Alleviation Of Metabolic Endotoxemia In Adults With Metabolic Syndrome With Milk Fat Globule Membrane
Phase: NA
Conditions: Metabolic Syndrome, Metabolic Endotoxemia
Sponsor: Ohio State University

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Alleviation of Metabolic Endotoxemia in Adults with Metabolic Syndrome with Milk Fat Globule 
Membrane 
Study Protocol NCT Number: NCT03860584 
Date: 10/18/2018 
 
I. 
Objectives 
 
Our preclinical evidence shows that phospholipid-rich milk fat globule membrane (MFGM) 
attenuates lipopolysaccharide-induced increases in gut permeability and pro-inflammatory 
cytokines. MFGM also attenuates inflammation in association with a prebiotic and/or antimicrobial 
activity that modulates microbiota composition. Our central hypothesis is that MFGM-enriched 
dairy milk compared with a matched milk beverage containing coconut and palm oil (control) 
decreases metabolic endotoxemia and improves glucose tolerance in metabolic syndrome (MetS) 
adults by increasing gut barrier integrity in association with alleviating gut dysbiosis and 
inflammation. To test this, we will fulfill the following objectives: 1) MFGM-mediated 
improvements in gut barrier function. We hypothesize that MFGM-enriched milk decreases MetS-
associated endotoxemia by alleviating gut dysbiosis and improving barrier integrity, and 2) 
Improvements in glucose tolerance by MFGM. Our hypothesis is that MFGM-enriched milk 
improves glycemic control in association with attenuating TLR4/NFκB-dependent inflammatory 
responses. 
 
II. 
Background and Rationale 
Endotoxemia is prognostic of metabolic health. Gut-derived bacterial endotoxins (i.e. LPS derived 
from the cell membrane of Gram-negative bacteria) trigger endotoxemia and induce NFκB 
inflammation following their ligation of TLR4. Key determinants of metabolic endotoxemia are 
intestinal bacteria overgrowth (i.e. gut dysbiosis) and diminished gut barrier integrity. Consequently, 
gut-derived endotoxin translocation triggers TLR4/NFκB-dependent inflammatory responses (e.g. 
TNFα, IL-1, IL-6) to provoke metabolic derangements (e.g. insulin resistance, lipogenesis).1 
Preventing gut-derived endotoxin translocation is therefore critical for alleviating endotoxemia. 
Although the mechanism by which dairy-rich diets protect against MetS is under investigation, our 
preclinical studies support gut-level antiinflammatory benefits of MFGM,2 a protein-lipid complex 
that surrounds the fat globules in milk. Thus, we propose a translational study in MetS persons using 
dairy milk formulated to be enriched with MFGM to alleviate endotoxemia. We expect that MFGM 
will suppress gut barrier dysfunction and endotoxin-inducing inflammatory responses in the host 
attributed to LPS-TLR4-NFκB activation. 
Dairy milk provides gut-level cardiometabolic benefit. Controlled studies aim to establish the 
cardiometabolic mechanisms of dairy foods that have been suggested by observational studies.3 
Chronic feeding studies generally support this concept, but many are difficult to interpret because of 
the complex composition of dairy milk. Likewise, displacement of “pro-disease” non-dairy foodstuffs 
by dairy foods also limits interpretation. The Bruno Lab has circumvented these challenges, in part, 
by studying the controlled administration of dairy milk or its constituents on postprandial metabolic 
health.3-6 For example, our recently completed randomized controlled trial in prediabetic adults 
attributes a portion of the cardiometabolic benefits of dairy directly to its casein and whey protein 
fractions (under review).7,8 Specifically, dairy proteins lower cardiovascular risk, likely by a 
mechanism involving the gut-vessel axis that increases cholecystokinin to limit hyperglycemia-
mediated oxidative stress that otherwise impairs nitric oxide-dependent vascular endothelial function. 
However, separate gut-level benefits of dairy milk are likely attributed to dairy fat-derived MFGM.2 
This is consistent with our studies in which MFGM exhibited antiinflammatory benefit in rodent 
models.2,9 Thus, through an innovative controlled feeding study with a matched placebo, we will have 

