The Effects of Diet and Lifestyle on Quality of Life and Methylation-related Biomarkers in Vivo.
Phase: NA
Conditions: Lifestyle, Diet, Healthy
Sponsor: National University of Natural Medicine

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
 
 
 
 
 
 
 
 
The effects of diet and lifestyle on quality of life and 
methylation-related biomarkers in vivo. 
 
Date: February 9th, 2018 
IRB Number: RB100217 
Version: 1.1 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
1 
 
PROTOCOL 
  
Study Title:  The effects of diet and lifestyle on quality of life and methylation-related 
biomarkers in vivo. 
 
Co-Principal Investigators: 
Ryan Bradley, ND, MPH  
(503) 552-1862   
 
 
 
 
Kara Fitzgerald, ND 
 
(203) 304-9502 
 
 
Clinical Investigator:  
Andy Erlandsen, ND  
(503) 552-1794 
  
 
 
 
  
Study Coordinators:  
Emily Stack 
 
 
(503) 552-1777 
 
 
 
 
 
Trina Soileau  
 
(503) 552-1746 
 
 
 
 
 
Site Monitor:  
 
Kim Koch, CMA 
 
(253) 853-7291 
 
Biostatistician: 
 
Doug Hanes, PhD 
 
(503) 552-1741 
 
Study Staff:     
 
Romilly Hodges, MS, CNS 
(203) 304-9502 xt 106 
Study  Coaches: 
 
Josette  Herdell 
 
(619) 718-1928 
 
 
 
 
Melissa Twedt  
 
(610) 324-9398 
 
 
 
 
Sally Logan 
 
 
(510) 409-1550 
 
 
 
 
Janine Henkel  
 
(206) 697-0568 
 
 
 
 
Sean Foss  
 
 
(203) 304-9502 x 113 
 
 
 
 
Despina Giannopoulou 
+44 07825427793 
 
Sponsor:  Metagenics, Inc. 
 
A. ABSTRACT  
The maintenance of health and the progression of disease are associated with an individual’s 
genetic make-up and environmental factors, including lifestyle choices (such as diet, exercise, 
behaviors, stressors, sleep, tobacco and alcohol use), environmental exposures and 
socioeconomic determinants.  Environmental factors have been shown to influence, sometimes 
rapidly, epigenetic processes thereby influencing genetic expression.1  Regulation of the human 
genome by the epigenome is now regarded as a cornerstone, heritable, physiologic process, 
playing a key role in phenotypic expression of health and disease.  
DNA methylation is a well-researched, primary epigenetic process.2  Aberrant DNA methylation 
resulting in hyper- or hypomethylated regions of the genome, generally results in inhibition or 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
2 
expression of certain genes and has been associated with the pathogenesis of numerous 
conditions, ranging from inflammation and accelerated aging, to cancer, autoimmunity, 
diabetes, heart disease, dementia, allergic disease, posttraumatic stress disease and others.3–6  
Likewise, certain healthy diet and lifestyle habits have been demonstrated to favorably 
influence DNA methylation patterns.7,8 
Understanding that environmental factors can potently and sometimes rapidly, favorably or 
negatively influence epigenetic expression, a short-term diet and lifestyle intervention may 
significantly augment DNA methylation expression.  
The purpose of this study is to evaluate a 9-week diet and lifestyle intervention on patient-
reported quality of life, symptoms, and DNA and biochemical methylation-related biomarkers in 
healthy males ages 50-72.  
 
B. SPECIFIC AIMS  
 
B.1 Primary Aim:  
To evaluate a 9-week diet and lifestyle intervention on patient-reported quality of life. 
 
Hypothesis: Quality of life will improve as a result a 9-week diet and lifestyle 
intervention, relative to a wait-list control condition. 
 
Outcome measure:  Changes in patient-reported quality of life, measured by the 
PROMIS-29 (Patient-Reported Outcomes Measurement Information System-29) 
questionnaire. 
 
B.2 Secondary Aim:  
To evaluate changes in patient-reported symptoms after a 9-week diet and lifestyle 
intervention.  
 
Hypothesis: Severity of patient-reported symptoms will decrease following a 9-week 
diet and lifestyle intervention.  
 
Outcome measures: Changes in patient-reported symptoms, measured by the MYMOP 
(Measure Yourself Medical Outcome Profile) questionnaire, NUNM Multi-system side 
effects/symptoms questionnaire, and the Medical Symptom Questionnaire (MSQ).  
 
 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
3 
B.3 Tertiary Aim:   
To assess changes in methylation at CpG sites of DNA after a 9-week diet and lifestyle 
intervention.  
 
Hypothesis: Methylation at CpG sites associated with chronic inflammation will change 
after the 9-week diet and lifestyle intervention. 
 
Outcome measure: Number of CpG methylation sites, measured by the Infinium 
MethylationEPIC Kit (Illumina, Inc.)  
 
B.4 Exploratory Aims: 
To evaluate changes in biomarkers associated with methylation. 
 
Outcome measures: Changes in the methylation related biomarkers as measured by 
Doctor’s Data Methylation Index Panel, Methylation Profile, and Folate Vitamer Panel.  
 
C. BACKGROUND AND SIGNIFICANCE  
 
C.1. Methylation background and significance 
 
Methylation, happening in every cell, virtually continuously, signifies the enzymatic addition of a 
single carbon compound on a substrate and regulates gene expression, protein function, and RNA 
processing.9  Folate vitamer, 5-methyltetrahydrofolate (5-MTHF) acts as key substrate within 
these pathways by transferring a methyl group to cobalamin by the enzyme 
methylenetetrahydrofolate reductase (MTHFR).  This new compound, methylcobalamin, is a 
coenzyme used by methionine synthase and transfers a methyl group to homocysteine, creating 
methionine.  Methionine undergoes a structural transformation under the presence adenosine 
triphosphate (ATP) to become S-adenosyl methionine (SAMe).  SAMe, the universal methyl 
donor, is essential for over a 100 methylation reactions by transferring a methyl group to DNA, 
proteins, phospholipids, amino acids and neurotransmitters.   
 
Research into the epigenome demonstrates that DNA methylation is a cornerstone regulator of 
gene expression. Genetic methylation is reliant on SAMe availability. Myriad environmental 
factors, as outlined above, also influence genetic methylation either directly or through altered 
SAMe availability.     
 
