Mechanisms of Obesity and Its Metabolic Complications in Youth
Phase: NA
Conditions: Obesity, Childhood
Sponsor: Yale University

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HRP-503B – BIOMEDICAL RESEARCH PROTOCOL (2017-1) 
 
 
 
 
Protocol Title: Mechanisms of Obesity and Its Metabolic Complications in Youth. 
 
 
Principal Investigator: Nicola Santoro 
Version Date: Amendments 10/30/2023 
 
(If applicable) Clinicaltrials.gov Registration #: NCT03454828 
 
 
INSTRUCTIONS 
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information needed by the IRB to determine whether a study meets approval criteria. Read the following 
instructions before proceeding: 
1. Use this protocol template for a PI initiated study that includes direct interactions with research 
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Library. 
2. If a section or question does not apply to your research study, type “Not Applicable” underneath. 
3. Once completed, upload your protocol in the “Basic Information” screen in IRES IRB system. 
SECTION I: RESEARCH PLAN 
 
1. Statement of Purpose: State the scientific aim(s) of the study, or the hypotheses to be tested. 
 
The overarching goal of this project is to determine whether the effect of gut microbiota on human metabolism 
might be mediated by the Short Chain Fatty Acid (SCFA) and whether the SCFA might modulate lipid 
metabolism. 
In fact, despite the evidence provided by animal studies, such studies have not been performed in humans. 
Moreover, this would be the first study determining the effect of SCFA synthesis on hepatic de novo lipogenesis 
and to assess whether and how isocaloric dietary changes (namely low carbohydrates) might modify the 
composition of the gut microbiota and reduce the synthesis of SCFA during adolescence, a sensitive period for 
the development of obesity. 

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Aim 1: Determine whether the gut microbiota of obese adolescents have greater ability to ferment 
carbohydrates (CHO) and synthetize SCFA compared to the microbiota of lean subjects, therefore 
obtaining a higher metabolizable energy yield from an equal amount of dietary CHO. Hypothesis: For 
an equal amount of dietary CHO, obese youth will synthesize SCFA at higher rates than lean youth. 
 
Aim 2: Determine whether the SCFA synthetic rates modulate hepatic de novo lipogenesis. 
Hypothesis: High rates of SCFA synthesis enhance hepatic de novo lipogenesis. 
 
Aim 3: Test the effect of an isocaloric low carbohydrate (CHO) diet on the bacterial synthesis of SCFA. 
Hypothesis: Lowering the amount of dietary CHO consumed will shift the composition of the gut microbiota 
towards species with a lower ability to ferment CHO and synthetize SCFA. 
 
Aim 4: To explore how COVID-19 lockdown has changed the dietary habits of participants enrolled in this 
study. In order to collect this information, we will ask for the participant to complete an online 3-day food 
record. This part of the study is non-invasive and there will be no in-person contact. 
 
Aim 5: To explore whether the colonic fermentation modulate gluconeogenesis. 
 
2. Probable Duration of Project: State the expected duration of the project, including all follow-up and 
data analysis activities. 7 years 
 
3. Background: Describe the background information that led to the plan for this project. Provide 
references to support the expectation of obtaining useful scientific data. 
 
Pediatric obesity: the growing epidemic. 
The prevalence of childhood obesity has progressively increased in the last four decades2. In 2010, 
approximately 43 million children were overweight worldwide and this number is expected to escalate up to 60 
million by 20201. Obese children and adolescents are at high risk of developing obesity complications (e.g. 
cardiovascular disorders and type 2 diabetes10-16) early in life. 
 
Furthermore, obesity overburdens the healthcare system by the high costs of such comorbidities17. Early onset 
obesity is the key risk factor for adult obesity2,18, but the only current treatment for it is represented by physical 
exercise and caloric restriction. Weight loss programs in children are especially challenging and show a very 
high attrition rate, approaching 30% in the first 12 months and 70% after 24 months19,20. Moreover, although 
some of these programs can be successful, a clinically significant weight loss is achieved for a short period of 
time given that only 1 in 6 overweight and obese report ever having maintained weight loss of at least 10% for 
1 year21. As long-term weight loss remains largely unachievable with current interventions, children who 
become obese are trapped in this phenotype for years, if not for life, therefore accumulating years of increased 
risk of cardio-metabolic complications. Why children and adolescents become obese and why the obese 
phenotype is so stable (irreversible in most cases) remain unanswered questions. This lack 
in knowledge represents a critical barrier towards our understanding of the pathophysiology underlying 
childhood obesity. 
 
Role of the gut microbiota in the development of obesity. 
The human gut microbiota is a complex community of 100 trillion archaeal and bacterial cells distributed over 
more than 1000 species5,22. The combined genomes of the gut microbiota – the microbiome – contain 100-fold 

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more genes than the human genome23 with significant biochemical capabilities to modulate human 
physiology5,24. Animal studies demonstrated that the gut microbiota may modulate both sides of the energy 
balance equation, namely energy harvest from the diet, energy storage and energy expenditure, therefore 
mediating diet induced obesity6,25. In fact, germ free animals show about 40% lower body fat than 
conventionalized animals26. Although most of the studies related to the gut microbiota and obesity has been 
carried out in animals, in the last few years it has been shown that obese individuals have a “predisposing” 
microbiota environment. Notably, studies in human twins discordant for obesity have shown that by 
transplanting fecal microbiota from adult obese human twin into germ-free mice caused an increase of total 
body fat mass3, while when mice harboring a lean twin's microbiota were housed with mice containing an obese 
co-twin's microbiota, the development of obesity and its complications were prevented in the cage mates3. 
More recently a human study has shown that subjects undergoing fecal microbiota transplantation for the 
treatment of Clostridium difficile develop new onset obesity if the transplant is received by a healthy but obese 
donor27. These data indicate that the composition of the gut microbiota may play a pivotal role in the 
development of the obese phenotype in humans, but the mechanisms by which the gut microbiota maybe 
causing weight gain remain unclear. 
 
Short Chain Fatty Acids as a link between gut microbiota and energy accumulation. 
Microbiota dysbiosis may influence the development of obesity through several mechanisms, including the 
production of bio-active metabolites5,28, in particular, the short chain fatty acids (SCFA) (acetate, butyrate and 
propionate)29. The SCFA are among the most abundant microbiota-derived metabolites and are produced 
through the fermentation of dietary carbohydrates not digested by the host. Direct evidence of the role of gut 
microbiota in energy harvest and fat deposition comes from germ-free rats, which have reduced intestinal levels 
of SCFA30, and twice as much urinary and fecal excretion of calories as that of conventional ones fed the same 
polysaccharide-rich diet31. Microbial energy harvest in obesity has been investigated in conventional genetically 
obese ob/ob mice, revealing increased amounts of SCFA in their caecum and reduced energy content in their 
feces compared to their lean littermates32. In vitro and animal studies suggest that, once produced, SCFA may 
enter the circulation and may be utilized for de novo lipid or glucose synthesis33,34. While the role of gut 
microbiota in promoting energy harvest has been shown in animal studies, the only evidence in humans derives 
from indirect investigations. For instance, people who are obese have higher levels of ethanol in their breath 
than lean35 and show greater amounts of fecal SCFA36, which suggests altered fermentation and increased 
microbial energy harvest. 
 
Mechanisms by which the SCFA can modulate the human metabolism. 
Microbial fermentation of polysaccharides may affect host adiposity through several complementary 
mechanisms. The main mechanism is related to the ability of the SCFA to be taken up by the liver and used as 
substrates for lipogenesis and gluconeogenesis, in states where their synthesis exceeds the intestinal metabolic 
needs. In fact, the liver metabolism of germ-free and colonized mice differs considerably, possibly because of 
the increased influx of SCFA into the liver of colonized mice. As a consequence, colonized mice have higher levels 
of stored triglycerides (TG) in the liver 37. Increased TG production in the liver of colonized mice is associated 
with reduced expression in the small intestine of the fasting-induced adipose factor, or ANGPTL44,6 which is a 
potent inhibitor of the enzyme lipoprotein lipase and mediates cellular uptake of TG. Germ-free Angptl4- 
deficient mice gain as much fat mass and body weight during high-fat feeding as colonized mice, indicating that 
ANGPTL4 may directly mediate microbial regulation of adiposity in mice6. In addition, SCFA can regulate gene 
expression by binding to the G-protein-coupled receptors (GPCRs) GPR41 and GPR4328. Signaling through these 
receptors affects several different functions depending on the cellular type. For example, SCFA suppress 
inflammation through GPR43 signaling and modulate the expression of PYY through a GPR41- dependent 

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mechanism38. Conventional Gpr41-deficient mice have reduced adiposity compared with conventional wild-type 
mice, whereas germ-free wild-type and Gpr41-deficient mice had similar adiposity, clearly indicating that the 
effect of the microbiota on fat deposition might be dependent on this SCFA receptor38. Altogether these data 
suggest that gut microbiota function as a metabolic organ fully integrated with the human metabolic pathways. 
 