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a novel opportunity to translate the benefits of MFGM to improve gut barrier function and attenuate 
pro-inflammatory responses at the gut and systemically. 
MFGM is a unique milk fat-derived antiinflammatory constituent. The MFGM is the membrane 
(trilayer) surrounding secreted milk fat droplets.10 It originates from the apical surface of mammary 
epithelial cells and is comprised of a unique triple phospholipid and cholesterol layer embedded with 
proteins and glycoproteins. The genes that synthesize MFGM are conserved across species and its 
composition differs little between-species. Notably, phospholipids account for 1% of total milk lipid, 
with ~one-third each from sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines. 
Although MFGM is present in most dairy foods, churn buttermilk is quite rich in this membrane 
fraction. Indeed, churning of milk fat disrupts the membrane surrounding the fat globules. This results 
in the coalescence of free fat that ultimately yields butter. In contrast, buttermilk is the aqueous phase 
(i.e. MFGM-containing portion). MFGM constituents (e.g. polar lipids, gangliosides, proteins) 
improve intestinal health9 and exert antiinflammatory activity systemically and at the gut.11-14  
MFGM protects against endotoxin-induced gut permeability. MetS is characterized by increased 
“gut leakiness” consistent with our preclinical studies in obese mice showing that endotoxemia occurs 
in association with decreased expression of intestinal tight junction proteins (e.g. occludin, zonula 
occluden, claudin).15,16 In contrast, our dietary interventions with a functional food (green tea) fully 
protected against the loss of gut barrier integrity and normalized serum endotoxin levels to attenuate 
hepatic TLR4/NFκB inflammation and oxidative stress.15,16 Similarly, the polar lipids present in 
MFGM provide resistance to epithelia stress.12,17-19 These bioactivities are important, because they 
are expected to support MFGM to alleviate metabolic endotoxemia in the proposed clinical nutrition 
intervention, consistent with a mechanism of improving gut barrier integrity (Obj 1) and attenuating 
TLR4/NFκB inflammation and glucose intolerance (Obj 2).  
Gut dysbiosis mediates endotoxemia in MetS. Inflamed intestine has high oxidant stress,20 which 
contributes to endotoxemia by permitting increased LPS absorption and transcytosis of enteric 
bacteria. Oxygen gradients also decrease steeply from the upper to lower gastrointestinal tract and 
increase from the lumen to the more vascularized epithelial layer. Oxygen regulates microbial 
colonization with more oxygen-tolerant bacteria in the mucosa (e.g. Helicobacter) whereas obligate 
anaerobes (e.g. Firmicutes) are luminal.21,22 Oxygen gradients can also contribute to microbial 
biogeography by influencing metabolite production and modulating redox effectors (e.g. nitric oxide, 
hydrogen sulfide, reactive oxygen species) of either bacteria or host origin.23 Importantly, reactive 
oxygen species generated by the intestinal epithelium24 function to exert redox-responsive pro-
inflammatory cell signaling and disrupt gut barrier integrity.25,26  
Our studies in obese mice demonstrate the importance of the microbiome to manage endotoxemia in 
relation to MetS.27 We show that endotoxemia is mediated by gut dysbiosis in association with 
increased intestinal inflammation that impairs gut integrity. In our first approach, we showed that 
obesity-associated endotoxemia is attributed to increased “gut leakiness” (based on FITC-dextran 
absorption) that causes higher levels of endotoxin in the portal vein circulation. We next showed that 
gut dysbiosis is associated with an obese phenotype. Specifically, obese mice had an increased ratio 
of Firmicutes:Bacteroidetes and decreased microbial diversity. This occurred in association with 
decreased predicted functions relating to the synthesis of short chain fatty acids (SCFAs; i.e. butyrate, 
propionate. These are important microbial metabolites that promote the expression of intestinal tight 
junction proteins28 by increasing the stability and transcriptional activity of hypoxia-inducible factor-
1α (HIF1α).29 Likewise, microbial functions relating to LPS biosynthesis were increased in obese 
mice. We also report that intestinal TNFα, iNOS, MCP-1, and TLR4 mRNA levels and other genes 
comprising this receptor (CD14, MD2) were all increased in obese mice compared with lean mice 
(data not shown). Together, especially in the context of the benefits demonstrated by our function 
food (green tea), targeting gut health is critical to ameliorating endotoxemia by improving microbiota 
composition and microbial functions that drive endotoxin synthesis and translocation.  

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MFGM can play a key role to improve microbiota composition and barrier function. Studies in rat 
pups show that MFGM supplementation improved gut development (e.g. intestinal growth, numbers 
of Paneth and Goblet cells, and expression of tight junction proteins) compared to control formula.30 
Importantly, the observed benefits of MFGM were similar to those among rat pups reared with 
mother’s milk. Likewise, both MFGM and mother’s milk similarly shifted gut microbiota populations 
compared with formula-fed controls, and they protected against Clostridium difficile toxin-induced 
intestinal inflammation. Microbiota analysis revealed that MFGM and mother’s milk pups had 
greater species richness and evenness compared with controls. In addition, MFGM supplemented 
pups had greater abundance of Lactobacilli compared with controls, but not to the extent observed in 
pups fed mother’s milk. This supports the potential of MFGM to enhance gut barrier integrity by 
increasing SCFA generation. In piglets, during the postnatal period, dietary supplementation of milk 
fat and MFGM was shown to modify intestinal protein digestion, modulate immune system 
maturation, and significantly alter gut microbial populations (e.g. increases in Proteobacteria and 
Bacteroidetes; decreases in Firmicutes).31 The latter supports MFGM-mediated improvements in 
microbiota composition to favorably improve gut physiology.  
In addition, based on these gut-level benefits that help to resolve gut dysbiosis by increasing 
proportions of “healthy” commensal bacteria, several important outcomes are also expected. For 
example, increased generation of microbial-derived SCFAs would not only ameliorate “gut 
leakiness” by upregulating tight junction proteins,28 but increased butyrate also improves barrier 
function by increasing the secretion of glucagon-like peptide-1 (GLP-1). This incretin helps to 
alleviate glucose intolerance by potentiating insulin sensitivity.32-34 Because pancreatic β-cell 
function, and hence insulin secretion, deteriorates beginning with prediabetes, MFGM would be 
expected to increase GLP-1 secretion, thereby alleviating dysregulated glycemic responses that 
provoke MetS risk. 
Summary. This overview demonstrates that: 1) low-grade inflammation in MetS is attributed to 
endotoxemia in association with poor gut barrier integrity, 2) endotoxemia is causally-related to the 
initiation of obesity and insulin resistance35 and circulating endotoxin is prognostic of progressive 
liver function deterioration,36 3) MFGM constituents provide antiinflammatory and prebiotic activity 
at the gut and attenuate endotoxemia, and 4) increased SCFAs from commensal bacteria of the gut 
not only improve gut integrity, but also promote intestinal incretin secretion to help manage glucose 
intolerance. Thus, our translational studies in MetS persons are expected to provide novel evidence 
that MFGM exhibits antiinflammatory activity to enhance glucose tolerance by attenuating 
endotoxemia. These findings would be of significance to public health because could they help to 
establish that dietary fat “quantity” is not necessarily detrimental to MetS risk, but rather fat “quality” 
potentiates host health due to enhanced gut barrier function. This would lead to evidence-based 
recommendations to help reverse course of dietary trends indicating that dairy consumption decreases 
in an age-dependent manner and that ~90% of Americans fail to meet the daily recommended intake 
of dairy.3  
Anticipated Results 
We expect the intervention to result in high compliance (>95%), consistent with our rigorous 
approach to provide all foods and test beverages.  
In Obj 1, MFGM is expected to time-dependently decrease serum endotoxin (i.e. d 0 to d 14), and 
lower day 14 endotoxin compared with coconut and palm oil-enriched milk. MFGM will also 
attenuate meal challenge-induced increases in gut-derived endotoxemia. These benefits will correlate 
with improved gut barrier function as indicated by: 1) decreased urinary recovery of sugar probes 
(i.e. increased gut integrity), 2) increased fecal SCFA levels; these potentiate intestinal junction 
protein expression,28 and 3) an alleviation of gut dysbiosis (e.g. microbial diversity, 
Firmicutes:Bacteroidetes ratio). Indeed, we expect reduced gut bacteria abundance. This is important 
because intestinal bacteria overgrowth provokes metabolic disease.37,38 Likewise, MFGM-mediated 