Imbalances in methylation pathways due to single nucleotide polymorphisms (SNPs) is a rapidly 
emerging area of investigation implicated in numerous disease processes.10 While genetic 
polymorphisms alter enzyme function, it may increase risk of certain diseases.10–12  Investigation 
into a single nucleotide polymorphism of MTHFR has gained popularity.  Individuals with a point 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
4 
mutation on MTHFR gene, result in higher plasma levels of homocysteine, a risk factor associated 
with atherosclerosis and coronary artery diseases.13,14  Hyperhomocysteinemia is significantly 
inversely correlated with DNA hypomethylation and exacerbated by folate deficiency.15   
 
C.2. Diet and lifestyle intervention as a means to support proper methylation  
 
Inadequate diet and physical inactivity are indicated as two of the top 5 modifiable risk factors 
in several chronic diseases as well as associated with all-cause mortality.16  A diet lacking 
adequate intake of essential nutrients and antioxidants, as well as lack of exercise increase 
biomarkers associated with oxidative stress, endothelial dysfunction, and DNA 
hypomethylation.17,18  Oxidative overload places stress on cellular metabolic function, and is 
correlated with an increased risk of chronic diseases, such as cancer, cardiovascular and 
neurodegenerative diseases.19–21   
 
With regard to the epigenome, poor diet has been associated with accelerated aging as 
evidenced by Horvath’s Epigenetic Clock looking at DNA methylation patterns.8  Oxidative stress 
has also been shown to limit methyl donor availability, thereby impacting DNA methylation, by 
promoting transsulfuration and glutathione synthesis while inhibiting the methylation cycle.  
Oxidative damage to the epigenome further contributes to aberrant DNA methylation 
patterns.22  
 
A nutrient-rich diet and regular, moderate, aerobic exercise reduces oxidative stress, and risk of 
disease, by increasing antioxidants and activity of antioxidant enzymes, while also altering gene 
specific DNA methylation levels.7,23,24   Phytochemicals in various plant foods have 
demonstrated the ability to modulate DNA methylation.  These include quercetin, found in red 
onion and apple skins, green tea, and red grapes; epigallocatechin-3-O-gallate (EGCG), found 
highest in green and white tea; genistein, found in soybeans; apigenin, found in parsley, 
artichoke, and celery; and curcumin, found in turmeric.25  In addition, regular physical activity 
has shown to induce epigenetic changes in DNA methylation and lower homocysteine 
levels.26,27 
 
To date, this would be the first known clinical trial investigating diet and lifestyle intervention 
on patient-reported quality of life, symptoms, and DNA and biochemical methylation-related 
biomarkers in healthy males ages 50-72. 
 
 
D. RESEARCH DESIGN AND METHODS STUDY FLOW  
 
 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
5 
D.1. Overview and Design 
 
This study is a preliminary randomized control trial evaluating a 9-week diet and lifestyle 
intervention on quality of life, patient-reported symptoms, and biomarkers associated with 
methylation.  We will recruit forty-eight participants with 24 randomized to each study group.  
Refer to Figure 1 for an overview of the study flow. 
 
Figure 1. Study Flow Overview  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
D.2 Study Intervention 
 
Study participants will be randomized into an intervention or control group.  The intervention 
group will undergo a 3-week washout period following the discontinuation of any non-
medically required nutrition or herbal supplements.  The intervention group will follow detailed 
diet and lifestyle recommendations over a 9-week period.  Two webinars at the start of the 
Randomization 
Control Group 
Intervention group 
Labs 
Infinium 
Methylation 
EPIC Index,  
Methylation 
Profile,  
Folate 
Vitamer 
panel  
 
Consent and Initial Screening Visit 
Telephone Screening 
DNA Methylation Pathway - SNP 
Visit 1 (week 1) 
Visit 2 (week 5) 
Visit 3 (week 9) 
Weekly: Phone contact (health 
coach) Weeks 1-4. 
Semi-weekly: Phone contact  
(health coach) Weeks 5-8 
Daily: Phytoganix, Ultraflora, 
MBody 360 
Measures 
Multisystem 
AE, MSQ, 
 MYMOP, 
 PROMIS-29, 
Lifestyle habits,  
Height, weight, 
 waist-hip ratio,  
blood pressure, 
VioScreen, FFQ 
Washout (3 week) 
Electronic technology webinar 
Introduction Webinar (week 1) 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
6 
intervention phase will provide information to participants on proper food and exercise 
choices, as well as how to access and use the required electronic resources (apps). 
 
The control group will undergo the same testing measures as the intervention group, but will 
not have access to the education information or be instructed to change diet or lifestyle factors.  
They will have access to the information after the study is complete.  
 
D.2.1 Diet Recommendations 
Specific dietary guidelines will be outlined for participants and should include the following:  
Per week 
Per day 
3 servings of liver  
 
(1 serving = 3 oz) 
 
Choose preferably organic 
liver 
5-10 eggs  
 
Ideally free-range, organic, 
omega-3 enriched  
 
2 cups of dark leafy greens  
 
Measured raw, chopped, and packed 
 
Including kale, Swiss chard, collards, spinach, 
dandelion, mustard greens 
 
Does not include salad greens such as romaine, iceberg, 
Spring mix 
2 cups cruciferous vegetables   
 
Measured raw, chopped, and packed 
 
Including broccoli, cabbage, cauliflower, Brussels 
sprouts, bok choy, arugula, kale, watercress, rutabaga, 
kohlrabi, radish, turnip 
3 cups colorful, low glycemic vegetables of your choosing 
1-2 medium beet  
4 tbsp (1/4 cup) pumpkin seeds (or pumpkin seed butter)   
4 tbsp (1/4 cup) sunflower seeds (or sunflower seed butter) 
1+ serving methylation adaptogens: 
 
1/2 cup berries (wild preferred) 
 
1/2 tsp rosemary 
 
1/2 tsp turmeric 
 
2 medium cloves garlic 
 
2 cups green tea (brewed 10 minutes) 
 
3 cups oolong tea (brewed 10 minutes) 
6 oz animal protein 
 
grass-fed, pastured, organic and hormone/antibiotic-
fee 
2 servings of low glycemic fruit 
 
General 
Choose organic at least half of the 
time. 
Stay hydrated  
Don’t eat between 7pm and 7am, 
and at least 2 hours before bed 
Include healthy oils  
 
Balance types of fat 
 
E.g. coconut, olive, flaxseed 
and pumpkin seed oil 
Avoid dairy, grains, legumes/beans 
Minimize plastic food containers 
 
These recommendations serve as a guidance, while individual intake may vary based on 
preference.  Throughout the study, participants will be provided 2-week menu plans with 
recipes for all meals, snacks, smoothies. 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
7 
Refer to Appendix A for detailed nutrition recommendations. 
D.2.2 Exercise Recommendations 
Participants will be encouraged to exercise a minimum of 30 minutes at least 5 days per week 
at an intensity of 60-80% of maximum perceived exertion.  Possible types of physical activity 
include, but not limited to, brisk walking, light jogging, hiking, dancing, yardwork, bicycling, and 
swimming.   
 
D.2.3 Stress Management Recommendations 
Participants will utilize the Cleveland Clinic’s Stress Free Now application to engage in a variety 
of guided stress reduction techniques, including meditation and mindful breathing.  
Recommended frequency is twice daily, preferably morning and evening. 
 
D.2.4. Lifestyle Recommendations 
Participants are encouraged to average a minimum of 7 hours of sleep per night. 
 
D.2.5. Study Supplements 
Participants will be provided with two supplements.  Details on the products are outlined in 
section D.12 of the protocol.  Participants will take 2 servings of PhytoGanix and 2 capsules of 
UltraFlora Intensive Care probiotic daily, in divided doses.   
 
D.2.6. Control group 
The control group will be waitlisted during the study intervention phase.  They will be provided 
with a copy of the intervention webinars and educational handouts after the study is 
concluded.   
 
