Gut-level Antiinflammatory Activities of Green Tea in Metabolic Syndrome
Phase: NA
Conditions: Dysbiosis, Endotoxemia, Metabolic Syndrome, Inflammation
Sponsor: Ohio State University

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Antiinflammatory Bioactivities of Green Tea Catechins at the Gut 
NCT Number: NCT03973996 
Date: 06/5/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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I. Objectives 
 
The objective of this application is to establish evidence-based recommendations for green tea 
caatechins, based on improvements in endotoxemia and restored gut barrier function, that promote 
optimal health in humans. The central hypothesis is that green tea extract (GTE) catechins function to 
limit metabolic endotoxemia by ameliorating microbial dysbiosis-mediated inflammation that 
otherwise provokes intestinal permeability. 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) translate intestinal-level benefits of GTE, and 2) define 
antiinflammatory catechin-microbiota interactions. Upon completing these studies, we expect to 
substantially advance a critically needed strategy to manage disease-promoting, systemic inflammatory 
responses that are driven by endotoxemia.  
 
II. Background and Rationale 
 
Limiting intestinal endotoxin translocation is an antiinflammatory strategy. Metabolic endotoxemia 
is defined by 2-3 times higher circulating endotoxin compared with healthy individuals, but is 
significantly less than that of levels observed in sepsis patients.1 Endotoxin is a component of Gram-
negative bacteria.2 Considerable evidence supports the premise that hepatic NFκB activation is 
mediated, at least in part, by the translocation of gut-derived endotoxin (e.g. LPS) to the portal 
circulation, and its ligation to hepatic Toll-like receptor 4 (TLR4). Indeed, nonalcoholic steatohepatitis 
(NASH) has well-recognized etiology mediated along the gut-liver axis.3 In particular, dietary patterns 
(e.g. HF diet) that provoke insulin resistance prevent the lowering of intestinal pH otherwise resulting 
from bacterial fermentation of carbohydrates.4 Consequently, the more neutral pH of the gut lumen 
favors the pathogenic bacteria overgrowth and inhibits the growth of beneficial bacteria (e.g. 
butyrogenic Bifidobacterium). Short chain fatty acids (SCFAs), especially butyrate, that are synthesized 
by resident microbes in the gut enhance barrier function.5 Indeed, butyrate-producing Clostridium 
butyricum mitigates NASH in rats (including NFκB p65 expression).6 These changes correlated with 
increased expression of intestinal tight junction proteins (TJPs), specifically zonula occluden (ZO) and 
occludin (OCC), and lowered circulating endotoxin. Thus, treating gut dysbiosis limits barrier 
permeability, and reduces hepatic exposure to gut-derived, and inflammation-inducing, bacterial 
products (i.e. LPS).3 This is consistent with NASH patients having intestinal bacteria overgrowth that 
correlates with circulating endotoxin and expression of hepatic TLR4, but not TLR2.7 Further, our 
evidence8 and that of others9 show that mice lacking TLR4/MyD88 signaling are protected from NASH 
despite increased portal vein endotoxin levels.9 Antibiotics also attenuate steatosis and endotoxemia in 
rodents,10 and probiotics in obese mice lower NFκB inflammation.11  
 
GTE Lowers NFκB Activation In A TLR4-Dependent Manner. NFκB centrally regulates 
inflammation and is activated in livers of patients12 and rodents with NASH.13-15 Despite 
pharmacokinetic studies16 showing that green tea catechins have limited bioavailability, short half-lives, 
and rapid biotransformation through phase II xenobiotic metabolism, our studies in a high-fat (HF)-
induced NASH model show that catechin-rich GTE lowers hepatic NFκB activation by decreasing IκBα 
phosphorylation.17,18 Consistent with our goal to translate dietary GTE as an antiinflammatory strategy, 
our research has focused on upstream targets by which GTE limits NFκB activation. Prominent targets 
to limit NFκB activation in NASH include those mediated intracellularly by reactive oxygen species 
(ROS) and extracellularly by signaling from TNF receptor-1 and TLR4.17  
Further study in Nrf2-KO mice demonstrated that GTE decreased hepatic TLR4 mRNA.17 This 
supported the alternative hypothesis that its antiinflammatory activities are mediated through a lowering 
of TLR4/NFκB signaling.17 This was corroborated in WT mice fed an HF diet prior to GTE treatment.19 
We reported that GTE ameliorates NASH while lowering hepatic mRNA of TLR4, its co-receptors 

