Effects of Choline From Eggs vs. Supplements on the Generation of TMAO in Humans (EGGS)
Phase: NA
Conditions: Cardiovascular Risk Factor
Sponsor: The Cleveland Clinic

 
 
EGGS Study, Version 1.2 
 
11-02-2017 
Protocol: Effects of Choline From Eggs vs. Supplements on the Generation of 
TMAO in Humans (EGGS) 
 
NCT # NCT03039023 
 
Version Date: 11/02/2017 

 
 
EGGS Study, Version 1.2 
 
11-02-2017 
Effects of choline from eggs vs. supplements on the generation of TMAO in 
humans (EGGS)  
 
Principal Investigator: W. H. Wilson Tang, MD  
Date: November 2, 2017; Version 1.2 
 
1.0 BACKGROUND AND SIGNIFICANCE 
The principal goal for the study is to examine whether there is a difference between the 
ingestion of choline through supplements versus choline found within eggs on plasma TMAO 
levels. We have previously shown that dietary intake of trimethylamines, including the choline 
group of phosphatidylcholine (PC), is mechanistically linked to cardiovascular disease risk and 
that the metabolism of these trimethylamine nutrients in humans is modulated by the intestinal 
microbes (gut microbes). Additionally, extensive animal studies link an essential role of gut 
microbiota to the metabolism of choline and the production of metabolites that promote / 
accelerate atherosclerotic processes (Wang et al, 2011, Nature). We have also recently shown 
a 10-fold increase in plasma TMAO levels following supplementation with choline bitartrate 
supplements. However, another pilot study by a collaborator did not show the same increase in 
plasma TMAO levels following the ingestion of whole eggs, a major dietary source of choline. 
Therefore, with this study we wish to examine the differences, if any, between the ingestion of 
an equivalent mass of total choline in the free form (as bitartrate salt) as a supplement vs. within 
whole eggs. We also wish to examine the ingestion of an equivalent mass of choline as a 
phosphatidylcholine (PC) supplement capsule and compare this to the ingestion of the choline 
bitartrate supplements and/or the whole eggs.  
 
Eggs, and specifically the egg yolk, contain a large amount of total choline (USDA Database for 
the Choline Content of Common Foods, 2008). However, egg white contains potential anti-
microbial peptides that could influence gut microbial composition and function, and therefore 
impact conversion of choline into TMA and TMAO observed in subjects. Therefore, we 
hypothesize that the consumption of whole eggs (hardboiled) will not elevate plasma TMAO 
levels to the same extent as a comparable amount of total choline ingested in capsule form as 
the choline bitartrate salt. We further hypothesize that the consumption of egg white with choline 
bitartrate tablets may result in less of a rise in TMAO levels than ingestion of the choline 
bitartrate supplement alone.  
 
The majority of the choline present in egg yolks is found as part of phosphatidylcholine. We 
therefore hypothesize that the ingestion of phosphatidylcholine supplements will not elevate 
plasma TMAO levels to the same extent as a comparable amount of total choline ingested in 
capsule form as the choline bitartrate salt, but will instead elevate plasma TMAO levels in a 
comparable fashion to the whole eggs (hardboiled). 
 
2.0 STUDY OBJECTIVES 
To compare the blood and urine levels of free choline, phosphatidylcholine (PC), 
trimethylamine-N-oxide (TMAO), trimethylamine (TMA), and gammabutyrobetaine in subjects 
before versus following the ingestion of choline provided in either the bitartrate salt (in 
supplement) form, within phosphatidylcholine (in supplement form), within eggs (whole or in 
part), or in combinations of these. 

 
 
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3.0 STUDY DESIGN 
This is a prospective non-blinded randomized study with 5 study arms as detailed below. For 
each study arm, subjects will be asked to consume choline bitartrate tablets, 
phosphatidylcholine capsules, and/or eggs or egg whites every day for 4 weeks. 
 