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prebiotic effects are anticipated to increase commensal bacteria (e.g. butyrate producers) and its anti-
microbial activities are expected to reduce pathogenic populations (e.g. pyrogenic bacteria, decreased 
LPS synthesis).  
In Obj 2, we expect that MFGM will improve glucose tolerance based on decreased fasting glucose 
(d 14) and attenuated postprandial glycemic responses following the test meal challenge. This will 
be attributed to antiinflammatory activities of MFGM at the gut (i.e. decreased calprotectin and 
myeloperoxidase) and on host expression of TLR4/NFκB genes (e.g. TLR4, IL-6). These 
antiinflammatory benefits are important because they potentiate intestinal GLP-1 and GIP secretion. 
These incretins improve insulin sensitivity and alleviate glucose intolerance by enhancing pancreatic 
β-cell function, and hence insulin secretion.32-34 Decreased TLR4/NFκB signaling will also help to 
limit metabolic derangements that mediate glucose intolerance in MetS.1 
 
Pitfalls and Alternatives 
Recruitment. We acknowledge the challenge to recruit MetS adults for a nutrition study. Consistent 
with our studies,39-41 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). If difficulty is encountered to recruit MetS persons, we can modify our approach to focus 
on persons with prediabetes. Endotoxemia and Gut Integrity. We plan to consider endotoxin levels 
as a covariate in relation to other study endpoints. Also, while we are experienced in measuring 
endotoxin,15,16,39,42,43 we are mindful of the experiences of others where these measurements were 
problematic.44 If necessary, we will use an alternative assay for LPS as we described,39 and/or 
measure surrogate markers of endotoxin exposure (LPS binding protein, soluble CD14) as we 
described.42,43 There is the possibility that MFGM may reduce circulating endotoxin without affecting 
gut integrity. This would indicate that MFGM regulates endotoxemia likely by reducing populations 
of Gram-negative bacteria or functions of bacteria relating to LPS biosynthesis (to be tested in Obj 
1) or alleviating transcellular absorption of excess LPS within chylomicrons. If MFGM does not alter 
taxonomic composition of the microbiota, metagenomic and metatranscriptomic analysis45,46 could 
be performed to define microbiota functionality. If MFGM does not improve glucose tolerance, we 
will assess triglyceridemia consistent with milk sphingolipid attenuating hepatic triglyceride in 
response to a high-fat diet.47 In the event that gut inflammation is unaffected, we will consider 
untargeted metabolomics of archived fecal samples to identify metabolite patterns in response to 
MFGM. Lastly, our approach to evaluate postprandial responses considers “net” AUC to reflect 
metabolite concentrations that increase and/or decrease relative to baseline. If this approach lacks 
sufficient sensitivity to detect between-treatment differences, we will calculate incremental AUC 
scores to only consider those changes to rise above baseline values. 
 
Significance of the Research and Potential Benefits 
Our “whole food” approach that matches nutrient profiles between study arms will maximize 
translation of the benefits of full-fat milk that are driven, at least in part, by MFGM. The dose of 
MFGM at 10-times that in whole milk also reflects that consumers obtain MFGM from dairy foods 
other than milk. As major outcomes, we expect to demonstrate novel evidence that MFGM-enriched 
dairy milk decreases metabolic endotoxemia and improves glucose tolerance in MetS adults by 
increasing gut barrier integrity in association with alleviating gut dysbiosis and inflammation. This 
will be of significance because MetS affects ~35% of Americans,48 and is driven by inflammation 
attributed to poor gut health that leads to excess absorption of bacterial endotoxins. Thus, dietary 
management of endotoxemia with dairy milk could significantly impact the incidence of MetS while 
helping to explain outcomes of observational studies suggesting benefits of full-fat milk for weight 
management.49 Ultimately, this study could help to debunk long-standing dogma that targets full-fat 

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dairy for energy reduction50 and support evidenced-based recommendations as part of forthcoming 
Dietary Guidelines for Americans. 
 
