Assessing Gut Microbiota Mediated Health Outcomes of Whole Wheat and Its Major Bioactive Components
Phase: NA
Conditions: PreDiabetes, Dysbiosis, Endotoxemia, Inflammation
Sponsor: Ohio State University

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Assessing Gut Microbiota Mediated Health Outcomes of Whole Wheat and Its Major Bioactive 
Components 
Study Protocol 
NCT Number: NCT05318183 
Date: 11/21/2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  

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Assessing gut microbiota mediated health outcomes of whole wheat and its major bioactive 
components  
  
I.  
Objectives  
  
The use of functional foods or their bioactive components to manage chronic disorders has become popular 
in recent years. One dietary product that can be tapped for its health benefits is whole grain. Observational 
studies suggest an inverse relationship between the intake of whole grain and obesity, prediabetes, some 
cancers, and all-cause mortality. A growing body of clinical evidence (mostly small randomized clinical 
trials) demonstrates positive effects of whole grain on certain risk factors for chronic diseases and 
cardiometabolic abnormalities; however, outcomes of clinical interventions have revealed substantial 
interpersonal variation in the health benefits, with a subset of individuals not exhibiting any measurable 
benefits. Whole grains/whole wheat contains significant amounts of bioactive phytochemicals in addition 
to its well-recognized fiber content. Outcomes of many studies support that whole grains/whole wheat 
provide favorably improve human health by the direct effects of these phytochemicals and/or their microbial 
metabolites. Thus, the objectives of this study are to investigate the gut microbiota-mediated effect of whole 
wheat consumption, the most widely consumed whole grain, on the fecal and plasma metabolomes, 
metabolic health, and gut health in a cohort of adults with prediabetes.  We hypothesize that gut microbial 
metabolism of whole wheat and its major bioactive components is a determining factor of human health 
benefits; and by understanding the gut microbiome--mediated benefits of whole grain, we can truly harness 
its benefits for metabolic health in humans.    
  
II.  
Background and Rationale  
A. 
Controversy in the benefits of whole grain/wheat bread consumption. Bread is consumed by 
billions of people worldwide. This key dietary component comprises ~10% of energy intake in adults.1 
Wheat is the most commonly used cereal for baking bread, and whole wheat contains germ and bran, which 
are rich in dietary fiber, proteins, phytochemicals, B-vitamins, lipids, iron, magnesium, and zinc.2, 3 While 
these components are considered ‘‘healthy,’’ they are often removed in the milling process of refined flour. 
It is well-known that dietary fiber in whole wheat plays an important role in human health. However, given 
the largely underestimated phytochemical contents in whole wheat, the role of phytochemicals during its 
consumption is understudied and therefore should be addressed.4, 5 The groups of reported phytochemicals 
in whole wheat include alk(en)ylresorcinols (ARs), benzoxazinoids, phytosteroids, sphingolipids, lignans, 
flavonoids, phenolic acids, fatty acids and glycolipids, tocols, carotenoids, oxylipins, and other minor 
components. Animal and in vitro studies6-8 have shown that diets containing whole wheat or wheat bran can 
reduce the fasting blood sugar by ~20 %; suppress high-sucrose diet-induced hyperinsulinemia by more 
than 50%; and wheat ARs (C15:0 – C25:0) may play important role in type II diabetes mellitus (T2DM) 
prevention by effectively inhibit glycerol-3-phosphate dehydrogenase and prevent triglyceride 
accumulation, as well as suppress adipocyte lipolysis and hormone-lipase activities. In addition, sinapic 
acid from whole wheat consumption has been shown to ameliorate hyperglycemia through inducing high 
GLUT4 expression and increased glucose uptake via the phospholipase C -protein kinase C signals in 
diabetic rats.9 Meanwhile, scientific evidence regarding the health benefits of whole-grain consumption in 
humans is contradictory despite accumulating observational studies and randomized control trials (RCTs). 
Several studies suggest that whole grain consumption is beneficial to human health based on associations 
that significantly reduce all-cause mortality;10 cancer risk, 11 cardiovascular diseases,12 T2DM, and 
metabolic syndrome; and an improvement in glycemic control. 1, 13, 14 Conversely, some studies showed 
improvement in only a handful of clinical markers 15-17. And, large-scale interventional crossover trials show 
no significant effect on these disease-risk markers.15, 18, 19  

