Achieving Nutritional Adequacy Of Vitamin E With An Egg/Plant-Based Food Pairing
Phase: NA
Conditions: Nutritional Requirements
Sponsor: Ohio State University

 
Cover Page  
Document: Study Protocol 
Official Study Title: Achieving Nutritional Adequacy Of Vitamin E With An 
Egg/Plant-Based Food Pairing 
 
ClinicalTrials.gov ID: NCT04287816 
 
Document Date: 12/07/2020 
 
 
 

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Achieving Nutritional Adequacy of Vitamins E and K With An Egg/Plant-Based Food Pairing 
 
Executive Summary 
 
The initial intention of this protocol was to conduct Study 1 and Study 2 as separate investigations, 
with each study entailing a cross-over design for 4 different 72-h pharmacokinetic (PK) trials. 
Prior to initiating these plans, the Sponsor provided additional support to conduct an ancillary 
investigation under Study 1, which resulted in a total of 6 different 72-h PK trials; no changes were 
made to Study 2 (i.e., 4 PK trials).  If participants consented separately to complete both Study 1 
and Study 2, this resulted in a total of 10 PK trials. Both during and post-pandemic, our progress 
to conduct and complete Study 1 and 2 has been impacted.  Thus, this revised protocol (submitted 
Aug 2022) reflects our efforts to gain efficiency in our work and address the study’s primary 
objectives by combining a subset of Study 1 and Study 2 PK trials into a new Study 3 that only 
involves 6 PK trials.  Our intent here is to complete all procedures in participants who are actively 
involved in Study 1 and Study 2.  Those who complete Study 1 will be invited to reconsent for 
enrollment in Study 2.  Thereafter, no new participants will be enrolled in Study 1 or Study 2; all 
future participants will be enrolled only in Study 3.  With this plan, we will enroll 5 participants in 
Study 1 to ensure fulfillment of primary and ancillary objectives. A maximum of 5 persons will 
complete Study 2 and up to 10 participants will complete Study 3. Thus, total enrollment across 
Study 1, 2, and 3 (n = up to 20) does not increase the number of total participants targeted for 
enrollment compared with our initial protocol that only involved Study 1 and Study 2. We can 
therefore achieve study all objectives without increasing overall enrollment, but will be able to 
reduce participant burden and potential risk. 
 
 
I. Objectives 
 
The objective of this study is to use deuterium-labeled spinach (containing stable isotopes of 
vitamin E (α-tocopherol; α-T) and vitamin K (phylloquinone; PQ)) to validate eggs as a dietary 
tool to improve α-T and PQ bioavailability directly from a model plant food, and hence achieve 
nutrient adequacy. Our hypothesis is that the bioavailability of α-T and PQ from deuterium-labeled 
spinach will be potentiated by egg intake in a dose- and time-dependent manner by increasing their 
secretion in intestinal-derived chylomicrons. Further, phospholipid-rich egg yolk lipid will 
enhance nutrient bioavailability compared with vegetable oil. To test this, our team with expertise 
in bionutrition, plant sciences, and advanced mass spectrometry will fulfill these specific aims:  
1) Study 1: Assess egg-mediated improvements in αT and PQ bioavailability 
The working hypothesis is that, compared with deuterium-labeled spinach alone, co-ingestion of 
eggs will dose- and time-dependently increase plasma bioavailability of spinach-derived 
deuterium-labeled αT and PQ (based on AUC0-72 h, Cmax, and % estimated absorption) without 
affecting time to maximal concentrations or half-lives. Further, eggs will dose-dependently 
improve chylomicron enrichment of deuterium-labeled αT and PQ in association with their greater 
absorption.  
2) Study 2: Potentiation of α-T and PQ bioavailability by egg yolk lipid  

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The working hypothesis is that phospholipid-rich whole eggs will enhance spinach-derived αT and 
PQ bioavailability compared with vegetable oil, and will be most functionally responsible for the 
benefits of eggs to enhance lipophilic nutrient absorption. We further hypothesize that egg whites 
will more greatly promote spinach-derived nutrient bioaccessibility compared with spinach alone. 
3) Study 3: Combination of primary objectives in Study 1 and 2 
This newly added Study 3 consists of select trials from Study 1 and Study 2 to allow us to assess 
dose-dependent effects of eggs and food matrix-dependent effects in a succinct study design 
involving only 6 pharmacokinetic trials. 
The rationale for these studies is that, by establishing the efficacy of eggs to potentiate the 
bioavailability of plant-derived fat-soluble nutrients, a strong framework will exist for an easily 
implementable health-promoting food pairing strategy to overcome malnutrition of α-T and PQ. 
The planned research is innovative because it will be the first study to establish a novel function 
of eggs to improve plant-derived α-T and PQ bioavailability by enhancing their intestinal 
absorption. These quantifiable outcomes, made possible with the use of a deuterium-labeled plant, 
are significant because they will establish: (1) critical knowledge for evidence-based dietary 
requirements of α-T and PQ, and (2) an egg-based food pairing that can enhance the health benefits 
of plant-centric dietary patterns. 
 
Study 1 
Study 2 
Study 3 
Arm 1 
Spinach + 0 hardboiled eggs 
(Time 0) 
Spinach + 0 hardboiled eggs 
(Time 0) 
Spinach + 0 hardboiled 
eggs (Time 0) 
Arm 2 
Spinach + 1 hardboiled egg 
(Time 0) 
Spinach + 2 hardboiled egg 
whites (Time 0) 
Spinach + 1 hardboiled 
egg (Time 0) 
Arm 3 
Spinach + 2 hardboiled eggs 
(Time 0) 
Spinach + 2 hardboiled eggs 
(Time 0) 
Spinach + 2 hardboiled 
eggs (Time 0) 
Arm 4 
Spinach + 3 hardboiled eggs 
(Time 0) 
Spinach + vegetable oil 
(Time 0) 
Spinach + 3 hardboiled 
eggs (Time 0) 
Arm 5 
Spinach + 0 eggs (Time 0) + 1 
egg (at 3 h) 
Not applicable 
Spinach + 2 hardboiled 
egg whites (Time 0) 
Arm 6 
Spinach + 1 egg (Time 0) + 1 
egg (at 3 h) 
Not applicable 
Spinach + vegetable oil 
(Time 0) 
 
 
 

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II. Background and Rationale 
 
Americans Have Poor Vitamin E and Vitamin K Status. Malnutrition, which manifests from 
nutrient deficiencies, excesses, or imbalances, impacts ~2.4 billion adults worldwide.1 In the US, 
inadequate status of the fat-soluble nutrients vitamin E (α-tocopherol; α-T) and vitamin K 
(phylloquinone; PQ) are problematic. Indeed, >92% of Americans fail to meet the Estimated 
Average Requirement (12 mg/d2) for α-T3 and up to 81% of adults have suboptimal circulating 
levels.4-5 Similarly, PQ intakes are poor with only 43% of men and 63% of women meeting gender-
specific Adequate Intakes (120 or 90 μg/d, respectively).6 Thus, a substantial proportion of 
Americans are at-risk for inadequate status of these health-promoting nutrients that alleviate 
oxidative distress and tissue injury (i.e. α-T) and post-translationally modify proteins essential for 
blood coagulation, bone metabolism, and lowering atherosclerotic risk (i.e. PQ).2, 7 Few foods are 
rich in α-T and/or PQ, and the leading sources are of plant origin. Thus, emphasis has been placed 
on effective food pairings to potentiate their bioavailability, which is essential for optimal health. 
Eggs: A Mediator of Nutrient Adequacy. Although some hypothesis-generating observational 
studies suggest that higher egg intakes increase cardiovascular-related risks,8-9 evidence from 
controlled interventions, including those from the Bruno Lab, directly support cardioprotective 
activities of eggs (e.g.10-14). While dense with micronutrients and phytochemicals, which directly 
promote favorable health outcomes, an often dismissed benefit of eggs is their yolk-derived lipids 
(e.g. emulsification-promoting phospholipids; ~1.3 g/egg15). These anti-inflammatory bioactive 
lipids15 can also help to address nutritional inadequacies when co-ingested with plant foods that 
are rich in lipophilic nutrients but limited in fat. Indeed, α-T and PQ are biosynthesized abundantly 
in certain plants (e.g. spinach, collards). Both nutrients require co-ingestion with fat to facilitate 
their absorption and bioavailability that is otherwise limited.2, 7 Nonesterified lipid, bile acids and 
pancreatic secretions are critical for α-T and PQ micellularization within the intestine. 
Subsequently, αT and PQ are transferred to enterocytes and secreted in chylomicrons. 
Chylomicron remnants are then taken up at the liver prior to secretion of α-T and PQ in VLDL for 
target tissue delivery. Thus, the co-ingestion of dietary fat is critical to α-T and PQ bioavailability, 
especially when α-T and PQ are derived from plants that are largely devoid of accessible lipid.  
Eggs have a highly digestible lipid matrix that potentiates the bioavailability of a lipophilic 
carotenoid.16 Separate from triglyceride and cholesterol, egg yolk is especially rich in 
phosphatidylcholine, which accounts for 72% of the total phospholipid content.15 While neutral 
lipids (e.g. triglyceride) are important for micelle formation, phosphatidylcholine- and bile acid-
containing micelles are demonstrated to enhance intestinal permeation of poorly absorbed 
compounds.17 Phospholipid micellar content is also critical for the binding to and dissociation from 
intestinal brush border scavenger receptors (e.g. cluster determinant-36; SR class B type I)18 that 
facilitate enterocyte uptake of αT and PQ.19-20 Eggs have been demonstrated to increase the 
absorption of α-T among adults who ingested a mixed-vegetable salad alone (prepared with 3 g 
canola oil) or the same salad with a high-egg or low-egg meal (150 vs 75 g eggs).21 Co-ingestion 
of the high-egg meal with salad increased α-T appearance in the triglyceride-rich lipoprotein 
fraction compared with the salad alone or the salad with the low-egg meal. PQ bioavailability also 
has been examined in relation to fat co-ingestion (0-32 g).22 Although the reported evidence 
supported dietary fat to dose-dependently increase chylomicron PQ, individual responses varied 
extensively and the predictive benefit of fat on PQ bioavailability was lost at lower fat intakes.  