III. 
Procedures 
 
A. Study Design 
We will enroll male and female MetS adults (n = 24; 18-65 y) to complete a 2-arm, double-blind, 
randomized controlled, crossover trial. They will be randomized in 4-unit blocks to receive, for 
14 d, a controlled diet with dairy milk (3.5% fat; 3 servings/d) enriched with MFGM-derived 
phospholipid or a matched dairy milk that instead contains coconut and palm oil (control). We 
will provide all foods during each study period to ensure weight maintenance and to increase 
homogeneity of gut and host responses (see Dietary Control).  
Anthropometrics and blood pressure will be assessed at d 0, 7, and 14. Prior to (d 0) and after 
each 2-wk arm (d 14), a fasting blood sample will be collected to assess serum endotoxin and 
metabolic chemistries (glucose, insulin), and TLR4/NFκB-dependent genes from isolated 
peripheral blood mononuclear cells (PMBCs). A breath sample will be collected to assess the 
correlation analysis of plasma metabolic biomarkers. After the 2-wk intervention, from fecal 
samples collected on d 13, we will assess microbiota composition and function, SCFAs, and 
intestinal inflammatory markers (calprotectin, myeloperoxidase). During this period, 
participants will also record daily stool characteristics using a 7-point Bristol Stool scale. On d 
14, participants in the fasted state will receive a high-fat/high-glucose meal challenge to induce 
gut-derived endotoxin translocation (described below). At 30-min intervals for 3-h, we will 
evaluate circulating endotoxin, glucose, and insulin; TLR4/NFκB-dependent genes will be 
assessed from PMBCs at 0 h and 3 h. Gut permeability probes will be co-administered with the 
test meal challenge, and 24-h urine will be collected to assess gut barrier integrity. Participants 
will then undergo a 2-wk washout prior to receiving the alternative treatment and completing 
all procedures in an identical manner. 
 
B. Study Subjects 
 
Enrollment Criteria. Participants will be recruited from Columbus, OH area with assistance from the 
OSU CCTS (see Facilities) consistent with our studies in MetS persons.41 Male and female adults 
with MetS (18-65 y) having no history of liver or cardiovascular disease or cancer will be enrolled. 
They will have ≥3 established criteria for MetS,51 with these specific parameters to improve study 
homogeneity: i) glucose (>100 mg/dL), ii) waist circumference (>89 or 102 cm for F/M), and iii) 
HDL-C (<50 or <40 mg/dL in F/M). Major exclusion criteria include: Blood pressure >140/90 
mmHg; unstable body mass (±2 kg over prior 3-mo) vegetarian; food allergies or lactose intolerance; 
user of dietary supplements or probiotics (within past 1-mo); pregnancy, lactation, changes in birth 
control (within 6-mo); any gastrointestinal disorders; chronic diarrhea; smoker; excess alcohol (>2 
drinks/d); excess aerobic exercise (>7 h/wk);  recent antibiotic or anti-inflammatory agent use;. 
 
Recruitment. We will recruit participants through posted flyers, e-mail, electronic and newsprint 
advertisements (e.g. campus student and faculty/staff newspapers, local and regional newspapers), 
word of mouth, and through use of ResearchMatch (OSU CCTS). The posted advertisements will 
instruct interested participants to call the study center (Bruno Laboratory, Department of Human 
Sciences) 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 

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acceptable study participant. If the caller and the interviewer agree that the caller should participate, 
the prospective participant will be invited to a screening meeting, where the study will be fully 
described, and the individual will be provided a consent form to complete prior to any involvement 
in the study procedures. Data collected during this phase will include participant’s age, health status, 
physical activity, and contact information (see Informed Consent attachment). Recruiting efforts 
through ResearchMatch will utilize a strategy whereby registered individuals in the ResearchMatch 
database can be searched for against their non-identifiable volunteer profile in the system. 
Unidentified individuals meeting search criteria will then be forwarded an electronic recruitment 
message (see ResearchMatch Recruitment Message attachment) that identifies them as a potential 
match for study participation. The secure ResearchMatch clearinghouse will route this standard 
notification that provides specific study content (i.e. content similar to that of a posted advertisement) 
to each of these potential ResearchMatch volunteers who will then have the option of replying “yes”, 
“no”, or not respond through a set of quick links available in this notification.  Note: This message 
will not include the study’s direct contact information (e.g. email, phone) as ResearchMatch will 
measure the response rate through the clearinghouse’s quick links made available in this electronic 
message. The response rate metrics will be made available to researchers through their 
ResearchMatch dashboard as well as the Institutional Liaison dashboards. By responding “yes”, the 
volunteer has authorized ResearchMatch to release their contact information to the researcher 
responsible for the study. This information will be made available on the researcher’s ResearchMatch 
study dashboard. The researcher will be responsible for managing this contact information as 
specified in the IRB-approved study protocol. ResearchMatch will also be collecting aggregate data 
regarding the status of ResearchMatch volunteers within the study. ResearchMatch volunteers 
consent to this within the ResearchMatch volunteer agreement. The ResearchMatch enrollment 
continuum will allow researchers to indicate where the volunteer currently stands within the 
recruitment process and thus helps researchers monitor the utility and effectiveness of this recruiting 
tool. Research access to recruit through ResearchMatch will last only as long as the duration of IRB-
study approval with the expiration date of ResearchMatch being identical to the end-date of OSU 
IRB approval. Researchers will be able to submit current IRB-approval letters for the lifetime of the 
study and thus provide evidence of successful continuing review applications. If an unintentional 
lapse in time occurs and the research is not able to submit this continuing review evidence via 
ResearchMatch, stored ResearchMatch data will not be deleted, but the researcher will not have 
access to this information until a current IRB-approval letter is uploaded and routed to the 
Institutional Liaison for review. A complete description of ResearchMatch along with the most 
current IRB approval from Vanderbilt University (i.e. this is the site where ResearchMatch was 
developed and its secure computer servers are housed) has been attached. 
 