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B. 
Gut microbes as a key mediating factor in individual phytochemical response. Food is generally 
processed by our gastrointestinal system primarily to supply energy and key functional elements to the 
body. Dietary components, such as carbohydrates, proteins, and fats are respectively catabolized into 
monosaccharides, amino acids, and fatty acids that serve as fuel to maintain organ function and promote 
cellular growth and recycling. Furthermore, the biological organization of the mammalian superorganisms, 
the gut microbiota, introduces a complex noneukaryotic compartment that also interplays with food 
components and key regulatory physiological processes of the host. It is now well-known that gut 
microbiota interacts with the diet and impacts host physiology and metabolism.20 However, there are many 
recent reports regarding a large inter-individual variation in the response to different dietary components 
including phytochemicals,21 and these findings have indicated that the gut microbial composition could be 
used to identify those subjects who would benefit from dietary interventions. 1, 13, 14 Circulating 
concentrations of phytochemicals, such as the flavonoids naringenin and hesperitin, psoralens, and lignans, 
can vary widely among individuals even in controlled feeding studies.22, 23 Previous reports have 
demonstrated that gut bacteria can hydrolyze glycosides, glucuronides, sulfates, amides, and esters,21  and 
they can also carry out reduction, ring-cleavage, demethylation, and dehydroxylation reactions.24-26 The 
hydrolysis of glycosides and glucuronides typically results in metabolites that are more biologically active 
than the parent compounds. In contrast, further bacterial degradation and transformation of aglycones can 
lead to the production of more or less active compounds, depending on the substrate being metabolized and 
the products formed. One of the most familiar examples of interindividual differences in the microbiome- 
dependent dietary effects relates to isoflavone metabolism. For instance, equol is a well-known microbial 
metabolite from the soy isoflavone daidzein, and the proportion of equol-producing individuals is reported 
to be approximately one-third to one-half of the human population.21 Host genetics may contribute to 
interindividual differences in equol production in humans; however, the reasons for such differences in the 
ability to harbor the equol-producing bacteria remain essentially unknown. Interindividual differences in 
gut microbial community composition also affect the metabolism of other phytochemicals and ultimately 
human exposure to specific bioactive compounds.  
C. 
Gut microbial diversity may explain the personalized protective effects of whole wheat against 
prediabetes.  As evident by the sharp incline in the prevalence of prediabetes, blood glucose levels are 
rapidly increasing in the worldwide population. It is estimated that impaired glucose tolerance will affect 
at least 37% of the adult population in the US alone.27 Prediabetes, characterized by chronically impaired 
blood glucose responses, is a significant risk factor for type II diabetes mellitus (T2DM), with up to 70% 
of prediabetics eventually developing the disease.28 It is also linked to other manifestations, collectively 
termed metabolic syndrome, including obesity, hypertension, non-alcoholic fatty liver disease, 
hypertriglyceridemia, and cardiovascular disease.29 Thus, maintaining normal blood glucose levels is 
considered critical for preventing and controlling metabolic syndrome.   
D. 
Summary. This overview demonstrates that consumption of whole grain foods has been associated 
with decreased risk of obesity, prediabetes, and type 2 diabetes.1, 30-32 However, mechanisms mediating the 
protective effect of whole grains are unclear, although glycemic response and insulin sensitivity are the 
most common mechanisms suggested by epidemiological studies. 33-36 An early report has raised the 
possibility of gut microbiome-mediated effects linking cereal fiber consumption with decreased risk of 
T2DM;37 this knowledge gap continues to persist. It was suggested that modifications in gut microbiota, 
gut fermentation, and influences of yet unknown molecular factors such as phytochemicals may affect 
insulin sensitivity and further development of type 2 diabetes. Recent results suggest a link between 
changed gut microbiota and varied glycemic responses.1, 13, 14 Furthermore, subjects with T2DM had 
depleted Bifidobacteria,38, and also the level of glucose tolerance was associated with certain gut bacteria.39 
Another review concluded that despite convincing epidemiological evidence, the lack of controlled clinical 
studies to support or explain the mechanisms underlying the protective effect of whole grains against type 
2 diabetes.40 Thus, this application aims to uncover the role of gut microbiota and their metabolites of 
whole wheat bread (WWB) components in mediating blood glucose excursions in prediabetes. We will 

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specifically compare the gut microbiota composition of prediabetes subjects who exhibited improved 
glucose metabolism following the WWB diet with those who responded least to this dietary intervention to 
determine if differences in gut bacteria could explain the beneficial effect of WWB in some individuals.   
III. Anticipated Results  
We expect to achieve >95% compliance (based on in-house observations of test meal consumption, returned 
foods, and circulating biomarkers of WWB intakes, e.g., alkylresorcinol concentrations of 19:0, 21:0, and 
23:0) without any adverse effects or changes in body mass or energy intakes.   
Based on prior studies,13 we expect a >25% within-trial decrease in postprandial blood glucose by WWB in 
high-responders compared to WB. While a statistically significant between-treatment effect is also 
expected, our study is designed to define ‘high-responders’ vs ‘low-responders’ towards precision nutrition 
rather than achieve group mean differences by bread treatment.14 In addition, we expect reduced serum 
endotoxin during WWB intervention, as metabolic endotoxemia is known to initiate insulin resistance in 
obese persons.41, 42 These benefits are expected to occur through an anti-inflammatory mechanism that 
attenuates gut-derived endotoxin translocation43 or an antimicrobial activity of WBB that depletes 
pyrogenic Gram-negative bacteria. In support of the former, we expect WWB will decrease small intestinal 
and colonic permeability,44 based on decreased urinary excretion of lactulose/mannitol and 
sucralose/erythritol. WWB may also alter, at least in a subset of the study participants, the gut microbial 
composition, as reported previously in both healthy adults as well as in persons with MetS (e.g. an increase 
of Lachnospira, Faecalibacterium, Prausnitzii, and Prevotella copri abundance and a decrease in 
Enterobacteriaceae abundance in the WWB group45, 46). It is also possible to detect selective antimicrobial 
activities of WWB (possibly due to its rich phytochemicals)16, 47 and consequentially decreased 
LPSproducing Gram-negative bacteria. If these findings are consistent in a subset group of study 
participants who have larger changes of glycemic response post WWB intervention, they will collectively 
suggest a mechanism, at least in part, that the multi-facet health benefit of WWB consumption is gut 
microbiome– dependent. Meanwhile, with our combined targeted and untargeted metabolomics approaches, 
we expect a group of known (e.g., energy metabolism metabolites and amino acids)48, 49, and unknown 
metabolic features may change significantly in fecal and blood metabolome between WWB and WB 
intervention. Taken together, these studies will establish a solid foundation for determining health and 
metabolic effects on the human host based on the composition and microbial metabolic capability of gut 
microbes.   
  