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Studies examining α-T and PQ bioavailability support the premise that fat, including that from 
eggs, is an important mediator of absorption. However, isotopes of α-T or PQ were not used. This 
precluded an understanding of: 1) whether eggs increased absorption of αT that was derived from 
a mixed-vegetable salad (2.1 mg), eggs themselves (1.14 mg/150 g eggs), or both;21 and 2) whether 
dietary fat at lower intakes dose-dependently increased PQ absorption.22 Addressing these 
knowledge gaps is a focus of our proposed study. We plan a pharmacokinetics trial in which eggs 
(0-3 large) will be ingested with deuterium-labeled spinach prior to assessing plasma and 
chylomicron deuterium-labeled αT and PQ. We will also address a secondary hypothesis that the 
“timing” of egg ingestion can potentiate nutrient bioavailability. Relative to the co-ingestion of 
eggs 
with 
deuterium-labeled 
spinach 
on 
nutrient 
bioavailability, we will also assess whether plasma and 
chylomicron deuterium-labeled αT and PQ bioavailability 
increases when an ‘egg snack’ is consumed 3 hours after the 
initial ingestion of spinach with or without an egg. This 
hypothesis is supported by reports detailing that dietary fat 
increases the bioavailability of plant-derived αT and PQ,7-8 
as well as recent evidence that a “second meal” a few hours 
post-ingestion of a “first meal” mobilizes αT that is 
otherwise “trapped” in enterocytes (Fig 1).23 Our 
investigative approach will provide the sensitivity needed to 
assess dose- and time-response benefits of eggs while also 
quantifying the extent to which eggs enhance absorption of 
these nutrients directly from a representative plant food. 
Thus, we expect to establish clear evidence to support eggs 
as part of “plant-centric” healthy eating patterns and 
recommendations (e.g. Dietary Guidelines for Americans;24 Mediterranean Diet25).  
Deuterium-labeled Plants: A Tool for Evaluating Nutrient Bioavailability. The food matrix, 
nutrient quantity, and other determinants of bioavailability have been examined in humans and 
animals. However, research tools have evolved in their sophistication to better interpret 
experimental outcomes. For example, when eggs were co-ingested with a mixed-vegetable salad, 
there was insufficient sensitivity to detect a dose-response effect of eggs (75 g vs 150 g) on α-T 
absorption.21 The use of unlabeled α-T also precluded a determination of which food source(s) 
accounted for the increase in α-T absorption, which was further confounded by endogenous α-T 
that is detectable in intestinal- and liver-derived chylomicrons and VLDL, respectively.26 Thus, an 
accurate measure of the proportion of the orally administered α-T that was absorbed and available 
for target tissue delivery could not be quantified. To address these limitations, the Bruno Lab and 
others use non-radioactive or stable isotopes of α-T and PQ to assess bioavailability in a 
scientifically rigorous manner. These tools enable precise nutrient assessments in plasma and 
isolated lipoproteins using LC-MS and detailed pharmacokinetic analyses to define gut-to-liver 
nutrient trafficking. Notable examples demonstrating this expertise of Dr Bruno (PI) include 
studies examining cigarette smoke-induced oxidative stress on dietary αT requirements,27-28 
vitamin C interactions that improve α-T status,29 the detriment of metabolic syndrome on gut-to-
liver trafficking of α-T,26, 30 and the dose-response effect of dietary fat on α-T absorption (Fig 2).31 
 
Fig 1. Putative benefits of egg phospholipid 
(PL) to improve intestinal trafficking of αT 
and PQ. PL from an Egg “First Meal” may 
promote: A) αT and PQ micellarization and B) 
transporter-mediated uptake of αT and PQ. PL 
from both an Egg “First Meal” C) and “Second 
Meal” D) may promote secretion of αT and PQ 
in chylomicrons (CM). TG, triglyceride. 

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We provided the most accurate evidence in humans that α-T absorption, when administered in an 
apple matrix, is only 10% when ingested in the absence of fat, and that its absorption increases in 
a dietary fat-dependent manner up to 33% when co-ingested with 11 g of fat (Fig 2).31 
Pharmacokinetic analysis indicated that the time to maximal plasma concentrations of αT (Tmax) 
and its half-life (t1/2) were unaffected regardless of the quantity of dietary fat ingested. Higher 
maximal concentrations (Cmax) of α-T in response to increasing dietary fat (Fig 2) were therefore 
attributed to greater intestinal absorption rather than altered gastric emptying. We also observed 
that fat intakes, within the range of 0-11 g ingested, linearly increased α-T bioavailability such that 
0.43 mg of α-T was absorbed for each additional gram of fat ingested (Fig 2C). Thus, our planned 
studies will exploit the highly digestible lipid content of eggs (4.8 g total fat/egg) to demonstrate 
that eggs dose- and time-dependently improve plant-derived αT and PQ bioavailability, and more 
substantially than phospholipid-free vegetable oil. Further, because co-PI Blakeslee is enlisted to 
hydroponically grow deuterium-labeled spinach, pharmacokinetic responses of both deuterium-
labeled α-T and PQ can be assessed independently of the confounding effects of the low-level 
amounts of unlabeled α-T and PQ present in egg yolks or those already present in intestinal or liver 
tissue. 
Deuterium-labeled (i.e. stable isotope) plants have distinct advantages for assessing nutrient 
bioavailability. While supplements containing isotopically labeled nutrients can be administered, 
α-T bioavailability from pills is poorer and has greater inter-subject variability than that from a 
food matrix.32 Further, studies involving plant-derived α-T and PQ are more physiologically 
relevant. This is because they examine the whole food matrix and low nutrient doses (compared 
with pills), which enable translational messages of greater public health importance. Indeed, we 
reported that a small dose of deuterium-labeled α-T was well-absorbed without affecting 
circulating unlabeled αT concentrations (Fig 2A).31 Because the total plasma α-T pool (i.e. sum of 
unlabeled and labeled α-T) was essentially unchanged during and between each pharmacokinetic 
trial, the absorbed hexadeuterium (d6-) α-T did not significantly change plasma vitamin E 
elimination kinetics. This outcome provided a novel opportunity to model the αT kinetics data and 
calculate evidence-based dietary α-T requirements that consider the amount of α-T absorbed 
relative to daily tissue needs and bodily elimination.  
Deuterium-labeled collard greens have been grown to study plasma PQ transport following PQ 
depletion and repletion, including its time-dependent incorporation into lipoprotein fractions.33 
Studies with deuterium-labeled collard greens also show that age-dependent increases in PQ 
bioavailability are attributed to increased circulating triglycerides in older adults.34 Using 
 
Fig 2. α-T bioavailability increases in 
a dietary fat-dependent manner. In a 
randomized, cross-over study, healthy 
adults ingested 80 g of apple pieces 
impregnated with 22 mg d6-αT alone (0 
g fat; 0% energy from fat), with a low-
fat breakfast (2.4 g fat; 6% fat), or with 
a regular fat breakfast (11.0 g fat; 21% 
fat). 
Plasma 
collected 
in 
timed 
intervals over 72 h was analyzed by 
LC-MS for unlabeled (d0) and labeled 
(d6)-αT. A-B) Co-ingestion of fortified 
apples with fat dose-dependently 
increased d6-αT concen- 
trations (Cmax
 and AUC0-72 h) without altering time to maximal concentration, rates of elimination, or half-lives. B) Dietary fat quantity ingested 
predicted d6-αT absorption such that Cmax increased by 0.33 μmol/L (equivalent to 0.43 mg absorbed) for each g of fat ingested. 