 
Arm 1: whole hardboiled egg  
 
 
Arm 2: choline bitartrate tablets  
 
 
Arm 3: whole hardboiled egg + choline bitartrate tablets 
 
 
Arm 4: egg whites + choline bitartrate tablets 
Arm 5: phosphatidylcholine capsules 
 
For arms 1-4, subjects will be randomly assigned to one of these study arms at the time of 
enrollment. For arm 5, the phosphatidylcholine arm, which we have added in version 1.2 of this 
protocol, subjects will not be randomized but will instead be assigned this arm until at least 10 
subjects have completed this arm. We expect to enroll at least 10 subjects into each of the 5 
arms of the study, up to a maximum of 100 total study participants. The large anticipated 
maximum enrollment accounts for any subjects who may withdraw early or who may need to be 
withdrawn because of antibiotic usage or non-compliance during the course of the study. 
 
At the first (baseline) study visit, subjects will have blood and urine samples collected. Subjects 
will then be randomized or assigned to one of the five study arms as described above. 
 
Following randomization, or assignment in the case of arm 5, subjects will be asked to pick up 
their assigned study food and/or supplements each week. Spot urine collections will be 
collected weekly. Subjects will also be asked to provide a stool sample at the time of the spot 
urine collection if they are able to. The stool samples will be optional, we will not require them. 
 
At the day 7 visit and the final visit they will have repeat blood and urine collections. After 4 
weeks of supplementation, subjects will have their final study visit. This visit will occur between 
26 and 30 days after their baseline visit. 
 
  
Day 1 
(Baseline) 
Day 2 
Day 7* 
Day 8 
Day 14* 
Day 21* 
Day 28* 
Final Visit 
(Day 29) 
Informed consent 
X 
 
 
 
 
 
 
 
Vital signs and brief 
medical history 
X 
 
X 
 
 
 
X 
 
Fasting Blood Draw 
X 
 
X 
 
 
 
X 
 
Pick up eggs and/or 
supplements 
 
X 
X 
 
X 
X 
 
 
Spot Urine sample 
X 
 
X 
 
X 
X 
X 
 
Start 24 hour urine 
X 
 
X 
 
 
 
X 
 
Return 24-Hour Urine  
 
X 
 
X 
 
 
 
X 

 
 
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Stool sample (optional) 
X 
 
X 
 
X 
X 
X 
 
*these study visits can occur +/- 2 days from day noted 
 
 
3.1 Study Endpoints 
 
Baseline plasma and urine fasting choline, phosphatidylcholine (PC), trimethylamine-N-
oxide (TMAO), trimethylamine (TMA), crotonobetaine and gammabutyrobetaine levels. 
o Changes in levels of non-labeled TMAO, TMA, crotonobetaine, betaine and 
gammabutyrobetaine over time measured by established techniques by mass 
spectrometry. 
o Baseline and subsequent (end of study blood draw) changes in platelet function 
during study. 
 
 
Serial changes in plasma and urine (area under curve and percent rise calculations). 
o Correlation with baseline fasting choline, phosphatidylcholine (PC), TMAO, TMA, 
crotonobetaine and gammabutyrobetaine levels. 
 
 
Changes in markers of metabolism (e.g. lipid profile, and other metabolic markers). 
 
 
4.0 SUBJECT SELECTION AND WITHDRAWAL 
Subjects must meet the following inclusion/exclusion criteria to be eligible for the study. 
4.1 Inclusion Criteria 
Inclusion criteria: 
 Men and women age 18 years or above. 
 Willing to remain on aspirin or stay off aspirin or aspirin products for 1 week prior 
to starting the study and throughout the study period. 
 Able to provide informed consent and comply with study protocol. 
 Able to be off all other supplements during the study period. 
 
4.2 Exclusion Criteria 
Exclusion criteria: 
 Significant chronic illness. 
 Active infection or received antibiotics within 1 month of study enrollment. 
 Use of OTC probiotic within 1 month of study enrollment. 
 Chronic gastrointestinal disorders, such as ulcerative colitis or Crohn’s disease. 
 Allergy to eggs or lactose. 
 Having undergone bariatric procedures or surgeries such as gastric banding or 
bypass. 
 Pregnancy. 
 Any condition that, in the judgment of the Investigator, would place a subject at 
undue risk by being enrolled in the trial or cause inability to comply with the trial. 
 