IV.  
Pitfalls and Alternatives  
Recruitment. We acknowledge the challenge to recruit adults with prediabetes for a nutrition study during 
this unusual time of pandemic. Consistent with our previous studies,50-52 we will consult the recruitment 
office of the OSU CCTS for assistance. They maintain a participant database (>126K registrants) and 
provide marketing support for study recruitment (e.g. social media, printed flyers, e-newsletters reaching 
~100K faculty/staff and students at OSU).   
All proposed procedures are well-established and frequently utilized by our research team. We, therefore, 
do not expect potential technical problems. However, we acknowledge the challenge of the potential 
interpersonal variability of dietary responses to WWB among the enrolled study participants, and there may 
be difficulties to identify sufficient “high-responders” and “low-responders” based solely on the glycemic 
responses. If needed, we will combine other health parameters, such as HOMA-IR, serum endotoxin, and 
select inflammatory biomarkers, to define response indicators for their gut microbiome dependency.   
  

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V.  
Significance of the Research and Potential Benefits  
Whole grain, especially whole wheat food, is a recognized, important part of healthy dietary patterns due to 
its high abundance of fibers, protein, and bioactive phytochemicals. However, from both clinical trials and 
observational studies, controversy persists to clearly define the advantage of having incorporating whole 
wheat into the diet to manage metabolic disorders including prediabetes. Gut microbial composition and 
their metabolism, as recently emerged in many exciting studies, may play a critical role in determining the 
health benefits of having whole wheat in a regular diet in diverse populations. Therefore, our study is 
significant because it will provide critical data to help resolve the controversy by studying the gut 
microbiome-mediated, personalized effects of whole wheat intake in persons with prediabetes. Specifically, 
the integration of our multi-omics workflow coupled with studies in humans and in vitro will advance an 
understanding of the metabolic signatures of gut bacteria function to control the risk of prediabetes/glucose 
intolerance  
  
VI. Procedures  
  
A. Overview of Study Design  
  
Figure 1. The schematic of the proposed clinical study with WWB intervention. Each dot represents a time point 
for its corresponding analysis. Measures for gut microbiome (red dots) involve participants collecting their 
own fecal samples during the preceding 24-hours. Investigators will then conduct 16S rRNA sequencing of 
fecal/gut bacteria to determine phylogenetic composition. Clinical and blood tests (green dots) entail measures 
of anthropometrics, blood pressure, routine blood chemistries (glucose, insulin, lipids), circulating inflammatory 
proteins (C-reactive protein, interleukin-6 and -8, tumor necrosis factor-α, myeloperoxidase, mRNA expression 
of pro-inflammatory genes from isolated peripheral blood mononuclear cells (Toll-like receptor-4, MyD88, p65, 
interleukin-6 and -8, tumor necrosis factor-α, myeloperoxidase, monocyte chemoattractant protein-1. Serum 
endotoxin will also be assessed. Measures of metabolomics (blue dots) will be performed from collected fecal 
and serum samples to quantify >200 metabolites from >25 metabolic pathways. In addition, short chain fatty 
acids will be evaluated from fecal samples. Gut health (purple dots) entails participants ingesting a solution 
containing non-digestible sugars and collecting their own urine for 24-hours. Urinary excretion of sugars is then 
measured to determine region-specific intestinal permeability.  
  