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isotopically-labeled kale, PQ bioavailability was determined to be only 4.7% with a plasma half-
time of 8.8 h.35 Deuterated collard greens have also been applied to the study of αT 
pharmacokinetics in younger and older persons.36 The outcomes showed that αT fractional 
disappearance rates (0.63 pools/d), half-lives (30.6 h), and absorption (24%) did not differ by age 
or gender, but that lipidemia slowed αT elimination without affecting αT absorption.  
In summary, α-T and PQ absorption, distribution, metabolism, and elimination are best evaluated 
with the use of stable isotope vitamers. A critical step towards human health is validating food 
pairings, and by extension dietary patterns, that can enhance the otherwise poor absorption of α-T 
and PQ. In the planned investigation, we expect that spinach-derived α-T and PQ bioavailability 
will be poor, but that eggs will dose-dependently and substantially improve intestinal α-T and PQ 
absorption and to a greater extent than that by vegetable oil. This novel evidence will support eggs 
as an important “dietary tool” to achieve nutrient adequacy in the context of health-promoting 
dietary patterns. Secondary outcomes will provide evidence, based on pharmacokinetic 
assessments, to help fill scientific gaps that preclude the ability to establish an RDA for PQ, in 
place of an Adequate Intake.7 Further, current vitamin E requirements are based on limited data 
from the 1950s from only 7 institutionalized men and do not consider the effects of food-nutrient 
interactions. Validated, modern technologies carried out with a high degree of scientific rigor, as 
proposed herein, are critical for moving the nutrition field forward, and supporting human health 
by identify health-promoting dietary strategies to overcome malnutrition.  
Anticipated Results and Significance of the Research 
Trials in Study 1 are expected to provide the first detailed evidence that eggs are an effective 
dietary tool to potentiate the bioavailability of plant (spinach)-derived α-T and PQ by mediating 
their intestinal absorption. Several novel outcomes are expected: (1) eggs co-ingested with 
deuterium-labeled spinach will dose-dependently increase the bioavailability of deuterium-labeled 
α-T and PQ (AUC0-72 h, Cmax, % absorption) without affecting half-lives or Tmax, and (2) eggs 
consumed 3 hours post-ingestion of a “first meal (spinach with or without an egg)” will improve αT 
and PQ bioavailability by mobilizing the α-T and PQ that is otherwise “trapped” in enterocytes (3) 
total egg lipid will strongly predict (P<0.05) improvements in bioavailability parameters, and (4) 
% absorption and chylomicron enrichment of deuterium-labeled α-T and PQ will increase with 
increasing egg intakes, which will indicate that eggs promote fat-soluble nutrient bioaccessibility 
from spinach. These outcomes will be utilized to define the extent to which egg-mediated 
improvements in αT bioavailability contribute to achieving adequate α-T status. Because a well-
defined cut-off for circulating PQ concentrations does not currently exist, the findings will be used 
to calculate daily turnover of PQ (based on elimination kinetics) in relation to bioavailability 
responses. This knowledge will complement anticipated outcomes of future studies that establish 
biomarkers of PQ status. Ultimately, our findings concerning both α-T and PQ will provide 
evidence to help redefine the Estimated Average Requirement (EAR) for α-T and the first-ever 
EAR for PQ, which is currently limited to an Adequate Intake. Thus, our outcomes will establish 
evidence-based dietary recommendations in relation to an effective food pairing that helps to 
achieve adequate status of these nutrients.  
Trials in Study 2 are expected to establish that phospholipid-rich egg lipid can potentiate α-T and 
PQ absorption and bioavailability. Because phospholipids are largely removed during oil 
refinement, the expected outcomes will support a novel benefit of an egg component that receives 

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limited attention. Further corroborating, pharmacokinetics assessments of α-T and PQ in response 
to their co-ingestion with whole egg vs. egg white are expected to demonstrate an added value of 
whole eggs (by way of its egg yolk lipids) to promote adequate nutrient absorption. In addition, 
provided that our secondary hypothesis is correct, that egg whites can increase the formation of 
simple micelles (i.e. bile salts without dietary fat), additional translational messages can be 
established to support a fat-free alternative in egg whites to improve lipophilic nutrient absorption 
and bioavailability. These findings, along with those under Study 1, would be of significance 
because lipophilic nutrients can be “trapped” in enterocytes and lost in the feces as enterocytes are 
sloughed off if not efficiently trafficked for secretion in chylomicrons. Thus, outcomes of these 
studies will serve as the foundation for easy-to-implement messages of public health importance 
in support of whole eggs and egg whites as part of a plant-based dietary pattern.  
The newly added Study 3 is hybrid approach to address the major objectives of Study 1-2 in an 
efficient manner. Thus, participants who complete Study 3 (instead of Study 1 and/or Study 2), 
will enable our understanding of X and Y simultaneously and avoid some scientific redundancies 
that were previously occurring under the separate conduct of Study 1 and Study 2. 
Lastly, our innovative approach with deuterium-labeled spinach will establish a new investigative 
tool to examine egg-mediated benefits on the bioavailability of other bioactive components present 
in spinach. For example, spinach is a leading dietary source of lutein (~12 mg/100 g). While the 
planned studies focus on α-T and PQ, which are dietary essential nutrients, future studies are 
immediately possible to examine lutein pharmacokinetics in relation to egg intake from the 
biospecimens (plasma and chylomicrons) that will be archived in the planned studies. Similarly, 
outcomes from the planned work will inform large scale food-based interventions regarding the 
appropriate delivery of α-T and PQ as part of a mixed-diet. This is important because, while current 
recommendations strongly encourage diets rich in fruits and vegetables, limited guidance is 
available concerning how consumers should effectively pair plant foods with other dietary 
matrices to potentiate micronutrient and phytochemical bioavailability to achieve optimal health. 
Pitfalls and Alternatives 
These studies utilize techniques that are well-established in our hands. Thus, we do not expect any 
technical problems to complete these analyses. Deuterated-spinach will be grown in several cycles. 
This may increase variability of the α-T and PQ content. If the dose is more variable than expected 
(±3%), or the 4:1 α-T:PQ ratio cannot be maintained, we will standardize the dose to PQ (500 µg). 
This is because α-T pharmacokinetics are unaffected by doses ranging from ~1-50 mg α-T (e.g.26, 
29, 31, 36). Should complex pharmacokinetic modeling be required to consider unanticipated 
influences of the gastrointestinal tract, plasma, and body tissue pools, we will consult the OSU 
Pharmacoanalytical Core on a fee-for-service basis.  
In light of the pandemic, which has significantly impacted the progress of the study, we are adding 
a new Study 3, which encompasses the primary objectives of Study 1 and 2. This newly added 
Study 3 will allow us to focus on the primary interest with a 1 × 6 trial rather than doing 2 separate 
trials having 1 × 6 and a 1 × 4 designs (i.e., 10 total pharmacokinetic trials). In this way, we will 
be able to comprehensively examine the initial proposed objectives and complete the project in a 
timely manner while also reducing participant burden.  

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We hypothesize that the lipid content of eggs (e.g. emulsification-promoting phospholipids) will 
substantially increase chylomicron enrichment of αT and PQ. However, we have considered the 
possibility that total bioavailability of α-T and PQ could be improved by whole eggs (compared 
with oil) without increasing their enrichment in chylomicrons. Should that occur, we will assess 
deuterium-labeled α-T and PQ from VLDL, as we described,26 to assess the extent to which eggs 
promote hepatic secretion of these vitamins. Studies in HepG2 cells could also be performed to 
define hepatocyte trafficking in response to egg lipids.   
 
 