Colonic fermentation and glucose metabolism. Human, in vivo and in vitro studies have shown that colonic 
fermentation has an effect on gluconeogenesis42-46. Although in vitro studies suggest that some SCFA deriving 
from colonic fermentation (such as propionate) might increase gluconeogenesis 42, studies in humans 
consistently suggest that colonic fermentation is associated with a better glycemic control44-46. In fact, previous 
studies on this topic have focused on adults and adolescents with type 1 and type 2 diabetes and had as main 
outcome postprandial changes of glucose or doses of insulin needed to maintain a good glycemic control45-47. 
Despite this evidence, to date there are no studies testing the direct effect of colonic fermentation on 
gluconeogenesis. 
 
 
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4. Research Plan: Summarize the study design and research procedures using non-technical language that 
can be readily understood by someone outside the discipline. Be sure to distinguish between standard of 
care vs. research procedures when applicable, and include any flowcharts of visits specifying their individual 
times and lengths. Describe the setting in which the research will take place. 
 
OGTT Visit 
 
During this visit the patients will undergo the OGTT 
Assessment of Glucose Tolerance Status 
Oral Glucose Tolerance Test (OGTT). The study nurse will do a nursing assessment, including measuring the 
patient’s height, weight, waist circumference, hip circumference, blood pressure, and pulse, along with 
evaluation of acanthosis nigricans and striae rubrae. The subject’s percent body fat, fat mass and lean mass will 
also be measured using a Tanita scale. The nurse will obtain a family and medical history from the patient and/or 
the patient’s parent/guardian. In addition, before starting the OGTT, the nurse will request a urine sample from 
the patient for analysis of microalbumin and creatinine, and to test for pregnancy. A consult may be provided by 
the YCCI bionutritionist at the time of the OGTT. 
 
The patient will receive 1.75 g/kg to a maximum of 75g of a sugar drink, orally (Glucola). The patient will have 
one intravenous line. “Emla” or a local anesthetic (0.1cc buffered lidocaine) will be applied before the placement 
of the IV catheter. Blood will be drawn 10 times over three hours. Should abnormal glucose results be found, 
appropriate referrals will be made. We will draw approximately 120 cc of blood during this study. The blood will 
be analyzed for metabolic parameters, including glucose, lactate, insulin, proinsulin, c-peptide, IL-6, TNF-α, GLP-1, 
glucagon, lipoproteins free fatty acids (FFAs), leptin, and adiponectin. 

Page 8 of 32 
 
 
 
As part of the 120 cc of blood drawn for the study, samples will be obtained via the IV line for genetic testing for 
the variants in the GCKR, TCF7L2, and PNPLA3 gene. This will be clearly explained in the Adolescent assent, Adult 
Consent and Parental Consent. 
 
Stool Specimen: After study consenting, the subject (or subject’s parent/guardian) will be provided with a kit to 
collect stool. If the subject cannot provide the sample at the given time, they can complete it at home. Once 
collected, the container will remain at 4 C and be brought back to researchers within 12 hours. The bacterial 
DNA will be extracted and the bacterial gene coding for the 16S RNA subunit will be characterized by Next 
Generation sequencing to assess the bacterial diversity. 
 
MRI VISIT 
 
Assessment of Body Composition: Imaging Studies 
 
Tanita. Percent body fat, fat mass, and fat-free mass will be measured via bioelectrical impedance analysis (BIA) 
using a Tanita scale. This method is non-invasive and, if found to be as accurate as the DEXA scan, may prove to 
be a more practical approach to measuring percent body fat. 
 
Abdominal Magnetic Resonance Imaging (MRI). MRI of the abdomen will be used to directly assess intra- 
abdominal fat deposition on a 1.5 Tesla magnet (Siemens). During the same session, we will acquire liver images, 
from which we will calculate the hepatic fat fraction and hepatic iron concentration. All patients will be screened 
for MRI safety before undergoing imaging of their abdomen. A localizing gradient echo sequence will be 
performed to allow accurate slice selection. Five true axial Fast Gradient Echo sections will be obtained with 
respiratory compensation through the abdomen, with the 3rd section at the L4/5 disc space. Saturation bands 
will be placed above and below the field of view in order to negate high signal in blood vessels. The largest field 
of view will be used to encompass the abdomen, although in larger patients this will be inadequate, and the 
sequence will need to be performed twice-once for the right side of the abdomen and once for the left. Each 
sequence should take approximately 6 minutes to acquire, and the entire scan should take no more than 45 
minutes. The images will be analyzed for the amount of subcutaneous and visceral fat present. The hepatic fat 
fraction will be calculated using a modified Dixon technique. This will be performed using a single breath-hold 
scan of about 15 seconds to obtain a section through the liver. A gradient echo pulse sequence is used with 
varying TR and TE parameters to provide in- and out-of-phase images, with TR kept constant. The signal from an 
appropriate area of hepatic tissue will be used to calculate the fat fraction. Importantly, an excellent correlation 
was reported between the liver fat content measured by FAST-MRI and liver biopsy (r2 = 0.853, p<0.001). The 
hepatic fat fraction will also be calculated using a three-point Dixon technique in combination with the iterative 
least-squares estimation method. As this method is implemented in a steady-state free precession sequence, it 
allows one to obtain multiple sections (typically 10-14 slices) through the liver within a single breath-hold scan of 
about 25-30 seconds. A scout image requiring a breath-hold of about 12 seconds will also be obtained. The 
hepatic iron concentration will be calculated using a T2 weighted and intermediate weighted gradient echo 
sequence obtained during two breath-hold sequences of about 26 seconds each. The signal from an appropriate 
area of hepatic tissue will be used to calculate the hepatic iron concentration. Subjects may be asked to repeat a 
scan if a sub-optimal image is obtained on the first attempt. 
 
When possible, the MRI scan will be on the same day as the OGTT as noted above, however it can be scheduled 
on a different day (either prior to or following completion of OGTT) based on ease of scheduling for the patient. 

APPROVED BY THE YALE UNIVERSITY IRB 12/1/2022 
APPROVED BY THE YALE UNIVERSITY IRB 12/1/2022 
 
 
Aim 1: Determine whether the gut microbiota of obese adolescents have greater ability to ferment 
carbohydrates 
(CHO) and synthetize SCFA compared to the microbiota of lean subjects, therefore obtaining a higher 
metabolizable energy yield from an equal amount of dietary CHO. Hypothesis: For an equal amount of dietary 
CHO, obese youth will synthesize SCFA at higher rates than lean youth. 
 
All subjects will be instructed about the benefit of a healthy diet and will meet with the dietician who will advise 
on healthy feeding behavior and lifestyle and will be given the option of enrolling, free of charge, in the Yale 
Pediatric Weight Management Program called “Bright Bodies.” The program includes the following 
components: exercise, nutrition education, behavior modification, and psychosocial support. 
We will enroll an equal number of obese (100) and lean (100). Lean adolescents will be recruited if at risk of 
developing obesity, prediabetes or diabetes due to their family history. The dietitian will instruct the subjects to 
follow a 3-day pre-intervention meal plan to ensure standardized calories and macronutrients intake up to the 
day of investigation in amounts to maintain weight in both groups. The pre-intervention diet will be based on 
the subjects’ age, weight and usual intake. The subjects will be admitted at the Hospital Research Unit (HRU) of 
the 
Yale New Haven Hospital at 7.00 AM. To 
determine SCFA synthetic rates we will 
give orally 20g of lactulose, an 
oligosaccharide naturally present in 
nature that is extracted from chicory. To 
account for hepatic uptake, isotope 
exchange in the TCA cycle in the liver 
and endogenous production by the liver, deuterated-SCFA will be infused from 7.30 AM, this will allow us to 
reach the quasi steady state for 2H-SCFA after ~1 hour. We will infuse 0.7 micromol/kg/min of labeled 
acetate after a labeled acetate priming dose of 1 mg/Kg. To assess SCFA synthesis blood samples will be 
collected every hour until 6.00 pm for a total of 150 ml (Figure 1). The subjects will be allowed to drink during 
the study, a meal will be served at the end of the study. 
 
We will also collect the breath hourly hydrogen breath test during the study to assess colonic fermentation by 
measuring hydrogen in the breath. Subjects blow into a small box to collect the breath. There are no risks 
related to this test. 
 
Rationale for using lactulose as substrate to measure SCFA synthesis. In order to study the ability of the gut 
microbiota to ferment carbohydrates, we have chosen lactulose because the SCFA synthesis and kinetics arising 
from lactulose ingestion has been widely studied and is well known. Due to the structural conformation of its 
linkages, lactulose resists hydrolysis in the human small intestine and is almost completely fermented by the 
colonic microbiota. Lactulose has low osmotic activity, however it may cause uncomfortable bowel movements, 
flatulence, loose stools, bloating, cramps, and diarrhea. 
 