We plan to conduct a 2x2 randomized, controlled, crossover trial in persons with prediabetes (n = 40; 1:1 
M/F) to assess 2-wk consumption of white bread (WB) vs whole wheat bread (WWB) on cardiometabolic 
and gut microbiome parameters (Figure 1). Participants will be recruited from the Columbus, OH area, 

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consistent with earlier successful studies conducted by the Bruno lab. Participants will be block-randomized 
in 4-unit blocks to receive WB or WWB (128 g/day; 4 bread slices/d, according to the USDA “My Plate” 
recommendation and other similar studies)14, 53, 54 for 2-wks. During each study arm, participants will be 
provided standardized test meals containing WB or WWB, these foods will be prepared and packaged in 
the Human Nutrition Metabolic Research Kitchen (Evans Laboratory) to be devoid of whole grain products, 
probiotic-containing foods (e.g., yogurt), or fermented foods that are known to alter gut bacteria 
populations. Before and after each 2-wk study arm, a fasting blood sample will be obtained for metabolomic 
and clinical measures. Blood pressure and anthropometrics also will be assessed on days 1, 7, 14, and 
participants will collect a fecal sample to provide to the study team during their visits on day 1 and day 14.  
Lastly, on day 14, participants will simultaneously complete an oral glucose tolerance test and gut 
permeability test, which involves ingesting a glucose solution that also contains non-digestible sugar probes 
prior to collecting complete 24-hour urine samples for assessments of upper and lower gut permeability 
(described below). Upon completing these procedures, participants will undergo >2-weeks wash-out before 
repeating the study identically, but with crossover to the alternate bread diet.  
  
B. Study Subjects  
  
1. 
Enrollment Criteria. Male and female adults with prediabetes (18-65 y), and having no history of 
liver or cardiovascular disease, overt diabetes, or cancer will be enrolled. They are expected to have fasting 
blood glucose between 100 to 125 mg/dL and not taking any medications to manage hyperglycemia. To 
improve participant homogeneity, we will target persons having a body mass index (BMI) of 30-35 kg/m2 
who are more likely to be afflicted with prediabetes. Major exclusion criteria: significant dietary restrictions 
(e.g. vegetarian, gluten-intolerant, lactose intolerant) or food allergies (e.g., nuts dairy, egg, soy, fish); user 
of dietary supplements, prebiotics, or probiotics; aerobic exercise >5 h/wk; smoker; alcohol consumption 
(≥2 drinks/d); pregnancy or fertility treatments; usage of antibiotics or antifungals within 3-mo prior to 
participation; chronically active inflammatory or neoplastic disease in the three years prior to enrollment; 
chronic gastrointestinal disorder, including diarrhea, inflammatory bowel disease, and Celiac disease; 
myocardial infarction or cerebrovascular accident in the 6 months prior to participation; coagulation 
disorders; chronic immunosuppressive medication usage.   
  
2. 
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; see 
attachment “Advertisements”), and word of mouth. The posted advertisements will instruct interested 
participants to call the study center (Bruno Laboratory, Department of Human Sciences) or complete an 
online pre-screening survey 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 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).   
  
3. 
Power Calculation and Data Analysis. Dr. Mo (biostatistician; co-PI and Dept of Biomedical 
Informatics) performed a power analysis. To ensure that the order by which each participant completes each 
trial is randomized, a random sequence generator (http://www.random.org/sequences/) will be used to 
determine the order by which each participant will undergo the two trials. We will primarily compare the 

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within-subject changes (WWB endpoint vs. WB endpoint) between high-responders and low-responders. 
We anticipate to have 6-12 high-responders and 5-10 low-responders of the total 40 study participants to 
achieve 80% power to detect a 1.6-fold difference in the changes of postprandial glucose levels (AUC from 
0 to 180 minutes) between high-responders and low-responders, at the expected coefficient of variation 
(CV) 0.5 at a significant level of 0.05.    
Mixed effect modeling will be performed, diet and time will be included as fixed factors while participant 
serves as a random factor. False discovery rate (FDR) will be used to control type I error rate for microbiome 
and metabolome data, while the Bonferroni method will be applied for other measures (e.g., inflammatory 
markers). Data will be first normalized before analysis. Spearman correlations will be calculated to define 
associations between microbes, inflammatory genes, metabolites that show significant changes in 
highresponders.   
To test whether the changes in microbes after WWB intake associate with the biological response, logistic 
regression will be fitted to each of the potential microbes or as a combination. We will also couple the 
logistic regression with receiver-operating characteristic (ROC) analysis to evaluate the specificity and 
sensitivity of the microbes that predict participants’ biological response, and the area under the curve (AUC) 
of the microbial model will be displayed. Data analysis will be performed in R (open resource) or SAS 
(SAS, Inc; Cary, NC). The sample size was calculated in PASS 20 (NCSS, LLC; Kaysville, Utah).   
C. Measurement/Instrumentation  
All procedures will take place in the Human Nutrition Program located in Evans Laboratory. Infrastructure 
that is present includes a metabolic research kitchen, a private office to meet with participants, a clinical 
research laboratory for performing phlebotomy and research procedures, and a separate biochemical 
laboratory for processing and preserving biological specimens.  
1. 
Anthropometric Parameters and Blood Pressure. At screening, day 1, 7, and 14, participants will 
rest for 15 minutes prior to determining blood pressure using an automated blood pressure cuff. BMI will 
be calculated from height determined from a wall-mounted stadiometer and weight from a calibrated scale.   
2. 
Blood Chemistries. Fasting plasma glucose and lipids (total cholesterol, HDL-C, triglyceride) will 
be measured by clinical assay (Pointe Scientific) at screening, day 1, and day 14. Fasting insulin will be 
measured by ELISA (ALPCO) at day 1 and day 14. Postprandial glucose (0-180 min at 30 min intervals) 
following an oral glucose tolerance test will also be measured on day 14. At the beginning and the end of 
each intervention arm, we will define the effects of WWB 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.52 We will also measure systematic inflammatory 
responses in both fasting and postprandial serum by ELISA (CRP, IL-6 and -8, TNFα, MPO) and by RTPCR 
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).   
3. 
Endotoxin. Serum endotoxin from the individual participant will be assessed on day 1 and day 14 
using a high-sensitivity fluorometric assay (PyroGene rFC; Lonza) and endotoxin-free consumables as 
reported in our previous studies.55 In our hands, inter- and intra-assay CV is 4.2-6.1%.  
4. 
Preparation of Whole Wheat Bread. To ensure uniformity in WB and WWB test products 
throughout this controlled study, both types of breads to be used will be prepared by co-Investigator Dr. 
Devin Peterson (Department of Food Science and Technology) and provided to the Bruno lab for clinical 
study.  WWB and WB will be prepared in the Peterson lab in the Parker Food Science and Technology 
Building as previously reported by Cong et al.,56 using the modified AACC straight-dough bread-making 
method.57 The formula, is 200 grams of flour, included 10.6 g yeast (active dry), 12.0 g sucrose, 3.0 g salt 
(NaCl), 6.0 g shortening, and 130.0 g of water. Ingredients will be mixed (KitchenAid, Benton Harbor, MI) 
until a sponge dough is formed, approximately 2.5 min. The dough will be placed into a 5-3/4” x 3-1/4” x 