D.2.7. Tools to measure study variables and assist with adherence: 
 
D.2.7.1. Mbody360 app  
 
MBODY360 is a HIPAA-Compliant Web-based complete health & wellness coaching platform 
that connects practitioners to their patients and clients in real-time and tracks patient’s 
engagement & compliance.  It includes secure video, text chatting, and group coaching tools.   
Participants will track their sleep, supplement usage, exercise, stress management.  Participant 
progress will be monitored daily by health coaches using the Mbody 360 app. 
Participants will be prompted to answer the following questions each day: 
 Did you eat according to program guidance? 
 What did you eat that was not according to the program? 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
8 
 Did you complete at least 30 minutes of moderate intensity physical activity today? 
 Did you complete the stress management program today?  For how long? 
 
How many hours of sleep did you get last night?  
 Did you take any new medications, prescription or over-the-counter, today? 
o *If yes, which new medication did you take? 
 Did you take your study probiotic supplement today?  
 Did you take your Phytoganix supplement today?  
 
D.2.7.2. VioScreen 
VioScreen is a validated food frequency questionnaire software program, which tracks food 
intake over the past 90 days.  It will take approximately 20 minutes for participants to input 
their dietary information.  Developed by the Nutrition Coordinating Center at the University of 
Minnesota, it utilizes a standardized food and nutrient database.   All participants will complete 
this at Visits 1, 2 and 3. 
 
D.2.7.3. Stress Free Now 
Stress Free Now by the Cleveland Clinic is an application that seven different relaxation 
techniques, including mindfulness and meditation.  Each technique lasts approximately 10 
minutes. 
 
D.2.7.4. Zoom 
A web-based communication program, Zoom will be utilized for group webinars and Q&A 
sessions throughout the study. 
 
D.2.7.5. Study Specific Food Frequency Questionnaire 
Not all foods recommended and consumed during the study intervention may be captured by 
VioScreen.  An additional food frequency questionnaire (FFQ), with approximately 250 foods 
and beverages, was created to assess consumption.  It has been designed to be consistent with 
the validated block Brief FFQ by NutritionQuest.  This will be administered electronically via 
RedCap online database to all study participants at Visits 1, 2, and 3.   
 
D.2.7.5. Adherence phone calls 
Participants will be contacted via phone once per week during the first 4 weeks of the study 
and every other week for the remainder of the study.   Designated health coaches will assess 
for adherence to the study intervention including dietary, stress management, and exercise 
recommendations.  Participants will be able to ask questions pertaining to the study 
intervention.   
 
Refer to Appendix B for detailed script of these phone calls. 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
9 
 
D.3 Description of Participant Population 
 
D.3.1. Participant Characteristics 
Sample size: n=48  
We will enroll and randomize up to 48 people which will allow for 20% attrition such that we 
have an evaluated sample size of 40 to allow for withdraws or dropouts.  
Age: 50-72 
Gender: Males only 
Minorities: all racial and ethnic backgrounds will be recruited from the general population 
without preference or discrimination. 
Vulnerable Populations (e.g. children, pregnant women, cognitively impaired): No vulnerable 
populations will be enrolled. 
 
D.3.2. Inclusion and Exclusion Criteria 
Individuals must meet the following participation criteria: 
 
Inclusion criteria: 
 Males, ages 50-72  
 Willing to adhere to 9 weeks of a dietary and lifestyle program including specific 
nutrition and exercise guidelines 
 Willing to avoid any over-the-counter medications, supplements or herbal products for 
the length of the study, except short-term use (<1 week) use at least 1 week before 
scheduled study visits  
 Willing to have blood drawn three times and abstain from food or beverage intake for 
10-12 hours before blood draws 
 Willing to provide saliva samples 
 Willing to track food intake, sleep, stress management techniques, and exercise daily 
 Willing to drink a nutrient-enriched beverage and take a encapsulated probiotic daily 
 Willing and able to use electronic devices and connect to the internet  
 Able to speak, read and understand English 
 
Exclusion criteria: 
 Currently taking any of the following prescription medications  
o Proton pump inhibitors: omeprazole (Prilosec, Prilosec OTC); aspirin and 
omeprazole (Yosprala); lansoprazole (Prevacid, Prevacid IV, Prevacid 24-Hour); 
;dexlansoprazole (Dexilent, Dexilent Solutab); rabeprazole (Aciphex, Aciphex 
Sprinkle); pantoprazole (Protonix) 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
10 
o H2-blockers: nizatidine (Axid, Axid AR, Axid Pulvules); famotidine (Pepcid, Pepcid 
AC); cimetidine (Tagamet, Tagamet HB); ranitidine (Zantac) 
o These classes of medications are excluded due to direct (due to nutrient 
requirements for metabolism) and indirect (through impaired digestion and 
assimilation of nutrients). 
 Use of nutrition supplements or herbal products not prescribed by a licensed healthcare 
provider for a medical condition 
 Currently following a prescribed dietary/lifestyle program or initiate within the 30 days 
prior to baseline 
 Initiation of or changes to an exercise regimen within 30 days prior to baseline 
 Use of nicotine, marijuana or cannabinoids (including CBD products) or recreational 
drugs/substances (such as but not limited to cocaine, phencyclidine [PCP], and 
methamphetamine) current/within the last 30 days or use during the study  
 Have a diagnosis of cardiovascular disease, kidney disease, liver disease, diabetes, 
autoimmune disease, high blood pressure, or cancer (does not include basal cell 
carcinoma, squamous cell carcinoma, and/or carcinoma in situ of the cervix). 
 Have a diagnosis of an immunodeficiency condition, such as Human Immunodeficiency 
Virus (HIV) infection or Acquired Immune Deficiency Syndrome (AIDS) 
 Have a diagnosis of neurodegenerative conditions such as, amyotrophic lateral sclerosis 
(ALS), Parkinson’s disease, Multiple Sclerosis, or Alzheimer’s disease. 
 Excessive alcohol consumption (more than 4 drinks per day or 14 per week on average) 
 Known sensitivity, intolerance or allergy to ingredients in the study supplements or in 
the recommended dietary therapy 
 Currently receiving intravenous nutrient therapy  
 Currently participating in another interventional research study or participated in 
another interventional study within the last 3 weeks prior to baseline 
 
D.4 Outcome Measures 
Table 1 below provides a summary of study outcomes, associated measures, and a brief 
description. More detailed descriptions of each measure are provided below Table 1. 
 
Table 1. 
 
OUTCOME 
MEASURE 
DESCRIPTION 
Primary Outcome 
Health-related 
Quality of Life 
Patient Reported Outcomes 
Measurement Information 
(PROMIS-29) 
PROMIS-29 is a validated, 29 question 
survey divided into seven sub-domains of 
function including physical functioning, 
social function, pain interference, pain 
intensity, sleep, depression, and anxiety. 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
11 
Secondary Outcome 
 