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CD14 and MD-2, and adapter protein MyD88.19 GTE also decreased circulating endotoxin that was 
otherwise increased in HF control mice.19 In our recent report,8 GTE in WT mice fed an HF diet 
inhibited NASH and hepatic NFκB activation to levels not different from those of loss-of-function 
TLR4 mutant mice fed an HF diet. GTE also lowered serum endotoxin independent of genotype. Thus, 
GTE protects against NASH consistent with a mechanism of inhibiting TLR4-mediated NFκB 
activation. 
Hepatic TLR4 signaling is mediated through the adaptor proteins MyD88 and TRIF, which regulate 
“early” and “late” NFκB activation, respectively.20 MyD88-dependent signaling occurs immediately 
after cell surface binding of LPS.21 In contrast, TRIF-dependent signaling first requires endocytosis of 
LPS,21 which facilitates endotoxin clearance.22 Neither GTE nor TLR4mut genotype affected TRIF,8 
whereas GTE lowered hepatic MyD88 and circulating endotoxin regardless of genotype. This suggests 
that GTE attenuates NFκB activation in a TLR4/MyD88-dependent manner and lowers endotoxemia, 
likely without affecting LPS clearance. These findings strongly support examining GTE to target 
endotoxin to mitigate TLR4 inflammation.  
GTE Improves Gut Health. Two mechanisms, acting independently or additively, likely explain GTE-
mediated decreases in endotoxemia. GTE potentially improves microbiota composition to decrease 
LPS-derived Gram-negative bacteria (e.g. E. coli) and/or it improves gut barrier function. 
Epigallocatechin gallate (EGCG), the predominant catechin in GTE prevents IFN-γ-induced increases 
in epithelial permeability in vitro.23 However, there are no reports in humans examining GTE on gut 
permeability. This is surprising considering that catechin levels in the intestinal lumen can easily reach 
2000 μM24 despite their low oral bioavailability (<5 μM).16 Thus, catechins potentially exert direct 
benefit at the intestine to improve gut barrier integrity.  
Our report supports that GTE alleviates endotoxemia by decreasing gut permeability.19 GTE treatment 
following HF-induced NASH resulted in decreases of circulating endotoxin and site-specific 
improvements of intestinal TJP mRNA levels. Although TJPs were unaffected in the jejunum, GTE 
increased duodenal OCC and ZO and ileal expression of OCC and claudin (CLDN). In WT and TLR4mut 
mice fed an HF diet, TJP mRNA was unaffected by genotype, whereas GTE increased duodenal and 
jejunal CLDN mRNA regardless of genotype.8 Because mRNA levels do not necessarily reflect a 
functional benefit in gut integrity,25 we assessed gut permeability in WT mice fed an HF diet containing 
GTE. Following oral gavage of FITC-dextran, GTE attenuated its serum levels that were otherwise 
increased in HF controls. Corroborating evidence also shows that GTE decreased endotoxin in the 
portal vein that was otherwise increased in HF mice. Serum FITC-dextran also correlated with portal 
vein endotoxin levels (r= 0.49; P<0.01). Further, GTE decreased ileal TNFα mRNA that was otherwise 
higher in HF mice. Thus, GTE alleviates endotoxemia, at least in part, by decreasing gut-derived 
endotoxin translocation, consistent with an antiinflammatory mechanism that enhances barrier function 
by improving intestinal TJP expression. 
A complementary line of evidence supports that catechins mitigate endotoxemia by improving 
microbiota composition. Microbial fermented green tea (FGT) attenuates HF-induced obesity in mice.26 
This is consistent with our works examining GTE in obese models.18,27,28 FGT decreased the ratios of 
Firmicutes/Bacteroidetes and Bacteroides/Prevotella. The Firmicutes phylum predominates in obese 
mice.29 Thus, obesity susceptibility is a potential consequence of increased energy absorption resulting 
from microbial fermentation. Conversely, decreased Firmicutes by FGT potentially explains the 
observed reduction in adiposity without affecting energy intake, which is similar to our works in mice 
fed GTE.17,18,27,28 Lowered Bacteroides/Prevotella also suggests a shift towards carbohydrate 
fermentation. This would enhance gut integrity by increasing SCFA availability to the host. Thus, GTE 
potentially alleviates dysbiosis by reducing Gram-negative bacteria (e.g. Bacteroidetes, 
Proteobacteria) that drive endotoxin-mediated inflammation while increasing SCFA from populations 
(e.g. Prevotella) to improve gut integrity.30 Indeed, similar to others,31 our underway studies indicate 
that GTE increases microbial functions relating to butanoate and propanoate metabolism. SCFA, both 