Power Calculation and Data Analysis. Dr. Brock (biostatistician; see Letter of Support) performed 
a power analysis using endotoxin (d 14) as the primary outcome. No human studies have examined 
MFGM on endotoxemia. Thus, we used our data from MetS and healthy persons in which fasting 
endotoxin was measured 5-times at 2-wk intervals. On average, endotoxin was >2-times higher in 
MetS (P<0.0001), and did not differ (P>0.4) by time or gender. We then calculated the mean 
difference in endotoxin between healthy and MetS subjects (16.4 ± 7.8 vs 32.4 ± 4.4 EU/mL, CV 
=48% vs 15%). Power analysis indicated that only 8 subjects/group would be required to obtain 95% 
power (α = 0.05) using the larger of the two CVs. For the proposed study in only MetS persons, we 
estimated that MFGM would result in a conservative improvement of 60% of the difference in 
endotoxin in the MFGM vs control phases: ((32.4-16.4) x 0.6 = 9.6 EU/mL); CV = 32% (SD = 10.4). 
Thus, we would have ~90% power (α = 0.05) with 14 MetS persons completing both study arms. Our 
targeted enrollment of 24 MetS persons (12F/12M) is therefore expected to be powered strongly to 
examine MFGM on endotoxemia. This will also account for attrition (estimated at 10%) and provide 
sufficient power to consider gender x treatment interactions.  

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Most data will be analyzed by linear mixed effects models with random effect for subjects (to account 
for repeated measures) and fixed effects for treatment, gender, time period, and their interactions. 
Multivariate regression will define correlations between variables with consideration of potential 
covariates. Significance for all analyses will be set at P<0.05. 
C. Measurement/Instrumentation 
 
Anthropometric Parameters and Blood Pressure. At screening, day 0, 7, and 14, 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. Fasting plasma glucose and lipids (total cholesterol, HDL-C, triglyceride) will 
be measured by clinical assay (Pointe Scientific) at screening, day 0, and day 14. Fasting insulin will 
be measured by ELISA (ALPCO) at day 0 and day 14. TLR4/NFκB-dependent genes from isolated 
peripheral blood mononuclear cells (PMBCs) will be assessed. For each arm of the study, we will 
measure circulating glucose and insulin at 0, 30, 60, 90, 120, 150, and 180 min following the ingestion 
of the glucose beverage. TLR4/NFκB-dependent genes will be assessed from PMBCs at 0 h and 3 h.  
 
Breath Analysis. Breath samples will be collected using a sterile, single use mouth piece into a 
sterile breath collection bag. Participants will be instructed not to brush their teeth with toothpaste, 
gargle with mouthwash, chew gum or eat any mints the morning of each test. Breath samples will 
be analyzed within one hour using a mass spectrometry-based breath analysis technique. 
 
 
Preparation of Milk Beverages. 
Isolation of MFGM Phospholipids. The main component for our formulation, the MFGM containing 
ingredient (i.e. buttermilk powder), will be prepared as described.52 Sweet cream will be prepared 
from milk obtained from the OSU Waterman Dairy Farm using a cream separator after pasteurization. 
After tempering (16 h, 4°C), cream is churned using a continuous pilot scale butter churn, and butter 
fines are then removed by filtration. A pilot plant-scale system using two spiral polymeric membranes 
fitted in parallel on the module (10 kDa, 11.33 m2 total surface area) will be used to concentrate 
buttermilk. This is performed at 15°C with transmembrane pressure of 500 kPa. Ultrafiltration is 
performed until a 10-fold volumetric concentration factor is achieved. Diafiltration (DF) is then 
performed by adding water continuously at 15°C to the feed tank to replace the removed permeate 
until a 5-fold DF factor is achieved. The final retentate is then spray-dried to obtain buttermilk powder 
having ~20-40 times the phospholipid content found in commercial milk.  
MFGM and Coconut/Palm Oil Beverages. Our approach entails formulating MFGM- and 
coconut/palm oil-enriched milks with 10-times the normal concentration of total phospholipids. 
MFGM (isolated above as buttermilk powder) contains ~2 g of phospholipid per 10 g of solid. Based 
on commercial whole milk containing 0.2-0.3 g/L phospholipid, the addition of 5-10 g MFGM (i.e. 
buttermilk powder) would yield a final concentration of 2-3 g/L of total phospholipid in our 
formulated beverage. Thus, milk beverage manufacture, based on our in-house pilot tests, would be 
as follows:  
MFGM Milk. A modified cream with milk phospholipids will be prepared using 40% butter fat, 10% 
MFGM ingredient (i.e. buttermilk powder), and 50% skim milk. Ingredients will be homogenized 
and added to skim milk to prepare milk containing 3.5% total fat (w/w).  
Coconut and Palm Oil Milk. A modified cream will be prepared by homogenizing 12.5% coconut 
oil, 37.5% palm oil, and 50% skim milk. This will yield a milk beverage matched to MFGM milk for 

