Dietary Green Tea Confection For Resolving Gut Permeability-Induced Metabolic Endotoxemia In Obese Adults
Phase: NA
Conditions: Obesity, Endotoxemia, Inflammation
Sponsor: Ohio State University

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Green tea confections to assess catechin pharmacokinetics and restore gut health in obese adults 
 
I. 
Objectives 
 
The objective of this application is to evaluate green tea catechin pharmacokinetic parameters and 
gut barrier function and gut microbiota composition in obese adults following chronic ingestion of a 
green tea extract (GTE)-rich confection. Our central hypothesis is that obese adults will have altered 
catechin pharmacokinetics compared to healthy adults and that chronic ingestion of a GTE-rich 
confection in obese adults will attenuate metabolic endotoxemia by limiting gut-derived endotoxin 
translocation and modulating gut microbiota composition. Metabolic endotoxemia is a condition 
characterized by elevated endotoxin in the blood, but at levels 10-50 times lower than during sepsis 
(1). To test this, we will complete the following objectives: 1) define alterations in catechin 
pharmacokinetics by health status (obese vs healthy adults), 2) evaluate gastrointestinal-level 
suppression of metabolic endotoxemia by GTE catechins, and 3) define changes in gut microbiota 
composition with GTE in relation to improvements in metabolic endotoxemia. Upon completing this 
study, we expect to provide translational evidence for the efficacy of a novel GTE-rich confection to 
attenuate metabolic endotoxemia and alter gut microbiota composition in obese adults. In addition, 
this study will provide preliminary evidence for future controlled trials investigating the beneficial 
impact of GTE in cohorts with underlying obesity, and other obesity-related metabolic disorders 
characterized by inflammation. 
 
II. 
Background and Rationale 
 
Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis. Nonalcoholic fatty 
liver disease (NAFLD) is the most prevalent liver disease in the U.S., affecting over 70 million 
American adults (2). NAFLD is the broad term used to describe a spectrum of progressive chronic 
liver conditions, which includes liver steatosis, nonalcoholic steatohepatitis (NASH) and cirrhosis. 
NASH is characterized by hepatic steatosis, hepatocyte ballooning, and inflammation (3). In the U.S., 
approximately 10-18 million Americans are diagnosed with NASH (4, 5). However, these statistics 
are likely underestimating the true prevalence since accurate diagnosis of the condition is complicated 
due to its asymptomatic presentation and requirement of a liver biopsy (5, 6). Further exacerbating 
this epidemic is that there are currently no Food and Drug Administration-approved treatments for 
NASH, other than weight management strategies. Major risk factors associated with NASH are 
obesity and type 2 diabetes (2, 7-9). Evidence suggests that between 25-94% (10-12) and 55-70% 
(13, 14) of obese and type 2 diabetics, respectively, have NASH. Therefore, intervening during 
obesity, in a prophylactic or preventative manner has the potential to mitigate the long-risk of 
developing NASH downstream of obesity. 
 
Green tea rich confections are a novel catechin delivery system. Green tea is safe when consumed 
as recommended (15). Epidemiological evidence suggests that approximately 10 servings/d of green 
tea (equivalent to 1 g/d of GTE) lowers the risk of liver injury and inflammation (16). Freshly brewed 
green tea is an effective catechin delivery system, and has shown to deliver maximum plasma 
concentrations of catechins (0.19-0.73 M) around 1.5-2.5 hours post-ingestion with levels rapidly 
declining after 3 hours (17, 18). To maintain a sustained plasma catechin level, it would require 
frequent ingestion of green tea, which is neither convenient nor practical for individuals. Previously, 
our lab, in collaboration with the Department of Food Science (Dr. Yael Vodovotz), developed a 
starch-based green tea extract (GTE)-rich confection that effectively delivered 1 g of decaffeinated 
GTE to adults (19). After ingestion of the GTE-rich confections, maximal plasma catechin 
concentrations (0.45-1.68 M) were reached about 2.5 hours post-ingestion. However, there was no 
apparent decline in plasma catechin levels during the 3-hour post-ingestion period (19). Chronic 
exposure of green tea catechins may lead to intestinal adaptations that enhances catechin absorption 

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and metabolism (20). Not only are GTE-rich confections able to adequately deliver catechins, but are 
more appropriate for frequent ingestion and are palatable, and more acceptable compared with 
brewed green tea that tends to be perceived by consumers as astringent or bitter (19, 21). For this 
application, our previously developed GTE-rich confection will be modified to be low-carbohydrate 
and low-calorie compared to our prior formulation that contained starch (50 g) (19). 
 
Catechins undergo extensive biotransformation with varying bioavailability. Green tea is a dietary 
rich source of catechins, comprising up to 30% of the dry weight (22). The four major catechins found 
in green tea are epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), 
and epicatechin (EC) (23, 24). Catechin pharmacokinetics in humans have been thoroughly studied 
(17, 18, 25-30). Maximum plasma catechin concentrations range from 0.07-1.8 M at approximately 
1-3 hours post-ingestion with half-lives of about 2.5-5.7 hours (17, 30). Additionally, catechins 
undergo extensive biotransformation, mainly through glucuronidation, sulfation, methylation, and 
ring-fission pathways in the liver and small and large intestines (31-35). EGCG appears primarily in 
the free form (unconjugated) in the blood (18, 26), while more than 80% of the catechins are in 
conjugated forms (25). Suggesting that the conjugated forms are more bioavailable than their parent 
compounds. Furthermore, unabsorbed catechins are metabolized by gut microbiota to ring fission 
products (e.g. valerolactones) and phenolic acids (e.g. hydroxybenzoic acid) that appear in the blood 
and urine of humans (32, 34, 36). Although these microbial metabolites are mostly prevalent in urine 
(34), maximal plasma concentrations have been found in levels greater than 0.2 M (37). The 
bioactivity of conjugated and catechin-derived microbial metabolites have been limitedly study, but 
evidence suggests potential antiinflammatory properties (33, 38, 39). Although the metabolism and 
pharmacokinetics of green tea catechins are well studied, most of these studies have been conducted 
in healthy adults. The extent to which catechin pharmacokinetics and metabolism are influenced by 
obesity and altered by chronic green tea ingestion remains unknown and will be addressed in this 
application. 
 
Green tea attenuates metabolic endotoxemia and improves gut barrier function. Obesity-mediated 
NASH is implicated through a mechanism involving the gut-liver axis. A key mechanism involves 
binding of the ligand endotoxin to Toll-like receptor 4 (TLR4), which in turn induces pro-
inflammatory signaling leading to NFB activation and hepatic inflammation. The gut microbiota is 
the major source of endotoxin, where it is found in the outer membrane of Gram-negative bacteria 
(40). Translocation of gut-derived endotoxin to the liver is primarily due to increased intestinal 
permeability, mediated in part by alterations in gut microbiota composition (41, 42). Our preclinical 
studies show that GTE treatment attenuates hepatic NFB activation and lowers systemic and portal 
vein endotoxin, otherwise increased in obese mice with NASH (43). Additionally, we show that GTE 
improves gut barrier function by restoring expression of small intestinal tight junction proteins 
(occludin, zonal occuldin-1, claudin) to similar levels of healthy controls (43). Studies in humans 
support our findings, showing obese and NASH patients have increased endotoxin and gut 
permeability compared to healthy individuals (44-46). Thus, rationale exists to examine GTE 
ingestion as a dietary strategy to mitigate metabolic endotoxemia by restoring intestinal barrier 
function.  
 