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straight chain and branched chain, will be assessed directly in our studies in humans and in vitro. 
Straight chain SCFA promote commensal bacteria growth and inhibit certain Gram-negative bacteria.32 
In contrast, branched chain SCFA, which are generated from branched chain amino acids by microbial 
metabolism, are associated with metabolic disorders involving insulin resistance.33,34  
Despite their similar metabolic benefits, and both being derived from the Camellia sinensis plant, GTE 
and FGT differ substantially in their compositions. GTE contains 30% total catechins that is highly 
enriched in EGCG (48%), and relatively lesser amounts of epigallocatechin (EGC) (31%), epicatechin 
gallate (ECG) (13%), and epicatechin (EC) (8%).27 In contrast, FGT has only 14% total catechins. It is 
comprised of mainly catechin (56%) and small proportions of EGCG (11%), EGC (15%), ECG (3%), 
EC (5%), and other low-level metabolites. That GTE and FGT elicit hepatoprotection, despite their 
unique compositions, suggests independent or additive benefits of catechins, or their metabolites arising 
from microbial metabolism, to ameliorate inflammation-inducing responses along the gut-liver axis. 
This concept is consistent with our preliminary studies in mice fed an HF diet formulated with GTE 
(2%), EGCG (0.3%; equivalent to the EGCG level in GTE), or catechin (CAT; 0.3%). EGCG and CAT 
similarly attenuated serum endotoxin and fecal calprotectin, a biomarker of intestinal neutrophil 
infiltration,35 to levels no different from the levels lowered by GTE. Also, MS-metabolomics studies 
detected >50 metabolites in fecal samples. Of those, there were only two metabolites common to the 
treatments with GTE, EGCG, and CAT that were not detected in the LF or HF control mice. These 
were 3’,4’-dihydroxyphenyl-γ-valerolactone and its microbial catabolite 3-hydroxyphenylacetic acid. 
Thus, while catechins provide direct antiinflammatory benefit to preserve barrier integrity, at least in 
vitro,23 these data support independent and/or additive bioactivities of microbial catechin metabolites.  
Further, a short-term study in adults shows that green tea improved microbiota composition (e.g. 
increased α-diversity, increased genera of SCFA producers) in association with reducing microbial 
functions relating to LPS biosynthesis.31 We recognize that our hypothesis contrasts with the findings 
of a clinical study36 showing that gut microbiota composition was unaffected in adults receiving EGCG-
enriched GTE (560 mg/d; 12 wk). However, critical study limitations likely precluded an observable 
benefit on gut health. Participants were normal weight (BMI = 23 kg/m2), and unlikely to have gut 
dysbiosis that occurs in obese or MetS persons.37-40 Dietary control consisted of encouraging 
participants to maintain their usual diets, which was not assessed rigorously nor were total polyphenol 
intakes considered. Indeed, diet accounts for >50% of microbiota changes and the responsiveness of 
the microbiota to shift rapidly (i.e. hours to days)41 in relation to its composition and functions. 
Compliance was evaluated based on pill counts without any biomarker measures. The parallel study 
design also limits statistical power for detecting a treatment effect. Finally, an intergenic spacer length 
polymorphism was used to profile and compare the microbiota among the groups. That technique is not 
based on DNA sequencing but based on comparing the length of the intergenic spacers among samples. 
Because different bacteria can and do have a similar length of the intergenic spacer, it cannot enable 
the detection or quantification of specific taxa (e.g. species, genera) of bacteria. In other words, even if 
some genera or species of bacteria were altered by EGCG, that technique would be unlikely to detect 
or quantify the changes. Actually, that technique is rarely used in recent years after next-generation 
DNA sequencing became available, which will be used in the proposed study. This will better identify 
and estimate the relative abundance of most bacteria, and treatment effects on gut microbiota (e.g. 
species composition and structure such as richness, evenness, change in relative abundance). Thus, the 
lack of prebiotic effect by EGCG in that study36 is not surprising, and contrasts evidence that tea 
phenolics inhibit pathogenic bacteria growth (e.g. Clostridium difficile;42 Staphylococcus spp43) while 
increasing the proportion of beneficial commensal bacteria (e.g. Bifidobacterium spp).43-45  
In conclusion, evidence shows that 1) limiting gut-derived endotoxin is an antiinflammatory strategy, 
2) GTE lowers TLR4/NFκB-dependent inflammation, and 3) GTE improves gut health through 
improving gut barrier function and alleviating gut dysbiosis. Our team has the expertise and experience 
to safely perform controlled crossover interventions to assess metabolic parameters, gut barrier 

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permeability, and gut microbiota composition. Thus, after completing these studies we expect to 
substantially advance a critically needed strategy to manage disease-promoting, systemic inflammatory 
responses that are driven by endotoxemia. 
 