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saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, but not enriched 
with phospholipids. 
Both beverages will be homogenized (2-stage at 1500 and 500 psi), pasteurized, bottled in single-use 
servings under aseptic conditions, and refrigerated until use. We anticipate preparing up to 250 
gallons of milk beverages to complete the study. Beverages are shelf-stable for 21 d and will be 
prepared in sufficient volume on a weekly basis proportional to the number of participants enrolled. 
Phospholipid content will be verified in the Jiménez-Flores Lab to be ±5% of expected concentrations 
with each prepared batch. Under the aforementioned manufacturing conditions, the energy and 
nutrient content of both beverages will be matched (3.2% protein and 3.5% fat, w/w). However, the 
MFGM beverage will contain 3 g/L MFGM phospholipid. The coconut/palm oil-based beverage will 
contain negligible levels of MFGM phospholipids (0.005% w/w), reflecting that skim milk is not 
fully devoid of these polar lipids. 
Dietary Control.  The intervention will be performed in the Human Nutrition Metabolic Kitchen 
under the auspice of a registered dietitian (PI Bruno). In each 2-wk intervention, participants’ diet 
will be rigorously controlled. All foods will be prepared, packaged, and provided every 3-4 d to 
supply a weight maintenance (i.e. eucaloric) diet. To assess compliance, participants will return 
MFGM/coconut/palm oil milk bottles for counting and any uneaten food portions for weighed 
measurement. Milk beverages will also be formulated to contain para-aminobenzoic acid (PABA; 
200 mg/milk serving). Spot urine samples will be collected 5 times during each study arm coinciding 
when participants pick up test foods. Urinary PABA will be measured by spectrophotometry 
according to procedures provided by Dr. Mario Ferruzzi (see Letter of Support). Separate from this, 
participants will also keep food logs to document any dietary deviation. Diets will be standardized at 
50-60% of energy from carbohydrate with low fiber intakes (~15 g/d) similar to Americans’ diets53 
to prevent potential masking of the benefits of MFGM, 15-20% from protein, and 25-30% from fat. 
Importantly, other than test beverages provided as part of the eucaloric diet, diets will be otherwise 
devoid of significant amounts of dairy foods, fermented products, and probiotics to prevent 
confounding effects.  
Gut Permeability. On d 14, postprandial endotoxemia and hyperglycemia will be induced by a high-
fat/high-glucose challenge meal,54,55 as described, while also assessing gut barrier integrity. 
Participants will ingest a controlled meal (~1200 kcal) consisting of 3 slices of toast with 50 g 
margarine along with a glucose tolerance beverage (75 g) that also contains gut permeability probes 
[lactulose (5 g), mannitol (1 g), sucralose (1 g), and erythritol (1 g)].56 In-house pilot tests indicate 
tolerability of the test meal. With this procedure, endotoxin increases by ~50% within 2 h54 and 
increased glucose provokes oxidative stress responses including lipid peroxidation.55,57 We will 
collect blood from 0-180 min at 30 min intervals and urine for 24 h to assess study outcomes. 
 
Endotoxin. Serum endotoxin will be measured from fasted participants (0 min) and during the 
postprandial period (30, 60, 90, 120, 150, 180 min) following ingestion of the sugar solution, using a 
fluorometric kit as we described.15,58  
 
Microbiota Composition. Microbiota composition will be assessed from fecal samples collected on 
d 13 of each study period. Samples will be extracted in the Bruno lab, and genomic sequencing will 
be performed in the OSU MCIC core lab (see Facilities) using routine procedures. In brief, effects 
due to MFGM on total bacterial abundance will be assessed using qPCR (i.e. to assess intestinal 
bacteria overgrowth).59 Microbiota of each sample will be characterized for diversity and community 
structure by sequencing 16S rRNA genes using MiSeq. Bioinformatic analysis will be performed 
using Qiime60 and bacteria identified by comparing each operation taxonomic unit (OTU) to RDP 
and Silva databases.61,62 α-Diversity (OTU richness, Shannon-Wiener diversity index, evenness) will 
be calculated using Qiime and β-diversity using the Bray-Curtis dissimilarity,63 followed by 
multivariate analysis (i.e. PCA, PERMANOVA, ANOSIM, PLS-DA) to determine MFGM effects. 