Green tea alters gut microbiota composition. Emerging evidence has highlighted the important role 
of the gut microbiota in the development of NASH (47, 48). Green tea catechins and the host 
microbiota exhibit a reciprocal relationship, where catechins are metabolized to more bioavailable 
metabolites by the microbiota and those catechins also alter the microbiota composition (49). Studies 
in mice showed that a green tea infusion and fermented green tea mitigate obesity and NASH 
symptoms (50, 51). Both studies showed distinct alterations in microbiota composition in the obese 
mice, specifically increases in the Firmicutes/Bacteroidetes ratio, typically associated with obesity 

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and type 2 diabetes. However, only the fermented green tea restored the Firmicutes/Bacteroidetes 
and Bacteroidetes/Prevotella ratios to that of control mice. Green tea catechins modulate the gut 
microbiota possibly through prebiotic (52-54) and/or antimicrobial (55-57) mechanisms. In vivo and 
in vitro studies have shown the beneficial impact of green tea in inhibiting and reducing pathogenic 
bacteria populations (e.g. Clostridium perfringens, E. coli) and promoting beneficial/commensal 
bacteria (e.g. Bifidobacterium spp., Lactobacillus sp.) growth (55, 56). These data support the health-
promoting effects of green tea on modulating gut microbiota composition.  
 
In conclusion, evidence shows that 1) obesity and type 2 diabetes are associated with NASH, 2) green 
tea-rich confections are an effective catechin delivery system, 3) catechins are considerably 
biotransformed, and 4) green tea is effective in attenuating metabolic endotoxemia, improving gut 
barrier function, and modulating gut microbiota composition. Our team has the expertise and 
experience to safely conduct pharmacokinetic trials and perform controlled interventions to assess 
metabolic parameters, gut barrier permeability, and gut microbiota composition. Thus, conducting 
this innovative study will further advance the scientific knowledge of green tea in humans and help 
to establish timely dietary recommendations that reduce inflammation in association with obesity.   
 
Anticipated Results 
Our clinical studies showed that a novel GTE-rich confection was an effective delivery system for 
catechins (19). We expect the GTE-rich confections to retain its effectiveness, even after modifying 
its composition to create a low carbohydrate and low calorie snack food product. Pharmacokinetic 
studies of green tea catechins have been extensively studied in healthy individuals. However, the 
extent to which catechin pharmacokinetics are altered in obese individuals has not been reported. We 
anticipate that obese individuals will have impaired catechin pharmacokinetic parameters (e.g. 
bioavailability, half-life) compared with healthy adults. Furthermore, prior studies in our lab show 
that GTE ameliorates hepatic inflammation in obese mice with NASH by improving gut barrier 
function and limiting gut-derived endotoxin translocation (43, 58). Based on these preclinical 
findings, we expect that obese individuals will have increased endotoxin levels and gut permeability 
compared to healthy controls and that chronic GTE ingestion will mitigate these parameters. Finally, 
evidence in animals (59, 60) and humans (61, 62) suggest a reduced microbial diversity and increased 
Firmicutes/Bacteroidetes ratio is associated with an obese phenotype. In addition, obese and NASH 
patients showed increased abundance of Gram-negative bacteria and higher levels of endotoxin 
compared to healthy individuals (63). Therefore, we expect that obese individuals will exhibit a less 
diverse microbiota composition, greater abundance of Firmicutes compared to Bacteroidetes, and 
increased abundance of Gram-negative bacteria and chronic GTE consumption will modulate these 
effects.  
 
Pitfalls and Alternatives 
Our lab has successfully conducted previous controlled intervention studies, and the techniques 
proposed for this study have been well established in our laboratory. Therefore, we do not expect to 
experience any technical problems or difficulties in this application.  
 
Significance of the Research and Potential Benefits 
NAFLD is the most prevalent liver disease in the U.S. affecting 80-100 million Americans (64), 
which is largely associated with obesity and type 2 diabetes (6, 10-14, 65). Although several 
mechanisms are involved in the development of NASH from NAFLD, metabolic endotoxemia due 
to increased gut barrier permeability and alterations in gut microbiota are highly implicated. Findings 
of this application are expected to provide the first evidence using a novel GTE-rich confection (i.e. 
snack food) to assess catechin pharmacokinetics in association with improvements in metabolic 
endotoxemia, gut permeability, and gut microbiota composition to improve gut health, otherwise 

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impaired in obese individuals. Thus, GTE-mediated improvements in obese subjects are expected to 
provide support for future translational studies in NASH patients.  
 
III. 
Procedures 
 
A. Research Design 
We will conduct a randomized, double-blind placebo controlled trial in obese and healthy individuals. 
We will enroll overweight/obese (BMI = 28-40 kg/m2) and age- and gender-matched healthy (BMI 
19-24 kg/m2) men and women (18-50 y; n = 20/group). Participants will be block randomized to 
receive GTE-rich (1 g GTE) or placebo confections and will be asked to consume the confections 
daily for 4 wk. Prior to (0 wk) and following 4-wk intervention, we will collect fasting blood, 24-
hour urine, and 3-d fecal samples. Additionally, at the beginning (0 wk), participants will perform a 
sugar probe test to assess gut permeability and a 12-h pharmacokinetics trial to assess green tea 
catechin absorption, metabolism, and elimination. At the end of the intervention (4 wk), participants 
will perform a second sugar probe test to assess gastrointestinal changes associated with chronic GTE 
supplementation. Blood collected at wk 0, 2, and 4 will be used to assess blood chemistries (i.e. liver 
function, glucose, insulin, insulin resistance, markers of inflammation). In addition, blood pressure, 
anthropometrics, and 3-d diet records will be measured and collected.  
 
GTE-rich Confections. The Bruno and Vodovotz labs have previously developed a starch-based 
GTE-rich confection (19) as a successful catechin delivery system. For this application, we will 
modify this confection to produce a GTE-rich confection that is low calorie and carbohydrate while 
delivering 1 g of decaffeinated GTE. Decaffeinated GTE will be used to minimize potential side 
effects such as excess caffeine consumption and caffeine/drug interactions that may occur. One 
confection will provide about 18 calories. Thus, 6 confections will provide about 110 calories per 
day, while providing 1 g of decaffeinated GTE. The catechin profile 82% catechins (w/w); 63% 
EGCG, 18% EGC, 10% ECG, 9% EC as we verified (19) is similar to that of freshly brewed green 
teas. All ingredients used will be food grade, meaning they are food certified safe for human 
consumption. The formulation consists of water, sucrose, gelatin, citric acid, and lime flavoring for 
treatment blinding. Sucrose is the main carbohydrate source because sugar alcohols alter microbiota 
composition (66). Placebo confections will be made identical to GTE-rich confections, but without 
GTE. Confections will have a similar sweet taste, soft texture, and consistency as commercially 
available lime-flavored Jell-O®. To deliver 1 g GTE, 6 confections (~1 cm3) will be consumed each 
day throughout the intervention; 2 with each meal. Confections will be packaged into single-serve, 
oxygen impermeable containers, and coded for study blinding. Participants will be instructed to 
refrigerate the confections to maintain freshness and quality.  
 
B. Sample 
 
Enrollment Criteria. Overweight/obese men and women are the ideal study population because of 
its close association with NASH (6, 10-14, 65). Obese/overweight participants will be required to 
meet these inclusion criteria: a) overweight/obese (BMI = 28-40 kg/m2), b) 18-50 y of age, c) fasting 
glucose < 126 mg/dL, d) normotensive (blood pressure < 140/90 mmHg), e) non-dietary supplement 
user, f) nonsmoker. We will also enroll age- and gender-matched healthy adults who meet the 
following criteria: a) normal weight (BMI 19-24 kg/m2), b) 18-50 y of age, c) normoglycemic (< 100 
mg/dL), d) normolipidemic (total cholesterol < 240 mg/dL and triglyceride < 150 mg/dL), e) 
normotensive (blood pressure < 140/90 mmHg). Those having any of these exclusion criteria will 
not be enrolled: a) regular tea drinkers (Camellia sinensis or herbal varieties; ≥ 2 cups/wk), b) 
vegetarians, c) use of any medications to manage diabetes, hypertension, or hyperlipidemia (e.g. 
statins, metformin, ACE inhibitors), d) use of any medications known to be contraindicated for use 
with green tea ingestion (e.g. antipsychotic medications [Clozapine, lithium, Diazepam]), blood 

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thinning medications [Warfarin]), e) user of dietary supplements, prebiotics or probiotics, f) recent 
use of antibiotics or antiinflammatory agents, g) women who are pregnant or lactating, or have 
initiated or changed birth control in the past 3-months, h) individuals with any gastrointestinal 
disorders or surgeries, i) hemochromatosis, j) alcohol intake > 3 drinks/day, k) any history of cancer.  
 