Anticipated Results 
Preclinical evidence supports the gut-level benefits of catechin-rich GTE to limit metabolic 
endotoxemia.8,19 We expect these preclinical findings to be translated to similar gut-level improvements 
in MetS persons utilizing a novel, catechin-rich GTE confection (i.e. snack food). We expect to achieve 
>95% compliance (based on returned confections and circulating catechins) without any adverse effects 
or changes in body mass or energy intakes. Based on our mouse studies,8,19 GTE is expected to decrease 
serum endotoxin in a time-dependent manner, and most substantially among MetS persons due to their 
endotoxemia. These benefits are expected to occur through an antiinflammatory mechanism that 
attenuates gut-derived endotoxin translocation. This would be consistent with GTE reducing endotoxin 
translocation to the portal vein by increasing intestinal expression of TJPs8,19 Thus, GTE will decrease 
small intestinal and colonic permeability, based on decreased urinary excretion of lactulose/mannitol 
(0-5 h) and sucralose (0-24 h), respectively.46  
Similar to our studies, GTE also will alleviate gut dysbiosis that is known to occur in MetS persons 
(e.g. depleted populations of SCFA-producing bacteria47). Selective antimicrobial activities of GTE 
will decrease LPS-derived Gram-negative bacteria (e.g. Bacteroides).42,44 GTE will also increase 
commensal bacteria, including Clostridia (Cluster XIV and IV) and other members of the Gram-
positive Phyla Firmicutes that generate SCFAs.48,49 This will be corroborated by greater fecal butyrate 
and other straight chain SCFAs. Thus, these findings would suggest a mechanism, at least in part, that 
the alleviation of gut dysbiosis restores butyrate generation.  
We have also considered antiinflammatory activities of GTE attributed to its catechins23 and/or catechin 
metabolites (e.g. γ-valerolactones).50,51 52 Indeed, fecal calprotectin (Fig 5) and myeloperoxidase, and 
systemic TLR4/NFκB pro-inflammatory genes in PMBCs and circulating proteins (i.e. p65, TLR4, 
MyD88, IL-6 and -10) are expected to be lowered by GTE.7,53 Measures of catechins/metabolites in 
feces and blood will be used to establish correlations with intestinal and systemic inflammatory 
biomarkers to suggest direct and/or indirect benefits of catechins/metabolites. Finally, GTE is not 
anticipated to affect liver function tests (i.e. ALT, AST), and their lack of change (and other clinical 
chemistries) would support the safe application of the GTE-rich confection to manage MetS risk. Thus, 
these translational studies will establish GTE to alleviate gut dysbiosis that otherwise provokes barrier 
dysfunction and host LPS-TLR4-NFκB inflammation to drive MetS risk.  
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 
The prevalence of MetS is at epidemic proportions in the US where it affects ~35% of all Americans 
and 50% of those >60 years in association with the high rates of obesity.54 Because MetS symptoms 
are often subclinical, most individuals are left unmanaged other than to encourage lifestyle 
modification. It is therefore not surprising that many MetS persons progress to more advanced disorders 
(e.g. NASH) and premature mortality.55,56 This is consistent with NASH being recognized as the hepatic 
manifestation of MetS.57,58 Indeed, NASH is the most prevalent liver disorder in the Western world 
where it affects ~20% of adults59 including 80-100 million Americans.60 Although MetS development 
and progression can be averted by improved diet,61,62 these recommendations have had little impact.63 
Correlative evidence supports that poor diet influences endotoxemia, gut barrier dysfunction, and 
glucose tolerance in humans.64-66 By contrast, green tea lowers risks for cardiovascular and liver 

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diseases.67,68 Evidence from our preclinical models also clearly show that GTE alleviates endotoxemia 
and TLR4/NFκB inflammation by improving gut health. However, no studies have targeted gut health 
with GTE to manage endotoxemia and alleviate MetS progression. Thus, our studies are expected to 
be of public health and scientific significance because they represent the first translational study in 
MetS persons examining GTE to manage disease-promoting, intestinal-level responses leading to 
endotoxemia and TLR4/NFκB inflammation. They will also establish independent benefits of 
catechins/catechin metabolites relative to altered microbial populations and functions. Thus, these 
studies are a critical step to advance a timely dietary recommendation to mitigate the growing burden 
of MetS, and by association NASH, by ameliorating endotoxin-TLR4 signaling. 
The potential impact of green tea to improve health is supported by data indicating that 4 out of 5 
Americans drink tea, with ~50% considered habitual consumers.69 The US is also agriculturally active 
in producing tea (Camellia sinensis) as part of a $17.6 billion global market, with annual sales of green 
tea outpacing black tea (11% vs 3.9%).70 Green tea is safe when consumed as recommended,71 but 
rigorous investigation is needed to establish evidence-based dietary recommendations that improve the 
health and well-being of Americans. In this regard, the status quo is that GTE decreases TLR4/NFκB 
inflammation and MetS criteria in preclinical models, in part, by limiting gut permeability to reduce 
endotoxemia.8,19 Endotoxin-TLR4 signaling is also implicated to initiate insulin resistance.1 However, 
no translational studies in MetS persons have examined GTE on gut barrier function despite it 
improving progressive pathologic responses of insulin resistance in humans, including NASH.72 In our 
opinion, the proposed research is innovative, because it is a substantial departure from the status quo 
by shifting the focus to establish antiinflammatory bioactivities of GTE acting at the intestinal-level to 
protect against an early insult mediating metabolic disorders. Our translational approach also will be 
the first to use a GTE-rich “healthy snack food”73 to reduce metabolic endotoxemia. This approach 
recognizes consumers’ shift in eating behaviors that show increased preference for convenient snack 
foods74 and circumvents a barrier in that most Americans consume green tea limitedly in lieu of 
catechin-deplete black tea.75-77 Thus, the outcomes of our nutritional approach that integrates clinical, 
biochemical, metagenomics, and metabolomics aspects is expected to vertically advance an easily 
implementable dietary approach that will define GTE bioactivities while also establishing timely 
recommendations to manage MetS, and alleviate this serious problem of public health concern. 
 
