test390 based on the proportional 
hazard rates with a significance level of 
0.05.(Table 4) Assuming that only one agent is 
effective, with 5 years of treatment and follow-
up, we will have 81% power to detect an 
observed RR of 0.70 (or 30% risk reduction) for 
the primary endpoint of autoimmune disease 
incidence. For the secondary endpoint of AITD, 
we will have 85% power to detect a 40% risk reduction. If both agents are effective in preventing 
autoimmune disease but act independently, power would be reduced slightly due to a somewhat 
smaller overall number of events. If the agents interact, however, power will be affected to the 
extent of the interaction. Should the agents act synergistically, power would increase. For 
example, if the effect of each agent alone is a reduction of 15%, but in combination the effect is 
stronger, with an additional 10% decrease, the RR comparing the combined group to the all 
placebo group would be 0.65, versus 0.72 with additive effects (on the multiplicative scale). 
Power for the total effects would increase to 91%. Power is shown for a given observed 
reduction in risk, assuming 80% compliance similar to that seen in trials such as WHS and PHS.  
b. Inflammatory biomarkers. Our power calculations are based on past changes in systemic 
inflammatory biomarkers in large cohort analyses and we have first assumed that only one 
agent is effective, that compliance is 80% and that the cohort of 2000 followed over two years 
for changes in biomarkers is randomly drawn from each of the four treatment groups. (Table 5) 
Based on the distribution of hsCRP, IL-6 and TNFR2, natural log-transformation may be used 
for power analysis if applicable. According to NHANES 1999-2002  
 data, median hsCRP level is 2.8 mg/L for men 
aged ≥60 and women aged ≥65391. Using a two-
sided type I error of 0.05, the sample size of 
500 patients with compliance of 80% in each 
group will achieve 89% power to detect a 25% 
reduction of hsCRP using a two-sided t test 
(based on log-transformed scale). Pai and 
colleagues examined plasma levels of TNFR2 and IL-6 as biomarkers for cardiovascular 
disease risk among participants in the Nurses’ Health Study and the Health Professional Follow-
up Study (mean age 60.3 for women, 65.2 for men)135. The mean TNFR2 was 2370 pg/ml (SD 
795 pg/ml) and the median IL-6 level was 1.6 pg/mL. A sample size of 500 per group with 
compliance of 80% will achieve more than 99% power to detect 20%-40% reduction of TNFR2 
with a two-sided significance level of 0.05. Based on a log-transformed scale, we will have 88% 
power to detect a 30% reduction in IL-6. 
c. Chronic knee pain. In 
the study by Angst and 
colleagues, the mean global 
score was 4.8 and the MCID 
for improvement in pain was 
0.75, for stiffness 0.72, for 
function 0.67 and for global 
Table 5. Statistical power for effects of a single agent on  
inflammatory biomarkers in a subcohort of 2000 individuals
Observed reduction %
hsCRP 
IL-6 
TNFR2 
                  20 
0.72 
0.55 
>0.99 
                  25 
0.89 
0.74 
>0.99 
                  30 
0.97 
0.88 
>0.99 
                  40 
0.99 
0.99 
>0.99 
†Assuming compliance to be 80%.  
Table 6. Statistical power for effects of a single agent on chronic knee pain in a 
factorial trial of 2000 (men aged ≥60 women aged ≥65), with 5 years of follow-up 
(80% compliance rate) 
 
Baseline Score 
SD 
MCID 
Power 
WOMAC knee pain 
4.83 
2.25 
0.75 
0.99 
WOMAC knee stiffness 
4.61 
2.67 
0.72 
0.96 
WOMAC knee function 
4.81 
2.18 
0.67 
0.99 
WOMAC Global 
4.80 
2.09 
0.67 
0.99 
SD= standard deviation per Angst, 2002 MCID= minimally clinically important difference 
Table 4. Statistical power for effects of a single agent on 
autoimmune disease in a factorial trial of 10,000 men aged ≥60 and 
10,000 women aged≥65 with 5 years of follow-up 
Observed RR† 
True RR 
Autoimmune Disease 
AITD 
0.70 
0.67 
0.81 
0.62 
0.65 
0.61 
0.91 
0.75 
0.60 
0.56 
0.97 
0.85 
†Observed RR=intent-to-treat RR assuming compliance 80%.  
True RR= with perfect compliance. AITD= autoimmune thyroid disease 

Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
16 
score 0.67 388. Assuming a type I error rate of 0.05 and a power of 0.80, this translated into 
sample sizes of 142, 216, 162, and 153 individuals respectively affording adequate statistical 
power to detect these changes in an RCT of an intervention using the WOMAC as an outcome. 
We will follow 2000 individuals with chronic daily knee pain longitudinally over up to 5 years. 
With estimated loss to follow-up and compliance of 80%, we will have generous statistical power 
to detect the MCID in improvement in WOMAC pain, stiffness, function and global  
scores. (Table 6) Moreover, repeated measurement of WOMAC scores will provide additional 
power to detect specified effect sizes. Again, unequal group sample sizes could reduce the 
power to some extent (although is unlikely to occur unless there is a strong chance imbalance). 
However, if we conservatively assume the intervention to placebo allocation ratio is 1:2, we will 
still achieve similar power to detect 25% reduction in both inflammatory biomarkers and the 
WOMAC MCID of knee pain. (The power to detect MCID for WOMAC knee pain would be > 
99%, for WOMAC knee stiffness 95%, and for WOMAC knee function 98%.)