Aim 1 Follow-up: After the completion of Aim 1, participants will be invited back for a one-year follow-up to 
measure height, weight, body mass composition with the Tanita scale, and to collect a 3-day food log. This 
follow-up is to assess if acetate production predicts change in BMI and body composition one year post study. 
 
 
 
 
Page 9 of 32 

APPROVED BY THE YALE UNIVERSITY IRB 12/1/2022 
APPROVED BY THE YALE UNIVERSITY IRB 12/1/2022 
 
 
Aim 2: Determine whether the SCFA synthetic rates modulate hepatic de novo lipogenesis. Hypothesis: High 
rates of SCFA synthesis enhance hepatic de novo lipogenesis. Subjects will assume 3ml/ Kg of body water of D20 
in three doses per os (at 7:00 PM, 10:00 PM and 3:00 AM) the night before the study. At the test day at 7:30 AM 
they will consume 2g of 
13C 
Inulin, 
13g 
of 
unlabeled inulin. Blood 
draws will be taken every 
hour until 8.00 PM. The 
total blood to be drawn is 
about 150 ml . The 
subjects will be allowed 
to drink during the study, a meal will be served at the end of the study. 
 
We will also collect the breath hourly hydrogen breath test during the study to assess colonic fermentation by 
measuring hydrogen in the breath. Subjects blow into a small box to collect the breath. There are no risks 
related to this test. 
 
 
Aim 3: Test the effect of an isocaloric low carbohydrate (CHO) diet on the bacterial synthesis of SCFA. 
Hypothesis: Lowering the amount of dietary CHO will shift the composition of the gut microbiota towards 
species with a lower ability to ferment carbohydrates and synthetize SCFA. Twenty-six obese subjects will be 
enrolled in a cross-over study and challenged with a low CHO or a normal CHO diet for a week. The subjects will 
switch diet after 4 weeks washout. Stool samples will be collected before and after the intervention to evaluate 
the gut microbiota. SCFA synthesis (as described in aim 1) will be measured at the beginning and at the end of 
the diet. Subjects will be enrolled after a 3 days run in period, in which they will try whether they could follow a 
low carbohydrates diet. 
 
 
Dietary intervention. 
a) 
Dietary Composition: The control “standard” diet composition will follow the American Dietary 
Guidelines of 55% CHO, 15% pro, and 30% fat. CHO content will be primarily complex CHO of high quality (14 g 
fiber/1,000 kcals and <10% of total kcals in the form of sugar). Fat content will be <7% saturated fats and 
primarily consist of monounsaturated fats. The experimental diet will consist of 30% CHO, 35% pro, and 35% 
fat. Since the experimental diet is lower in CHO, the fiber and sugar content will be calculated based on total 
CHO in the same percentage as the control (0.25 g fiber per each kcal of CHO and 18.2% sugar for total CHO). 
This will ensure the same quality of CHO in both diets (for example, <10% of total kcals from sugar would be a 
higher sugar intake in the low CHO diet). Kcal levels will be determined based on subject’s usual food intake. 
The subjects will be instructed to keep a 3-day food record (2 weekdays and 1 weekend day) prior to the study 
and the average kcal intake will be calculated using the Nutrition Data Systems for Research (NDSR) (University 
of Minnesota, 2016 version). Kcal levels will be assigned to the closest 200 kcals (1800, 2000, 2200, 2400, 2600, 
2800, 3000). Utilizing the formulas to derive the same quality of CHO in both the standard- and low-CHO diets, 
the 2000 kcal standard diet, for example, will consist of 1100 kcals of CHO (55% total kcals), 27 g fiber (.025 g 
fiber per each kcal of CHO), and 50 g sugar (18.2% total CHO), while the low-CHO diet will consist of 600 kcals of 
CHO (30% total kcals), 15 g fiber (.025 g fiber 
per each kcal of CHO), and 27 g sugar (18.2% 
total CHO). A 3-day rotating menu will be 
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provided for both groups to ensure variety and increase compliance. 
b) 
Run-in period: To screen for adherence, the eligible subjects will undergo a 3 day run-in period. During 
this period all the subjects will consume a low CHO diet and will complete a food record every day. Participants 
assessed to be compliant to the diet (defined as following the diet for all the five days without any deviation) 
will be randomized to the intervention (low CHO) first or the control (standard CHO) diet. During the run in 
period the food will be provided by the Metabolic Kitchen of Yale New Haven Hospital and subjects will 
complete a daily food record. Moreover, we will discuss the study expectation at length with the subjects. 
c) 
Instruction of Isocaloric Diets: Four weeks after the run-in period (wash out), the subjects enrolled will 
undergo each dietary intervention for 1 week. Subjects will be instructed on the meal plan by the dietitian prior 
to the intervention and will be taught how to keep a simplified food record daily to validate the comprehension 
of the meal plan and compliance. This simplified food record will allow adolescents to check off foods consumed 
and write in any foods they may have consumed that are not on the meal plan. Foods will be provided to the 
subjects twice during the 7 days from the metabolic kitchen. The dietitian will meet with the child and family at 
mid-week food pick up to obtain the food record, assess compliance, and review the plan. 
d) 
Retention Strategy: Non-compliance of the diet might be a problem in this very challenging population of 
adolescents, although it is unlikely due to the short nature of the diet intervention period. At Yale we have 
extensive experience using both dietary interventions and/or pharmacologic interventions as treatments for 
weight management, insulin resistance and diabetes in obese adolescents. Our retention strategies include 
developing rapport with each child and family involved in the study, through phone calls, sending holiday 
greeting cards and birthday cards to the child involved in the study, making sure that test results such as lipids, 
glucose and insulin levels are well explained to the family, and being appreciative of the time and effort subjects 
and families give to the study. With a short intervention such as this, we plan to build rapport and have frequent 
phone calls with the subject/family. The research staff is composed of experienced personnel who have 
serviced many subjects enrolled in studies through the Yale Pediatric Obesity Clinic. The PI and some of the 
study personnel live in New Haven and are very familiar with the population. A “hot line” by which study 
participants may reach the study personnel after work hours will be available. The subjects will be reminded of 
the visit at least three days before and will be scheduled at the best convenience for the families. 
e) 
Compliance Assessment: To enforce compliance the food for the whole study will be provided by the 
Metabolic Kitchen of the Yale New Haven Hospital. To assess compliance we will ask the patient to complete a 
simplified food record daily. Also, should they choose to eat something different from what is given to them by 
the Metabolic Kitchen, they have the choice of writing the food in the blank space provided for that day or to 
take a picture of the products and indicate the amount eaten. The latter choice may be of more interest to this 
technologically savvy age group and will be more accurate for the dietitian to assess. The mid-week (mid 
intervention) visit for food pick up and food record review will allow the dietitian to problem solve, if necessary, 
before the final four days of the intervention. Moreover, subjects will also be recruited among a pool of 
subjects who have been previously recruited in our studies and have shown a high rate of adherence. 
 
Aim 4 – Exploratory 
 
To assess how COVID-19 lockdown has changed the dietary habits of participants enrolled in this study. In order 
to collect this information, we will ask for the participant to complete an online 3-day food record. This part of 
the study is non-invasive and there will be no in-person contact. 
 
 
 
Aim 5 – Exploratory 

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To assess whether the colonic fermentation modulate gluconeogenesis. Hypothesis: Glucose fermentation 
induced by lactulose is associated with a drop in gluconeogenesis. Subjects will assume 3ml/ Kg of body water of 
D20 in three doses per os the night before the study and at 7:30 AM they will consume 20g of lactulose. Blood 
draws will be taken every hour until 8.00 PM. The total blood to be drawn is about 150 ml. The subjects will 
be allowed to drink during the study, a meal will be served at the end of the study. 
 
We will also collect the breath hourly hydrogen breath test during the study to assess colonic fermentation by 
measuring hydrogen in the breath. Subjects blow into a small box to collect the breath. There are no risks 
related to this test. 
 
 
Aim 6 - Measurement of TCA cycle in vivo in the brain and in the liver by using Deuterium metabolic imaging. 
 
 
Deuterium metabolic imaging (DMI) is a simple MR-based technique to map metabolism with high temporal 
and/or spatial resolution. The metabolic fate of deuterated substrates, including glucose and acetate, can be 
monitored with deuterium MR methods. Recent preliminary data obtained with this protocol suggest that youth 
with obesity have higher TCA cycle compounds in plasma than lean. This is probably due to an increased 
metabolic rate related to insulin resistance. To determine if changes observed in plasma mirror metabolic 
changes in the tissue (brain and liver), we will use a technique that combines stable isotopes and MRI. To 
measure TCA cycle in the brain we will use 6,6-2H2-glucose, a compound that has been used for many years in 
clamp studies, while to measure TCA cycle in the liver we will use D3-Acetate, the compound that we are 
currently using to measure acetate production (Aim 1 and 3). 
 