III. Procedures 
 
A. Research Design 
We will conduct a randomized double-blind, placebo-controlled crossover trial in metabolic syndrome 
(MetS) and healthy individuals. We will enroll adults with (n = 20) and age- and gender-matched 
healthy (n = 20) men and women (18-65 y). 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, while 
instructed to abstain from all tea varieties and follow a low-polyphenol diet.78 Blood will be collected 
at day 0, 14, and 28 for metabolic assessments and safety monitoring by assessing a serum chemistry 
panel [e.g. liver function (aminotransferases), renal function (creatinine, BUN), CBC, glucose, insulin, 
insulin resistance79]. At the same time points, blood pressure and anthropometrics will be assessed, and 
dietary nutrient and flavonoid intakes will be evaluated from 3-d food records using NDSR dietary 
analysis software and the NCC Flavonoid and Proanthocyanidin database as we described.73,80 On day 
28, participants will provide a 3-d fecal sample. Following blood collection, they will complete a gut 
permeability test and collect urine through day 29 (described below). Upon completing these 
procedures, participants will undergo ~1-mo wash-out before repeating the study identically, but with 
crossover to the alternate treatment.  
GTE-rich Confections. The Bruno and Vodovotz labs have previously developed a starch-based GTE-
rich confection73 as a successful catechin delivery system. For this application, we will modify this 

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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 
8 calories. Thus, 6 confections will provide about 51 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 verified73 is similar to about 5 servings/day of green tea. 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 81. 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. Male and female adults with MetS (18-65 y) having no history of liver, 
cardiovascular disease, or cancer will be enrolled. They are expected to meet ≥3 established criteria for 
MetS,82 and we will enroll those fulfilling these specific inclusion criteria to improve participant 
homogeneity: fasting glucose (100-126 mg/dL), waist circumference (>89 cm or 102 cm for F/M), 
HDL-C (<50 or <40 mg/dL in F/M), triglyceride >150 mg/dL. Although elevated blood pressure is a 
symptom/criteria for MetS, only those individuals having blood pressure <140/90 mmHg will be 
enrolled to avoid those having with Stage 2 hypertension.83 Age- and gender-matched healthy adults 
(19-25 kg/m2) will also be enrolled. They will be normoglycemic (glucose <100 mg/dL), normotensive 
(<120/80 mmHg) and normolipidemic (HDL-C >50 mg/dL or >40 mg/dL in F/M; triglyceride <150 
mg/dL). Major exclusion criteria for all participants include: concurrent tea consumption (Camellia 
sinensis or herbal varieties); vegetarian; user of dietary supplements, prebiotics, or probiotics within 
past month; hemochromatosis; any gastrointestinal disorders or surgeries; chronic diarrhea; Parkinson’s 
disease; smoker; alcohol consumption (>2 drinks/d); recent use of antibiotics or antiinflammatory 
agents. We will also not enroll any participants using any medications to manage diabetes, 
hypertension, or hyperlipidemia (e.g. statins, metformin, ACE inhibitors), any medications known to 
be contraindicated for use with green tea ingestion (e.g. antipsychotic medications [Clozapine, lithium, 
Diazepam], blood thinning medications [Warfarin], high blood pressure medications [nadolol], and 
monoamine oxidase inhibitors [selegiline]), women who are pregnant or lactating, or have initiated or 
changed birth control in the past 3 months. 
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 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).  

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Power Calculation and Data Analysis. We performed a power analysis using serum endotoxin (day 
28) as the primary outcome. Mean serum endotoxin of MetS and healthy adults was used to predict a 
conservative 30% improvement by GTE in MetS persons; this corresponds to a 10 EU/mL decrease in 
endotoxin in MetS. Simulations were used to estimate power based on 20 healthy and 20 MetS adults 
randomized to treatment vs control in a 50/50 ratio, respectively. Based on a linear model with treatment 
and health status, and within-subject measures, we will have >90% power (α=0.05) to detect the 
primary contrast of interest (GTE vs placebo in MetS adults) and additionally, >85% power (α=0.05) 
to detect an interaction between treatment and health status. Most data will be analyzed by linear mixed 
effects models (LMMs) with random effect for subjects (to account for repeated measures) and fixed 
effects for health status, gender, GTE treatment, time period, and their interactions. Multivariate 
regression analysis will define correlations between study variables with consideration of potential 
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 Chemistries. At screening, fasting plasma glucose, triglyceride, and HDL-cholesterol will be 
measured by spectrophotometry utilizing validated clinical assays (Pointe Scientific). At day 0, 14, and 
28 of the interventions, collected blood will be sent to The Ohio State University Medical Center for 
metabolic evaluations by assessing a serum chemistry panel [e.g. [e.g. liver function 
(aminotransferases), renal function (creatinine, BUN), CBC, glucose, insulin, insulin resistance79]. 
 