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 through use of ResearchMatch (OSU CCTS). The posted advertisements will 
instruct interested participants to 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 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). Recruiting efforts through ResearchMatch will utilize a 
strategy whereby registered individuals in the ResearchMatch database can be searched for against 
their non-identifiable volunteer profile in the system. Unidentified individuals meeting search 
criteria will then be forwarded an electronic recruitment message (see ResearchMatch Recruitment 
Message attachment) that identifies them as a potential match for study participation. The secure 
ResearchMatch clearinghouse will route this standard notification that provides specific study 
content (i.e. content similar to that of a posted advertisement) to each of these potential 
ResearchMatch volunteers who will then have the option of replying “yes”, “no”, or not respond 
through a set of quick links available in this notification.  Note: This message will not include the 
study’s direct contact information (e.g. email, phone) as ResearchMatch will measure the response 
rate through the clearinghouse’s quick links made available in this electronic message. The 
response rate metrics will be made available to researchers through their ResearchMatch dashboard 
as well as the Institutional Liaison dashboards. By responding “yes”, the volunteer has authorized 
ResearchMatch to release their contact information to the researcher responsible for the study. This 
information will be made available on the researcher’s ResearchMatch study dashboard. The 
researcher will be responsible for managing this contact information as specified in the IRB-
approved study protocol. ResearchMatch will also be collecting aggregate data regarding the status 
of ResearchMatch volunteers within the study. ResearchMatch volunteers consent to this within the 
ResearchMatch volunteer agreement. The ResearchMatch enrollment continuum will allow 
researchers to indicate where the volunteer currently stands within the recruitment process and thus 
helps researchers monitor the utility and effectiveness of this recruiting tool. Research access to 
recruit through ResearchMatch will last only as long as the duration of IRB-study approval with the 
expiration date of ResearchMatch being identical to the end-date of OSU IRB approval. 
Researchers will be able to submit current IRB-approval letters for the lifetime of the study and 
thus provide evidence of successful continuing review applications. If an unintentional lapse in time 
occurs and the research is not able to submit this continuing review evidence via ResearchMatch, 
stored ResearchMatch data will not be deleted, but the researcher will not have access to this 
information until a current IRB-approval letter is uploaded and routed to the Institutional Liaison 
for review. A complete description of ResearchMatch along with the most current IRB approval 
from Vanderbilt University (i.e. this is the site where ResearchMatch was developed and its secure 
computer servers are housed) has been attached. 
 

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Power Calculation and Data Analysis. A power calculation was performed using serum endotoxin 
(lipopolysaccharide; LPS) as the primary outcome. Studies in obese (44, 67, 68) adults and 
individuals with metabolic syndrome (69, 70) show increased serum LPS or LPS-binding protein 
compared to healthy controls. The known difference in metabolic endotoxemia between obese and 
healthy adults was used to predict an expected 50% improvement by GTE treatment. Based on a 
linear model with treatment and health status, we will have >90% power to detect the primary 
contrast of interest (GTE vs. placebo in obese individuals) and additionally 80% power to detect an 
interaction between treatment and health status (α=0.05). Thus, we plan to recruit 20 obese and 20 
healthy adults for our study. Data from this investigation are expected to be normally distributed or 
achieved through appropriate transformation. Most data will be analyzed by repeated measures 
ANCOVA to consider effects due to health status, gender, GTE treatment, and their interactions. 
Multivariate regression analysis will be performed to define pairwise correlations between study 
variables in the presence or absence of potential study covariates. Statistical significance for all 
analyses will be set at P < 0.05. 
 
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/Urine Chemistries. Fasting plasma triglyceride, total cholesterol, and glucose will be 
measured by clinical assay (Pointe Scientific) at screening and throughout the study. Fasting insulin 
will be measured by ELISA (ALPCO) during the 4-wk study. Serum alanine aminotransferase 
(ALT) and urinary creatinine will also be measured throughout the study (Pointe Scientific).  
 
Diet Assessment. Participants will be instructed to maintain a low-polyphenol diet throughout the 
study. Participants will record any consumption of polyphenol-rich foods throughout the study (a 
record sheet will be provided, see Polyphenol Intake Record attachment). Participants will be 
instructed to abstain from polyphenol-rich foods including tea of any kind, coffee, fruits, fruit 
juices, chocolate, nuts, legumes, and wine (a list will be provided, see List of Polyphenol-Rich 
Foods attachment). Food records (3-d) will be evaluated at wk 0, 2, and 4 using NDSR software 
and the NCC Flavonoid and Proanthocyanin database to assess energy, nutrient, and flavonoid 
intakes as we described (19, 71). 
 
Plasma and Urine Catechin and Catechin-metabolites. To assess the absorption, metabolism, and 
excretion of green tea catechins during the pharmacokinetics trial (wk 0), plasma and urine catechin 
and catechin-metabolites will be measured by liquid chromatography-mass spectrometry (LC-MS). 
Green tea catechins (EGCG, ECG, EGC, EC) and their metabolites (e.g. valerolactones) will be 
measured using an adapted LC-MS method (72).  
 
Metabolic Endotoxemia. Serum endotoxin will be measured at wk 0, 2, and 4 using a commercially 
available fluorometric assay that assesses endotoxin-mediated activation of recombinant Factor C 
and subsequent cleavage of a flurogenic substrate (PyroGene rFC; Lonza) as we detailed (43).  
 
Gastrointestinal (GI) Permeability. A 4-sugar probe procedure appropriate for human clinical 
study will be performed as described (73). In brief, at wk 0 and 4, participants in the fasted state 
will ingest a 500-mL solution of sucrose (40 g), lactulose (5 g), mannitol (1 g) and sucralose (1 g) 
dissolved in water. Urine will be collected over a 24-hour period to assess GI permeability. 
Standardized sucrose-free meals (i.e. plain bagel, cream cheese, scrambled eggs, potato chips) will 

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be provided during the urine collection duration. Urinary sugars will be measured by LC-MS and 
permeability calculated as urinary excretion (%) and excretion ratios for sugars from 0-5 hours and 
6-24 hours to reflect upper and lower GI permeability, respectively (74-77).  
 
Inflammation. During the 4-wk intervention, we will define systemic and intestinal inflammation 
and the effects of GTE consumption. Circulating C-reactive protein and myeloperoxidase will 
provide information as to systemic inflammation, and fecal calprotectin will provide information 
regarding intestinal inflammation (78). These inflammatory markers will be measured using 
commercially available ELISA kits as we described (70). Additionally, we plan to perform 
polymerase chain reaction experiments to assess pro-inflammatory gene expression of white blood 
cells. 
 
Microbiota Composition. Microbiota composition will be assessed from feces collected from 3 
days during week 0 and 4 using previously described procedures (79-83). Total bacterial abundance 
will be determined by qPCR (84). Microbiota diversity and community structure will be determined 
for each sample by sequencing 16S rRNA genes using MiSeq. Bacteria will be identified by 
comparing sequences representing each operation taxonomic unit (OTU) with databases. 
Multivariate analysis techniques will be used to determine differences in obese and healthy 
individuals the effects of GTE.  
 