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III. Procedures 
 
A. Research Design 
Our initial objective was to conduct two separate trials having 1 × 4 study designs (Study 1 (4-
arms) & Study 2 (4-arms). Arms 5 and 6 in Study 1 were added to address a secondary objective 
through an ancillary study. Due to the significant delay in progress caused by the global pandemic, 
we plan focus the remainder of work on the primary objectives by enrolling only 5 participants, 
instead of 10, for Study 1 and create a new Study 3 that combines select arms of Study 1 and Study 
2 into a succinct 1 × 6 study design. This will avoid the need to re-consent persons to complete 
Study 2 and overall reduce participant burden. 
For the planned studies, we will conduct three separate cross-over studies. The first study consists 
of a 6-arm cross-over study (Study 1), the second in a separate cohort will be 4-arm crossover 
study ( Study 2), and the third study consists of a 6-arm cross-over study (Study 3). For all studies, 
healthy adults who fulfill the enrollment criteria will be enrolled from the Columbus, OH area. 
Studies will be completed in the OSU Human Nutrition Research Center.  
For Study 1, fasted participants will ingest ~100 g of deuterium-labeled steamed spinach with 0, 
1, 2, or 3 hardboiled eggs (Study 1 Arms 1-4, respectively). In Study Arm 5, participants will 
ingest spinach alone followed by 1 egg 3-hours later. In Study Arm 6, participants will ingest 
spinach with 1 egg followed by another egg 3-hours later. Thus, Study Arms 1-4 will test the dose-
dependent effects of eggs on nutrient bioavailability and Study Arms 5-6 (with comparison to 
Study Arms 1 and 2) will test the ‘timing’-dependent effects of eggs on nutrient bioavailability. 
For all studies, ~100 g of deuterium-labeled steamed spinach (to provide 2 mg α-T and 500 µg 
PQ37) will be administered. Hardboiled eggs will be administered at a dose of 0-3 eggs (~50 g 
each) to provide 0-14.4 g fat. For each cross-over trial, blood will be collected prior to (0 h) and at 
3, 4.5, 6, 7.5, 9, 12, 24, 36, 48, and 72 h post-test meal ingestion to ensure that blood samples are 
collected at times corresponding to the Tmax of α-T between 10-12 h23 and Tmax of PQ at either 3-
5 h22, 38 or 6-8 h34-35. Plasma and chylomicrons will be collected and archived as we described.26  
For Study 2, a separate cohort of healthy adult participants will ingest ~100 g of deuterium-labeled 
steamed spinach alone (0 g fat), or with 2 egg whites (0 g fat), 2 whole eggs (9.6 g fat), or vegetable 
oil (9.6 g fat) (Study 2 Arms 1-4, respectively). Blood will be collected from 0-72 h as indicated 
above, processed, and archived until analyzed.  
For Study 3, a separate cohort of healthy adult participants will ingest ~100 g of deuterium-labeled 
steamed spinach alone (0 g fat), or 1, 2, or 3 hardboiled eggs (Study 1 Arms 1-4), or with 2 egg 
whites (0 g fat), or vegetable oil (9.6 g fat) (Study 2 Arms 2 & 4). Blood will be collected from 0-
72 h as indicated above, processed, and archived until analyzed.  
For each study, a 7-d washout period between study arms is sufficient, based on our reports, to 
fully eliminate labeled compounds and prevent “carry-over” to the subsequent trial.26, 31 To 
standardize postprandial responses and accurately define pharmacokinetic parameters under 
Studies 1-3, all foods will be controlled for 3-d preceding each trial and for the initial 24-h of each 
trial. The controlled diet will be eucaloric and standardized for α-T and PQ intakes. For all of these 
planned studies, only blood will be collected for the isolation of plasma and chylomicrons.  No 
fecal samples, urine samples, or genetic material is being collected. 

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B. Sample 
 
Total Number of Participants For Studies 1-3. For both planned studies, up to 40 persons in total 
will be screened in order to identify 15 persons (n = 5 per study) who meet the below 
inclusion/exclusion criteria. 10 persons in each study are needed to enhance statistical power and 
consider potential effects due to gender as described below. The newly added Study 3 will allow 
us to still have 10 persons under the arms that fulfill the primary objectives. Maximum 15 persons 
will be formally enrolled in the study.  
Inclusion/Exclusion Criteria. Participants (18-65 y) will be required to meet the following 
inclusion criteria: a) BMI = 19-25 kg/m2; b) normolipidemic (total cholesterol <240 mg/dL; 
triglyceride <150 mg/dL); c) glucose <100 mg/dL; d) normal hematocrit level (41%-50% for men 
and 36%-48% for women); e) normal hemoglobin level (13.5-17.5 g/dL for men and 12.0-15.5 
g/dL for women); f) no use of dietary supplements for >1 mo; g) no use of medications that affect 
lipid or glucose metabolism; h) non-smoker; and i) no history of gastrointestinal disorders. 
Participants having any of these criteria will be excluded: a) egg allergy; b) >2 alcoholic drinks/d; 
c) >7 h/wk of aerobic activity; d) >2 kg body mass change in the past 1 mo; e) women who are 
pregnant, lactating, or initiated or changed birth control in the past 3 mo, f) history of iron 
deficiency anemia; g) vegetarian. We will also not enroll any participants using any medications 
to manage diabetes, hypertension, or hyperlipidemia (e.g. statins, metformin, ACE inhibitors) or 
any blood thinning medications [Warfarin]). 
Power Calculation. The quantity of egg-derived fat is similar to that in our report demonstrating 
a dose-response increase in d6-α-T bioavailability when ingested with 0-11 g of fat derived from 
cream cheese.31 Using the smallest difference in d6-α-T Cmax, which occurred between the 0 g and 
2.4 g fat levels (Fig 2A), our power analysis indicated that only 5 participants would be needed to 
detect statistically significant differences (90% power, P<0.05; PS Power and Sample Size). 
Similar powering is also obtained when using d6-α-T AUC0-72 h or absorption (%). Studying so few 
participants is impractical for establishing dietary recommendations. Thus, for studies 1-3 separate 
cohorts of  5 adults will be enrolled. By combining participants from all 3 studies, we will have 10 
adults under each primary objectives to consider egg-dependent increases in primary outcomes 
(Cmax, AUC, estimated absorption of α-T or PQ). This increases statistical power to >97% (P<0.01) 
while allowing for consideration of potential differences between men and women. 
Recruitment. We will recruit participants through posted flyers, e-mail, electronic and newsprint 
advertisements (e.g. campus student and faculty/staff newspapers, local and regional newspapers), 
word of mouth, and social media (e.g. Facebook). The posted advertisements will instruct 
interested participants to complete an online eligibility survey or call the study center (Bruno 
Laboratory, Department of Human Sciences) to obtain further information. During the phone-in 
hours, a trained individual (i.e. project coordinator or graduate assistant) will be available to 
describe the study and determine preliminary qualification by conducting a scripted phone 
interview (e.g. do you take dietary supplements?, do you smoke?; see Phone Script attachment). 
The individual will record answers and assess whether or not the person calling is likely or not to 
be an acceptable study participant. If the caller and the interviewer agree that the caller should 
participate, the prospective participant will be invited to a screening meeting, where the study will 
be fully described and the individual will be provided a consent form to complete prior to any 

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involvement in the study procedures. Data collected during this phase will include participant’s 
age, health status, physical activity, and contact information (see Informed Consent attachment).  
Anticipated Data Analysis Plan. Primary outcomes (Cmax, AUC0-72 h, absorption) will be analyzed 
initially using 2-way repeated measures ANOVA to assess effects due to treatment, gender, and 
their interaction. If no gender effects are observed, consistent with prior reports,29, 31, 39 we will 
reassess outcomes using 1-way RM ANOVA and Bonferonni's post-test, as appropriate, to focus 
on egg-dependent effects on pharmacokinetics. Multiple linear regression, controlling for within-
subject repeated measures,40 also will be performed to define relations among study variables. 
C. Measurement/Instrumentation 
Anthropometric Parameters and Blood Pressure. At screening, participants will rest for 15 
minutes prior to determining blood pressure using an automated cuff. BMI will be calculated from 
height determined from a wall-mounted stadiometer and weight from a calibrated scale. Waist 
circumference will be assessed at the level of the umbilicus using a nonflexible measuring tape.  
Blood Chemistries. At screening, fasting plasma glucose, triglyceride, cholesterol, hematocrit, and 
hemoglobin will be measured by spectrophotometry utilizing validated clinical assays (Pointe 
Scientific). In our hands, intra- and inter-assay coefficient of variance (CV) for these assays is 
<8%. 
Diet Assessment/Control. To improve diet homogeneity and limit potential confounding effects 
of the basal diet on study outcomes, participants will be provided all foods and beverages 
consistent with our prior studies.26 These controlled diets will be provided for the 3-days prior to 
each pharmacokinetics trial and during the initial 12-hours of each pharmacokinetics trial. All 
foods will be prepared in the Human Nutrition Metabolic Kitchen, packaged, and provided to 
participants in a storage cooler. A registered dietitian (PI Bruno) will prescribe the 3-d eucaloric 
diet to deliver 50-60% of energy from carbohydrate, 15-20% from protein, and 25-30% from fat. 
Dietary α-T will be standardized at 7 mg/d and PQ at 80 µg/d based on typical Americans intakes.2, 
7 Vitamin C will be provided at the gender-specific RDA2 based on our reports that vitamin C 
status regulates α-T bioavailability.28-29, 41 To control for between-trial physiological differences, 
participants will repeat the prescribed diet in an identical manner. For compliance assessment, 
participants will return all empty containers and/or uneaten portions to assess actual energy and 
nutrient intakes using NDSR dietary analysis software. 
Deuterium-labeled Spinach. Spinach has been selected for all  studies as a plant-rich source of α-
T and PQ.37 Spinach biosynthesizes α-T and PQ in quantities appropriate for pharmacokinetic 
studies, and is a member of the Brassicaceae family known for its diverse health benefits.42 These 
features and its rapid maturation cycle (~6 wk) make it highly amenable to isotopic labeling for 
studies examining αT and PQ bioavailability.33-34, 36 It also can be steamed and preserved frozen 
without any appreciable loss of αT or PQ, and is a vegetable commonly paired with eggs in the 
diets of Americans (e.g. salads, omelets), Europeans, and Asians.  