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The functional metagenome will be predicted using PICRUSt64 and PanFP65 based on reference 
databases and the KEGG Orthology classification scheme to obtain a functional gene abundance 
matrix.  
We will validate preclinical findings showing increased microbiota potential to generate SCFAs in 
response to MFGM.30,31,47 These gut health-promoting metabolites (butyrate, acetate, propionate) will 
be quantified by LC-MS as we described,57 with modification,66 from fecal samples collected on d 
13. Quantification will be performed relative to 13C-butyrate, -acetate, or -propionate (internal 
standards), and normalized to fecal dry matter. 
D. Detailed Study Procedures 
 
Overview of Study Procedures. Potential participants who call the study center in an anonymous 
manner for more information as well as those identified through ResearchMatch will be given a brief 
description about the study and asked a few questions to determine their eligibility (see Phone Script 
attachment). If they meet the eligible criteria, they will be invited to the study center for a screening 
meeting. During the meeting, the Informed Consent (see Informed Consent attachment) will be 
explained and provided for them to review. The participant will then be given the opportunity to 
review the Informed Consent form. If he/she chooses to participate in the study, they will then be 
asked to provide written consent. 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. A breath sample will also be collected. If they are not 
fasted at least 10 hours, they will be asked to come back in the fasted state at time of mutual 
convenience. These blood results in combination with anthropometric parameters will determine the 
participant’s eligibility. Eligible participants who agree to proceed with the study will then complete 
two 2-week intervention periods in a randomized order, each followed by a 3-h postprandial trial. We 
estimate that completion of all study procedures will take ~6-8 weeks per participant. Each step of 
the study procedure will be discussed in detail below: 
 
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, 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 (10 mL; 
2 tubes) so that we may measure blood chemistries (glucose, HDL-cholesterol, triglyceride). A breath 
sample will also be collected for the purpose of the correlation analysis of human plasma metabolic 
biomarkers. 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 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 

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own physician. Those having body measurements, blood pressure, and blood values within 
acceptable limits (see Inclusion/Exclusion criteria), will be invited to participate in the study. Subjects 
will be included or excluded based on a best fit of the inclusion and exclusion criteria (an example of 
best fit would be if a potential subject says he/she exercises 6 h/wk, which is close to our exclusion 
criteria of 7 h/wk, they might still be included in the study if they meet all other inclusion and 
exclusion criteria more closely than other potential subjects). Participants will be read one phone 
script if they qualify and another phone script if they do not qualify (see Participant Eligibility Phone 
Script attachment). Potential participants who qualify for the study will be communicated a message 
as follows: “Congratulations! You have been selected for participation in our study based on your 
blood testing results. A study investigator will be telephoning you to invite you to the testing session. 
Would you like to know your blood chemistry results?” Subjects not selected for study will be told 
the following message: “You have not been chosen for our study, but thank you for your interest.”  
This message will be followed by an explanation why they were not chosen, such as lab values outside 
of the range we are looking for: “Your blood testing data is…..we were looking for participants who 
had levels less than …..”.  “We can provide you a copy of your results if you would like…..how 
would you like us to provide them to you?”. “You should also consider sharing these results with 
your physician.”  
 
If a participant is telephoned and is unavailable, a message will be left requesting a callback at a 
convenient time or that at a member of the study team will try calling again at a later time. No 
confidential or sensitive information will be shared with third parties or left on answering machines. 
 
Intervention timeline. Each participant will complete a 2-arm (MFGM-Enriched Milk; 
Coconut/Palm Oil-Enriched Milk), randomized, cross-over study. For this study, each participant will 
visit the study center a total of 12 times (6 times per study arm, including 1) baseline measurements 
of anthropometrics, serum glucose, insulin, endotoxin, and pick-up of meals for days 1-3, 2) pick-up 
of meals for days 4-6, 3) pick-up of meals for days 7-10 and measurement of anthropometrics, 4) 
pick-up of meals for days 11-14 and stool collection kit, 5) 3 h postprandial testing, and 6) return 
samples from 24-h urine collection. Interventions will be separated by two weeks (for women, 
interventions will be separated by 2, 6, 10 etc. weeks to ensure testing will be performed at the same 
time of the menstrual cycle). 
 
Baseline visit. On day 0 of each trial arm, participants will report in the fasted state (10-12 hours) to 
the study center for assessment of height, weight, waist circumference, blood pressure, serum 
glucose, serum insulin, and serum endotoxin. They will receive 3-4 days of meals plus MFGM-
enriched milk or coconut/palm oil-enriched milk. 
 
Dietary intervention. Participants will be provided all foods for the 14-d intervention period. After 
the baseline visit, participants will report back to the study center on days ~6 and 10 to pick up their 
meals for the proceeding 3-4 days. On day 7, anthropometrics (height, weight, and waist 
circumference) will also be measured to ensure weight maintenance.  
 
Test trials.  At the end of the intervention period (day 14), participants will report back to the 
laboratory in the fasted state (10-12 hours) for assessment of height, weight, waist circumference, 
blood pressure, plasma glucose, insulin, gastric inhibitory peptide (GIP), glucagon-like peptide-1 
(GLP-1), and serum endotoxin. Participants will return a stool sample collected on day 13 of the 
intervention period at home. Participants will ingest a sugar solution containing 75 g glucose, which 
we and others have routinely shown to provoke oxidative stress responses including lipid 
peroxidation. This will be co-ingested with 1 g sucralose, 1 g erythritol, 5 g lactulose, and 1 g mannitol 
for assessment of gut permeability. Then, at 30 min intervals during the 3 h postprandial period, we 
will assess blood markers. During each study visit, participants will have access to drinking water 

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and use of the restroom as needed. Urine will be collected during the 3 h postprandial period in the 
laboratory, and participants will be instructed on urine collection for when they return home following 
their laboratory visit. Participants will return to the study center the following day to drop off their 
24-h urine collection tubes. 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. 
 