D. Detailed Study Procedures 
 
Overview of Study Procedures. Potential participants who call the study center in an anonymous 
manner for more information as well as those identified through ResearchMatch 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. Women choosing to participate will also be asked to 
self-administer a urinary pregnancy test. If women self-report that the pregnancy test is negative, 
they will continue with the screening process. 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 be invited to meet with a registered 
dietitian or trained research personnel who will instruct them on how to complete a dietary food 
record, explain the details regarding following a low-polyphenol diet throughout the intervention, 
and provide instruction of foods to avoid prior to the pharmacokinetic trial at wk 0. Prior to the 
initial pharmacokinetics trial, participants will be asked to collect 3 days of fecal samples and return 
to the study center to participate in a sugar probe test. After completing the sugar probe test, 
participants will return to the study center at least 3 days after to complete a 12-h pharmacokinetics 
trial. Afterwards, participants will be provided a 2-wk supply of GTE-rich or placebo confections. 
After 2 wk, fasting blood will be collected from the participants and the other 2-week supply of 
confections will be provided for the remainder of the study. Upon completing the 4-week 
intervention, participants will complete a second sugar probe test. We estimate that completion of 
all study procedures will take 6-8 wk per participant. Each step of the study procedure will be 
discussed in detail below. See Study Design attachment for an overview of the study. 

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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 
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 have the opportunity to 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, blood pressure, or plasma 
chemistries (see Enrollment Criteria) do not meet the study criteria. All female participants will be 
asked to undergo a self-administered pregnancy test using a pregnancy urine test strip to ensure that 
only non-pregnant women are included in the study. 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 (10 mL; 1 tube) so that we may measure blood chemistries (glucose, cholesterol, 
triglyceride). All samples will be coded to maintain participant anonymity. If the participant’s 
anthropometrics, blood pressure, or plasma chemistries do not meet the study criteria, they will be 
told that 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, blood pressure, 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’s BMI is 27 kg/m2, which is outside 
the specified BMI inclusion range for overweight/obese participants 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: I have your screening results. 
“Congratulations, you meet the eligibility criteria for our clinical trial. Your body measurements, 
blood pressure, and fasting blood chemistries of glucose, triglyceride, and cholesterol were all 
within the specified range. Your glucose falls in the [category], triglyceride falls in the [category], 
and cholesterol falls in the [category]. A summary of your results has been prepared for you, which 
you will receive when you come in for your trials. We look forward to and are thankful for your 
participation.” Potential participants who do not qualify will be provided the following message: “I 
have your screening results. Unfortunately, you do not meet the eligibility criteria for our clinical 
trial. Your body measurements, blood pressure, glucose, triglyceride, and/or cholesterol were not 
within the eligible range. Your glucose falls in the [category], triglyceride falls in the [category], 
and cholesterol falls in the [category]. A summary of your results has been prepared for you. Please 
let me know if you would like to drop by the study center to collect a hard copy of your information 
or if you would like them emailed to you. Thank you so much for your time and effort.” All 
individuals regardless of study eligibility will be encouraged to consider sharing these results with 
their physician. 
 
In the event that 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 

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time. No confidential or sensitive information will be shared with third parties or left on answering 
machines. 
 
Dietary Meeting. Eligible participants who meet the study inclusion criteria, a dietary meeting will 
be scheduled with a registered dietitian or trained individual (i.e. nutritional sciences graduate 
student) from the research team. During the dietary meeting, the dietitian/trained individual will 
show the participant how to properly complete the 3-d diet record (see Diet Record Form 
attachment). Participants are expected to follow a low-polyphenol diet throughout the intervention 
to avoid potential confounding effects on the study outcomes. The dietitian/trained individual will 
clearly identify polyphenol-rich foods/drinks participants should avoid during the study and inform 
them to record if they accidently consume any polyphenol-rich foods/drinks during the study (see 
List of Polyphenol-Rich Foods and Polyphenol Intake Record attachments). The dietitian/trained 
individual will inform the participant of key foods to avoid, which includes any type of tea, coffee, 
fruits, fruit juices, chocolate, nuts, legumes, and wine. These foods are known to be rich in 
polyphenols, and consumption may confound the interpretation of the pharmacokinetics trial and 
intervention. In addition to dietary advice, the participant will be informed of the proper way to 
collect their urine and feces using the provided sterile collection containers. Fecal samples will be 
collected by the participant from 3 days prior to the pharmacokinetics trial. Participants will be 
provided a cooler with ice to store their fecal samples for up to 24 hours. The participant will then 
return their samples to the study center or coordinate with study personnel to meet at a public and 
mutual location and return their samples within 24 hours from collection.  
 
The participant will be instructed to abstain from exercise for 24 hours prior to their sugar probe 
test and pharmacokinetics trial. For each visit to our study center, participants will be instructed to 
be fasted for 10-12 hours to ensure reliable blood sampling. Participants will be instructed to 
complete the dietary record for 3-d before the pharmacokinetics trial and second sugar probe test 
(wk 0 and 4) and their wk 2 study center visit. 
 
Test Trials. Participants will be block randomized into two groups to receive either a GTE-rich or 
placebo (without GTE) confection. Participants will complete a pharmacokinetics trial prior to 
starting the intervention. Prior to (wk 0) and following 4-wk intervention, participants will collect 3 
days of fecal samples and complete a sugar probe test. For this study, each participant will visit the 
study center a total of 6 times, including the screening and dietary meetings, to complete individual 
test trials. For each study center visit, participants will report in a fasted state (10-12 hours).  
 
Prior to the first study visit, participants will complete a 3-d diet record. At the first study visit, 
participants will perform a sugar probe test. This test will consist of participants ingesting a test 
beverage of 40 g sucrose, 5 g lactulose, 1 g mannitol, and 1 g sucralose dissolved in water (500 
mL). After ingestion, the participants will be provided a standardized sucrose-free meal within the 
first 5 hours and water throughout. Urine will be collected from 0-5 and 6-24 hours in provided 
containers.  
 
The second study visit will be scheduled at least 3 d after the sugar probe test. The second study 
visit will consist of a 12-h pharmacokinetics trial. Participants will ingest one-half the daily GTE-
rich confections (0.5 g GTE). Blood will then be collected at the following hourly time points: 0 
(before confection ingestion), 0.25, 0.5, 1, 2, 3, 5, 8, 10, 12 h. Urine will be collected between 0-4, 
4-8, 8-12, and 12-24 h post-ingestion into provided urinary collection containers. During the 
pharmacokinetics trial, participants will be allowed free access to water and provided a 
standardized-polyphenol free meal.  
 

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Upon completing the pharmacokinetics trial, the intervention phase will begin. Participants will be 
provided a 2-week supply of GTE-rich or placebo confections. They will be advised to consume 6 
confections/ every day (2 confections/meal; 1 g GTE/d) for the following 4-wk. After 2 wk of the 
intervention phase, participants will complete a 3-day diet record and return for their third study 
visit. At this visit, fasting blood will be collected. Participants will then receive their final 2-wk 
supply of confections. After 4 wk, participants will complete a final 3-d diet record and perform the 
sugar probe test once again as described above. During each study visit, participants will have 
access to drinking water and use of the restroom as needed.  
 
Sugar Probe Test. On the morning of testing at wk 0 and 4, participants will arrive at the study 
center after abstaining from food and only consuming water for 10-12 hours. A sugar probe test will 
be conducted as described (73). Upon arrival, participants will be asked to empty their urinary 
bladder. Subsequently, they will then be asked to ingest a drink containing 40 g sucrose, 5 g 
lactulose, 1 g mannitol, and 1 g sucralose dissolved in water (500 mL). Within 2-hours after 
ingesting the sugar test beverage, participants will be asked to consume an additional 500 mL of 
water. Additionally, participants will be provided with standardized sucrose-free meals during the 
24-h period. All foods consumed during the 24-hour period will be free of artificial sweeteners. 
Urine will be collected from 0-5 and 6-24 hours in sterile containers. For each urinary collection 
period, participants will be provided 2-L urine collection bottles and a Styrofoam cooler with ice 
packs to store collected urine until they return their samples the next day. 
 