4.3 Subject Recruitment and Screening 
Subjects will be recruited from the Cleveland, OH area. Subjects will be recruited by 
several methods, including (but not limited to) posted advertisements, chart review, and 
personal interactions with patients in the cardiology outpatient departments at the 

 
 
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Cleveland Clinic Foundation. All advertising materials used for this study will be 
approved by the Cleveland Clinic IRB prior to dissemination.  
 
4.4 Early Withdrawal of Subjects 
Subjects may withdraw from the trial at any time and for any reason. Some possible 
reasons for early withdrawal include the following: 
 
Development of a medical condition or need for concomitant treatment that 
precludes further participation in the trial 
 
The investigator removes the subject from the trial in the best interests of the 
subject 
 
Development of an active infection or starting an antibiotic treatment regimen 
 
Study completion or discontinuation prior to participant completion 
 
Subject withdraws consent to continued participation in the trial 
 
5.0 STUDY DRUG/BIOLOGIC 
5.1 Description 
Choline bitartrate and phosphatidylcholine supplements will be used in this study.  These 
will be obtained from a commercial source and stored as below.  Whole, hardboiled, pre-
peeled eggs and egg whites will also be obtained from a commercial source and stored 
as below. 
 
5.2 Treatment Regimen 
For subjects in arms 1, 3, and 4, 500 mg choline bitartrate tablets will be given to be 
taken daily by mouth. Subjects will be asked to take two tablets each day, one in the 
morning and one in the evening, for a total of 1,000 mg daily. Subjects in arm 2 will not 
receive any choline tablets.  
For subjects in arms 2 and 3, four pre-peeled hardboiled eggs will be given to be taken 
daily by mouth, two in the morning and two in the evening. Subjects in arm 3 will be 
asked to eat the hardboiled eggs in addition to taking the choline bitartrate tablets. 
For subjects in arm 4, four pre-peeled hardboiled eggs will be given and subjects will be 
instructed to eat only the egg whites while discarding the egg yolks. Subjects will be 
asked to eat two egg whites in the morning and two egg whites in the evening. Subjects 
in arm 4 will be asked to eat the egg whites in addition to taking the choline bitartrate 
tablets. 
For subjects in arm 5, 420 mg phosphatidylcholine capsules will be given to be taken 
daily by mouth. Subjects will be asked to take 6 capsules each day, 3 in the morning and 
3 in the evening, for a total of 2,520 mg daily. Subjects in arm 5 will not receive any 
hardboiled eggs. 
 
5.3 Subject Compliance Monitoring 
Subjects will be observed taking their assigned treatment, either eggs or choline 
supplements, by a study coordinator or other study personnel during their first few visits 
to ensure the subjects are able to comply with the study procedures. Following this, 
weekly spot urine collections will be analyzed for choline, phosphatidylcholine (PC), 

 
 
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TMAO, TMA, crotonobetaine, and gammabutyrobetaine levels to ensure that subject 
levels do not drop from baseline, which would indicate non-compliance with the study 
treatments. 
 
5.4 Receiving & Storage 
Choline bitartrate and phosphatidylcholine supplements will be obtained from a 
commercial source and stored at room temperature in a dry location. They will be kept 
locked in a secure location. Temperature will be monitored and recorded weekly to 
make sure that the temperature remains between 15° and 30° Celsius. 
Whole, hardboiled, peeled eggs will be obtained from a commercial source and stored 
in a refrigerator, which will be monitored to ensure that the temperature remains under 
5°C. Eggs will not be stored longer than two weeks. 
 
5.5 Dispensing Study Drug 
The supplements will be dispensed directly to the subject by the study coordinators or 
other study personnel. The number of supplements given at each visit will be recorded 
on a drug accountability record. This form will contain a running log of supplements 
received and given out. Any supplements inadvertently dropped or damaged will be 
logged and destroyed. All entries in the log will be dated and initialed by study staff. 
 