Change(s) in 
patient-reported 
symptoms 
Measure Yourself Medical 
Outcome Profile (MYMOP) 
MYMOP is a patient-centered outcome 
measure that allows for the participant to 
self-select their top health concern(s) or 
symptom(s) they are dealing with in their 
own words. The participant can also 
choose an activity that is limited by that 
health concern. 
NUNM Multi-system AE 
questionnaire 
The NUNM Multi-system 
Symptom/Adverse Event Questionnaire is 
a standardized ninety-one point 
monitoring form that requires asking 
questions pertaining to the following 
organ systems:  eyes/ears/nose/throat, 
gastrointestinal, neurological/ 
musculoskeletal, psychological/general, 
cardiopulmonary, skin, genitourinary and 
whole body systems. 
Tertiary Outcome 
Methylation status 
of CpG sites 
Infinium Methylation EPIC 
Index by Illumina 
Epigenetic profile that assesses 
methylation on over 850,000 CpG DNA 
sites 
Exploratory Outcomes 
Changes in the 
methylation 
related biomarkers 
Methylation Profile by 
Doctor’s Data 
Functional assessment of the phenotypic 
expression of common SNPs and provides 
a "methylation index," a ratio of SAM to 
SAH. 
Folate Vitamer Panel by 
Doctor’s Data 
Measures folate congeners affecting 
enzyme function in methylation pathways 
including unmodified Folic Acid (UMFA), 
5-Methyltetrahydrofolate (5-MTHF), 
tetrahydrofolate (THF), folinic Acid (5-
CHO-THF) 
Change(s) in 
patient-reported 
symptoms 
Medical Symptoms 
Questionnaire (MSQ) 
The MSQ is a 71-question measure that 
measures various health areas including: 
Digestive tract, Eyes, Energy/Activity, 
Emotions, Head, Heart, Joint/Muscles, 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
12 
Lungs, Mind, Mouth/Throat, Nose, Skin, 
Weight, and Other. 
Measurement of 
Potential 
Confounders 
DNA Methylation Pathway 
Profile 
Assesses changes to SNPs - DNA genotype 
which may affect enzymatic function 
which include, methionine metabolism, 
detoxification, hormones, and vitamin D.    
 
D.4.1. Primary Outcome: Patient-reported Quality of Life 
Patient Reported Outcomes Measurement Information - PROMIS – 29 Profile 
The Patient Reported Outcomes Measurement Information System (PROMIS) is a collection of 
patient-reported health status inventories funded by the NIH.  PROMIS provides a standardized, 
reliable, and valid measure of health status that assesses physical, mental, and social well–
being from the patient perspective. The PROMIS-29 Profile is a collection of self-report short 
forms containing items from 7 PROMIS domains (Physical Function, Anxiety, Depression, 
Fatigue, Sleep Disturbance, Ability to Participate in Social Roles and Activities, Pain Interference, 
and Pain Intensity).  The PROMIS-29 Profile consists of 4 questions per domain rated on a 5-
point rating scale along with a 1 question Pain Intensity section rated on an 11-point scale.  The 
domains are assessed over the past 7 days except for the Physical Function domain which has 
no specified time frame. 
 
PROMIS-29 
Table 2. Grading of PROMIS-29 
Score 
Severity  
29-52 
None-minimal 
51-75 
Mild 
76-100 
Moderate 
101-125 
Moderately severe 
126-150 
Severe 
 
PROMIS-29 provides a continuous symptom severity score (7 categories).  Total raw scores range 
from 29 to 150.  A raw score is created from each subscale (except Pain Intensity) that makes up 
the Profile except the Pain Intensity item.  Scores are translated into T-scores which are 
referenced to mean score levels in the general U.S. population.  Lower PROMIS scores have been 
shown to predict reduced health related quality of life for multiple conditions. 
 
D.4.2. Secondary Outcome: Patient-reported Symptom Change 
 
D.4.2.1. Primary Measure: Measure Yourself Medical Outcome Profile (MYMOP) 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
13 
Measure Yourself Medical Outcome Profile (MYMOP) is a patient centered outcome measure 
that allows for the participant to self-select their top health concern(s) or symptom(s) they are 
dealing with in their own words. The participant can also choose an activity that is limited by 
that health concern. The participant is then asked about their own general wellbeing. Each of 
these questions is scored on a 0-6 scale. The medications that the participant is taking related 
to the specific symptoms and their desire to cut down the medication is also recorded.  
 
Scoring of the MYMOP is an average of all of the profile questions answered. Participants only 
have to choose one symptom and answer the well-being profile question. If a second related 
symptom and activity level are identified all of the scores are included in the average final 
score. 
 
MYMOP has been shown to be an effective outcome measurement for health concerns, 
especially in a diverse population and has more sensitivity to change compared to the SF-
36.29,30   
 
D.4.2.2. Secondary Measure: NUNM Multi-system Symptom/Adverse Events Questionnaire 
The NUNM Multi-system Symptom/Adverse Event Questionnaire is a standardized ninety-one 
point monitoring form that requires asking questions pertaining to the following organ systems:  
eyes/ears/nose/throat, gastrointestinal, neurological/ musculoskeletal, psychological/general, 
cardiopulmonary, skin, genitourinary and whole body systems. 
  
Table 3. Grading of Items on the NCNM Adverse Event Monitoring Form 
Grade 
Definition 
0=Not Present 
No AE, Function within normal limits 
1=Mild; asymptomatic or mild symptoms; 
clinical or diagnostic observations only; no 
intervention 
No intervention; asymptomatic lab or 
radiographic findings; marginal clinical 
significance 
2=Moderate; minimal, local or noninvasive 
intervention indicated 
AE limited activities of daily living (ADLS) 
3=Severe or medically significant but not 
immediately life-threatening 
AE significantly limited basic self-care, AE 
required initial hospitalization or 
prolongation of hospitalization 
4=Life-threatening 
Life-threatening consequences AE 
5=Fatal 
Fatal AE 
 
Changes in a current symptom(s) by a grade of 2 or higher, or a new symptom(s) graded 2 or 
higher will be included in analysis to compare symptom changes between groups.  
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
14 
D.4.3. Tertiary Outcome: Changes in methylation at CpG sites of DNA 
The tertiary outcome will be the assessment of methylation changes at “CpG” sites on DNA 
using a commercial available assay kit. CpG sites are sites on DNA in which a cysteine base is 
separated from a guanine base by only one phosphate; CpG sites are known sites for regulation 
by methylation. The Infinium MethylationEPIC Kit by Illumina uses the latest technology to 
assess methylation status of over 850,000 DNA sites across the genome.  Using DNA provided 
from a saliva sample, this assay measured changes in methylation at “CpG” sites of DNA, i.e., 
whether or not sites for methylation are, or are not, methylated. For a given time point, the 
degree of methylation at each site (i.e., β) is presented as a color ranging from “green” (i.e., β 
=0, low methylation) to “red” (i.e., β =1, high methylation). We will compare the methylation 
state of participants at baseline to their status at the end of the study.  “Differential” 
methylation is considered a change in CpG site methylation of Δβ ≥0.66. See Figure 2 below for 
a typical representation of the results of the assay.28 
 
Figure 2: Typical Demonstration of Results Applying the Infinium MethylationEPIC Kit 
 
 
A saliva sample will collect approximately 2 mL (~1 teaspoon) into a cryovial. Saliva samples will 
be labeled only with a study ID, date of collection, and visit number and frozen to -70 °C.  
 
At the conclusion of the study, all samples will be batched shipped to: Yale University Center for 
Genome Analysis Attn: Guilin Wang, PhD, Associate Director of Microarray; 300 Heffernan Dr. 
B-36, West Haven, CT 06516.  
 