Pharmacokinetics Trial. Within 3-d after completing the first sugar probe test, participants will 
return to the study center to perform a 12-h pharmacokinetics trial. At least 3-d prior to the 
pharmacokinetics trial, participants will abstain from polyphenol-rich foods and drinks, including 
any type of tea, coffee, fruits, fruit juices, any chocolate (dark, milk), nuts, legumes (soy), and 
wine. Participants will arrive to the study center after abstaining from food and only consuming 
water for 10-12 hours. Participants will be provided a 15-min rest period, prior to the first blood 
collection. During the rest period, the participants will sit in a phlebotomy chair and a flexible 
catheter will be inserted into a forearm vein by a trained phlebotomist. After a 15-min sitting rest 
period, a blood sample will be obtained from a 3-way stopcock connected to the end of the 
catheter. Blood will be collected with a syringe and transferred to appropriate tubes for processing 
and subsequent determination of biochemical measurements. Following the first blood collection, 
participants will consume 3 GTE-rich confections (0.5 g GTE). Blood samples will be collected at 
0 (prior to ingestion), 0.25, 0.5, 1, 2, 3, 5, 8, 10, and 12 h. Additionally, urine will be collected 
between 0-4, 4-8, 8-12, and 12-24 h. During the 12 h trial period, participants will be able to 
freely move and walk around if they choose to, as long as they stay in close proximity to the study 
center. We will provide a dedicated office space for participants to sit down in between blood 
draws. If the participant chooses to move around, we will wrap their arm with a bandage to 
minimize any risks and ensure the catheter stays in place to prevent the need to insert a needle 
again. Additionally, a person of the research team will escort and monitor the participant if they 
choose to move around the study center. After each blood sample is obtained, the catheter will be 
flushed with saline to prevent the formation of clots and to minimize the likelihood of having to 
insert a needle again. At 4 hours, each participant will be provided with a standardized 
polyphenol-free meal. A trained phlebotomist will perform all cannula insertions, needle sticks, 
and blood collections. After the 12 h trial, participants will be provided with a light snack (e.g. 
granola bar) and prepared standardized polyphenol-free meals to consume for the remainder of the 
24-h urine collection period. Participants will also be provided with a 2-wk supply of GTE-rich or 
placebo confections. Participants will be advised to consume 6 confections each day (2 per meal; 
1 g GTE/d) for the entire 4-week intervention study. Participants will be contacted periodically by 
email, text, or phone, by research personnel to provide polite reminders to enhance study 
compliance. 

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Intervention Phase / Test Confections. After completing the pharmacokinetics trial, participants 
will partake in a 4 wk intervention. Participants will be block randomized to receive a GTE-rich or 
placebo (no GTE containing) confection. Confections will be low carbohydrate and low calorie. 
Each serving (6 g) provides <15 kcal, while delivering 1 g of decaffeinated GTE. The catechin 
profile (82% catechins (w/w); 63% EGCG, 18% EGC, 10% ECG, 9% EC) is similar to that of 
freshly brewed green teas. Sucrose will be the main carbohydrate source because sugar alcohols 
alter microbiota composition (66) and would confound interpretation. Decaffeinated GTE was 
chosen to minimize potential side effects to prevent excess caffeine consumption or caffeine/drug 
interactions that could result during comorbidity management of metabolic conditions. Placebo 
confections will be identical to GTE-rich confections, but without GTE. Confections will be 
packaged in single-serve, oxygen impermeable containers, and coded for study blinding. 
Participants will be asked to store the confections in the refrigerator to maintain quality and 
freshness.  
 
Participants will be asked to consume 6 confections each day (2 with each meal) for 4 weeks. 
Consumption of 6 GTE-rich confections is equivalent to delivering 1 g GTE/d. Participants will 
receive a 2-wk supply of confections after completing the pharmacokinetics trial. Halfway through 
the intervention (wk 2), participants will visit the study center in a fasted state (10-12 h). 
Participants will return any leftover confections. After a 5 min stabilization period, a single fasting 
blood sample will be collected following procedures stated above. After collecting blood, the 
participants will be provided the final 2-wk supply of confections. At the conclusion of the 
intervention phase (wk 4), participants will perform a second sugar probe test. Compliance will be 
evaluated based on the number of confections returned throughout the study.  
 
Sampling Handling. During the pharmacokinetics trial, at each time point, a blood sample (20 mL; 
1.4 tablespoons x 10 time points = 200 mL or 0.85 cups) will be collected into evacuated blood 
collection tubes. Collecting a total of 200 mL or 0.85 cups at the pharmacokinetics trial is necessary 
to ensure adequate amount of plasma for each time point to accurately analyze the green tea 
catechin metabolites over the 12-hour period. During blood collection, participants may feel an 
initial pain when inserting the needle, bruising around the insertion area, lightheadedness, or 
fainting, which are common when donating blood. However, we do not foresee any additional 
significant risks for collecting this amount of blood over 12 hours, other than the risks stated 
previously.  
 
Throughout the span of the study (~6-8 weeks in duration depending on participant/investigator 
availability) which consists of one acute ingestion of GTE-rich confections, a blood collection at 
wk 2 and 4, and 1 screening day, we will be collecting a total of ~255 mL or ~1.08 cups) of blood. 
Urine will be collected in provided containers (VWR) containing 10% thymol to inhibit bacterial 
growth. Feces will be collected using a commercial commode specimen collection system (Fisher 
Scientific). Briefly, the collection kit consists of the necessary materials (e.g. disposable spatula, 
gloves, waste bag) for participants to easily and hygienically collect their stool without 
contaminating the sample or themselves. We will provide participants with coolers with ice to store 
their fecal samples for up to 24-h after collection. We will then have the participants return their 
fecal samples to the study center or coordinate with study personnel to meet at a public and mutual 
location to return their samples within 24-h after collection. Volumes of urine will be recorded and 
fecal mass and observations will be recorded based on the Bristol Stool Chart (85).  
 
During each blood collection, plasma and white blood cells will be obtained by centrifugation, 
and then transferred to cryogenic storage tubes. Serum samples will be obtained by allowing the 
blood to clot, followed by centrifugation and transfer to cryogenic storage tubes. Tubes will be 

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stored at -80° C until analysis can be completed. Analyses will include plasma glucose, insulin, 
triglyceride, total cholesterol, catechins (EGCG, ECG, EGC, EC), catechin-metabolites (e.g. 
valerolactones, catechin-derivatives), C-reactive protein, myeloperoxidase, pro-inflammatory 
gene expression and serum endotoxin and ALT. Urine will be stored at -80ºC until analysis can be 
completed. Analysis from urine will include catechins, catechin-metabolites, sucrose, mannitol, 
lactulose, sucralose, and creatinine. Fecal samples will be stored at -80ºC until analysis can be 
completed. Feces analysis will include microbiota composition and calprotectin. Remaining 
plasma, serum, urine, and fecal samples not used for these analyses will be archived for 5 years at 
-80 C in the event we decide to measure additional inflammatory, antioxidant, or microbiota 
related markers. 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 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 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. Test confections will be assigned a code as well. This will minimize measurement bias 
when performing analysis on dietary records, and biochemical markers because all samples/records 
will be coded. The codes will only be broken once data analysis has been completed and verified by 
the PI. 
 
IV. 
Literature Cited 
 
1. 
Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, 
Chabo C, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 
2007;56(7):1761-72. doi: 10.2337/db06-1491. 
2. 
Angulo P. Obesity and nonalcoholic fatty liver disease. Nutr Rev 2007;65(6 Pt 2):S57-63. 
3. 
Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol 
2013;10(11):686-90. doi: 10.1038/nrgastro.2013.171. 
4. 
Vizuete J, Camero A, Malakouti M, Garapati K, Gutierrez J. Perspectives on Nonalcoholic Fatty 
Liver Disease: An Overview of Present and Future Therapies. J Clin Transl Hepatol 2017;5(1):67-
75. doi: 10.14218/JCTH.2016.00061. 
5. 
Ahmed A, Wong RJ, Harrison SA. Nonalcoholic Fatty Liver Disease Review: Diagnosis, 
Treatment, 
and 
Outcomes. 
Clin 
Gastroenterol 
Hepatol 
2015;13(12):2062-70. 
doi: 
10.1016/j.cgh.2015.07.029. 