Sugar Probe Test. On the morning of testing at day 14, participants will arrive at the study center 
after abstaining from food and only consuming water for 10-12 hours. A sugar probe test will be 
conducted as described.56 Upon arrival, participants will be asked to empty their urinary bladder. 
Subsequently, they will then be asked to ingest a drink containing 1 g sucralose, 1 g erythritol, 1 g 
mannitol, and 5 g lactulose dissolved in 75 g glucose (240 mL). Within 2-hours after ingesting the 
sugar test beverage, participants will be asked to consume an additional 500 mL of water. 
Additionally, participants will be provided with standardized sucrose, erythritol, mannitol, and 
lactulose-free meals (e.g. bagel, peanut butter, scrambled eggs) during the 24-h period. All foods 
consumed during the 24-hour period will be free of artificial sweeteners. Urine will be collected from 
0-5 and 6-24 hours in sterile containers. For each urinary collection period, participants will be 
provided 2-L urine collection bottles and an insulated reusable bag with ice packs to store collected 
urine until they return their samples the next day. 
 
Sampling Handling. Once at baseline (d 0), once at d 7, and at each time point of the postprandial 
trial a blood sample (14 mL, 4 mL, and 20 mL x 7 time points = 140 mL, respectively) will be 
collected into evacuated blood collection tubes. Collecting a total of 158 mL or 0.67 cups once at 
baseline, once at d 7, and during the postprandial trial is necessary to ensure adequate amount of 
plasma and serum for each time point to accurately analyze levels of glucose, insulin, endotoxin, 
gut hormones, and TLR4/NFκB signaling 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. Breath samples will be collected in a clean breath collection bag supplied with a 
sterilized disposable mouth piece. Participants need to breathe into the bag at their normal breathing 
speed. Generally, three to five regular breaths will be sufficient to fill the breath bag, and there is no 
risk associated with this procedure. 
 
 
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, 7 
and 14 and then repeated following a ≥2 week washout period, and 1 screening day, we will be 
collecting a total of ~324 mL or ~1.37 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.67  
 

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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, 
TLR4, CD14, MD2, MyD88, p65, IL-1, IL-6, IL-8, TNF-α, MCP-1, GLP-1, GIP, 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 short chain fatty acids (butyrate, acetate, propionate). 
Remaining plasma, serum, urine, and fecal samples not used for these analyses will be archived for 
5 years at -80 C in the event we decide to measure additional inflammatory, antioxidant, or 
microbiota related markers. Appropriate notation has been made in the informed consent to alert 
participants that we will be archiving specimens and that they have the right to refuse our use of 
these specimens for future analyses. Lastly, approval from OSU IRB will be sought via a protocol 
amendment prior to the analysis of any additional biomarkers not specified herein. 
 
Privacy/Confidentiality. For all data and records that are a part of this study, a number (i.e. code) 
will be assigned to each participant and will only be available to research personnel. Any records 
containing the names of participants will be stored in a locked filing cabinet or on a password 
protected computer in the PI’s laboratory or office. Research personnel under the supervision of the 
PI and the PI himself will be the only individuals that have access to this information. The names of 
participants will not be used for publication in any form. The records will be maintained until the 
data are published, up to a maximum of five years. All archived samples will be coded, but the key 
linking the code to each participant’s identifiable information will have been destroyed. In addition, 
participants will be instructed that their participation in this study is voluntary and that they may 
withdraw at any time without prejudice. Data (biochemical values) obtained from this study will 
stored on a computer in the PI’s laboratory. In addition, a backup of digital data will be stored on the 
PI’s computer in his office. Both computers are password protected and both doors are locked when 
work areas are not in use. 
 
E. Internal Validity 
 
For all data and records that are a part of this study, a number (i.e. code) will be assigned to each 
participant. 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. 
 
F.   Medical Safety Plan 
All aspects of the clinical study will be conducted in Dr. Bruno’s clinical lab located in in 262D 
Campbell Hall. Participants will be fasted for 10-12 hours prior to each study visit. We recognize that 
certain risks associated with fasting include: hypoglycemia, weakness, and fainting. This duration of 
fasting is consistent with guidelines set forth by the American Diabetes Association to minimize risk 
to the individual62 when determining fasting blood glucose concentrations.  
 
Risks related to hypoglycemia are anticipated to be low due to many participants’ already 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 Sudeep Shrestha, Emily Shaw and 
Geoffrey Sasaki (Postdoctoral Fellow and Doctoral Students), all of them are members of the research 
team. 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. Emily 

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Shaw is certified by the Red Cross in CPR and basic first aid. Additionally, Dr. Bruno (PI; 325 
Campbell Hall) has an academic office in close proximity to the study center and has significant 
experience coordinating clinical research studies involving overnight fasting, and other dietary- 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 procedures 
throughout each study visit will occur while positioned on a hospital bed in the prone position. In the 
event that a participant was to become weak, dizzy, or faint, they would already be ideally positioned 
to minimize risks associated with these symptoms. In the event that hypoglycemia-related symptoms 
occur, the study would be terminated to allow the participant to recover.  We are prepared to provide 
pre-packaged beverages and snacks containing simple carbohydrates (e.g. Gatorade, fruit juice, apple 
sauce, crackers) that will allow for rapid restoration of blood glucose. These food items will be stored 
in a refrigerator, located in close proximity (262 Campbell Hall), 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. 
 
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. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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