D.4.4. Exploratory Outcomes: Changes in biomarkers associated with methylation 
 
D.4.4.1. Methylation Profile – Doctor’s Data 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
15 
Methionine is an important amino acid involved in the methylation of DNA, proteins, and 
neurotransmitters.  Impaired methionine metabolism may be caused by genetic changes, 
nutritional deficiencies, aging and environmental toxins and associated with various health 
conditions such as cardiovascular disease.  This test measures various intermediates in 
methionine metabolism, including cystathionine, cysteine, homocysteine, methionine, S-
Adenosylhomocysteine (SAH), S-Adenosylmethionine (SAM), SAM/SAH ratio, and adenosine.  
Abnormal levels may indicate single nucleotide polymorphisms to specific enzymes. 
 
A venous blood draw will collect 6 mL of blood (~1 tablespoon) into an EDTA tube (purple top).   
 
 
D.4.4.2. Folate Vitamer Panel – Doctor’s Data 
Folate exists in several different chemical forms which are most commonly involved in one 
carbon metabolism.    Abnormal levels of folate vitamers may indicate single nucleotide 
polymorphisms affecting enzyme function in methylation pathways.  This test measures folate 
congeners including unmodified Folic Acid (UMFA), 5-Methyltetrahydrofolate (5-MTHF), 
tetrahydrofolate (THF), folinic Acid (5-CHO-THF). 
 
A venous blood draw will collect 6 mL of blood (~1 tablespoon) into an EDTA tube (purple top).   
 
D.4.4.3. Tertiary Measure: Medical Symptoms Questionnaire (MSQ) 
The MSQ is a 71 question survey that measures various health areas including: Digestive tract, 
Eyes, Energy/Activity, Emotions, Head, Heart, Joint/Muscles, Lungs, Mind, Mouth/Throat, Nose, 
Skin, Weight, and Other.  Each question is measured on a 5 point rating scale ranging from 
Never or almost never have symptom to frequently have it and effect is severe.  Scoring: 
Optimal scoring is less than 10, Mild Toxicity: 10-50, Moderate Toxicity: 50-100, Severe Toxicity: 
over 100. 
 
This instrument is a non-validated tool utilized in the functional medicine community to assess 
health.  The current study may have the potential to validate the MSQ by comparing results to 
the PROMIS-29 and NUNM Multi-system Symptom/Adverse Event Questionnaire. 
 
D.4.5. Measurement of Potential Confounders 
 
DNA Methylation Pathway Profile – Doctor’s Data 
This test assesses changes to DNA genotype, called single nucleotide polymorphisms (SNPs), 
which may affect enzymatic function.  Potential biochemical pathways impacted include, 
methionine metabolism, detoxification, hormones, and vitamin D.   This is a blood spot test and 
requires collection of 5 drops of blood after a finger stick. 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
16 
D.5 Participant Schedule 
 
D.5.1 Overview 
Individuals will be screened over the phone and at one in-person visit.  Eligible participants who 
choose to enroll in the study will attend a total of 4 in-person visits.  Participants will be 
randomized into either the intervention or control group after the screening visit.  All study 
visits will be conducted at the Helfgott Research Institute in Portland, OR.  Refer to Table 4 for 
an outline of the study visits. 
 
Table 4: Study Procedures 
 
Phone 
Screening 
Screening 
Visit 
Study 
Visit 1 
(Week 1) 
Study 
Visit 2 
(Week 5) 
Study 
Visit 3 
(Week 9) 
Eligibility screening 
X 
 
 
 
 
Consent for study 
participation 
 
X 
 
 
 
Eligibility verification  
 
X 
 
 
 
Demographic form 
 
X 
 
 
 
W-9 
 
X 
 
 
 
Anthropometrics/Vitals: 
height, weight, waist-hip 
ratio, heart rate, blood 
pressure, temperature, 
respiratory rate 
 
X 
X 
X 
X 
Questionnaires: 
 
NUNM Multi-system 
Symptom/Adverse Event 
 
MSQ 
 
MYMOP 
 
PROMIS-29 
 
Lifestyle Habits 
 
VioScreen 
 
Study specific FFQ 
 
 
X 
X 
X 
Blood Draw, blood spot card 
and saliva sample 
 
 
X 
X 
X 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
17 
 
D.5.2. Recruitment  
Recruitment will be conducted in the general community surrounding Portland, OR using flyers, 
online, newspaper, and electronic newsletters.   
 
D.5.3. Telephone Screening 
Potential participants will be screened over the phone prior to coming in for the first visit. A 
standardized telephone script will be used. The telephone screening call will take approximately 
10-15 minutes. Participants who meet eligibility criteria over the phone will be scheduled for a 
screening visit.  The consent form will be provided to participants prior to the screening visit via 
email; they will be instructed to review the form ahead of time but not to sign it until the 
screening visit where they can ask questions about the study.  If an individual is eligible for the 
study, enrollment will happen on a rolling basis. 
 
D.5.4. Screening Visit 
All study visits will take place at the Helfgott Research Institute in Portland, OR. 
The objective of the Screening Visit is to explain the study procedures, secure informed 
consent, and verify eligibility for the trial.  Eligibility will be verified by reviewing responses to 
the eligibility questionnaire.  The screening visit is estimated to take 45 minutes. 
 
The following will be performed/obtained at screening: 
 Signed and dated informed consent 
 Eligibility Questionnaire 
 Verification that the participant meets the inclusion and exclusion criteria; 
 W-9 – once determined eligible 
 Demographics (including age, gender, ethnicity, and race); 
 Anthropometrics and vitals (including height, weight, pulse rate, blood pressure); 
 
DNA Methylation Pathway 
Profile - SNP Panel 
 
 
X 
 
 
Infinium Methylation EPIC 
Index  
 
 
X 
 
X 
Methylation Profile 
 
 
X 
X 
X 
Folate Vitamer Panel 
 
 
X 
X 
X 
Estimated Time to Complete 
15 
minutes 
45 
minutes 
90 
minutes 
90 
minutes 
90 
minutes 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
18 
Sample half size servings of the study supplement drink will be provided while participants are 
completing the eligibility questionnaire. Upon sampling the shakes, participants will be asked if 
they are willing to take the product twice per day for the study period.  
 
 
D.5.5. Informed Consent 
Consent will be obtained from participants at the beginning of the screening visit. The consent 
form will include the purpose of the study, a description of the procedures, a list of risks and 
benefits, an assurance of confidentiality, an assurance of withdrawal without prejudice, a 
statement about payment policies for research related injuries, study incentives, and a telephone 
number for answering questions about the research, all explained at an 8th grade reading level. 
Participants will also be given information on their right to privacy for personal health 
information based on HIPAA regulations. Procedures for data sharing using de-identified data will 
be explained later in this protocol. 
 
D.5.6. Randomization 
Once a participant is deemed eligible and provided signed consent, the study staff will 
determine randomization order using a random number generator (randomization.com). After 
enrollment, the study coordinator will select the next sequential envelope from the locked filing 
cabinet within the study site office. The study staff will write the current date and participant 
initials on the bottom right corner of the envelope and open the envelope and retrieve its 
contents. The envelope contents will show which cohort the participant is assigned to, with 
intervention or control.  This will be assigned to the participant ID number and entered into 
REDCap.  
 
D.5.7. Washout Period 
Study participants assigned to the intervention group will be asked to refrain from taking any 
over-the-counter supplements, medications or herbal products, except short-term use (<1 
week) and at least 1 week before scheduled study visits, alcohol, nicotine, marijuana or 
cannabinoids (including CBD products) or recreational drugs/substances for 3 weeks before the 
baseline visit.  Written instructions will be provided. 
 