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 13
6. 
Lopez-Velazquez JA, Silva-Vidal KV, Ponciano-Rodriguez G, Chavez-Tapia NC, Arrese M, Uribe 
M, Mendez-Sanchez N. The prevalence of nonalcoholic fatty liver disease in the Americas. Ann 
Hepatol 2014;13(2):166-78. 
7. 
Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural history of 
non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol 
Ther 2011;34(3):274-85. doi: 10.1111/j.1365-2036.2011.04724.x. 
8. 
Boza C, Riquelme A, Ibanez L, Duarte I, Norero E, Viviani P, Soza A, Fernandez JI, Raddatz A, 
Guzman S, et al. Predictors of nonalcoholic steatohepatitis (NASH) in obese patients undergoing 
gastric bypass. Obes Surg 2005;15(8):1148-53. doi: 10.1381/0960892055002347. 
9. 
Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ. The 
diagnosis and management of non-alcoholic fatty liver disease: practice Guideline by the American 
Association for the Study of Liver Diseases, American College of Gastroenterology, and the 
American 
Gastroenterological 
Association. 
Hepatology 
2012;55(6):2005-23. 
doi: 
10.1002/hep.25762. 
10. 
Dixon JB, Bhathal PS, O'Brien PE. Nonalcoholic fatty liver disease: predictors of nonalcoholic 
steatohepatitis and liver fibrosis in the severely obese. Gastroenterology 2001;121(1):91-100. 
11. 
Crespo J, Fernandez-Gil P, Hernandez-Guerra M, Cayon A, Mayorga M, Dominguez-Diez A, 
Fernandez-Escalante JC, Pons-Romero F. Are there predictive factors of severe liver fibrosis in 
morbidly obese patients with non-alcoholic steatohepatitis? Obes Surg 2001;11(3):254-7. doi: 
10.1381/096089201321336548. 
12. 
Adams LA, Lindor KD. Nonalcoholic fatty liver disease. Ann Epidemiol 2007;17(11):863-9. doi: 
10.1016/j.annepidem.2007.05.013. 
13. 
Bazick J, Donithan M, Neuschwander-Tetri BA, Kleiner D, Brunt EM, Wilson L, Doo E, Lavine 
J, Tonascia J, Loomba R. Clinical Model for NASH and Advanced Fibrosis in Adult Patients With 
Diabetes and NAFLD: Guidelines for Referral in NAFLD. Diabetes Care 2015;38(7):1347-55. doi: 
10.2337/dc14-1239. 
14. 
Targher G, Bertolini L, Padovani R, Rodella S, Tessari R, Zenari L, Day C, Arcaro G. Prevalence 
of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 
diabetic patients. Diabetes Care 2007;30(5):1212-8. doi: 10.2337/dc06-2247. 
15. 
Sarma DN, Barrett ML, Chavez ML, Gardiner P, Ko R, Mahady GB, Marles RJ, Pellicore LS, 
Giancaspro GI, Low Dog T. Safety of green tea extracts : a systematic review by the US 
Pharmacopeia. Drug Saf 2008;31(6):469-84. 
16. 
Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and 
liver diseases. BMJ 1995;310(6981):693-6. 
17. 
Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, Schantz SP. Blood and urine levels of tea 
catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol 
Biomarkers Prev 1998;7(4):351-4. 
18. 
Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, Lambert G, Mohr S, Yang CS. 
Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by 
humans: formation of different metabolites and individual variability. Cancer Epidemiol 
Biomarkers Prev 2002;11(10 Pt 1):1025-32. 
19. 
Sapper TN, Mah E, Ahn-Jarvis J, McDonald JD, Chitchumroonchokchai C, Reverri EJ, Vodovotz 
Y, Bruno RS. A green tea-containing starch confection increases plasma catechins without 
protecting against postprandial impairments in vascular function in normoglycemic adults. Food 
Funct 2016;7(9):3843-53. doi: 10.1039/c6fo00639f. 
20. 
Redan BW, Chegeni M, Ferruzzi MG. Differentiated Caco-2 cell monolayers exhibit adaptation in 
the transport and metabolism of flavan-3-ols with chronic exposure to both isolated flavan-3-ols 
and enriched extracts. Food Funct 2017;8(1):111-21. doi: 10.1039/c6fo01289b. 
21. 
Lee J, Chambers DH. Descriptive analysis and U.S. consumer acceptability of 6 green tea samples 
from China, Japan, and Korea. J Food Sci 2010;75(2):S141-7. doi: 10.1111/j.1750-
3841.2009.01503.x. 

THE OHIO STATE UNIVERSITY 
 
Institutional Review Board 
Version 3 – November 22, 2017 
 
 14
22. 
Chacko SM, Thambi PT, Kuttan R, Nishigaki I. Beneficial effects of green tea: a literature review. 
Chin Med 2010;5:13. doi: 10.1186/1749-8546-5-13. 
23. 
Balentine DA, Wiseman SA, Bouwens LC. The chemistry of tea flavonoids. Crit Rev Food Sci 
Nutr 1997;37(8):693-704. doi: 10.1080/10408399709527797. 
24. 
Sano M, Tabata M, Suzuki M, Degawa M, Miyase T, Maeda-Yamamoto M. Simultaneous 
determination of twelve tea catechins by high-performance liquid chromatography with 
electrochemical detection. Analyst 2001;126(6):816-20. 
25. 
Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, Balentine DA, Yang CS. Analysis of plasma 
and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 1995;4(4):393-
9. 
26. 
Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R, Shahi F, Crowell JA, Yang CS, Hara Y. Phase I 
pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin 
gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 2001;10(1):53-8. 
27. 
Stalmach A, Troufflard S, Serafini M, Crozier A. Absorption, metabolism and excretion of Choladi 
green tea flavan-3-ols by humans. Mol Nutr Food Res 2009;53 Suppl 1:S44-53. doi: 
10.1002/mnfr.200800169. 
28. 
Renouf M, Guy P, Marmet C, Longet K, Fraering AL, Moulin J, Barron D, Dionisi F, Cavin C, 
Steiling H, et al. Plasma appearance and correlation between coffee and green tea metabolites in 
human subjects. Br J Nutr 2010;104(11):1635-40. doi: 10.1017/S0007114510002709. 
29. 
Fung ST, Ho CK, Choi SW, Chung WY, Benzie IF. Comparison of catechin profiles in human 
plasma and urine after single dosing and regular intake of green tea (Camellia sinensis). Br J Nutr 
2013;109(12):2199-207. doi: 10.1017/S0007114512004370. 
30. 
Renouf M, Marmet C, Guy PA, Beaumont M, Lepage M, Williamson G, Dionisi F. Dose-response 
plasma appearance of green tea catechins in adults. Mol Nutr Food Res 2013;57(5):833-9. doi: 
10.1002/mnfr.201200512. 
31. 
Vaidyanathan JB, Walle T. Glucuronidation and sulfation of the tea flavonoid (-)-epicatechin by 
the human and rat enzymes. Drug Metab Dispos 2002;30(8):897-903. 
32. 
Meng X, Sang S, Zhu N, Lu H, Sheng S, Lee MJ, Ho CT, Yang CS. Identification and 
characterization of methylated and ring-fission metabolites of tea catechins formed in humans, 
mice, and rats. Chem Res Toxicol 2002;15(8):1042-50. 
33. 
Lu H, Meng X, Li C, Sang S, Patten C, Sheng S, Hong J, Bai N, Winnik B, Ho CT, et al. 
Glucuronides of tea catechins: enzymology of biosynthesis and biological activities. Drug Metab 
Dispos 2003;31(4):452-61. 
34. 
Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N, Brighenti F. Bioavailability and 
catabolism of green tea flavan-3-ols in humans. Nutrition 2010;26(11-12):1110-6. doi: 
10.1016/j.nut.2009.09.021. 
35. 
Schantz M, Erk T, Richling E. Metabolism of green tea catechins by the human small intestine. 
Biotechnol J 2010;5(10):1050-9. doi: 10.1002/biot.201000214. 
36. 
Roowi S, Stalmach A, Mullen W, Lean ME, Edwards CA, Crozier A. Green tea flavan-3-ols: 
colonic degradation and urinary excretion of catabolites by humans. J Agric Food Chem 
2010;58(2):1296-304. doi: 10.1021/jf9032975. 
37. 
Li C, Lee MJ, Sheng S, Meng X, Prabhu S, Winnik B, Huang B, Chung JY, Yan S, Ho CT, et al. 
Structural identification of two metabolites of catechins and their kinetics in human urine and blood 
after tea ingestion. Chem Res Toxicol 2000;13(3):177-84. 
38. 
Lambert JD, Rice JE, Hong J, Hou Z, Yang CS. Synthesis and biological activity of the tea catechin 
metabolites, M4 and M6 and their methoxy-derivatives. Bioorg Med Chem Lett 2005;15(4):873-6. 
doi: 10.1016/j.bmcl.2004.12.070. 
39. 
Kim YH, Won YS, Yang X, Kumazoe M, Yamashita S, Hara A, Takagaki A, Goto K, Nanjo F, 
Tachibana H. Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell 
and Natural Killer Cell Activities. J Agric Food Chem 2016;64(18):3591-7. doi: 
10.1021/acs.jafc.6b01115. 