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2-1/4” (14.6 × 8.3 × 5.7 cm) loaf pan and fermented for 52 min at 30 °C and 85 % relative humidity, then 
proofed for 25 min, again for 33 min and punched between proofs. Finally, the dough will be baked at 215 
°C (Doyon convection oven, Menominee, MI) for 17 min. The bread in this study will be made with limited 
ingredients to focus on the influence of endogenous chemistry in whole wheat flour on health activity of 
whole wheat bread.   
5. 
Dietary Control.  To improve diet homogeneity and limit potential confounding effects of whole 
grain/wheat intakes on study outcomes, participants’ basal diets will be rigorously controlled during each 
2-wk intervention by a registered dietitian (PI Bruno) to provide 50-60% energy from carbohydrate, 2535% 
fat, and 15-25% protein consistent with the typical American dietary pattern. Participants will be required 
to consume bread-containing test meals at breakfast and lunch each day; these will be consumed at the 
study center 4 d/wk during each 2-wk study arm to establish rapport and ensure compliance to study 
procedures. The WB and WBB used in this study will be prepared in the pilot lab in the Department of 
Food Science and Technology under the auspice of Dr. Peterson (co-investigator of the project) to ensure 
compositional consistency.  
  
Participants’ basal diets, including bread-containing test meals, will be administered in a eucaloric manner 
by estimating energy requirements using the Harris-Benedict equation,58 with appropriate adjustment for 
physical activity.45 During each 2-wk study arm, participants will return any uneaten foods, which will be 
weighed to determine actual energy and nutrient intakes using NDSR dietary analysis software. All foods 
and beverages will be identical between study arms with the exception of WBB and WB. Participants will 
also complete a food diary to record the consumption of any non-prescribed foods. Non-fasting body weight 
will be measured 3 times/week throughout the study to monitor weight stability. If body mass fluctuates >1 
kg, the participant’s prescribed energy intake will be adjusted to prevent additional weight loss or gain. 
Thus, our approach will minimize participant burden while applying sufficient dietary rigor to test the 
hypothesis that WBB consumption, principally through its bioactive phytochemicals, improves glucose 
tolerance in a microbiota-mediated manner.  
  
6. 
Gut Permeability. On day 14 of each intervention, fasted participants will ingest a glucose solution 
(75 g; oral glucose tolerance test) containing 4-sugar probes [lactulose (5 g), mannitol (1 g), sucralose (1 
g), and erythritol (1 g)] as described.59, 60 Urinary sugars will be assessed by LC-MS as we established.59 
Sugar excretion (%), and excretion ratios of sugars from 0-5 h (lactulose/mannitol) and 6-24 h 
(sucralose/erythritol) will be calculated to reflect upper and lower gastrointestinal permeability, 
respectively.61-64 This approach is based on lactulose being absorbed in the small intestine, but not the colon, 
whereas sucralose is absorbed in the colon.65 Thus, these studies will define site-specific gut permeability, 
and the benefit of WWB treatment.  
  
7. 
Microbiota Composition. Microbiota composition and the predicted functional metagenome will 
be assessed from fecal samples provided on day 14 of each intervention arm as previously described.66-70 
Effects of WWB (vs WB) on total bacterial abundance will be determined by qPCR.71 Microbiota will be 
characterized for diversity and community structure by sequencing 16S rRNA genes using MiSeq. Bacteria 
will be identified by comparing sequences representing each operation taxonomic unit (OTU) with RDP 
and Silva databases. 72, 73 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,74 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 PICRUSt75 and PanFP76 based on reference 
genome databases and the KEGG Orthology classification scheme to obtain a functional gene abundance 
matrix.  