Diet Assessment/Control. To improve diet homogeneity and limit potential confounding effects of 
polyphenol intakes on study outcomes, participants will follow a low-polyphenol diet during each study 
phase, as described by Dr. Vodovotz.78 In brief, participants will receive weekly dietary education under 
the auspice of a registered dietitian (PI Bruno) to omit or limit polyphenol-rich foods. Participants will 
be instructed to abstain from foods containing >50 mg/serving of total polyphenols and limit those 
having 10-50 mg/serving of polyphenols to a maximum of 2 servings/d. Foods containing <10 
mg/serving will be unrestricted. This results in a decrease of dietary polyphenols by 45% compared to 
usual intakes (1568 mg/d), and with 95% adherence.78 Participants will be provided a list of common 
polyphenol-rich foods to avoid (see List of Polyphenol-Rich Foods attachment) as well as a form to 
record any consumption of these foods/drinks. Fiber intakes with this dietary approach are also similar 
to the low intakes of the typical American diet (~15 g/d).84 Food records (3-d) will be evaluated on 
three occasions during the intervention using NDSR software and the NCC Flavonoid and 
Proanthocyanin database will be used to assess energy, nutrient, and flavonoid intakes as we 
described.73,80 
 
Metabolic Endotoxemia. Serum endotoxin will be measured using a fluorometric kit from fasting blood 
samples at day 0, 14, and 28 as we described.19 Briefly, the fluorometric assay assesses endotoxin-
mediated activation of recombinant Factor C and subsequent cleavage of a fluorogenic substrate 
(PyroGene rFC; Lonza). In our hands, inter- and intra-assay CV is 4.2-6.1%. To facilitate data 
interpretation, the following complementary markers of metabolic endotoxemia will be measured in 
plasma collected at day 0, 14, and 28: trimethylamine N-oxide (TMAO), soluble cluster of 
differentiation 14 (sCD14), and lipopolysaccharide-binding protein (LBP). Trimethylamine N-oxide is 
produced by certain gut bacteria and is reflective of gut dysbiosis. Soluble CD14 and LBP guide 
bacterial toxins to immune cells for recognition and are reflective of increased gut barrier permeability. 

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Trimethylamine N-oxide will be quantified by LC/MS whereas LBP and sCD14 will be quantified by 
ELISA using commercial kits.  
 