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Spinach will be grown hydroponically by Dr. Joshua Blakeslee (co-PI, Department of Horticulture 
and Crop Sciences, CFAES, OSU) in growth chambers located in Rightmire Hall as described for 
deuterium-labeled collard greens,34, 36 with minor modifications. Plants will be grown using 
standard hydroponics techniques appropriate to generate cruciferous vegetable plants (e.g. 
spinach) for human consumption in other research efforts. Prior to spinach cultivation, controlled-
environment plant growth chambers will be washed and sterilized. Briefly, chambers will be 
washed twice with soap, a bleach solution, and water prior to wiping down twice with 75% alcohol 
treating for 3 hours with UV light. Seeds will be sown on rock wool in a hydroponic growth system, 
and plants grown at 22ºC under fluorescent lights (200-220 µm/m/s), with an 18-h photoperiod. 
Spinach will be grown using a hydroponic nutrient solution supplemented with deuterated water 
(31 atom%; obtained from Sigma-Aldrich), as described.34, 36 The plant nutrient solution is 
obtained commercially (MSSP06-50LT; Caisson Labs) and contains all the essential nutrients for 
plants (e.g. nitrogen, vitamins, minerals) to stimulate rapid growth. Under these growth conditions 
with this concentration of deuterated water, 100% of the α-T and PQ molecules present in leafy 
tissues become isotopically labeled. To generate sufficient spinach for the proposed studies, we 
plan 4-5 lots (growth cycles) of ~6-wk each to yield ~15 kg total spinach. For each cycle, spinach 
will be grown in 3-4 controlled-environment growth chambers, harvested, pooled, and analyzed 
for total α-T and PQ and their isotopomer distributions for use in the proposed clinical trial.  
Spinach will be harvested at 6-wk post-germination, pooled, and analyzed by HPLC and LC-MS 
prior to steaming, preparing individual portions containing ~2 mg αT and 500 µg PQ (anticipated 
to be ~100 g pre-steamed spinach37), and freezing in a refrigeration/freezer unit dedicated for foods 
for human consumption. Each pooled sample will undergo LC-MS/MS analyses to assess the 
isotoper profile of both αT and PQ.34-36 MS analyses are expected to reveal a normal distribution 
range of mass-to-charge ratios of labeled α-T and PQ (see example in Fig 2).36, 43 The most 
abundant ions representing >70% of each deuterated vitamer (referred herein as dx-αT and dx-PQ) 
will inform the specific tracers assessed in the clinical trial. Total αT and PQ from liquid nitrogen-
pulverized spinach will be quantified by routine HPLC 
procedures in the OARDC Metabolite Analysis Cluster (OMAC, 
located in the Blakeslee Lab). LC-MS/MS analyses will be 
performed by Dr. Blakeslee at both the OMAC (targeted 
metabolomics) and the OSU Campus Chemical Instrumentation 
Center if broad-spectrum metabolomics become needed. 
Plasma and Lipoprotein (chylomicron, VLDL, LDL, HDL) 
Vitamin E. Labeled (dx-) and unlabeled (d0-) αT will be measured 
in samples obtained at 0-72 h using our established LC-MS 
method,26, 28, 31 with minor modification to use tocol as an internal 
standard. In brief, plasma is saponified, extracted with hexane, 
dried under nitrogen, reconstituted, and injected on the LC-MS 
in ESI mode at appropriate m/z ratios (informed in Section 3.vi). 
Scientific Rigor – Intra- and inter-assay CV is <6% in our hands. NIST-traceable standards are 
used for quantification, and quality controls of known plasma αT concentration are included in 
each batch of analyzed samples to validate accuracy and precision.  
Plasma and Lipoprotein (chylomicron, VLDL, LDL, HDL) Vitamin K. Labeled (dx-) and 
unlabeled (d0-) PQ will be assessed from samples obtained from the 72-h pharmacokinetics study, 
Fig 
3. 
Example 
of 
isotopomer 
distribution of deuterium-labeled α-T 
derived from deuterium-labeled collard 
greens (data from REF 35). The mass-to-
charge ratio (m/z) of unlabeled (d0)-α-T is 
429. As shown, no d0-α-T is detected and 
the isotopes comprising the majority of total 
α-T are at m/z of 438-442 (d9- to d13-α-T). 

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as described,35 with minor modifications. In brief, plasma is spiked with β-apo-carotenal (internal 
standard) prior to precipitating proteins with ethanol. Following hexane extraction, the extract is 
dried under nitrogen and reconstituted in 1:1 methyl-tert-butyl-ether (MTBE):methanol. The 
sample is then injected on the LC-MS/MS in APCI mode and separated on a YMC C30 column 
using a binary gradient of 1 mM ammonium acetate in methanol (mobile phase A) and MTBE 
(mobile phase B). Scientific Rigor – Procedures have been established using reference serum of 
known PQ concentration. Intra- and inter-assay CV is <9.2%. Quality control plasma has been 
procured, aliquoted in single-use vials, stored at -80°C, and is included with each analysis to ensure 
accurate quantification. 
Pharmacokinetic Analysis. Plasma triglyceride and total cholesterol will be measured at each 
time point from each subject using enzymatic assays. Pharmacokinetics will be calculated with 
and without dx-α-T and dx-PQ normalized to plasma lipids (i.e. sum of triglyceride and total 
cholesterol) to exclude the possibility of lipidemia influencing the analysis.36, 44 Area under the 
time-concentration curve (AUC0-72 h) and other pharmacokinetic parameters (i.e. fractional rates 
of absorption and elimination, Cmax, absorption Tmax) will be determined as we described.26, 29, 31  
Antioxidant Status & Oxidative Distress. Although the proposed studies are confined to healthy 
persons, and diet will be controlled, measures of antioxidant status and oxidative distress will 
define the independent benefits of eggs on α-T and PQ bioavailability. Thus, at baseline (0 h) of 
each 72-h trial, we will measure plasma vitamin C with our HPLC-ECD method45 and carotenoids 
(lycopene, α- and β-carotene, lutein/zeaxanthin) by HPLC-PDA as we reported.46 The lipid 
peroxidation biomarker malondialdehyde will be measured by HPLC-FL as we described.47 Intra- 
and inter-assay CV for each of these assays is 6-14% 
D. Detailed Study Procedures 
Overview of Study Procedures. Potential participants who call the study center in an anonymous 
manner for more information will be given a brief description about the study and asked a few 
questions to determine their eligibility (see Phone Script attachment). If they meet the eligible 
criteria, they will be invited to the study center for a screening meeting. During the meeting, the 
Informed Consent (see Informed Consent attachment) will be explained and provided for them to 
review. The participant will then be given the opportunity to review the Informed Consent form. 
If he/she chooses to participate in the study, they will then be asked to provide written consent. 
After receiving informed consent, the participants’ height, weight, waist circumference, and blood 
pressure will be measured. Additionally, a small fasting blood sample will be collected to blood 
chemistry analysis. If they are not fasted at least 10 hours), they will be asked to come back in the 
fasted state at time of mutual convenience. These blood results in combination with anthropometric 
parameters will determine the participant’s eligibility. Eligible participants who agree to proceed 
with the study will then be formally enrolled to complete one of the three planned cross-over 
studies (i.e. Study 1 or Study 3 with 6 cross-over periods; or Study 2 with 4 cross-over periods). 
Participants may complete both cross-over trials provided that they wash-out between studies for 
>6 weeks. We estimate that all procedures to complete Study 1 and Study 3will require ~12 weeks 
per participant and that Study 2 will require ~8 weeks per participant. 
Screening Meeting. Potential participants who have met the initial criteria of the study (based on 
the telephone interview) will be invited to the study center at a mutually convenient time. During 