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8. 
MS-Metabolomics & Short Chain Fatty Acids. We will perform rigorous metabolomics analysis 
of serum samples on day 1 and day 14 and fecal samples provided at day 1 and day 14 from study 
participants use multiplex MS-based metabolomics with the following techniques: (1). Targeted LC-
MS/MS metabolic profiling (TMP) methods using both regular and stable isotope-labeled chemical 
standards to achieve accurate, specific, and quantitative metabolite measurements of >200 metabolites from 
more than 25 metabolic pathways.  77 This method can provide accurate pathway-by-pathway measurement 
and validated metabolite identification for further biological system analysis, as demonstrated by our recent 
publications 78-80. (2). We will also use a newly developed metabolomics approach, Globally Optimized 
Targeted (GOT)MS 81 to analyze broader coverage of metabolites. GOT-MS combines many of the 
advantages of targeted detection and global profiling in metabolomics analysis, including the capability to 
detect unknowns, broad metabolite coverage, and excellent quantitation. (3) We will also use untargeted 
metabolomics for exploratory studies: Briefly, a Thermo LC-Q-Exactive Hybrid MS system will be used 
for untargeted analysis. Compound Discoverer 3.1 software will be used with customized workflows for 
different types of samples. Compound identification will be achieved by multiple database searches 
(HMDB, m/z cloud, METLIN, mass bank, etc.) 82-84 and MS/MS fragmentation pattern comparisons to 
authentic standards. In addition, using our targeted LC-MS/MS-based panel of >50 frequently reported 
phytochemicals in whole wheat, such as representative alkylresorcinols, benzoxazinoids, phenolics (e.g., 
ferulic acid),4, 5  we will quantify these phytochemicals and metabolites in serum and fecal samples 
following enzymatic hydrolysis with β-glucuronidase/sulfatase.85 Analyte identification and quantification 
will be confirmed by comparing fragmentation patterns relative to purified standards or reference databases. 
In addition to defining the effect of WWB treatment on host metabolism, time-dependent serum measures 
of alkylresorcinol concentrations of 19:0, 21:0, and 23:0 and total alkylresorcinols in study participants who 
consume WWB will objectively establish subject compliance to the dietary intervention.45, 86  
Separate LC-MS/MS procedures that are established by our research team, based on our published report,87 
and will be used to evaluate a panel of 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 is often depleted with gut dysbiosis whereas the 
latter is 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 negative ESI mode for quantitative 
analysis.  
  
D. Detailed Study Procedures  
1. 
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. Prospective participants will be provided the Informed 
Consent form via email for review prior to an in-person discussion at the study center. During the in-person 
meeting, the Informed Consent (see Informed Consent attachment) will be explained and a hardcopy 
provided for them to review. If he/she chooses to participate in the study, they will then be asked to provide 
written consent. After receiving informed consent, the participants’ height, weight, 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 then complete two 2-
week intervention periods in a randomized order, each followed by a 3-h postprandial trial where a glucose 
tolerance test will be administered (Figure 1). We estimate that completion of all study procedures will take 
~6-8 weeks per participant. Each study procedure will be discussed in detail below:  

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2. 
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 review it, and if they choose to participate in the study, they will be 
asked to provide written consent. Although the participant will be asked to sign the informed consent, the 
participant will be told that they will not be asked to participate if their body measurements or plasma 
chemistries (see Enrollment Criteria) do not meet the study criteria. If the participant has provided consent, 
we will then measure the participant’s height, weight, 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; 2 tubes) so 
that we may measure blood chemistries (glucose, total and HDL-cholesterol, triglyceride). All samples will 
be coded to maintain participant anonymity. If the participant’s anthropometrics or plasma chemistries do 
not meet the study criteria, they will be told that they do not meet the study criteria.   
  
Potential participants who meet the criteria will be contacted within a few days after their screening meeting 
to provide them with their blood and body measurement results and inform them of their eligibility to 
participate in the study. Consistent with our CLIA exemption, blood results will be provided in a categorical 
manner (i.e. “normal”, “marginally high”, “high”) rather than providing actual blood concentrations of lab 
values (see Subjects Results Sheet). Potential participants having any blood values outside of the “normal 
range” will be directed/encouraged to follow-up with their own physician. Those having body 
measurements and blood values within acceptable limits (see Inclusion/Exclusion criteria), will be invited 
to participate in the study. While only blood glucose and BMI will be used as the major inclusion/exclusion 
criteria, other blood chemistries and blood pressure measurements will also be assessed to characterize 
participants’ general health status. Subjects will be included or excluded based on a best fit of the inclusion 
and exclusion criteria (an example of best fit would be if a potential subject says he/she exercises 4 h/wk, 
which is close to our exclusion criteria of 5 h/wk, they might still be included in the study if they meet all 
other inclusion and exclusion criteria more closely than other potential subjects). Participants will be read 
one phone script if they qualify and another phone script if they do not qualify (see Participant Eligibility 
Phone Script attachment). Potential participants who qualify for the study will be communicated a message 
as follows: “Congratulations! You have been selected for participation in our study based on your blood 
testing results. A study investigator will be telephoning you to invite you to the testing session. Would you 
like to know your blood chemistry results?” Subjects not selected for study will be told the following 
message: “You have not been chosen for our study, but thank you for your interest.”  This message will be 
followed by an explanation why they were not chosen, such as lab values outside of the range we are looking 
for: “Your blood testing data is…..we were looking for participants who had levels less than …..”.  “We can 
provide you a copy of your results if you would like…..how would you like us to provide them to you?”. 
“You should also consider sharing these results with your physician.”   
  