THE OHIO STATE UNIVERSITY 
 
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Version 3 – November 22, 2017 
 
 15
40. 
Ramachandran G. Gram-positive and gram-negative bacterial toxins in sepsis: a brief review. 
Virulence 2014;5(1):213-8. doi: 10.4161/viru.27024. 
41. 
Hausmann M. How bacteria-induced apoptosis of intestinal epithelial cells contributes to mucosal 
inflammation. Int J Inflam 2010;2010:574568. doi: 10.4061/2010/574568. 
42. 
Teixeira TF, Collado MC, Ferreira CL, Bressan J, Peluzio Mdo C. Potential mechanisms for the 
emerging link between obesity and increased intestinal permeability. Nutr Res 2012;32(9):637-47. 
doi: 10.1016/j.nutres.2012.07.003. 
43. 
Li J, Sapper TN, Mah E, Moller MV, Kim JB, Chitchumroonchokchai C, McDonald JD, Bruno 
RS. Green tea extract treatment reduces NFkappaB activation in mice with diet-induced 
nonalcoholic steatohepatitis by lowering TNFR1 and TLR4 expression and ligand availability. J 
Nutr Biochem 2017;41:34-41. doi: 10.1016/j.jnutbio.2016.12.007. 
44. 
Ruiz AG, Casafont F, Crespo J, Cayon A, Mayorga M, Estebanez A, Fernadez-Escalante JC, Pons-
Romero F. Lipopolysaccharide-binding protein plasma levels and liver TNF-alpha gene expression 
in obese patients: evidence for the potential role of endotoxin in the pathogenesis of non-alcoholic 
steatohepatitis. Obes Surg 2007;17(10):1374-80. doi: 10.1007/s11695-007-9243-7. 
45. 
Luther J, Garber JJ, Khalili H, Dave M, Bale SS, Jindal R, Motola DL, Luther S, Bohr S, Jeoung 
SW, et al. Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal 
Permeability. 
Cell 
Mol 
Gastroenterol 
Hepatol 
2015;1(2):222-32. 
doi: 
10.1016/j.jcmgh.2015.01.001. 
46. 
Giorgio V, Miele L, Principessa L, Ferretti F, Villa MP, Negro V, Grieco A, Alisi A, Nobili V. 
Intestinal permeability is increased in children with non-alcoholic fatty liver disease, and correlates 
with liver disease severity. Dig Liver Dis 2014;46(6):556-60. doi: 10.1016/j.dld.2014.02.010. 
47. 
Abdou RM, Zhu L, Baker RD, Baker SS. Gut Microbiota of Nonalcoholic Fatty Liver Disease. Dig 
Dis Sci 2016;61(5):1268-81. doi: 10.1007/s10620-016-4045-1. 
48. 
Haque TR, Barritt ASt. Intestinal microbiota in liver disease. Best Pract Res Clin Gastroenterol 
2016;30(1):133-42. doi: 10.1016/j.bpg.2016.02.004. 
49. 
Ozdal T, Sela DA, Xiao J, Boyacioglu D, Chen F, Capanoglu E. The Reciprocal Interactions 
between Polyphenols and Gut Microbiota and Effects on Bioaccessibility. Nutrients 2016;8(2):78. 
doi: 10.3390/nu8020078. 
50. 
Liu Z, Chen Z, Guo H, He D, Zhao H, Wang Z, Zhang W, Liao L, Zhang C, Ni L. The modulatory 
effect of infusions of green tea, oolong tea, and black tea on gut microbiota in high-fat-induced 
obese mice. Food Funct 2016;7(12):4869-79. doi: 10.1039/c6fo01439a. 
51. 
Seo DB, Jeong HW, Cho D, Lee BJ, Lee JH, Choi JY, Bae IH, Lee SJ. Fermented green tea extract 
alleviates obesity and related complications and alters gut microbiota composition in diet-induced 
obese mice. J Med Food 2015;18(5):549-56. doi: 10.1089/jmf.2014.3265. 
52. 
Tzounis X, Vulevic J, Kuhnle GG, George T, Leonczak J, Gibson GR, Kwik-Uribe C, Spencer JP. 
Flavanol monomer-induced changes to the human faecal microflora. Br J Nutr 2008;99(4):782-92. 
doi: 10.1017/S0007114507853384. 
53. 
Axling U, Olsson C, Xu J, Fernandez C, Larsson S, Strom K, Ahrne S, Holm C, Molin G, Berger 
K. Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and 
inflammation in high-fat fed C57BL/6J mice. Nutr Metab (Lond) 2012;9(1):105. doi: 
10.1186/1743-7075-9-105. 
54. 
Jin JS, Touyama M, Hisada T, Benno Y. Effects of green tea consumption on human fecal 
microbiota 
with 
special 
reference 
to 
Bifidobacterium 
species. 
Microbiol 
Immunol 
2012;56(11):729-39. doi: 10.1111/j.1348-0421.2012.00502.x. 
55. 
Okubo T, Ishihara N, Oura A, Serit M, Kim M, Yamamoto T, Mitsuoka T. In Vivo Effects of Tea 
Polyphenol Intake on Human Intestinal Microflora and Metabolism. Biosci Biotechnol Biochem 
1992;56(4):588-91. doi: 10.1271/bbb.56.588. 
56. 
Lee HC, Jenner AM, Low CS, Lee YK. Effect of tea phenolics and their aromatic fecal bacterial 
metabolites 
on 
intestinal 
microbiota. 
Res 
Microbiol 
2006;157(9):876-84. 
doi: 
10.1016/j.resmic.2006.07.004. 