 
TCA cycle measurement in the brain. 
To measure TCA cycle measurement in the brain we will infuse 6,6-2H2-glucose for 2 hours and we will detect the 
changes in TCA derived compounds in the brain during the infusion. A total of 50 ml of blood will be obtained 
during this study. 
 
The labeled glucose is prepared by the Investigational Drug Service of the Yale Pharmacy, as a solution of 20% 
(w/v) in water. The labeled glucose is infused following a protocol used for decades in the Yale Magnetic 
Resonance Research Center (MRRC), consisting of a ramp phase followed by a plateau phase. The amount of 
glucose infused is 0.75g/kg body weight (BW); the volume of glucose solution infused over a 120 min study is 3.6 
ml/kg BW. 
 
TCA cycle measurement in the liver. 
To measure TCA cycle measurement in the liver we will infuse d3-acetate for 2 hours and we will detect the 
changes in TCA derived compounds in the liver during the infusion. A total of 50 ml of blood will be obtained 
during this study. 
 
The labeled acetate is prepared by the Investigational Drug Service of the Yale Pharmacy, as a solution of 350 mM 
2H-labeled sodium-acetate in water. The labeled acetate is infused following a protocol used for decades in the 
Yale MRRC, consisting of a ramp phase followed by a plateau phase. The amount of acetate infused is 0.73g/kg 
body weight (BW); the volume of acetate solution infused over a 120 min study is 14.2 ml/kg BW. 

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Calendar of activities for each aim (visits, frequency, assessments, blood draw collection, week number). 
 
Visit 
number 
Frequency 
Assessment 
Amount of blood to be collected 
Week 
number 
AIM 1 
1 
once 
OGTT 
120 
1 
2 
once 
Acetate synthesis 
94 
2 
AIM 2 
1 
once 
DNL assessment 
150 
3 
AIM 3 
1 
once 
Acetate synthesis 
94 
4 
2 
once 
Acetate synthesis 
94 
5 
3 
once 
Acetate synthesis 
94 
6 
4 
once 
Acetate synthesis 
94 
7 
AIM 4 
1 
once 
One year follow-up 
0 
8 
AIM 5 
1 
once 
gluconeogenesis 
150 
9 
AIM 6 
1 
once 
MRI/Infusion - Brain 
50 
10 
2 
once 
MRI/Infusion - Liver 
50 
11 
Each visit is one day. If a subject is recruited for more than one aims, then at least 12 weeks will be allowed 
between the visits 
 
 
 
 
5. Genetic Testing ☒ 
 
The genetics portion of the study is optional. Subjects who do not wish to have additional blood drawn for 
genetic analysis may still have the OGTT. We may wish to contact family members of the subject for the genetic 
study as well. However, no family member will be contacted without the permission of the index subject, and 

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informed signed consent or assent will be obtained from all participants. Refusal to participate will in no way 
influence the subjects’ relationship with the physicians, nurses or researchers, or with Yale University or Yale- 
New Haven Hospital, nor will it influence their receipt of treatment. An opportunity will be given to the 
parent/subject to indicate if the genetic sample results can be used for recruitment for current and future 
studies. Once a participant turns 18, if he/she has not completed the study, will be reconsented in order to 
complete the study. 
 
6. Subject Population: Provide a detailed description of the types of human subjects who will be recruited into 
this study. 
We will enroll an equal number of 100 obese and 100 lean, between the age of 15 and 25, lean (BMI <85th 
percentile) and obese (BMI > 95th percentile). The two groups will be matched by age, gender, pubertal stage 
and ethnicity. As controls we will recruit only lean adolescents at risk of developing obesity, prediabetes or 
diabetes due to their family history. 
 
7. Subject classification: Check off all classifications of subjects that will be specifically recruited for enrollment 
in the research project. Will subjects who may require additional safeguards or other considerations be 
enrolled in the study? If so, identify the population of subjects requiring special safeguards and provide a 
justification for their involvement. 
☐ Children 
☐ Healthy 
☐Fetal material, placenta, or dead fetus 
☐ Non-English Speaking 
☐ Prisoners 
☐Economically disadvantaged persons 
☐ Decisionally Impaired 
☐ Employees 
☐Pregnant women and/or fetuses 
☐ Yale Students 
☐ Females of childbearing potential 
NOTE: Is this research proposal designed to enroll children who are wards of the state as potential subjects? 
Yes ☐ NoX ☐ 
8.  Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion or exclusion? 
Inclusion criteria: Good general health, taking no medication on a chronic basis, age 15 to 25 years, in 
puberty (girls and boys: Tanner stage III – V), BMI >25th and <85th for lean cohort; BMI >95th for obese 
cohort, girls who begin menstruating must have a negative pregnancy test during the study. Exclusion 
criteria: hematocrit below 32%, baseline creatinine >1.0 mg, food allergies, pregnancy, presence of 
endocrinopathies (e.g. Cushing syndrome), significant chronic illness. 
 
The hematocrit will be measured in all the patients at the research unit the day of the visit before the beginning 
of the study. During the assessment of vital signs the patients will be asked about signs of anemia (fatigue, 
malaise, weakness, dizziness). The study will be performed only if the hematocrit is above 32% and the patients 
does not refer any symptoms of anemia. The hematocrit will be measured using the hematocrit analyzer available 
at the research unit (HemataSTAT II Analyzer Serial). 
 
 
9. How will eligibility be determined, and by whom? 
Before the enrollment, the clinical characteristics will be assessed through a clinical evaluation during a 
screening visit in which the PI and the study coordinator will assess the eligibility through a comprehensive 
evaluation (height, weight, disease history, current medications etc.). 

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10. Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, discomforts, or 
inconveniences associated with subjects participating in the research. 
 
A potential complication of the indwelling catheters used in these studies is thrombophlebitis. The incidence of 
this complication in our hands is less than 0.1%. Local hematoma is uncommon and on occasion there is short- 
lived local pain occurring at the site of venipuncture. The risk of stable isotopes appears to be negligible. In fact, 
13C inulin and labeled water will be given orally, therefore there will not be any risk related to sterility and 
pirogenicity. Lactulose is occurring in the normal diet and is benign, though it may cause uncomfortable bowel 
movements, flatulence, loose stools, bloating, cramps, and diarrhea. The dose of D20 used in this study are in 
the range of 1-2 mg/Kg of weight, which is what is commonly used in clinical studies. Also, in the past infusions 
of compounds labeled with stable isotopes have been used to evaluate the SCFA turnover in newborn without 
any side effect. Acetate infusion is low risk. Acetate is largely present in our body. Labeled acetate has been 
used for years in medical research all over the world including Yale (Petersen KF Cell Metabolism 2016) without 
any side effects. 
 
 
11. Minimizing Risks: Describe the manner in which the above-mentioned risks will be minimized. 
As stable isotopes are given orally there is no risk for infection related to stable isotopes infusion. We have 
been using isotope infusions in conjunction with clamping and lipogenesis studies in children who have not 
had any adverse events. The risks of acetate infusion are the same as stable isotope infusion. 
 
Magnetic resonance (MR) is a technique that uses magnetism and radio waves, not xrays, to take pictures 
and measure chemicals of different parts of the body. The United States Food and Drug Administration 
(FDA) has set guidelines for magnet strength and exposure to radio waves, and we carefully observe those 
guidelines. 
 
You will be watched closely throughout the MR study. Some people may feel uncomfortable or anxious. If 
this happens to you, you may ask to stop the study at any time and we will take you out of the MR scanner. 
On rare occasions, some people might feel dizzy, get an upset stomach, have a metallic taste or feel tingling 
sensations or muscle twitches. These sensations usually go away quickly but please tell the research staff if 
you have them. 
 
There are some risks with an MR study for certain people. If you have a pacemaker or some metal objects 
inside your body, you may not be in this study because the strong magnets in the MR scanner might harm 
you. Another risk is the possibility of metal objects being pulled into the magnet and hitting you. To lower 
this risk, all people involved with the study must remove all metal from their clothing and all metal objects 
from their pockets. We also ask all people involved with the study to walk through a detector designed to 
detect metal objects. It is important to know that no metal can be brought into the magnet room at any 
time. Also, once you are in the magnet, the door to the room will be closed so that no one from outside 
accidentally goes near the magnet. 
 