D.5.8. Study Visits  
There will be 3 in-person study visits for all participants during the 9-week study. 
 
Baseline/Study Visit 1 (Week 1) 
The following procedures, evaluation and/or assessments will be obtained at Study Visit 1:  
 Height and weight 
 Vital signs including heart rate, blood pressure, respiration rate and temperature   

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
19 
 Questionnaires including Lifestyle Habits, PROMIS-29, MYMOP, MSQ, and NUNM 
Adverse Events, VioScreen 
 Blood draw to collect 2 tubes of blood 
 Finger stick to collect 5 drops of blood 
 Saliva sample 
 If in intervention group, will receive study supplements for first 4 weeks and instructions 
on how and when to take 
 Case report form completed by study staff 
 Waist-to-hip ratio will be calculated using the following methods: 
o Participant with stand up straight and breathe out.  Use a tape measure to check 
the distance around the smallest part of the waist, just above the umbilicus. This 
is the waist circumference. 
o Measure the distance around the largest part of the hips — the widest part of 
the buttocks. This is the hip circumference. 
o Calculate the waist-to-hip ratio by dividing the waist circumference by the hip 
circumference. 
 
Study Visit 2 (Week 5) 
The following procedures, evaluation and/or assessments will be obtained at Study Visit 2:  
 Weight 
 Vital signs including heart rate, blood pressure, respiration rate and temperature  
 Questionnaires including Lifestyle Habits, PROMIS-29, MYMOP, MSQ, and NUNM 
Adverse Events, VioScreen 
 Blood draw to collect 2 tubes of blood 
 Saliva sample 
 If in intervention group, will receive study supplements for remaining 4 weeks of study 
 Case report form completed by study staff 
 
Study Visit 3 (Week 9) 
The following procedures, evaluation and/or assessments will be obtained at Study Visit 3:  
 Weight  
 Waist-to-hip ratio will be calculated as previous defined in Study Visit 1 
 Vital signs including heart rate, blood pressure, respiration rate and temperature.   
 Questionnaires including Lifestyle Habits, PROMIS-29, MYMOP, MSQ, and NUNM 
Adverse Events, VioScreen 
 Blood draw to collect 2 tubes of blood 
 Saliva sample 
 If in intervention group, return unused supplements 
 Case report form completed by study staff  

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
20 
 
D.5.9. Educational Webinars 
Weekly, live webinars will provide information on the diet, exercise, stress management, and 
lifestyle recommendations.  Prior to initiating any of these changes, an introductory webinar 
will provide an overview of the study intervention.  An additional webinar will provide detailed 
instruction on how to access and utilize two electronic applications – MBody360 and Stress 
Free Now.  All webinars will be delivered via Zoom, a free online communication application. 
 
Refer to Appendix C for detailed information on webinar content 
 
D.6. Study Withdrawal and/or Early Termination 
Participants will be informed that they have the right to withdraw from the study at any time, 
without affecting their relationship with NUNM. Participants may also be discontinued from the 
study if the investigators determine it is in the best interest of the participant.  
 
Information about study withdrawal or premature discontinuation from the study will be 
captured throughout the study. Study withdrawal or premature discontinuation will be 
categorized as follows: 
 Participant began taking an excluded medication or supplement 
 The Principal/Clinical Investigator decided that withdrawal/discontinuation was in the 
best interest of the participant due to an adverse event, non-compliance, or another 
reason 
 The participant requests withdrawal from the study for reasons other than above 
 The participant failed to return for study visits 
 
If a participant withdraws before the second visit, they will be replaced, however participants 
who withdraw between the second and third visits will not be replaced. Every effort will be 
made to collect a final salivary sample and all final labs from all participants, even if they 
withdraw from the study early. 
 
D.7. Study Products 
 
D.7.1. PhytoGanix (tropical fruit flavor)  
Organic Superfruits & Greens Powder Drink Mix is a food-based phytonutrient blend designed 
to support intestinal health and immune function. 
 
 
 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
21 
Serving Size: 1 Scoop (10.4 grams) 
 
 
 
Ingredient 
Amount per serving 
Calories  
40  
Total Carbohydrate  
9 g  
Dietary Fiber  
1 g  
Sugars  
1 g  
Protein  
1 g  
Vitamin C (as ascorbic acid)  
100 mg  
Calcium  
20 mg  
Iron  
1.4 mg  
Sodium  
30 mg  
Organic Seed and Prebiotic Blend:  
5.53 g  
Isomalto-Oligosaccharide, Flax Seed Powder, Chia Seed Powder, Quinoa 
(Chenopodium quinoa Willd.) Sprout Powder  
  
Organic Veggie Blend:  
2.35 g  
Spirulina Whole Algae Powder, Carrot Root Powder, Broccoli Head 
Powder, Cauliflower Head Powder, Spinach Leaf Powder, Parsley Leaf 
Powder, Bamboo Shoot Extract, Beet Root Powder  
  
Organic Fruit Blend:  
1.2 g  
Apple Fruit Powder, Pineapple Fruit Powder, Strawberry Fruit Powder, 
Raspberry Fruit Powder, Blackberry Fruit Powder, Blueberry Fruit Powder  
  
Herbal Support Blend:  
300 mg  
Milk Thistle Seed Extract, Turmeric Rhizome Extract, Green Tea Leaf 
Extract, Reishi Mushroom Whole Plant Powder, Maitake Mushroom 
Whole Plant Powder, Shiitake Mushroom Whole Plant Powder, Japanese 
Knotweed Root Extract, Quercetin Dihydrate, Organic Maca Root Powder, 
Organic Cassia Bark Powder, Dong Quai Root Powder, Ginger Root 
Powder, Garlic Bulb Powder  
  
Enzyme Blend (including protease, amylase, lipase, and cellulase)  
200 mg  
Lactobacillus acidophilus VK3 Strain  
200 mg  
 
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
22 
 
 
D.7.2. UltraFlora Intensive Care  
 
Probiotic designed to help maintain healthy flora and bowel function during dietary changes, 
promote the integrity of the GI intestinal barrier, and support a healthy immune response. 
 
Ingredient 
Amount per serving 
Lactobacillus plantarum 299v Strain 
20 billion live Strain organisms††  
 
Ingredients: Lactobacillus plantarum 299v Strain, Potato starch, capsule 
(hydroxypropylmethylcellulose), Lactobacillus plantarum 299v (soy), and magnesium stearate 
(vegetable). 
 
D.7.2. Product Descriptions 
PhytoGanix contains organic fruit, vegetables, chia, quinoa sprouts, herbs, plant enzymes, 
prebiotics and probiotics.  PhytoGanix Intensive will be provided and packed by Metagenics, 
Inc. in individual serving packets. It should be stored at room temperature in a cool, dry place.  
 
UltraFlora Intensive Care contains the L. plantarum 299v strain of probiotics to help relieve 
intestinal irritation and bowel discomfort, promote the integrity of the GI intestinal barrier, and 
support a healthy immune response. UltraFlora Intensive Care will be provided and packed by 
Metagenics, Inc. in glass jars containing 30 capsules. It should be stored at room temperature in 
a cool, dry place or keep refrigerated.  
 
The study products will be labeled with the following information: the product name, the study 
title, the IRB #, dosing information, the phone # of the Study Coordinator and Clinical 
Investigator. 
 