Gut Permeability. At wk 4 (d 28), fasted participants will ingest a sugar probe solution [sucrose (40 g), 
lactulose (5 g), mannitol (1 g), sucralose (1 g), and erythritol (1 g)] as described.85 Sucrose-free meals 
will be provided for 24 h (through d 29). Urinary sugars will be assessed using our established LC-MS 
method.85 Sugar excretion (%), and mannitol-normalized excretion ratios of sugars from 0-5 h and 6-
24 h will be calculated to reflect upper and lower GI permeability.86-89 This approach is based on 
lactulose being absorbed in the small intestine, but not the colon, whereas sucralose is absorbed in the 
colon; sucrose is used to reflect gastric permeability.46 Thus, these studies will define site-specific gut 
permeability, and benefit of GTE treatment. 
Microbiota Composition and Function. Microbiota composition and the predicted functional 
metagenome will be assessed from fecal samples collected on 3 days at wk 4 (d 25-27), as Dr. Yu (co-
PD) described.90-94 Effects of GTE and MetS status on total bacterial abundance will be determined by 
qPCR.95 Microbiota will be characterized for diversity and community structure by sequencing 16S 
rRNA genes using MiSeq. Briefly, one amplicon library (targeting the V3-V4 hypervariable region 
using bacteria-specific primers) will be prepared by pooling PCR products from three replicate PCR 
reactions for each sample. Amplicon libraries will be sequenced using the 2x300 pair-ended sequencing 
reactions. Sequencing reads (≥50,000/sample) will be subjected to quality control to filter out artificial 
sequences prior to bioinformatics analysis using Qiime.96 Bacteria will be identified by comparing 
sequences representing each operation taxonomic unit (OTU) with RDP and Silva databases. 97,98 α-
Diversity (OTU richness, Shannon-Wiener diversity index, evenness) will be calculated using Qiime. 
Comparisons of diversity among samples (β-diversity) will be performed using the Bray-Curtis 
dissimilarity,99 followed by multivariate analyses (e.g. principle coordinate analysis, permutational 
multivariate analysis of variance, analysis of similarity, partial least square discriminant analysis). The 
functional metagenome will be predicted using PICRUSt100 and PanFP101 based on reference genome 
databases and the KEGG Orthology classification scheme to obtain a functional gene abundance matrix. 
Plasma and Fecal Catechins and Catechin-Metabolites. Using our targeted LC-MS/MS-based panel 
of >50 metabolites, we will quantify GTE catechins and metabolites in fecal samples (d 27) and plasma 
(d 0, 14, 28) following enzymatic hydrolysis with β-glucuronidase/sulfatase.73 Analyte identification 
and quantification will be confirmed by comparing fragmentation patterns relative to purified standards 
or reference databases. In addition to defining the interactive effects of MetS status and GTE treatment 
on catechin metabolism, time-dependent plasma measures will objectively establish subject compliance 
to the dietary intervention. 
Fecal Short Chain Fatty Acids. Separate LC-MS/MS procedures that are established based on a 
published report,33 will evaluate ten C2-C6 straight chain SCFAs (i.e. butyrate, acetate, propionate) and 
branched chain SCFAs (e.g. isobutyric acid, isovaleric acid). Straight chain SCFAs are predominantly 
from bacterial fermentation of fibers whereas branched chain SCFAs are derived mainly from bacteria 
metabolism of branched chain amino acids. The former are often depleted with gut dysbiosis whereas 
the latter are often increased. In brief, fecal SCFAs are derivatized with 3-nitrophenylhydrazine to 
increase their stability prior to injecting on the LC-MS/MS system operated in ESI mode and 
quantifying against area ratios of authentic standards relative to 13C6-3NPH. 
Host and Intestinal Inflammation. At wk 4, we will define the effects of GTE on inflammatory 
responses. We will assess intestinal inflammation by measuring fecal calprotectin and myeloperoxidase 
(i.e. indices of neutrophil inflammatory responses) by ELISA as we described.102 We will also measure 
systematic inflammatory responses in plasma by ELISA (CRP, IL-6 and -8, TNFα, MPO) and by RT-
PCR analysis of peripheral blood mononuclear cells (PMBCs; isolated at blood collection) to assess 
the expression of genes involved in TLR4/NFκB signaling (TLR4, MyD88, p65, IL-6 and -8, TNFα, 
MPO, MCP-1).  

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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 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 for blood chemistry analysis. If they are not fasted at 
least 10 hours), they will be asked to come back in the fasted state at a 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 at wk 0 (d 0), who will instruct them on how to complete a dietary 
food record and explain the details regarding following a low-polyphenol diet throughout the 
intervention. At this first visit, a fasting blood sample will be collected and the participants will be 
provided 2-wk supply of confections. They will be instructed to consume 6 confections per day. After 
2 weeks, the participant will come in for a mid-intervention visit (d 14). A fasting blood sample will be 
collected and the participant will be provided another 2-wk supply of confections to complete the 4-wk 
intervention. Prior to the participants’ last study visit, they will collect 3-d stool samples and return the 
samples or meet with study personnel to return their stool samples upon collection. At the end of the 
intervention (d 28), participants will visit the study and a fasting blood sample will be collected. 
Following this, participants will perform a gut permeability test. After completing the gut permeability 
test, participants will then follow about a 1-month wash-out period. Following the wash-out period, 
participants will repeat these procedures, except receiving the alternative study treatment (GTE, 
placebo) than what they previously received. We estimate that completion of all study procedures (2 4-
wk interventions and 1-month wash-out period) will take 10-12 weeks per participant. Each step of the 
study procedures will be discussed in detail below. See Study Design attachment for an overview of the 
study. 
 
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. 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 (15 mL; 1.5 
tubes) so that we may measure blood chemistries (glucose, HDL, 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 

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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 male subject’s waist circumference is 101 cm, which is outside the specified waist 
circumference inclusion range for MetS 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 fall 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 
next visit. 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 fall 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 e-mailed 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 time. 
No confidential or sensitive information will be shared with third parties or left on answering machines. 
 
Intervention Timeline. Each participant will complete a 2-phase (GTE vs placebo) randomized, 
crossover study. Each study intervention will consist of 3 study visits (Day 0, 14, 28) over a 4-wk 
period. After completing the first intervention, participants will undergo a wash-out period (1 month), 
and afterwards, begin the alternative intervention.  
 
On Day 0, participants will come to the study center in a fasted state and provide a blood sample (45 
mL) to measure endotoxin, TMAO, LBP, sCD14, a panel of clinical chemistries, and catechins. 
Following the blood collection, participants will be instructed on how to complete a 3-d diet record as 
well as be informed about maintaining a low-polyphenol diet throughout the duration of the 
intervention. Participants will then be provided a 2-wk supply of confections, for which they will be 
instructed to consume 6 confections each day (2 confections/meal; 1 g GTE/d).  
 