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this time, the participant and a member of the research team will meet in a private, quiet conference 
room or office. The individual will be provided the informed consent form, and its contents will 
be described to the potential participant. The participant will then review it, and if they choose to 
participate in the study, they will be asked to provide written consent. Although the participant 
will be asked to sign the informed consent, the participant will be told that they will not be asked 
to participate if their body measurements or plasma chemistries (see Enrollment Criteria) do not 
meet the study criteria. If the participant has provided consent, we will then measure the 
participant’s height, weight, waist circumference, and blood pressure. Next, if the participant is 
fasted for at least 10 hours, we will ask if a trained individual can draw a small blood sample (8 
mL) so that we may measure blood chemistries (glucose, cholesterol, triglyceride, hematocrit, and 
hemoglobin). All samples will be coded to maintain participant anonymity. If the participant’s 
anthropometrics or plasma chemistries do not meet the study criteria, they will be told that they do 
not meet the study criteria.  
Potential participants who meet the criteria will be contacted within a few days after their screening 
meeting to provide them with their blood and body measurement results and inform them of their 
eligibility to participate in the study. Consistent with our CLIA exemption, blood results will be 
provided in a categorical manner (i.e. “normal”, “marginally high”, “high”) rather than providing 
actual blood concentrations of lab values (see Subjects Results Sheet). Potential participants having 
any blood values outside of the “normal range” will be directed/encouraged to follow-up with their 
own physician. Those having body measurements and blood values within acceptable limits (see 
Inclusion/Exclusion criteria), will be invited to participate in the study. Subjects will be included 
or excluded based on a best fit of the inclusion and exclusion criteria (an example of best fit would 
be if a potential subject says he/she exercises 8 h/wk, which is close to our exclusion criteria of >7 
h/wk, they might still be included in the study if they meet all other inclusion and exclusion criteria 
more closely than other potential subjects). Participants will be read one phone script if they qualify 
and another phone script if they do not qualify (see Participant Eligibility Phone Script 
attachment). Potential participants who qualify for the study will be communicated a message as 
follows: “Congratulations! You have been selected for participation in our study based on your 
blood testing results. A study investigator will be telephoning you to invite you to the testing 
session. Would you like to know your blood chemistry results?” Subjects not selected for study 
will be told the following message: “You have not been chosen for our study, but thank you for 
your interest.”  This message will be followed by an explanation why they were not chosen, such 
as lab values outside of the range we are looking for: “Your blood testing data is…..we were 
looking for participants who had levels less than …..”.  “We can provide you a copy of your results 
if you would like…..how would you like us to provide them to you?”. “You should also consider 
sharing these results with your physician.”  
If a participant is telephoned and is unavailable, a message will be left requesting a callback at a 
convenient time or that at a member of the study team will try calling again at a later time. No 
confidential or sensitive information will be shared with third parties or left on answering 
machines. 
Intervention timeline. Each participant who meets formal study entry criteria will be expected to 
complete either Study 1 or Study 3 (6-arm cross-over study) or Study 2 (4-arm cross-over study). 
Both studies are designed identically with the exception that deuterium-labeled spinach will be 
consumed with different test meals to assess fat-nutrient bioavailability.  

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To complete the pharmacokinetics studies, each participant will visit the study center a total of 31 
times (Study 1 or Study 3) or 21 times (Study 2) to complete all procedures of the respective cross-
over trial. This includes 1 visit for screening and 5 visits (1 visit to pick-up food and 4 visits for 
blood collection) during each 72-h pharmacokinetics trial (x 6 cross-over trials = 30 visits; x 4 
cross-over trials = 20 visits). Each 72-h cross-over study arm will be separated by at least 1-week. 
Food Pick-Up Visit. On day -3 of each trial arm, participants will visit the study center to pick-up 
a cooler containing their foods and beverages. To ensure that foods meet participants’ preferences, 
a study member will contact the participant by phone or email to share the menu of the 3-day diet. 
If specific foods or daily meals are unacceptable, the study team will substitute foods or complete 
daily meals from one of the other days on the diet.  
Test trials. On day 0 of each trial arm, participants will report to the study center in the fasted state 
(10-12 hours with no food or beverage except for water). They will return all empty food containers 
and/or uneaten portions. Arthrometric measurements will be performed. Then, a trained 
phlebotomist will collect a blood sample. Participants will then receive, in random order 
(determined with a random sequence generator; http://www.random.org/sequences/), one of the 
test meals to consume (see below table). Thereafter, blood samples will be collected at 3, 4.5, 6, 
7.5, 9, and, 12 hours post-meal ingestion to isolate plasma and circulating lipoproteins for 
biochemical measurements (e.g. vitamin E, vitamin K). During each study visit, participants will 
have access to drinking water and can use the restroom as needed. A standardized lunch (after the 
4.5-h blood draw) and dinner (after the 9-h blood draw) also will be provided.  
For all test meals, deuterium-labeled spinach will be generated by Dr. Blakeslee as described 
above. Harvested spinach will be stored in vacuum-sealed bags in a freezer dedicated for human 
foods. For each trial ~100 g of spinach will used. In the Human Nutrition Metabolic Kitchen, 
spinach will be rinsed consistent with safe food handling practices and then steamed. Hardboiled 
eggs will also be prepared in egg cooker. These whole eggs in their entirety, or their egg white 
portion will be used to prepare the test meals. Whole eggs along with vegetable oil will be 
purchased from a local supermarket (e.g. Kroger). 
  
Study 1 
Study 2 
Study 3 
Test Meal 1 
Spinach + 0 hardboiled eggs 
(Time 0) 
Spinach alone (Time 0) 
Spinach + 0 hardboiled 
eggs (Time 0) 
Test Meal 2 
Spinach + 1 hardboiled egg 
(Time 0) 
Spinach + 2 eggs whites 
(Time 0) 
Spinach + 1 hardboiled 
egg (Time 0) 
Test Meal 3 
Spinach + 2 hardboiled eggs 
(Time 0) 
Spinach + 2 whole eggs 
(Time 0) 
Spinach + 2 hardboiled 
eggs (Time 0) 
Test Meal 4 
Spinach + 3 hardboiled eggs 
(Time 0) 
Spinach + vegetable oil 
(Time 0) 
Spinach + 3 hardboiled 
eggs (Time 0) 
Test Meal 5 
Spinach + 0 eggs (Time 0) + 1 
egg (at 3 h) 
Not applicable 
Spinach + 2 hardboiled 
egg whites (Time 0) 

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Teat Meal 6 
Spinach + 1 egg (Time 0) + 1 
egg (at 3 h) 
Not applicable 
Spinach + vegetable oil 
(Time 0) 
 
On day 1 of each trial arm, they will report to the study center in the fasted state for blood collection 
at 24-h post-meal ingestion. They will return again for a non-fasting blood draw at 36-h post-meal 
ingestion.  
On day 2 of each trial arm, they will report to the study center in the fasted state for blood collection 
at 48-h post-meal ingestion.   
On day 3 of each trial arm, they will report to the study center in the fasted state for blood collection 
at 72-h post-meal ingestion. This is the terminal blood draw. Participants will then undergo at least 
1-week washout before completing the remaining 3 trials within either study (see Table) in random 
order and in a manner identical to procedures described above.  
Sampling Handling. Once at screening (8 mL), and 11-times during each pharmacokinetic trial 
(0, 3, 4.5, 6, 7.5, 9, 12, [16 mL/time point]; 24, 36, 48, and 72 h [8 mL/time point]), a blood sample 
will be collected. During each trial, a total volume of 144 ml (~0.6 cup) will be collected. Thus, 
872 mL (~3.6 cups) will be collected in Study 1 (or Study 3) (screening + 6 trials) that spans at 
least 12 weeks. For Study 2, we calculate that 584 ml (~2.4 cups) of blood will be collected 
(screening + 4 trials) over a span of at least 8 weeks. 
During each trial, plasma (0-72 h) and lipoproteins (0-72 h; chylomicrons, VLDL, LDL, HDL)  
will be obtained by centrifugation, and then transferred to appropriate storage tubes. Tubes will be 
stored at -80˚C until analysis can be completed. Analyses of blood samples will include labeled 
and unlabeled α-T and PQ cholesterol, triglycerides, vitamin C, carotenoids, and oxidative stress 
biomarkers (i.e. malondialdehyde). No genetic materials are being collected or archived as part of 
this study. However, remaining blood sample aliquots not used for these analyses will be archived 
for 5 y at -80˚C in the event we decide to measure additional biomarkers related to the study 
objectives. Archival of blood samples also includes those obtained at screening, both for persons 
meeting eligibility requirements as well as those who do not meet eligibility requirements. 
Appropriate notation has been made in the informed consent to alert participants that we will be 
archiving specimens and that they have the right to refuse our use of these specimens for future 
analyses. Lastly, approval from the OSU IRB will be sought via a protocol amendment prior to the 
analysis of any additional biomarkers not specified herein. 
Privacy/Confidentiality. For all data and records that are a part of this study, a number (i.e. code) 
will be assigned to each participant, and will only be available to research personnel. Any records 
containing the names of participants will be stored in a locked filing cabinet or on a password 
protected computer in the PI’s laboratory or office. Research personnel under the supervision of 
the PI and the PI himself will be the only individuals that have access to this information. The 
names of participants will not be used for publication in any form. The records will be maintained 
until the data are published, up to a maximum of five years. All archived samples will be coded, 
but the key linking the code to each participant’s identifiable information will have been destroyed. 
In addition, participants will be instructed that their participation in this study is voluntary and that 