We want you to read and answer very carefully the questions on the MR Safety Questionnaire related to 
your personal safety. Take a moment now to be sure that you have read the MR Safety Questionnaire and 
be sure to tell us any information you think might be important. This MR study is for research purposes 
only and is not in any way a complete health care imaging examination. The scans performed in this study 
are not designed to find abnormalities. The principal investigator, the lab, the MR technologist, and the 

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Magnetic Resonance Research Center are not qualified to interpret the MR scans and are not responsible 
for providing a health care evaluation of the images. If a worrisome finding is seen on your scan, a 
radiologist or another physician will be asked to review the relevant images. Based on his or her 
recommendation (if any), the principal investigator or consulting physician will contact you, inform you of 
the finding, and recommend that you seek medical advice as a precautionary measure. The decision for 
additional examination or treatment would lie only with you and your physician. The investigators, the 
consulting physician, the Magnetic Resonance Research Center, and Yale University are not responsible for 
any examination or treatment that you receive based on these findings. The images collected in this study 
are not a health care MR exam and for that reason, they will not be routinely made available for health care 
purposes. 
 
A member from the study staff will accompany the patient to the MRRC and stay for the entire duration of 
the MRI scan 
 
DNA Analysis. The analysis of blood for genetic research raises special issues of confidentiality. Variation in 
some genes is known to be directly related to risk for certain illnesses. In some cases, knowledge of genetic 
information could have negative psychological consequences or could affect access to or retention of 
certain benefits or entitlements. For example, the information could potentially be used against an 
individual if it were revealed to insurance companies or potential employers. We will take precautions to 
ensure that confidentiality is maintained and that the genetic information is not unintentionally disclosed to 
inappropriate third parties. 
 
12. Data and Safety Monitoring Plan: Include an appropriate Data and Safety Monitoring Plan (DSMP) based on 
the investigator’s risk assessment stated below. (Note: the HIC will make the final determination of the risk 
to subjects.) 
a. 
What is the investigator’s assessment of the overall risk level for subjects participating in this 
study? GREATER THAN MINIMAL RISK 
b. 
If children are involved, what is the investigator’s assessment of the overall risk level for the 
children participating in this study? presenting a minor increment over minimal risk with no 
prospect of direct benefit to individual subjects, but likely to yield generalizable knowledge 
about the subject's disorder or condition (per 45 CFR § 46.406), with permission obtained from 
both parents. 
c. 
Include an appropriate Data and Safety Monitoring Plan. Examples of DSMPs are available here 
http://your.yale.edu/policies-procedures/forms/420-fr-01-data-and-safetymonitoring-plans- 
templates for 
i. Included at the end of the document. 
 
d. 
For multi-site studies for which the Yale PI serves as the lead investigator: 
i. 
How will adverse events and unanticipated problems involving risks to subjects or others be 
reported, reviewed and managed? Write here 
ii. What provisions are in place for management of interim results? Write here 
iii. What will the multi-site process be for protocol modifications? Write here 
 
13. Statistical Considerations: Describe the statistical analyses that support the study design. 
Statistical Plan for aims 1 and 2. Justification of Sample size: Sample size was estimated in PASS 

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V2012 (Kayesville, UT) in order to enroll a sufficient number of obese children to detect differences in SCFA 
synthesis for aim 3. Based on this sample size, the number of lean children was then determined to detect 
differences between lean and obese (aim 1). For aim 1, given a standard deviation of 1.0 for SCFA, 
100 lean children (compared to 100 obese children) will be required to detect a difference of 0.65 with 90% 
power at the two-sided 0.05 significance level. This sample size will also provide 80% power to detect an rsquare 
of 0.07 (aim 2). Statistical analyses: Analysis for Aim 1: the primary outcome will be the peak percent change 
from fasting in total SCFA estimated by dividing the difference between total SCFA at fasting and the peak total 
SCFA by the total SCFA at fasting. Total SCFA is calculated by summing the synthetic rates of the single SCFAs. 
Peak percent change will be compared between the lean and obese groups using a mixed model to account for 
the pair matching. A fixed effect will be included for group (obese/lean) and a random effect for an indicator of 
the matched pair. Differences in means along with 95% confidence intervals will be estimated. We will also 
explore between group differences in SCFA across all post-inulin time-points by estimating the area under the 
curve following inulin. Since the pre-intervention diet will be based on the subjects’ usual intake, we will adjust 
for dietary intake to try to overcome the possibility that dietary patterns during the study might affect SCFA 
synthesis. Since the two groups will be matched by age, gender, Tanner stage and ethnicity the analyses will not 
need to be adjusted for these variables. Nevertheless, we will determine whether differences between lean and 
obese groups are modified by gender by adding an interaction of gender by group. Analysis for Aim 2: To 
evaluate the association between total SCFA and de novo lipogenesis, peak percent changes in de novo 
lipogenesis will be regressed on peak percent changes in total SCFA using a mixed model. The mixed model will 
include a fixed effect for peak percent total SCFA and a random effect for the matched pair indicator. To explore 
whether the magnitude of the associations may be dependent on other variables (such as gender), interactions 
between percent peak total SCFA and these variables will be included to the models in order to describe the 
degree of effect modification. An additional exploratory analysis using a doubly multivariate repeated measures 
mixed model will be evaluated to examine the correlation of total SCFA and de novo lipogenesis across all post- 
inulin time-points. 
 
Statistical Plan for Aim 3. Justification of Sample size: In our preliminary data from obese children we observed a 
standard deviation for percent peak total SCFA synthesis of 1.0 and an absolute difference of 1.5% between 
those with the lowest and highest BMI. Given the following: 1) a power of 90%, 2) a standard deviation of 1.0, 
and 3) a two-sided 0.05 significance level a sample size of 21 obese children receiving both low CHO vs control 
diet will be required to detect a difference of 0.75 in peak total SCFA synthesis. The conservative 0.75 difference 
was selected based on being 50% of that estimated between the highest and lowest BMI in our preliminary 
work. We’ll enroll 26 obese children for aim 3 to accommodate up to a 20% dropout. Statistical Analyses: The 
primary objective of the analysis is to demonstrate that a low CHO diet will lower SCFA synthetic rate more than 
control at follow-up. The percent change in peak total SCFA rate, calculated as the follow-up peak total SCFA 
minus the baseline peak total SCFA divided by the baseline peak total SCFA, will be compared between the low 
CHO period and the control period using a repeated measures mixed model. The mixed model will included 
fixed effects for treatment and period as well as the baseline covariates of SCFA, age, sex, pubertal stage and 
ethnicity. A random effect will be included for subject to allow for correlation between repeated measures. The 
treatment by period interaction will be evaluated and if significant (p<0.10) examination of the primary aim will 
focus on the interventions received during the first period (i.e. to exclude the possibility of carryover). Linear 
contrasts and two-sided 95% confidence intervals will be estimated comparing mean percent peak total SCFA 
change for low CHO to control. Sex-specific effects of low CHO will be assessed by inclusion of interactions of 
treatment with sex. Given the assumption required for missing data, we will evaluate patterns of missing data as 
well as determine baseline characteristics that are predictive of dropout. If identified, these characteristics will 
be included as covariates in supportive sensitivity analysis. 

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SECTION II: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND DEVICES 
 
Statistical Plan for Aim 4. Since aims 4 is an exploratory studies for which we do not have preliminary data it is 
difficult to determine the sample size needed to detect differences in dietary changes during the lockdown. The 
main outcome for this arm of the study will be changes in Kcal intake. Secondary aims will be changes in the 
intake of macro- and micro nutrients. This arm of the study will be limited to ~40 subjects for which we have 
pre-lockdown dietary information. Data analysis. Descriptive statistics, such as means and standard deviations 
(or medians and interquartile range, IQR), counts and percent will be used to describe characteristics of subjects 
before the study. A paired non-parametric test will be used to compare the outcomes before and after the 
lockdown within groups, while a general linear model will be used to compare changes in the outcomes 
between groups. Statistical significance will be established at the two-sided alpha of 0.05. Analyses will be 
performed using SAS 9.4 (Cary, NC), Prism and R statistical software. 
 
Statistical Plan for Aim 5. In our preliminary data, we observed changes in glucose of 89.3 mg/dl at baseline and 
78.8 mg/dl at the end of the study (7 hours after lactulose ingestion). Given the following: 1) a power of 95%, 2) 
a standard deviation of 8.0, and 3) a one-sided 0.05 significance level a sample size of 14 subjects will be 
required to detect the above shown difference in percent changes with 95% power at the one-sided 0.05 
significance level within each group. Since we will explore also whether percent changes in gluconeogenesis are 
different between lean we will recruit about 20 subjects per group. Statistical Analyses: Median and 
interquartile ranges will be calculated. The primary outcome will be gluconeogenesis changes after lactulose 
ingestion within groups (lean and obese). Non-parametric one sided test will be used to compare 
gluconeogenesis between baseline and the end of the study. We will also explore the possibility that changes in 
gluconeogenesis might occur to different extents between the groups, therefore we will compare percent 
changes gluconeogenesis between the groups using a general linear model using basal gluconeogenesis as 
covariate. 
 