On Day 14, participants will return to the study center in the fasted state and return any uneaten 
confections. Another blood sample (45 mL) will be collected for measurement of biomarkers as 
described above. Participants will then be asked to complete another 3-d diet record and be reminded 
to keep maintaining a low-polyphenol diet.  
 
Prior to the final visit on Day 28, participants will collect a stool sample during the preceding 3 days. 
Participants will collect their stool samples in the comfort of their home and will be asked to return the 
samples on ice or meet with study personnel to return the samples to the study center within 24 h upon 

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collection. On Day 28, participants will come to the study center in the fasted state. A fasting blood 
sample (60 mL) will be collected to measure the biomarkers mentioned above along with analysis of 
white blood cell inflammatory genes. Following blood collection, participants will perform a gut 
permeability test. Participants will consume a test beverage that contains sucrose (40 g), lactulose (5 
g), mannitol (1 g), sucralose (1 g), and erythritol (1 g) dissolved in water (250 mL). After ingestion of 
the beverage, participants will be instructed to collect their urine from 0-5 and 6-24 hours in the 
provided containers.  
 
After completing the first intervention, participants will partake in a wash-out period (1 month) and 
then repeat the same study procedures (i.e. Day 0, Day 14, Day 28), but will receive the alternative 
confection (i.e. GTE vs placebo). During each study visit, participants will have access to drinking 
water and use of the restroom as needed. Details about each study visit and associated test trials that 
will occur is explained below: 
 
Intervention Phase / Test Confections. During the 4-wk intervention, participants will receive a GTE-
rich or placebo (no GTE containing) confection. Confections will be low-carbohydrate and low-calorie. 
Each serving (6 g) provides <8 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 
composition81 and would confound interpretation. Decaffeinated GTE was chosen to minimize 
potential side effects such as excess caffeine consumption or caffeine/drug interactions that could result 
from 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. At the first visit, 
participants will provide a fasting blood sample and then receive a 2-wk supply of confections. Halfway 
through the intervention (d 14), 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 (d 28), participants will perform a gut permeability test and provide another fasting blood sample. 
Compliance will be evaluated based on the number of confections returned throughout the study as well 
as objectively by measuring catechin/catechin-metabolites in the plasma. To ensure randomization, a 
random number generator will be used to randomly assign the sequence by which the participant will 
complete each of the interventions. 
 
Gut Permeability Test. On the morning of testing at d 28 of the intervention, participants will arrive at 
the study center after abstaining from food and only consuming water for 10-12 hours. A gut 
permeability test will be conducted as described85. 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, 1 g sucralose, and 1 g erythritol dissolved in water (250 mL). Within 2-
hours after ingesting the sugar test beverage, participants will be asked to consume an additional 500 
mL of water. Participants will be instructed to abstain from foods that contain artificial sweeteners 
during the 24-hour period. Urine will be collected from 0-5 and 6-24 hours in sterile containers with 
10% thymol (inhibit bacterial growth). For each urinary collection period, participants will be provided 
2-L urine collection bottles and a cooler with ice packs to store collected urine until they return their 
samples the next day. 

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Sampling Handling. For the entire study (~3 months), including the screening phase and completion 
of both interventions (GTE vs placebo), we plan to collect a total of 315 mL or 1.3 cups of blood. At 
screening, 15 mL of blood will be obtained to ensure adequate sample to perform biochemical measures 
of eligibility. During each 4-wk intervention, we will collect a total of 150 mL or 0.6 cups. At day 0 
and 14 we will collect 45 mL (0.2 cups) and at day 28 we will collect 60 mL (0.25 cups). All blood will 
be collected into evacuated blood collection tubes. The greater volume of blood collected at day 28 is 
necessary to ensure that there is adequate blood to perform the same analyses as for day 0 and 14 as 
well as for additional analyses (e.g. white blood cell inflammatory markers). 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. 
 
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 stool samples 
for up to 24 h after collection. We will then have the participants return their stool 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.103  
 
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 stored at -80°C 
until analysis can be completed. Analyses will include triglyceride, HDL, liver function 
(aminotransferases), renal function (creatinine, BUN), CBC, glucose, insulin, pro-inflammatory gene 
expression, catechins (EGCG, ECG, EGC, EC), catechin-metabolites (e.g. valerolactones, catechin-
derivatives), serum endotoxin, TMAO, LBP, and sCD14. Urine will be stored at -80ºC until analysis 
can be completed. Analysis from urine will include sucrose, mannitol, lactulose, sucralose, and 
erythritol. Fecal samples will be stored at -80ºC until analysis can be completed. Feces analysis will 
include microbiota composition, myeloperoxidase, calprotectin, and SCFAs. 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, which 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 be stored 
on a computer in the PI’s laboratory. In addition, a backup of digital data will be stored on the PI’s 

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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 of 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. 
 
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