D.7.3. Product Dispensing 
Participants assigned to the intervention group will be given ~60 packets of PhytoGanix 
(adequate to provide 2 servings per day until Visit 2) and 2 bottles of UltraFlora Intensive Care 
at Study Visit 1. They will be instructed to wait to take the supplements until after the 
Introduction Webinar during the first week of the study.  This will cover the approximately 56 
dosages needed until Visit 2, plus extra dosages to allow for any loss of product.  Participants 
will be provided with written instructions on taking the study product. At Visit 2, participants 
will return any unused study product in exchange for 2 additional bottles of each study product 
to cover the approximately 56 doses needed from Visit 2 until Visit 3. At Visit 3, participants will 
return any unused study product. 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
23 
 
D.7.4. Product Administration 
Participants will be instructed to blend, shake, or briskly stir 1 level scoops of PhytoGanix into 8 
ounces of water or juice two times daily.  Participants will be instructed to take 2 capsules of 
UltraFlora intensive Care with 8 ounces of water once a day. 
 
D.7.5. Product Adherence 
Participants will be contacted by phone weekly Weeks 1-4 and semi-weekly Weeks 5-8 by the 
health coach team to check in on their progress.  If participants report issues with product 
adherence product, the health coach team will discuss strategies with the participant to 
increase adherence. (For example, they may suggest posting note on the refrigerator or keeping 
the study product jar in a location such as a kitchen counter or dining table in order to 
incorporate taking the study product into daily routines.) 
 
Adherence will be quantified by the study investigators who will count unused capsules and 
weigh unused study product after it is returned at the 5 week and 9 week study visits. 
 
D.8. Blood and Saliva Collection and Analysis 
Fasting blood samples and saliva samples will be obtained at the baseline, 5 week and 9 week 
visits. All study participants will be assigned a unique study ID number, and all blood samples, 
saliva samples, and laboratory paperwork will be identified with that number.   
 
Blood collection will consist of a venous blood draw into of approximately 12 mL of blood ~2 
tablespoons) into two EDTA (purple top) tubes and one finger stick collecting 5 drops of blood 
for a blood spot test. 
 
These samples will be picked up by courier on the same day they are collected and transported 
to Doctor’s Data, Inc for analysis.  
 
Saliva sample collection will include approximately 2 mL collected from each participant. 
Participants will have abstained from food or beverages, except water, overnight prior to the 
collection.  Participants will simply spit into a screw topped cryovial labeled with their study ID, 
date of collection, study time point, and approximate volume. If necessary, participants will be 
allowed up to 8 oz., of water in order to provide the sample, waiting at least 15 minutes 
between water intake and the sample collection. If necessary, cotton buccal cushions/swabs 
will be provided for participants to chew on to induce salivation.  
 
 
E. HUMAN SUBJECTS RESEARCH  
 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
24 
E.1. Potential Risks 
 
E.1.1. Blood Draws 
There will be three blood draws during this study. The main risks associated with drawing blood 
are infection, bruising, redness, pain, discomfort, or bleeding at the needle puncture site. 
Occasionally, individuals may feel lightheaded or faint during or immediately after a blood 
sample is collected. There is also a rare risk of swelling around the vein. 
 
E.1.2. Study Product Ingestion 
Most of the ingredients are essential macronutrients and micronutrients. Risks associated with 
any dietary supplement include gastrointestinal symptoms such as gas, bloating, digestive upset 
and change in bowel movement frequency or consistency.  Individuals with known sensitivities, 
intolerances, and allergies to any of the product ingredients will be excluded from the study. 
 
 
The product ingredients are approved for human use by the FDA under GRAS (Generally 
Recognized as Safe) status and are frequently consumed in commonly eaten foods, spices, 
flavorings, and beverages. The study products are described in sections D.7.1. and D.7.2.   
 
Lactobacillus species are usually well-tolerated when taken orally.  Lactobacillis actobacillus 
acidophilus and the Lactobacillus plantarum 299v strain have been used safely in studies lasting 
from one week to six months. 
 
E.1.3. Confidentiality 
There is a small risk that information about a study participant could be inadvertently disclosed 
to non-study personnel. 
 
E.2. Protection Against Risks 
 
E.2.1. Blood Draws 
Study protocol and procedures to minimize risks associated with blood draws are in place. 
Blood samples will be obtained by certified phlebotomists, or licensed physicians in medical 
facilities, which includes procedures and staff to handle medical emergencies. 
 
E.2.2. Study Product Ingestion  
Prospective participants will be screened for eligibility to minimize potential contraindications 
to the study product as well as determine general health status.  Adverse event monitoring will 
occur throughout the study. 
 
E.2.3. Confidentiality 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
25 
Throughout the study, measures to ensure the privacy of information on study participants will 
be maintained.  Details of Confidentiality and Data security are outlined in section G.1. 
 
Participants will be told about any new information that may affect their willingness to 
participate in the study. 
 
E.3. Potential Benefit 
Participants may not receive a direct benefit from their participation in this research. 
Participants may experience improved quality of life through adherence to the lifestyle 
recommendations included in the proposed intervention, however their composite effects are 
unknown. Participants may experience improved cardiovascular health through adherence to 
the physical activity and stress management recommendations in the proposed intervention.  
 
F. Importance of Knowledge to be Gained 
The proposed research will benefit society by producing proof-of-concept data on the efficacy 
of diet and lifestyle factors to alter epigenetic signaling after a short-term intervention. 
Demonstrating alterations in methylation sites in pathways with known associations with 
disease risk may lead to improvements in disease prevention initiatives and interventions 
toward the improved health of the public.  
 
G. DATA COLLECTION, SOURCES AND ANALYSIS 
 
G.1. Confidentiality and Data Security 
Throughout the study, measures to ensure the privacy of information on study participants will 
be maintained. All study investigators and staff have been certified in human subjects research 
and have received training in HIPAA regulations. Participants and staff will be informed of the 
confidentiality of information and assured that data will be used only for statistical purposes 
and group analyses in which the individual cannot be identified. No data beyond what is stated 
in the Informed Consent will be sought without authorization from the participant. Information 
on illnesses and hospitalizations will not be sought from hospitals or doctors without a signed 
medical release from the subject. Conversely, no information on any individual will be released 
to anyone other than study personnel without a signed medical release from the participant, or 
where appropriate, the next of kin or a physician in case of a life threatening emergency to the 
subject. All study personnel will be instructed not to discuss any participants with persons other 
than study personnel. 
 
Each participant will be assigned a unique alpha-numeric ID upon screening. All blood tubes, 
and associated paperwork will be marked using the unique ID to protect patient confidentiality. 
All data resulting from study visits will be collected on standardized case report forms (CRFs). 
Data will be transferred from CRFs to a secure REDCap database for data management. REDCap 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
26 
is a secure, web-based application that supports electronic data capture for research 
studies. REDCap features include: 1) intuitive data entry features; 2) audit trails for tracking 
data manipulation and export; 3) user-based privileges that support HIPAA compliance; 4) 
seamless data export to common statistical packages; and 5) procedures for importing data 
from external sources. The REDCap database will be accessible only by the study team. Data will 
be destroyed 3 years after completion of the study.  Computer files will be deleted from the 
server and paper files will be destroyed using a professional document shredding service.   
 