5.6 Return and Destruction of Study Drug  
At the completion of the study or, if a subject withdraws early, at their final visit, any 
remaining choline supplements will be returned to the study coordinator. The 
supplements returned, if any, will be recorded on the drug accountability record. Any 
discrepancies will be investigated and resolved prior to the destruction of any 
supplements. Returned supplements will be destroyed on site and their destruction will 
be documented. 
 
6.0 STUDY PROCEDURES 
This is a prospective pilot study consisting of 5 arms. Up to 100 subjects will be enrolled across 
all arms in this pilot study, with a goal of having at least 10 subjects complete each arm of the 
study. 
 
Arm 1: Choline Bitartrate Tablets Alone 
Subjects in this arm will be asked to take two 500mg choline bitartrate tablets (for a total 
of 1,000mg) daily by mouth for four weeks. They will be asked to take one capsule in the 
morning and one in the evening. We aim to have at least 10 subjects complete this arm. 
 
Arm 2: Whole Hardboiled Eggs 
Subjects in this arm will be asked to eat four hardboiled eggs daily for four weeks. They 
will be asked to eat 2 eggs in the morning and 2 eggs in the evening. We aim to have at 
least 10 subjects complete this arm. 
 
Arm 3: Whole Hardboiled Egg + Choline Bitartrate Tablets 

 
 
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Subjects in this arm will be asked to eat four hardboiled eggs and take two 500mg 
choline bitartrate tablets (for a total of 1,000mg) by mouth daily for four weeks. They will 
be asked to eat 2 eggs and take 1 capsule in the morning, and then eat 2 eggs and take 
1 capsule in the evening. We aim to have at least 10 subjects complete this arm. 
 
Arm 4: Egg Whites + Choline Bitartrate Tablets 
Subjects in this arm will be asked to eat the equivalent of four egg whites and take two 
500mg choline bitartrate tablets (for a total of 1,000mg) by mouth daily for four weeks.  
They will be asked to eat 2 egg whites and take 1 capsule in the morning, and then eat 2 
egg whites and take 1 capsule in the evening. We aim to have at least 10 subjects 
complete this arm. 
 
Arm 5: Phosphatidylcholine Capsules Alone 
Subjects in this arm will be asked to take six 420 mg choline bitartrate tablets (for a total 
of 2,025 mg) daily by mouth for four weeks. They will be asked to take three capsule in 
the morning and three in the evening. We aim to have at least 10 subjects comlete this 
arm. 
 
 
6.1 Study Visit Procedures 
 
Blood Draw 
 
Spot Urine 
 
Medical History 
 
Medication Review 
 
Physical Exam including blood pressure, height, weight, and waist circumference 
 
24-hour Urine Collection 
 
Stool Sample Collection (optional) 
  
 
7.0 STATISTICAL PLAN 
7.1 Sample Size Determination 
Based on previous research, we calculate that a sample size of 5 in each arm will 
provide us with a power of 0.9 to detect a significant difference between groups at a 
significance level of 0.05. However, we plan to enroll at least 10 subjects in each arm in 
order to compensate for any unexpected occurrences or subject dropouts. 
 
7.2 Statistical Methods 
We plan to use standard descriptive statistics to characterize the overall study 
population and subgroups of interest both at baseline and during follow-up. Summary 
statistics such as means, medians, standard deviations, and ranges will be produced for 
measured variables, and compared within and between groups with chi-square, Student 
t-test/Wilcoxon test, or ANOVA analysis as appropriate. Graphical methods will be used 
extensively to examine distributions, identify potential influential points, and guide in data 
transformations if warranted.  
 
8.0 SAFETY AND ADVERSE EVENTS 

 
 
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8.1 Recording of Adverse Events  
At each contact with the subject, the investigator will seek information on adverse 
events by specific questioning and, as appropriate, by examination. Information on all 
adverse events will be recorded immediately in the source document. All clearly 
related signs, symptoms, and abnormal diagnostic procedures results will also be 
recorded in the source document.  
All adverse events will be immediately reported to the primary investigators and to the 
IRB, per their procedures. The clinical course of each event will be followed until 
resolution, stabilization, or until it has been determined that the study treatment or 
participation is not the cause. Serious adverse events that are still ongoing at the end 
of the study period will be followed up to determine the final outcome. Any serious 
adverse event that occurs after the study period and is considered to be possibly 
related to the study treatment or study participation will be recorded and reported 
immediately. 
 