Statistical plan for aim 6. Sample size was estimated with the clinicalc online software (Sample Size Calculator 
(clincalc.com). Since lactate serves as a precursor of TCA cycle, we based our power calculations on the lactate 
levels measured during the fasting state in 12 lean and 18 obese children. Based on the mean+/-SD of lactate 
levels in non-obese group of 0.78+/-0.22 and in the group with obesity 1.023 +/- 0.36, a sample size of 23 
subjects per group (46) is needed for an alpha of 0.05 and a power of 95%. Statistical Analyses: Median and 
interquartile ranges will be calculated. The primary outcome will be TCA cycle after 6,6 D glucose ingestion 
during the steady state. Non-parametric two sided test will be used to compare TCA cycle between the groups. 
 
 
 
 
If this section (or one of 
its parts, A or B) is not applicable, check off N/A and delete the rest of the section. 
 
A. RADIOTRACERS 
 
B. DRUGS/BIOLOGICS 
Does the study do any 
 
of the following: 
Specify the use of an approved drug or biologic? 
Use an unapproved drug or biologic? 
☐ N/A 
☒N/A 

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Use a food or dietary supplement to diagnose, cure, treat, or mitigate a disease or condition? No 
 
This study uses: 
13C inulin (labeled) (isotope) 
deuterated SCFA/labeled acetate 
Deuterium labeled water (heavy water D2o) 
6,6-2H2-glucose (isotope) 
Lactulose is a dietary supplement that is approved to treat constipation. We will use lactulose to stimulate 
production of short chain fatty acids by the gut flora. 
 
 
Cold Isotopes: 
FDA allows the use of cold isotopes in research without an IND when certain conditions apply (see below). 
Confirm that the following are true: 
 
1. The research is intended to obtain basic information regarding the metabolism (including kinetics, 
distribution, and localization) of a drug labeled with a cold isotope or regarding human physiology, 
pathophysiology, or biochemistry. 
2. The research is not intended for immediate therapeutic, diagnostic, or preventive benefit to the study 
subject. 
3. The dose to be administered is known not to cause any clinically detectable pharmacologic effect in 
humans based on clinical data from published literature or other valid human studies. 
4. The quality of the cold isotope meets relevant quality standards. 
5. The investigation [will be] conducted in compliance with the requirements for review by an IRB (21 CFR 
part 56) and the requirements for informed consent (21 CFR part 50). 
 
I confirm all of the 5 points above. In fact, the study is intended to obtain basic information regarding the 
metabolism, not to provide therapeutic or diagnostic benefit. 
The dose of isotopes to be used have been shown to be safe. In particular, D20 and 13C inulin have shown to be 
safe at the dose that we are providing. We and others used D20 at the proposed dose in previous studies 
without any side effects (Goffredo M et a. JCEM 2017). For the 13C inulin other investigators have used up to 
15grams of labeled Inulin without any side effect (Deroover L et al. Nutrients 2017). 
 
 
1. If an exemption from IND filing requirements is sought for a clinical investigation of a drug product that is 
lawfully marketed in the United States, review the following categories and complete the category that 
applies (and delete the inapplicable categories): 
 
Exempt Category 1: The clinical investigation of a drug product that is lawfully marketed in the United States 
can be exempt from IND regulations if all of the following are yes: 
1. The intention of the investigation is NOT to report to the FDA as a well-controlled study in support 
of a new indication for use or to be used to support any other significant change in the labeling 
for the drug. 
☒ 

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2. The drug that is undergoing investigation is lawfully marketed as a prescription drug product, and 
the intention of the investigation is NOT to support a significant change in the advertising for 
the product. 
☒ 
3. The investigation does NOT involve a route of administration or dosage level or use in populations 
or other factor that significantly increases the risks (or decreases the acceptability of the risks) 
associated with the use of the drug product 
☒ 
4. The investigation will be conducted in compliance with the requirements for institutional (HIC) 
review and with the requirements for informed consent of the FDA regulations (21 CFR Part 50 
and 21 CFR Part 56). 
☒ 
5. The investigation will be conducted in compliance with the requirements regarding promotion 
and charging for investigational drugs. 
☒ 
Exempt Category 2 (all items i, ii, and iii must be checked to grant a category 2 exemption) 
☐ i. The clinical investigation is for an in vitro diagnostic biological product that involves one or 
more of the following (check all that apply): 
☐ Blood grouping serum 
☐ Reagent red blood cells 
☐ Anti-human globulin 
☐ ii. The diagnostic test is intended to be used in a diagnostic procedure that confirms the 
diagnosis made by another, medically established, diagnostic product or procedure; and 
☐ iii. The diagnostic test is shipped in compliance with 21 CFR §312.160. 
Exempt Category 3 
☐ The drug is intended solely for tests in vitro or in laboratory research animals if shipped in accordance with 
21 CFR 312.60 
Exempt Category 4 
☐ A clinical investigation involving use of a placebo if the investigation does not otherwise require submission 
of an IND. 
2. Background Information: Provide a description of previous human use, known risks, and data addressing 
dosage(s), interval(s), route(s) of administration, and any other factors that might influence risks. If this is 
the first time this drug is being administered to humans, include relevant data on animal models. 
Write here 
 
3. Source: Identify the source of the drug or biologic to be used. The stable isotopes will be purchased from 
the Cambridge Isotope laboratories Inc. or from the Sigma Aldrich. 
 
a) 
Is the drug provided free of charge to subjects? ☐YES ☐NO If 
yes, by whom? Write here 

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Note: If the YNHH IDS (or comparable service at CMHC or WHVA) will not be utilized, explain in detail how the PI 
will oversee these aspects of drug accountability, storage, and preparation. 
☒N/A 
If Yes, please be aware of the following requirements: 
A YNHH New Product/Trial Request Form must be completed via EPIC: Pull down the Tools tab in the EPIC 
Banner, Click on Lawson, Click on “Add new” under the New Technology Request Summary and fill out the forms 
requested including the “Initial Request Form,” “Clinical Evidence Summary”, and attach any other pertinent 
documents. Then select “save and submit” to submit your request; AND 
Your request must be reviewed and approved in writing by the appropriate YNHH committee before 
subjects/subjects may be scheduled to receive the investigational device or investigational procedure. 
4. Storage, Preparation and Use: Describe the method of storage, preparation, stability information, and for 
parenteral products, method of sterilization and method of testing sterility and pyrogenicity. 
The compounds will be handled from and stored at the Investigational Drug Service. The IDS provides 
handling and packaging of the stable isotopes. 
Check applicable Investigational Drug Service utilized: 
☐ YNHH IDS 
☐ CMHC Pharmacy 
☐ West Haven 
VA 
☐ PET Center 
 
☐ None 
☐ Other: 
 
5. Use of Placebo: ☒Not applicable to this research project 
 
 
6. 
Continuation of Drug Therapy After Study Closure ☒Not applicable to this 
project 
 
B. DEVICES 
 
1. 
Are there any investigational devices used or investigational procedures performed at Yale-New Haven 
Hospital (YNHH) (e.g., in the YNHH Operating Room or YNHH Heart and Vascular Center)? ☐Yes ☐No 
 
 
2. Background Information: Provide a description of previous human use, known risks, and any other factors 
that might influence risks. If this is the first time this device is being used in humans, include relevant data 
on animal models. Write here 
3. Source: 
a) Identify the source of the device to be used. Write here 
b) Is the device provided free of charge to subjects? ☐Yes ☐No 
 
4. Investigational device accountability: State how the PI, or named designee, ensures that an investigational 
device is used only in accordance with the research protocol approved by the HIC, and maintains control of 
the investigational device as follows: 

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SECTION III: RECRUITMENT/CONSENT AND ASSENT PROCEDURES 
a) Maintains appropriate records, including receipt of shipment, inventory at the site, dispensation or use 
by each participant, and final disposition and/or the return of the investigational device (or other 
disposal if applicable): Write here 
b) Documents pertinent information assigned to the investigational device (e.g., date, quantity, batch or 
serial number, expiration date if applicable, and unique code number): Write here 
c) Stores the investigational device according to the manufacturer's recommendations with respect to 
temperature, humidity, lighting, and other environmental considerations: Write here 
d) Ensures that the device is stored in a secure area with limited access in accordance with applicable 
regulatory requirements: Write here 
e) Distributes the investigational device to subjects enrolled in the IRB-approved protocol: Write here 
 
1. Targeted Enrollment: Give the number of subjects: 200 
a. Targeted for enrollment at Yale for this protocol: Children and adolescents between 15 and 25 years of 
age. 
b. If this is a multi-site study, give the total number of subjects targeted across all sites: NA 
 