THE OHIO STATE UNIVERSITY 
 
Institutional Review Board 
Version 3 – November 22, 2017 
 
 16
57. 
Reygaert WC. The antimicrobial possibilities of green tea. Front Microbiol 2014;5:434. doi: 
10.3389/fmicb.2014.00434. 
58. 
Li J, Sapper TN, Mah E, Rudraiah S, Schill KE, Chitchumroonchokchai C, Moller MV, McDonald 
JD, Rohrer PR, Manautou JE, et al. Green tea extract provides extensive Nrf2-independent 
protection against lipid accumulation and NFkappaB pro- inflammatory responses during 
nonalcoholic steatohepatitis in mice fed a high-fat diet. Mol Nutr Food Res 2016;60(4):858-70. 
doi: 10.1002/mnfr.201500814. 
59. 
Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut 
microbial 
ecology. 
Proc 
Natl 
Acad 
Sci 
U 
S 
A 
2005;102(31):11070-5. 
doi: 
10.1073/pnas.0504978102. 
60. 
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated 
gut microbiome with increased capacity for energy harvest. Nature 2006;444(7122):1027-31. doi: 
10.1038/nature05414. 
61. 
Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, Sogin ML, Jones WJ, 
Roe BA, Affourtit JP, et al. A core gut microbiome in obese and lean twins. Nature 
2009;457(7228):480-4. doi: 10.1038/nature07540. 
62. 
Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated 
with obesity. Nature 2006;444(7122):1022-3. doi: 10.1038/4441022a. 
63. 
Yuan J, Baker SS, Liu W, Alkhouri R, Baker RD, Xie J, Ji G, Zhu L. Endotoxemia unrequired in 
the pathogenesis of 
pediatric 
nonalcoholic 
steatohepatitis. J 
Gastroenterol 
Hepatol 
2014;29(6):1292-8. doi: 10.1111/jgh.12510. 
64. 
Sass DA, Chang P, Chopra KB. Nonalcoholic fatty liver disease: a clinical review. Dig Dis Sci 
2005;50(1):171-80. 
65. 
Younossi ZM, Stepanova M, Afendy M, Fang Y, Younossi Y, Mir H, Srishord M. Changes in the 
prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 
2008. Clin Gastroenterol Hepatol 2011;9(6):524-30 e1; quiz e60. doi: 10.1016/j.cgh.2011.03.020. 
66. 
Tamura M, Hoshi C, Hori S. Xylitol affects the intestinal microbiota and metabolism of daidzein 
in adult male mice. Int J Mol Sci 2013;14(12):23993-4007. doi: 10.3390/ijms141223993. 
67. 
Sun L, Yu Z, Ye X, Zou S, Li H, Yu D, Wu H, Chen Y, Dore J, Clement K, et al. A marker of 
endotoxemia is associated with obesity and related metabolic disorders in apparently healthy 
Chinese. Diabetes Care 2010;33(9):1925-32. doi: 10.2337/dc10-0340. 
68. 
Gonzalez-Quintela A, Alonso M, Campos J, Vizcaino L, Loidi L, Gude F. Determinants of serum 
concentrations of lipopolysaccharide-binding protein (LBP) in the adult population: the role of 
obesity. PLoS One 2013;8(1):e54600. doi: 10.1371/journal.pone.0054600. 
69. 
Liu X, Lu L, Yao P, Ma Y, Wang F, Jin Q, Ye X, Li H, Hu FB, Sun L, et al. Lipopolysaccharide 
binding protein, obesity status and incidence of metabolic syndrome: a prospective study among 
middle-aged and older Chinese. Diabetologia 2014;57(9):1834-41. doi: 10.1007/s00125-014-3288-
7. 
70. 
Mah E, Sapper TN, Chitchumroonchokchai C, Failla ML, Schill KE, Clinton SK, Bobe G, Traber 
MG, Bruno RS. 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. doi: 10.3945/ajcn.115.118570. 
71. 
Guo Y, Mah E, Davis CG, Jalili T, Ferruzzi MG, Chun OK, Bruno RS. Dietary fat increases 
quercetin bioavailability in overweight adults. Mol Nutr Food Res 2013;57(5):896-905. doi: 
10.1002/mnfr.201200619. 
72. 
Misaka S, Kawabe K, Onoue S, Werba JP, Giroli M, Kimura J, Watanabe H, Yamada S. 
Development of rapid and simultaneous quantitative method for green tea catechins on the 
bioanalytical 
study 
using 
UPLC/ESI-MS. 
Biomed 
Chromatogr 
2013;27(1):1-6. 
doi: 
10.1002/bmc.2740. 
73. 
Mitchell CM, Davy BM, Halliday TM, Hulver MW, Neilson AP, Ponder MA, Davy KP. The effect 
of prebiotic supplementation with inulin on cardiometabolic health: Rationale, design, and methods 

THE OHIO STATE UNIVERSITY 
 
Institutional Review Board 
Version 3 – November 22, 2017 
 
 17
of a controlled feeding efficacy trial in adults at risk of type 2 diabetes. Contemp Clin Trials 
2015;45(Pt B):328-37. doi: 10.1016/j.cct.2015.10.012. 
74. 
Camilleri M, Nadeau A, Lamsam J, Nord SL, Ryks M, Burton D, Sweetser S, Zinsmeister AR, 
Singh R. Understanding measurements of intestinal permeability in healthy humans with urine 
lactulose 
and 
mannitol 
excretion. 
Neurogastroenterol 
Motil 
2010;22(1):e15-26. 
doi: 
10.1111/j.1365-2982.2009.01361.x. 
75. 
Dastych M, Dastych M, Jr., Novotna H, Cihalova J. Lactulose/mannitol test and specificity, 
sensitivity, and area under curve of intestinal permeability parameters in patients with liver 
cirrhosis and Crohn's disease. Dig Dis Sci 2008;53(10):2789-92. doi: 10.1007/s10620-007-0184-
8. 
76. 
Farhadi A, Gundlapalli S, Shaikh M, Frantzides C, Harrell L, Kwasny MM, Keshavarzian A. 
Susceptibility to gut leakiness: a possible mechanism for endotoxaemia in non-alcoholic 
steatohepatitis. Liver Int 2008;28(7):1026-33. doi: 10.1111/j.1478-3231.2008.01723.x. 
77. 
Hilsden RJ, Meddings JB, Sutherland LR. Intestinal permeability changes in response to 
acetylsalicylic acid in relatives of patients with Crohn's disease. Gastroenterology 
1996;110(5):1395-403. 
78. 
Wang L, Llorente C, Hartmann P, Yang AM, Chen P, Schnabl B. Methods to determine intestinal 
permeability and bacterial translocation during liver disease. J Immunol Methods 2015;421:44-53. 
doi: 10.1016/j.jim.2014.12.015. 
79. 
Xia Q, Williams T, Hustead D, Price P, Morrison M, Yu Z. Quantitative analysis of intestinal 
bacterial populations from term infants fed formula supplemented with fructo-oligosaccharides. J 
Pediatr Gastroenterol Nutr 2012;55(3):314-20. doi: 10.1097/MPG.0b013e3182523254. 
80. 
Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal 
samples. Biotechniques 2004;36(5):808-12. 
81. 
Kim M, Morrison M, Yu Z. Evaluation of different partial 16S rRNA gene sequence regions for 
phylogenetic analysis of microbiomes. J Microbiol Methods 2011;84(1):81-7. doi: 
10.1016/j.mimet.2010.10.020. 
82. 
Stiverson J, Williams T, Chen J, Adams S, Hustead D, Price P, Guerrieri J, Deacon J, Yu Z. 
Prebiotic Oligosaccharides: Comparative Evaluation Using In Vitro Cultures of Infants' Fecal 
Microbiomes. Appl Environ Microbiol 2014;80(23):7388-97. doi: 10.1128/AEM.02200-14. 
83. 
Galley JD, Yu Z, Kumar P, Dowd SE, Lyte M, Bailey MT. The structures of the colonic mucosa-
associated and luminal microbial communities are distinct and differentially affected by a 
prolonged murine stressor. Gut Microbes 2014;5(6):748-60. doi: 10.4161/19490976.2014.972241. 
84. 
Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time 
PCR using a broad-range (universal) probe and primers set. Microbiology 2002;148(Pt 1):257-66. 
doi: 10.1099/00221287-148-1-257. 
85. 
Lewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand J 
Gastroenterol 1997;32(9):920-4. doi: 10.3109/00365529709011203.