If a participant is telephoned and is unavailable, a message will be left requesting a callback at a convenient 
time or that at a member of the study team will try calling again at a later time. No confidential or sensitive 
information will be shared with third parties or left on answering machines.  
  
3. 
Study timeline and activities occurring at each study visit. As indicated in Figure 1, each 
participant will complete a 2-arm (whole wheat bread; white bread), randomized, cross-over study. For this 
study, each participant will visit the study center a total of 13 times (screening visit plus 6 times per study 
arm), including:  
  

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Informed Consent & Screening Visit.  Potential participants will visit the study center to meet 
with a designated study team member. At this meeting, the informed consent will be reviewed with 
the potential participant and be allotted time to address any questions. If the participant agrees, the 
informed consent form will be signed for enrollment.  If the participant is fasted, anthropometrics 
and blood pressure will be measured, and a fasting blood sample will be collected to determine 
whether circulating chemistries are within acceptable limits. If the participant is not fasted, these 
procedures will be scheduled at a time of mutual convenience.  If the participant fulfills study 
inclusion criteria, the participant will proceed to complete Trial 1 procedures.  
  
• 
Trial 1 Procedures (2-week intervention to WWB or WB) o Day 1: Participant will visit the 
study center for measures of anthropometrics and blood pressure, the collection of a fasting blood 
sample, and to pick-up daily meals for Days 13. All foods and beverages will be provided, and daily 
test meals will include WWB or WB. The participant will also provide a fecal sample that was self-
collected within the preceding 24-hours.  
  
o Day 3: Participant will visit the study center briefly to pick-up meals that will be consumed 
on Days 4-6. Participant will return all uneaten foods and containers to the study team to 
determine actual food consumption during the preceding period.  
  
o Day 7: Participant will visit the study center briefly to pick-up meals that will be consumed 
on Days 7-10. Participant will return all uneaten foods and containers to the study team to 
determine actual food consumption during the preceding period. Measures of 
anthropometrics will be conducted and a urine sample will be collected to assess 
compliance to test meals.    
  
o Day 10: Participant will visit the study center briefly to pick-up meals that will be 
consumed on Days 11-14. Participant will return all uneaten foods and containers to the 
study team to determine actual food consumption during the preceding period. A stool 
collection kit will also be provided.   
  
o Day 14: Participant will visit the study center in the fasting state. The participant will 
provide a fecal sample that was self-collected during the preceding 24-hours. A urine 
sample will be collected to assess compliance to test meals.  Participant will also return all 
uneaten foods and containers to the study team to determine actual food consumption 
during the preceding period. Anthropometrics and blood pressure will be assessed. Blood 
will be collected (0 min) and then an oral glucose tolerance test (75 g) and gut permeability 
test as described.59 will be completed simultaneously. After emptying their bladder, the 
participant will ingest a 75 g glucose solution containing non-digestible sugars. Blood 
samples will be collected at 30-min intervals for 3-hours. Participant will be provided urine 
collection containers to self-collect urine for the subsequent 24-hours. During this 24-hour 
period, participants will also be provided controlled meals and beverages.  
  
o Day 15: Participant will return to the study center to provide collected urine. All procedures 
for Trial 1 are deemed complete.  
  
• 
Wash-out Period.   
Trial 1 and Trial 2 will be separated by at least two-weeks for men. However, the washout period for women 
may be extended to enable testing during the same phase (± 3 days) of the menstrual cycle.  
  

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Trial 2 Procedures (2-week intervention to WWB or WB).  
All procedures for Trial 2 will be identical to those of Trial 1 with the exception that the participant will 
receive the alternative test meals. If the participant was assigned WWB in Trial 1, WB will be assigned in 
Trial 2. If WB was assigned in Trial 1, then WWB will be provided in Trial 2.  
  