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they may withdraw at any time without prejudice. Data (food records and biochemical values) 
obtained from this study will stored on a computer in the PI’s laboratory. In addition, a backup of 
digital data will be stored on the PI’s computer in his office. Both computers are password 
protected and both doors are locked when work areas are not in use. 
E. Internal Validity 
For all data and records that are a part of this study, a number (i.e. code) will be assigned to each 
participant. All collected blood samples will also be coded. This will minimize measurement bias 
when performing analysis on biochemical markers. The codes will only be broken once data 
analysis has been completed and verified by the PI. 
F. Medical Safety Plan 
All aspects of the clinical study will be conducted in Dr. Bruno’s clinical lab located in Campbell 
Hall. Participants will be fasted for 10-12 hours prior to each study visit. We recognize that certain 
risks associated with fasting include: hypoglycemia, weakness, and fainting. This duration of 
fasting is consistent with guidelines set forth by the American Diabetes Association to minimize 
risk to the individual when determining fasting blood glucose concentrations.  
Risks related to hypoglycemia are anticipated to be low due to their underlying “healthy” health 
status and a prescribed diet for the three preceding days that will supply ~50% carbohydrate. In 
addition, shortly upon arrival to our study center, they will be ingesting an plant/spinach-based test 
meal. Throughout each visit to the study center, participants will be closely monitored a member 
of the research team. These persons will monitor the safety and well-being of participants for any 
signs and symptoms of hypoglycemia including: confusion, dizziness, irritability, weakness, 
headaches, and fainting. Additionally, Dr. Bruno (PI; 325 Campbell Hall) has an academic office 
in close proximity to the study center and has significant experience coordinating clinical research 
studies involving overnight fasting, and other dietary-related meal challenges, thereby supporting 
the competency of our research team in managing potential adverse events relating to fasting 
glucose and glycemic responses.  
Consistent with our prior studies of similar design, and to ensure participant safety, all blood 
collection procedures throughout each study visit will occur while positioned on a hospital bed in 
the prone position. In the event that a participant was to become weak, dizzy, or faint, they would 
already be ideally positioned to minimize risks associated with these symptoms. In the event that 
hypoglycemia-related symptoms occur, the study would be terminated to allow the participant to 
recover. We are prepared to provide pre-packaged beverages and snacks containing simple 
carbohydrates (e.g. Gatorade, fruit juice, apple sauce, crackers) that will allow for rapid restoration 
of blood glucose. These food items will be stored in a refrigerator that is located in close proximity 
and dedicated for foods used in research studies. Participant status (e.g. attentiveness, skin color) 
will be monitored in our clinical laboratory to ensure recovery. The clinical area where participants 
undergo blood draws is also equipped with first-aid measures (e.g. smelling salt) and all study 
team members are trained to assist with basic first-aid application if needed. Due to the amount of 
blood required for the study, participants will be advised not to donate blood and inform their 
doctor about the study if they need a blood draw at the doctor’s office while they are on the study.  

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Any adverse hypoglycemic response that might occur during this study will receive care 
commensurate to the symptoms.  For example, if a participant were to faint, then smelling salts 
would be administered along with a carbohydrate-containing food upon regaining consciousness. 
The study PI would also be contacted.  Alternatively, should a more severe hypoglycemic response 
occur (e.g. contusion or laceration relating to fainting), the research team would immediately 
contact medical services (i.e. 911). For non-life threatening emergencies, any OSU students 
participating in these studies would be directed to the Wilce Student Health Center.  For other 
emergencies or those participants who are not OSU students, individuals would be directed to the 
Wexner Medical Center either by transporting them directly or requesting ambulance service. 
Regardless of the complexity of the adverse events, the research team would monitor the 
participant in the interim, provide palliative care as appropriate, and follow-up after any medical 
care has been provided. 
IV. Literature Cited 
 
1. 
World 
Health 
Organization 
Malnutrition. 
https://www.who.int/news-room/fact-
sheets/detail/malnutrition (accessed April 4). 
2. 
Institute of Medicine (U.S.). Panel on Dietary Antioxidants and Related Compounds., Dietary 
reference intakes for vitamin C, vitamin E, selenium, and carotenoids : a report of the Panel on Dietary 
Antioxidants and Related Compounds, Subcommittees on Upper Reference Levels of Nutrients and of 
Interpretation and Use of Dietary Reference Intakes, and the Standing Committee on the Scientific 
Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. National 
Academy Press: Washington, D.C., 2000; p xx, 506. 
3. 
Maras, J. E.; Bermudez, O. I.; Qiao, N.; Bakun, P. J.; Boody-Alter, E. L.; Tucker, K. L., Intake of 
alpha-tocopherol is limited among US adults. J Am Diet Assoc 2004, 104 (4), 567-75. 
4. 
McBurney, M. I., Majority of Americans not consuming vitamin E RDA. J Nutr 2011, 141 (10), 
1920. 
5. 
McBurney, M. I.; Yu, E. A.; Ciappio, E. D.; Bird, J. K.; Eggersdorfer, M.; Mehta, S., Suboptimal 
serum alpha-tocopherol concentrations observed among younger adults and those depending exclusively 
upon food sources, NHANES 2003-2006. PloS one 2015, 10 (8), e0135510. 
6. 
Harshman, S. G.; Finnan, E. G.; Barger, K. J.; Bailey, R. L.; Haytowitz, D. B.; Gilhooly, C. H.; 
Booth, S. L., Vegetables and mixed dishes are top contributors to phylloquinone intake in US adults: data 
from the 2011-2012 NHANES. J Nutr 2017, 147 (7), 1308-1313. 
7. 
Institute of Medicine (US) Panel on Micronutrients., Dietary Reference Intakes for vitamin A, 
vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, 
vanadium, and zinc. National Academy Press: Washington, D.C., 2001. 
8. 
Djousse, L.; Gaziano, J. M., Egg consumption in relation to cardiovascular disease and mortality: 
the Physicians' Health Study. Am J Clin Nutr 2008, 87 (4), 964-9. 
9. 
Zhong, V. W.; Van Horn, L.; Cornelis, M. C.; Wilkins, J. T.; Ning, H.; Carnethon, M. R.; 
Greenland, P.; Mentz, R. J.; Tucker, K. L.; Zhao, L.; Norwood, A. F.; Lloyd-Jones, D. M.; Allen, N. B., 
Associations of dietary cholesterol or egg consumption with incident cardiovascular disease and mortality. 
Jama 2019, 321 (11), 1081-1095. 
10. 
McDonald, J. D.; Chitchumroonchokchai, C.; Li, J.; Mah, E.; Labyk, A. N.; Reverri, E. J.; Ballard, 
K. D.; Volek, J. S.; Bruno, R. S., Replacing carbohydrate during a glucose challenge with the egg white 
portion or whole eggs protects against postprandial impairments in vascular endothelial function in 
prediabetic men by limiting increases in glycaemia and lipid peroxidation. Br J Nutr 2018, 119 (3), 259-
270. 
11. 
McDonald, J. D.; Mah, E.; Chitchumroonchokchai, C.; Reverri, E. J.; Li, J.; Volek, J. S.; Villamena, 
F. A.; Bruno, R. S., Co-ingestion of whole eggs or egg whites with glucose protects against postprandial 

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Institutional Review Board 
Version: 2 (Dec 7, 2020) 
 