2. Indicate recruitment methods below. Attach copies of any recruitment materials that will be used. 
☒Flyers 
☐ Internet/web postings 
☐ Radio 
☐ Posters 
☐ Mass email solicitation 
☐ Telephone 
☐ Letter 
☐ Departmental/Center website 
☐ Television 
☐ Medical record review* 
☐ Departmental/Center research boards 
☐ Newspaper 
☐ Departmental/Center newsletters 
☐ Web-based clinical trial registries 
☐ Clinicaltrails.gov 
☒ YCCI Recruitment database 
☒ Other: Yale Pediatric Obesity Clinic 
☐ Social Media (Twitter/Facebook): 
* Requests for medical records should be made through JDAT as described at 
http://medicine.yale.edu/ycci/oncore/availableservices/datarequests/datarequests.aspx 
 
3. Recruitment Procedures: 
a. Describe how potential subjects will be identified. The subjects will be also recruited from a sample of ~1000 
obese children and adolescents, constituting the Yale Pediatric Obesity Cohort, who agreed to being 
contacted for recruitment purposes . This cohort of lean and obese youth has been carefully phenotyped for 
fat distribution as well as for lipid and glucose metabolism (including the assessment of insulin sensitivity by 
the oral glucose tolerance test)70. If needed, more subjects will be recruited through the Yale Pediatric 
Obesity Clinic as well as from the CTSA-funded Yale Center for Clinical Investigation Database, comprised of 
subjects in the community. 
 
b. Describe how potential subjects are contacted. Potential subjects will be contacted by the PI or the study 
personnel by phone, mail or email. 
 
 
c. Who is recruiting potential subjects? The PI and the research assistant will be recruiting the subjects. 
 
4. Assessment of Current Health Provider Relationship for HIPAA Consideration: 

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The investigator assures that the protected health information for which a Waiver of Authorization has been 
requested will not be reused or disclosed to any person or entity other than those listed in this application, 
except as required by law, for authorized oversight of this research study, or as specifically approved for use in 
another study by an IRB. 
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale HIPAA-Covered 
entity must be accounted for in the “accounting for disclosures log”, by subject name, purpose, date, recipients, 
and a description of information provided. Logs are to be forwarded to the Deputy HIPAA Privacy Officer. 
Does the Investigator or any member of the research team have a direct existing clinical relationship with any 
potential subject? 
☐ Yes, all subjects 
☒Yes, some of the subjects 
☐ No 
 
If yes, describe the nature of this relationship. Dr Caprio, a co-investigator on this study, is the physician 
leading the Yale obesity clinic, through which some of the subjects will be recruited. 
5. Request for waiver of HIPAA authorization: (When requesting a waiver of HIPAA Authorization for either 
the entire study, or for recruitment purposes only. Note: if you are collecting PHI as part of a phone or 
email screen, you must request a HIPAA waiver for recruitment purposes.) Choose one: 
☒ X☐ For entire study 
☒ For recruitment/screening purposes only 
☐ For inclusion of non-English speaking subject if short form is being used and there is no translated HIPAA 
research authorization form available on the University’s HIPAA website at hipaa.yale.edu. 
 
i. Describe why it would be impracticable to obtain the subject’s authorization for use/disclosure of 
this data: The PI has moved to a new institution and is asking to transfer samples and data to the 
new institution, and since recruitment occurred over the past years it would be difficult to reach the 
subjects and re-consent them to ask permission to transfer data and samples. 
 
ii. If requesting a waiver of signed authorization, describe why it would be impracticable to obtain the 
subject’s signed authorization for use/disclosure of this data: Some of the subjects will be 
responding to a flyer and may call us by phone for a pre-screening. 
 
6. Process of Consent/Assent: Describe the setting and conditions under which consent/assent will be 
obtained, including parental permission or surrogate permission and the steps taken to ensure subjects’ 
independent decision-making. 
Each person will be contacted by the P.I. or member of the research team to determine his or her 
eligibility for the study. The purpose, nature and potential complications of the study will be 
explained to each potential subject in detail. The P.I. or designated researcher will set up a screening 
session during which the study will be reviewed with the subject and she will be asked to read the 
human consent form which has been previously approved by the Yale Human Investigations 
Committee. All parties involved will be given time to ask questions. The researcher obtaining 
consent will ask the subject a series of questions to ensure that they have understood the main 
procedures involved in the study, along with the risks and commitment. Only after this they will be 

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Note* If more than 2 study participants are enrolled using a short form translated into the same language, then 
the full consent form should be translated into that language for use the next time a subject speaking that 
language is to be enrolled. 
Several translated short form templates are available on the HRPP website (yale.edu/hrpp) and translated HIPAA 
Research Authorization Forms are available on the HIPAA website (hipaa.yale.edu). If the translation of the short 
form is not available on our website, then the translated short form needs to be submitted to the IRB office for 
approval via modification prior to enrolling the subject. Please review the guidance and presentation on use of 
the short form available on the HRPP website. 
If using a short form without a translated HIPAA Research Authorization Form, please request a HIPAA waiver in 
the section above. 
For a waiver of signed consent, address the following: 
• Would the signed consent form be the only record linking the subject and the research? YES ☐ NO ☐ 
• Does a breach of confidentiality constitute the principal risk to subjects? YES ☐ NO ☐ 
OR 
• Does the research pose greater than minimal risk? YES ☐  NO☐ 
asked to give informed consent to participate. The signed form (and a copy) will be kept as a 
permanent record. 
7. Evaluation of Subject(s) Capacity to Provide Informed Consent/Assent: Indicate how the personnel 
obtaining consent will assess the potential subject’s ability and capacity to consent to the research being 
proposed. The researcher obtaining consent will ask the subject a series of questions to ensure that 
they have understood the main procedures involved in the study, along with the risks and 
commitment. Only after this they will be asked to give informed consent to participate. 
8. Non-English Speaking Subjects: Explain provisions in place to ensure comprehension for research involving 
non-English speaking subjects. If enrollment of these subjects is anticipated, translated copies of all 
consent materials must be submitted for approval prior to use. Write N/A 
As a limited alternative to the above requirement, will you use the short form* for consenting process if you 
unexpectedly encounter a non-English speaking individual interested in study participation and the translation 
of the long form is not possible prior to intended enrollment? YES ☐ NO ☐ 
 
 
9. 
Consent Waiver: In certain circumstances, the HIC may grant a waiver of signed consent, or a full waiver 
of consent, depending on the study. If you will request either a waiver of consent, or a waiver of signed consent 
for this study, complete the appropriate section below. 
 
☐ Not Requesting any consent waivers 
 
☒Requesting a waiver of signed consent: 
☒ Screening only (if for recruitment, the questions in the box below will apply to recruitment 
activities only) 
☐ Entire Study (Note that an information sheet may be required.) 
 

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SECTION IV: PROTECTION OF RESEARCH SUBJECTS 
For a full waiver of consent, please address all of the following: 
• 
Does the research pose greater than minimal risk to subjects? ☐ Yes If you answered yes, stop. A waiver 
cannot be granted. 
☐ No 
• 
Will the waiver adversely affect subjects’ rights and welfare? YES ☐  NO☐ 
• 
Why would the research be impracticable to conduct without the waiver? Write here 
• 
Where appropriate, how will pertinent information be returned to, or shared with subjects at a later 
date? Write here 
 
 
 
☐ Requesting a waiver of consent: 
☐ Recruitment/Screening only (if for recruitment, the questions in the box below will apply to 
recruitment activities only) 
☐ Entire Study 
 
 
Confidentiality & Security of Data: 
1. What protected health information (medical information along with the HIPAA identifiers) about subjects 
will be collected and used for the research? Age, gender, height, weight, BMI, previous and current diseases, 
medications, family history of diseases. NA 
 
2. How will the research data be collected, recorded and stored? 
De-identified plasma samples will be collected and SCFA and DNL will be measured in plasma. 
 
3. How will the digital data be stored? ☐CD ☐DVD ☐Flash Drive ☐Portable Hard Drive ☐Secured Server 
☐ Laptop Computer ☒Desktop Computer ☐Other 
4. What methods and procedures will be used to safeguard the confidentiality and security of the identifiable 
study data and the storage media indicated above during and after the subject’s participation in the study? 
All personal information will be handled in confidence and in accordance with local data protection laws. All 
research materials will be held in locked cabinets and stored on password-protected computers. Only study 
personnel will have access to the database to record results as they accrue. While being analyzed samples will be 
stored in locked areas in the secured laboratories of the investigator. When the results of the research are 
published or discussed in conferences, no information will be included that would reveal a subject's identity 
unless specific consent for this is obtained. The subjects will be informed of those parties who may have access 
to their data as listed on the Research Authorization form. The samples will be stored long term in the facilities 
of the Core laboratories of the Yale Center for Clinical Investigation. 
 