• 
Study Completion  
On Day 16 of Trial 2, the participant’s involvement in the entire study is completed. A check will be provided 
to the participant in accordance with the payment schedule (up to $350) that is outlined in the consent form.   
4. 
Blood Collection and Sampling Handling. Once at baseline (day 0; 14 mL), and at each time point 
of the postprandial trial (20 mL * 7 timepoints over 3-h), a blood sample will be collected into evacuated 
blood collection tubes. Collecting a total of 154 mL or 0.65 cups once during each study arm is necessary 
to ensure an adequate volume of plasma and serum for each time point to accurately analyze levels of 
glucose, insulin, endotoxin, and pro-inflammatory genes. 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 3 hours, other than the risks stated previously.   
Throughout the span of the study (~8-12 weeks in duration depending on participant/investigator 
availability) which consists of four weeks total of controlled feeding, a blood collection at day 0 and 14 and 
then repeated following a ≥2-week washout period, and 1 screening day, we will be collecting a total of 
~318 mL or ~1.34 cups) of blood. Urine will be collected in provided containers (VWR) containing 10% 
thymol to inhibit bacterial growth. We will then have the participants return their urine 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. Feces will be collected using a commercial commode specimen collection 
system (Fisher Scientific). Briefly, the collection kit consists of the necessary materials (e.g., gloves, waste 
bag) for participants to easily and hygienically collect their stool without contaminating the sample or 
themselves. Volumes of urine will be recorded and fecal mass and observations will be recorded based on 
the Bristol Stool Chart.88   
  
During each blood collection, plasma 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 plasma glucose, insulin, triglyceride, HDL cholesterol, proinflammatory genes 
and/or proteins (e.g., TLR4, CD14, MD2, MyD88, p65, IL-1, IL-6, IL-8, TNF-α, MCP-1), and serum 
endotoxin. Urine will be stored at -80ºC until analysis can be completed. Analysis from urine will include 
sucralose, erythritol, lactulose, and mannitol. Fecal samples will be stored at -80ºC until analysis can be 
completed. Feces analysis will include microbiota composition, calprotectin, myeloperoxidase (MPO), 
and fecal metabolomics analysis. 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 biomarkers. 
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.  
  
E. 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. 

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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 (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.  
  
F. 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. 
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.  
  
G.   Medical Safety Plan  
All aspects of the clinical study will be conducted in Dr. Bruno’s clinical lab located in 1013 Evans 
Laboratory. Participants will be fasted for 10-12 hours prior to each study visit. We recognize that certain 
risks associated with fasting include: hypoglycemia, weakness, and fainting. This duration of fasting is 
consistent with guidelines set forth by the American Diabetes Association to minimize risk to the 
individual62 when determining fasting blood glucose concentrations.   
  
Risks related to hypoglycemia are anticipated to be low due to participants’ already marginally elevated 
fasting blood glucose (study entry criteria = 100-126 mg/dL). They will also be ingesting a glucose beverage 
containing 75 g of glucose immediately following the fasting period. Throughout each visit to the study 
center, participants will be closely monitored by a member of the study team (PI, postdoctoral scholar, or 
graduate student). They will monitor the safety and well-being of participants for any signs and symptoms 
of hypoglycemia including: confusion, dizziness, irritability, weakness, headaches, and fainting.  
Additionally, Dr. Bruno (PI; 1013 Evans Laboratory) has an academic office in close proximity to the study 
center and has significant experience coordinating clinical research studies involving overnight fasting, and 
other dietary- and carbohydrate-challenges, thereby supporting the competency of our research team in 
managing potential adverse events relating to fasting glucose and glycemic responses.   
  
Consistent with our prior studies of similar design, and to ensure participant safety, all blood collection 
procedures throughout each study visit will occur while positioned on a hospital bed in the prone position 
or while seated in a phlebotomy chair. In the event that a participant was to become weak, dizzy, or faint, 
they would already be in a safe position to minimize risks associated with these symptoms. In the event that 
hypoglycemia-related symptoms occur, the study would be terminated to allow the participant to recover.  
We are prepared to provide pre-packaged beverages and snacks containing simple carbohydrates (e.g. 
Gatorade, fruit juice, apple sauce, crackers) that will allow for rapid restoration of blood glucose. These 
food items will be stored in a refrigerator, located in the adjacent metabolic research kitchen, that is 
dedicated for foods used in research studies. Participant status (e.g. attentiveness, skin color) will be 
monitored in our clinical laboratory to ensure recovery. The clinical area where participants undergo blood 
draws is also equipped with first-aid measures (e.g. smelling salt) and all study team members are trained 
to assist with basic first-aid application if needed.  
  

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Any adverse hypoglycemic response that might occur during this study will receive care commensurate to 
the symptoms.  For example, if a participant were to faint, then smelling salts would be administered along 
with a carbohydrate-containing food upon regaining consciousness. The study PI would also be contacted.    
  
Alternatively, should a more severe hypoglycemic response occur (e.g. contusion or laceration relating to 
fainting), the research team would immediately contact medical services (i.e. 911). For non-life-threatening 
emergencies, any OSU students participating in these studies would be directed to the Wilce Student Health 
Center.  For other emergencies or those participants who are not OSU students, individuals would be 
directed to the Wexner Medical Center either by transporting them directly or requesting ambulance service. 
Regardless of the complexity of the adverse events, the research team would monitor the participant in the 
interim, provide palliative care as appropriate, and follow-up after any medical care has been provided.  
  
  
  
VII.  
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52. 
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