 
19
hyperglycaemia-induced oxidative stress and dysregulated arginine metabolism in association with 
improved vascular endothelial function in prediabetic men. Br J Nutr 2018, 120 (8), 901-913. 
12. 
Katz, D. L.; Gnanaraj, J.; Treu, J. A.; Ma, Y.; Kavak, Y.; Njike, V. Y., Effects of egg ingestion on 
endothelial function in adults with coronary artery disease: a randomized, controlled, crossover trial. Am 
Heart J 2015, 169 (1), 162-9. 
13. 
van der Made, S. M.; Kelly, E. R.; Berendschot, T. T.; Kijlstra, A.; Lutjohann, D.; Plat, J., 
Consuming a buttermilk drink containing lutein-enriched egg yolk daily for 1 year increased plasma lutein 
but did not affect serum lipid or lipoprotein concentrations in adults with early signs of age-related macular 
degeneration. J Nutr 2014, 144 (9), 1370-7. 
14. 
Fuller, N. R.; Caterson, I. D.; Sainsbury, A.; Denyer, G.; Fong, M.; Gerofi, J.; Baqleh, K.; Williams, 
K. H.; Lau, N. S.; Markovic, T. P., The effect of a high-egg diet on cardiovascular risk factors in people 
with type 2 diabetes: the Diabetes and Egg (DIABEGG) study-a 3-mo randomized controlled trial. Am J 
Clin Nutr 2015, 101 (4), 705-13. 
15. 
Andersen, C. J., Bioactive egg components and inflammation. Nutrients 2015, 7 (9), 7889-913. 
16. 
Chung, H. Y.; Rasmussen, H. M.; Johnson, E. J., Lutein bioavailability is higher from lutein-
enriched eggs than from supplements and spinach in men. J Nutr 2004, 134 (8), 1887-93. 
17. 
Zhao, Y.; Cui, Y.; Li, Y.; Li, L., Stable phosphatidylcholine-bile salt mixed micelles enhance oral 
absorption of paclitaxel: preparation and mechanism in rats. J Drug Target 2014, 22 (10), 901-12. 
18. 
Goncalves, A.; Gontero, B.; Nowicki, M.; Margier, M.; Masset, G.; Amiot, M. J.; Reboul, E., 
Micellar lipid composition affects micelle interaction with class B scavenger receptor extracellular loops. 
J Lipid Res 2015, 56 (6), 1123-33. 
19. 
Reboul, E., Vitamin E intestinal absorption: Regulation of membrane transport across the 
enterocyte. IUBMB life 2019, 71 (4), 416-423. 
20. 
Goncalves, A.; Margier, M.; Roi, S.; Collet, X.; Niot, I.; Goupy, P.; Caris-Veyrat, C.; Reboul, E., 
Intestinal scavenger receptors are involved in vitamin K1 absorption. J Biol Chem 2014, 289 (44), 30743-
52. 
21. 
Kim, J. E.; Ferruzzi, M. G.; Campbell, W. W., Egg consumption increases vitamin E absorption 
from co-consumed raw mixed vegetables in healthy young men. J Nutr 2016, 146 (11), 2199-2205. 
22. 
White, W. S.; Zhou, Y.; Crane, A.; Dixon, P.; Quadt, F.; Flendrig, L. M., Modeling the dose effects 
of soybean oil in salad dressing on carotenoid and fat-soluble vitamin bioavailability in salad vegetables. 
Am J Clin Nutr 2017, 106 (4), 1041-1051. 
23. 
Traber, M. G.; Leonard, S. W.; Ebenuwa, I.; Violet, P.-C.; Wang, Y.; Niyyati, M.; Padayatty, S.; 
Tu, H.; Courville, A.; Bernstein, S.; Choi, J.; Shamburek, R.; Smith, S.; Head, B.; Bobe, G.; Ramakrishnan, 
R.; Levine, M., Vitamin E absorption and kinetics in healthy women, as modulated by food and by fat, 
studied using 2 deuterium-labeled α-tocopherols in a 3-phase crossover design. Am J Clin Nutr 2019, 110 
(5), 1148-1167. 
24. 
U.S. Department of Health and Human Services and U.S. Department of Agriculture 2015–2020 
Dietary Guidelines for Americans. 8th Edition. http://health.gov/dietaryguidelines/2015/guidelines/ 
(accessed Jan 13). 
25. 
Garcia-Fernandez, E.; Rico-Cabanas, L.; Rosgaard, N.; Estruch, R.; Bach-Faig, A., Mediterranean 
diet and cardiodiabesity: a review. Nutrients 2014, 6 (9), 3474-500. 
26. 
Mah, E.; Sapper, T. N.; Chitchumroonchokchai, C.; Failla, M. L.; Schill, K. E.; Clinton, S. K.; 
Bobe, G.; Traber, M. G.; Bruno, R. S., alpha-Tocopherol bioavailability is lower in adults with metabolic 
syndrome regardless of dairy fat co-ingestion: a randomized, double-blind, crossover trial. Am J Clin Nutr 
2015, 102 (5), 1070-80. 
27. 
Bruno, R. S.; Leonard, S. W.; Li, J.; Bray, T. M.; Traber, M. G., Lower plasma alpha-carboxyethyl-
hydroxychroman after deuterium-labeled alpha-tocopherol supplementation suggests decreased vitamin E 
metabolism in smokers. Am J Clin Nutr 2005, 81 (5), 1052-9. 

THE OHIO STATE UNIVERSITY 
 
Institutional Review Board 
Version: 2 (Dec 7, 2020) 
 
 
20
28. 
Bruno, R. S.; Ramakrishnan, R.; Montine, T. J.; Bray, T. M.; Traber, M. G., {alpha}-Tocopherol 
disappearance is faster in cigarette smokers and is inversely related to their ascorbic acid status. Am J Clin 
Nutr 2005, 81 (1), 95-103. 
29. 
Bruno, R. S.; Leonard, S. W.; Atkinson, J.; Montine, T. J.; Ramakrishnan, R.; Bray, T. M.; Traber, 
M. G., Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. 
Free Radic Biol Med 2006, 40 (4), 689-97. 
30. 
Traber, M. G.; Mah, E.; Leonard, S. W.; Bobe, G.; Bruno, R. S., Metabolic syndrome increases 
dietary alpha-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-
blind, crossover clinical trial. Am J Clin Nutr 2017, 105 (3), 571-579. 
31. 
Bruno, R. S.; Leonard, S. W.; Park, S.; Zhao, Y. Y.; Traber, M. G., Human vitamin E requirements 
assessed with the use of apples fortified with deuterium-labeled alpha-tocopheryl acetate. Am J Clin Nutr 
2006, 83 (2), 299-304. 
32. 
Leonard, S. W.; Good, C. K.; Gugger, E. T.; Traber, M. G., Vitamin E bioavailability from fortified 
breakfast cereal is greater than that from encapsulated supplements. Am J Clin Nutr 2004, 79 (1), 86-92. 
33. 
Erkkilä, A. T.; Lichtenstein, A. H.; Dolnikowski, G. G.; Grusak, M. A.; Jalbert, S. M.; Aquino, K. 
A.; Peterson, J. W.; Booth, S. L., Plasma transport of vitamin K in men using deuterium-labeled collard 
greens. Metabolism 2004, 53 (2), 215-221. 
34. 
Ellis, J. L.; Fu, X.; Al Rajabi, A.; Grusak, M. A.; Shearer, M. J.; Naumova, E. N.; Saltzman, E.; 
Barger, K.; Booth, S. L., Plasma response to deuterium-labeled vitamin K intake varies by TG response, 
but not age or vitamin K status, in older and younger adults. J Nutr 2019, 149 (1), 18-25. 
35. 
Novotny, J. A.; Kurilich, A. C.; Britz, S. J.; Baer, D. J.; Clevidence, B. A., Vitamin K absorption 
and kinetics in human subjects after consumption of 13C-labelled phylloquinone from kale. Br J Nutr 2010, 
104 (6), 858-62. 
36. 
Traber, M. G.; Leonard, S. W.; Bobe, G.; Fu, X.; Saltzman, E.; Grusak, M. A.; Booth, S. L., alpha-
Tocopherol disappearance rates from plasma depend on lipid concentrations: studies using deuterium-
labeled collard greens in younger and older adults. Am J Clin Nutr 2015, 101 (4), 752-9. 
37. 
U.S. Department of Agriculture; Agricultural Research Service; Nutrient Data Laboratory USDA 
National Nutrient Database for Standard Reference, Release 28. https://ndb.nal.usda.gov/ndb/ (accessed 
Apr 27, 2019). 
38. 
Marinova, M.; Lütjohann, D.; Breuer, O.; Kölsch, H.; Westhofen, P.; Watzka, M.; Mengel, M.; 
Stoffel-Wagner, B.; Hartmann, G.; Coch, C.; Oldenburg, J., VKORC1-dependent pharmacokinetics of 
intravenous and oral phylloquinone (vitamin K1) mixed micelles formulation. Eur J Clin Pharmacol 2013, 
69 (3), 467-75. 
39. 
Leonard, S. W.; Paterson, E.; Atkinson, J. K.; Ramakrishnan, R.; Cross, C. E.; Traber, M. G., 
Studies in humans using deuterium-labeled alpha- and gamma-tocopherols demonstrate faster plasma 
gamma-tocopherol disappearance and greater gamma-metabolite production. Free Radic Biol Med 2005, 
38 (7), 857-66. 
40. 
Bland, J. M.; Altman, D. G., Calculating correlation coefficients with repeated observations: Part 
1--Correlation within subjects. BMJ 1995, 310 (6977), 446. 
41. 
Traber, M. G.; Buettner, G. R.; Bruno, R. S., The relationship between vitamin C status, the gut-
liver axis, and metabolic syndrome. Redox Biol 2018, 21, 101091. 
42. 
Roberts, J. L.; Moreau, R., Functional properties of spinach (Spinacia oleracea L.) phytochemicals 
and bioactives. Food Funct 2016, 7 (8), 3337-53. 
43. 
Fu, X.; Peterson, J. W.; Hdeib, M.; Booth, S. L.; Grusak, M. A.; Lichtenstein, A. H.; Dolnikowski, 
G. G., Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid 
chromatography/mass spectrometry. Anal Chem 2009, 81 (13), 5421-5. 
44. 
Gross, M.; Yu, X.; Hannan, P.; Prouty, C.; Jacobs, D. R., Jr., Lipid standardization of serum fat-
soluble antioxidant concentrations: the YALTA study. Am J Clin Nutr 2003, 77 (2), 458-66. 
45. 
Bruno, R. S.; Dugan, C. E.; Smyth, J. A.; DiNatale, D. A.; Koo, S. I., Green tea extract protects 
leptin-deficient, spontaneously obese mice from hepatic steatosis and injury. J Nutr 2008, 138 (2), 323-31. 

THE OHIO STATE UNIVERSITY 
 
Institutional Review Board 
Version: 2 (Dec 7, 2020) 
 
 
21
46. 
Guo, Y.; Mah, E.; Bruno, R. S., Quercetin bioavailability is associated with inadequate plasma 
vitamin C status and greater plasma endotoxin in adults. Nutrition 2014, 30 (11-12), 1279-86. 
47. 
Mah, E.; Noh, S. K.; Ballard, K. D.; Matos, M. E.; Volek, J. S.; Bruno, R. S., Postprandial 
hyperglycemia impairs vascular endothelial function in healthy men by inducing lipid peroxidation and 
increasing asymmetric dimethylarginine:arginine. J Nutr 2011, 141 (11), 1961-8.