All completed forms will be kept in locked files to which only project personnel have access. 
These files will also be in locked rooms. Data from paper forms will be manually entered into 
REDCap and kept as electronic files. Electronic files will be password protected with access 
given to study personnel only. All analyses will be conducted by study staff at the Helfgott 
Research Institute.  The study biostatistician will only access de-identified data exported from 
REDCap to Excel or SPSS. Only de-identified data will be presented in any reports, presentations 
or manuscripts. 
 
G.2. Informed Consent 
Consent will be obtained from the participants for the study protocol. The Consent Form will 
include the purpose of the study, a description of the procedures, a list of risks and benefits, an 
assurance of confidentiality, an assurance of withdrawal without prejudice, study incentives, 
and telephone numbers for answering questions about the research. Participants will also be 
given information on their right to privacy for personal health information based on HIPAA 
regulations. Procedures for data sharing using de-identified data will be explained. 
 
 
  
G.3. Adverse Event Monitoring  
If an Adverse event (AE) is reported at Study Visit 1 or Study Visit 2, the Study Coordinator will 
contact the CI and/or the PI as soon as possible, i.e., during the appointment, for AEs graded as 
“moderate” or more severe (Grade 3 or higher). If the Study Coordinator is unable to reach the 
CI/PI within the appointment, the participant will be contacted by the PI as soon as possible 
within the same day. The Study Coordinator and CI will complete the “Standardized Adverse 
Events Questionnaire: Spontaneous Reporting” form (Appendix A) in order to capture details 
regarding the adverse event and any action taken.  
 
If a participant calls the Study Coordinator or the Principal/Clinical Investigator to report AEs, a 
similar procedure will be followed, which will include completion of the “Standardized Adverse 
Events Questionnaire: Spontaneous Reporting” form. 
 
G.3.1. Risk Classification: Low  
All procedures are considered “low risk” as per the NUNM Helfgott Research Institute 
Procedure/Intervention Risk Classification list. These low-risk procedures include administering 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
27 
questionnaires, and venipuncture/blood draws on non-pregnant adults. Participants will be told 
about any new information that may affect their willingness to participate in the study.  
 
G.3.2. Reporting to the NUNM Institutional Review Board 
All adverse events will be recorded in the Adverse Event Log.  Anticipated adverse events will 
be reported to IRB during continuing review. Unanticipated problems will be reported to IRB 
within 10 working days of the investigators being notified of the event. Serious adverse events 
will be reported to IRB within 24 hours of the investigators being notified of the event. 
 
G.3.3. Adverse Event Definitions 
Adverse Event (AE):  Any untoward medical occurrence in a clinical investigation subject. An AE 
can therefore be an unfavorable and unintended sign (including abnormal laboratory findings), 
symptoms (nausea and loose stools), or disease temporally associated with the study, whether 
or not related to the study procedure, or a worsening of a pre-existing condition or illness. 
 
Serious Adverse Event (SAE):  Any untoward medical occurrence that results in death, is life-
threatening, requires inpatient hospitalization or prolongation of existing hospitalization, 
results in persistent or significant disability or incapacity, or results in a congenital anomaly or 
birth defect. Other important medical events requiring medical or surgical intervention to 
prevent a serious outcome would also be considered SAEs. 
 
Unanticipated Problems (UP):  Any incident, experience, or outcome that meets all of the 
following criteria: 1) unexpected (in terms of nature, severity, or frequency) given (a) the 
research procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the characteristics of the 
subject population being studied; 2) related or possibly related to participation in the research 
and 3) suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
G.4. Statistical Analysis 
 
PROMIS 29 T-scores will be presented as mean (SD), for each group at each of the three data 
collection time points.  For the primary aim, we will compare changes in PROMIS-29 T-scores 
between groups, using a linear mixed model with random intercept to test for a Group*Visit 
interaction.   If there is a significant main effect, we will also present uncorrected contrasts 
comparing changes from baseline to Week 5, and from baseline to Week 9, between groups. 
 
Although PROMIS T-scores are not particularly susceptible to extreme outliers, data will be 
examined for any outlying PROMIS scores or changes in PROMIS scores.  If these scores are 
deemed to be in error, then they will be treated as missing in all analyses.  Otherwise, scores 

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
28 
will be retained in the main analysis; but we will conduct separate sensitivity analyses to test 
for effects of outlying data on results. 
 
Mixed models incorporate all available data, without excluding any participants from analysis.  
Nevertheless, any participants who have baseline data only will be excluded from analysis.  As 
an exploratory follow-up analysis, we will re-analyze changes from Week 1 to Week 9, using 
data from completers only. 
 
For the secondary aim, the total score of the MSQ will be analyzed similarly to PROMIS-29.  
Since MYMOP asks for two symptoms without distinction, we will classify each problem as 
either the Primary problem (the problem with the highest symptom rating at baseline; or, in the 
case of a tie, the first problem listed) or the Secondary problem.  We will also calculate the 
average of the two problem scores, at each collection time point.  If only one problem is listed, 
then this will be used as both the Primary and Average, for that individual, with Secondary 
score missing.  Changes in MYMOP severity scores will be compared between groups using a 
non-parametric Mann-Whitney test.  Separate comparisons will be made between changes 
over the first 4 weeks of the study and changes over the entire 9-week intervention period. 
 
 
G.5 SPONSOR RESPONSIBILITIES FOR INTERVENTIONAL PRODUCT(S) 
 
The sponsor, Metagenics, is responsible for supplying the interventional product(s) in 
accordance with Good Manufacturing Practices (GMP). These products will be fully 
characterized, properly coded and suitably packaged in a way to provide protection against 
deterioration with appropriate labelling.  Sufficient samples of each batch and a record of 
analyses and characteristics will be kept by the sponsor for reference so that, if necessary, an 
independent laboratory is able to re-check the interventional product(s), e.g. for quality control 
or bioequivalence. Records of the quantities of interventional products supplied will be 
maintained with batch or serial numbers by sponsor. The sponsor will ensure that the 
investigator is able to establish a system within their institution for adequate and safe handling, 
storage, use during the course of the study and, if appropriate, return or destruction of the 
interventional product(s) upon completion of the study.  
 
G.6 SPONSOR MONITORING 
 
The sponsor, Metagenics, will appoint suitable and appropriately trained monitor(s) and clinical 
research support personnel to conduct regular site visits starting with recruitment and 
continuing on a schedule that is mutually agreed upon by the study coordinator and sponsor. 
Follow up information from these visits will be provided to site, sponsor and to Principal 
Investigators in a timely manner for their reference purposes.  

  
 
Protocol  
Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
29 
 
Sponsor will ensure to provide ongoing training to their study personnel so that they are 
suitably qualified and to keep them up to date with new developments in clinical research. 
 
H. Incentives 
Participants will earn $50.00 for each week they participate in the study, up to $450 if they 
participate for the full 9 weeks.  Checks will be mailed after visit 2 (week 5) and visit 3 (week 9), 
compensated for each week they participated. 
 
 
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Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
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Study Title: Methylation Diet and Lifestyle (MDL) Study 
PI: Ryan Bradley, ND, MPH and Kara Fitzgerald, ND 
IRB #: RB100217 
Approval Date: 2.9.18 
Ver. 1.1 
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