8.2 Serious/Unexpected Adverse Events 
Serious and unexpected adverse events will be reported to the Ethical Committee in 
accordance with institutional guidelines. In the case there would be any adverse 
events or unanticipated problems, an appropriate adverse event or unanticipated 
report form will be made and the Ethical Committee notified immediately. The study 
will also be stopped immediately if a subject would experience any unanticipated 
discomfort or adverse cardiac events (e.g. hospitalization). There will be no DMC and 
no interim analyses. 
 
9.0 DATA HANDLING AND RECORD KEEPING 
9.1 Confidentiality and Privacy 
Information about study subjects will be kept confidential and managed according to the 
Health Insurance Portability and Accountability Act of 1996 (HIPAA). Subjects will be 
informed of their rights and the privacy safeguards in place during the consent process. 
 
9.2 Data Management 
Study data will be collected and managed using REDCap (Research Electronic Data 
Capture).  REDCap is a secure, web application designed to support data capture for 
research studies, providing user-friendly web-based case report forms, real-time data 
entry validation (e.g. for data types and range checks), audit trails and a de-identified 
data export mechanism to common statistical packages (SPSS, SAS, Stata, R/S-Plus).   
The system was developed by a multi-institutional consortium which includes Cleveland 
Clinic and was initiated at Vanderbilt University.  The database is hosted at the 
Cleveland Clinic Datacenter.  The system is protected behind a login and Secure 
Sockets Layer (SSL) encryption. There is an audit trail tracking all logins and activities in 
the database. Data collection is customized for each study or clinical trial based on a 
study-specific data dictionary defined by the research team with guidance from the 
REDCap administrator in Quantitative Health Sciences at the Cleveland Clinic.        
 

 
 
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9.3 Records Retention 
All source documents, including the case report forms, will be kept for at least 6 years 
following the completion of the study. Documents will be stored in a secure location with 
limited access.  
 
10.0 STUDY MONITORING, AUDITING AND INSPECTING 
10.1 Study Monitoring 
The Investigator will ensure that any monitor or other compliance or quality assurance 
reviewer is given access to all study-related documents and study related facilities, 
and that they have adequate space to conduct the monitoring visit. 
 
10.2  Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the 
IRB, the sponsor, government regulatory bodies, and Institutional compliance and 
quality assurance groups of all study related documents (for example, source 
documents, regulatory documents, data collection instruments, study data etc.). The 
investigator will ensure the capability for inspections of applicable study-related 
facilities (for example, pharmacy, diagnostic laboratory, etc.). Participation as an 
investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable Institutional compliance and quality assurance 
offices. 
 
11.0 ETHICAL CONSIDERATIONS 
This study will be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312 and International Conference on Harmonization guidelines), 
applicable government regulations and Institutional research policies and procedures. 
This protocol and any amendments will be submitted to a properly constituted independent 
Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal approval 
of the study conduct. The decision of the IRB concerning the conduct of the study will be made 
in writing to the investigator before commencement of this study. 
Prior to enrollment, all subjects for this study will be provided a consent form describing this 
study and providing sufficient information for subjects to make an informed decision. 
 
12.0 
REFERENCES 
1. Patterson K, Bhagwat S, Williams J, Howe J, and Holden J. USDA Database for the Choline 
Content of Common Foods, Release Two. January 2008. 
2. Wang et al, Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. 
Nature 2011 Apr 7;472(7341):57-63 
3. Rebouche et al, Metabolic Fate of Dietary Carnitine in Human Adults: Identification and 
Quantification of Urinary and Fecal Metabolites. The Journal of Nutrition 1991, 121:4. 
4. Bidulescu et al, Repeatability and measurement error in the assessment of choline and 
betaine dietary intake: the Atherosclerosis Risk in Communities (ARIC) Study.  Nutrition 
Journal 2009, 8:14 
5. Wang et al, Metabolomics studies reveal gut flora dependent lipid metabolism as novel 
pathway in cardiovascular disease pathogenesis.  In preparation.