The research data and specimen will be shared with Kansas University Medical Center. 
• Does the research include any activities that would require signed consent in a non-research context? YES ☐ 
NO ☐ 

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SECTION V: POTENTIAL BENEFITS 
SECTION VI: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS 
 
 
 
5. What will be done with the data when the research is completed? Are there plans to destroy the identifiable 
data? If yes, describe how, by whom and when identifiers will be destroyed. If no, describe how the data 
and/or identifiers will be secured. 
Participant permission is requested to retain research data. This data will be stored for research purposes after 
the research is completed. This is outlined in the protocol and in all consent/assent forms. Subjects who opt 
not to store their data long term, data will be anonymized within three years after the end of the study. 6. If 
appropriate, has a Certificate of Confidentiality been obtained? Yes 
 
Dr. Santoro will be the point of contact should a participant wish to withdraw their samples 
 
 
Potential Benefits: All the subjects will be instructed about the benefit of a healthy diet and will meet with the 
dietician who will advise on healthy feeding behavior and lifestyle. This will be a direct benefit for those children 
that are already obese and overweight and for those who are at risk of becoming obese, given their family 
history. All the subjects enrolled in the study will also be given the option of enrolling, free of charge, in the Yale 
Pediatric Weight Management Program called “Bright Bodies.” The program includes the following 
components: exercise, nutrition education, behavior modification, and psychosocial support. Some obese 
individuals will undergo a low carbohydrates diet during the study. This study will take place under the 
supervision of Mary Savoye, who is also the director of the Bright Bodies program a weight loss program 
designed for obese children and adolescents. In addition to the benefits to the subjects, the study will increase 
our understanding of the role of gut microbiome in the development of obesity in children and adolescents. 
This is the first study in human exploring the possibility that the products of microbial metabolism in the gut 
might determine fat accumulation and lead to obesity during childhood. The study about changes in dietary 
pattern during the lockdown will inform on whether and how adolescents’ diet has changed during that time. If 
we observe an unhealthy dietary behavior characterized by the consumption of more junk food, our dietician 
will advise on how to revert this tendency and how to consume a healthier food. Moreover, learning about the 
effect of colonic fermentation on glucose production will be important to understand if modifying the diet of 
youth with obesity, that represent a group at high risk for pre-diabetes and diabetes, a better glucose control 
after a meal of high fermentable food could be reached. In fact, there are studies in children and adults with 
type 1 and type 2 diabetes that have demonstrated that consuming food made with highly fermented dough 
improves the glycemic control after the meal so that the patients need lower insulin to control glycemic 
excursion after the meal (Zanfardino A et al Diabetes Technol Ther. 2019 Dec;21(12):721-726 ; Cavagnuolo L et 
al Diabetes Care. 2019 Oct;42(10):e157-e158; Giacco R et al. Br J Nutr. 2001 Jan;85(1):33-40). 
 
 
 
 
 
 
All portable devices must contain encryption software, per University Policy 5100. If there is a technical reason 
a device cannot be encrypted please submit an exception request to the Information Security, Policy and 
Compliance Office by clicking on url http://its.yale.edu/egrc or email it.compliance@yale.edu 

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1. Alternatives: What other alternatives are available to the study subjects outside of the research? 
The study subjects will be given the chance to attend the Yale pediatric Obesity Clinic available at the 
Department of Pediatrics of Yale University regardless of their decision to participate in the study. 
2. Payments for Participation (Economic Considerations): Describe any payments that will be made to subjects, 
the amount and schedule of payments, and the conditions for receiving this compensation. 
 
Subjects who will enroll in the study will receive a reimbursement of $150 for every full day study, $150 for 
each MRI/Infusion study, $60 for the MRI, $50 for the glucose tolerance test, $20 for completion of a 3-day 
food log, and $20 for a stool sample collection. We will pay for parking for coming to the study visits. 
 
We will use a pre-paid debit card to provide payment for taking part in the study. We will have to share your 
name, address, and telephone number with the banking institution issuing the debit card for ePayments. You 
may receive a card in the mail with the first payment following completion of the first visit. You will need to 
activate the card over the phone. Payments for additional visits will be automatically added to your card after 
complete on of each following visit. You are responsible for paying state, federal, or other taxes for the 
payments you receive for being in this study. Taxes are not withheld from your payments.” 
 
Compensation made to U.S. human research study participants is considered taxable income to the 
participant, regardless of how the participant is paid, and is reported by Yale University. Jonathan 
Andrejczyk from Yale Shared Services, will determine which subjects have payments over $600 for whom 
Yale does not have a Social Security number on file and will work with the coordinator to collect the Social 
Security numbers that are required. If a coordinator knows in advance that a subject will receive $600 or 
more in a calendar year for a study, then they should request the Social Security number upfront. For 
Subjects where social security numbers are required they should complete W-9 Form which the coordinator 
should submit to Jonathan. 
 
3. Costs for Participation (Economic Considerations): Clearly describe the subject’s costs associated with 
participation in the research, and the interventions or procedures of the study that will be provided at no 
cost to subjects. There are no costs for the subjects associated with the participation in the study. The low 
carbohydrates diet will be provided by the Metabolic Kitchen of the HRU free of charge for the subjects. 
4. In Case of Injury: This section is required for any research involving more than minimal risk, and for minimal 
risk research that presents the potential for physical harm (e.g., research involving blood draws). 
 
a. Will medical treatment be available if research-related injury occurs? Write here 
 
b. Where and from whom may treatment be obtained? Write here 
c. Are there any limits to the treatment being provided? Write here 
d. Who will pay for this treatment? Write here 
e. How will the medical treatment be accessed by subjects? Write here 
 
If injury should occur during any of the procedures, the overseeing physician and the study nurse will 
attend to the immediate needs of the patient. Should any acute care visit (i.e. emergency room or 
outpatient clinic), hospital admission or chronic care be necessary, the subject’s medical insurance will be 
responsible for covering any incurred charges. 

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A billable service is defined as any service rendered to a study subject that, if he/she was not on a study, would 
normally generate a bill from either Yale-New Haven Hospital or Yale Medical Group to the patient or the 
patient’s insurer. The service may or may not be performed by the research staff on your study, but may be 
provided by professionals within either Yale-New Haven Hospital or Yale Medical Group (examples include x-rays, 
MRIs, CT scans, specimens sent to central labs, or specimens sent to pathology). Notes: 1. There is no distinction 
made whether the service is paid for by the subject or their insurance (Standard of Care) or by the study’s funding 
mechanism (Research Sponsored). 2. This generally includes new services or orders placed in EPIC for research 
subjects. 
If answered, “yes”, this study will need to be set up in OnCore, Yale’s clinical research management system, for 
Epic to appropriately route research related charges. Please contact oncore.support@yale.edu 
If you answered “no” to question 4a, or "yes" to question 4b or c, please contact the YNHH Department of 
Physician Services (688-2615) for prior approval before commencing with your research protocol. 
Please note that if this protocol includes Yale-New Haven Hospital subjects, including subjects at the HRU, the 
Principal Investigator and any co-investigators who are physicians or mid-level practitioners (includes PAs, 
APRNs, psychologists and speech pathologists) who may have direct patient contact with subjects on YNHH 
premises must have medical staff appointment and appropriate clinical privileges at YNHH. If you are uncertain 
whether the study personnel meet the criteria, please telephone the Physician Services Department at 203-688- 
2615. By submitting this protocol as a PI, you attest that you and any co-investigator who may have patient 
contact has a medical staff appointment and appropriate clinical privileges at YNHH. 
 
 
 
Will this study have a billable service? Yes ☐ 
No☒ 
 
 
Are there any procedures involved in this protocol that will be performed at YNHH or one of its affiliated 
entities? 
Yes ☐ No ☐ 
If Yes, please answer questions a through c and note instructions below. 
a. Does your YNHH privilege delineation currently include the specific procedure that you will perform? Yes ☐ 
No 
☐ 
b. Will you be using any new equipment or equipment that you have not used in the past for this procedure? Yes 
☐ No ☐ 
c. Will a novel approach using existing equipment be applied? Yes ☐ No ☐ 
 
 
 
IMPORTANT REMINDER ABOUT RESEARCH AT YNHH 
IMPORTANT REMINDERS 

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Data and Safety Monitoring Plans (DSMP) 420 
FR.1 
 
 
 
Greater Than Minimal Risk DSMP 
1. Personnel responsible for the safety review and its frequency: 
 
The principal investigator will be responsible for monitoring the data, assuring protocol compliance, and 
conducting the safety reviews at the specified frequency, which must be conducted at a minimum of 
every 6 months (including when reapproval of the protocol is sought). During the review process, the 
principal investigator (monitor) will evaluate whether the study should continue unchanged, require 
modification/amendment, or close to enrollment. Either the principal investigator, the IRB and Safety 
Monitoring Committee (DSMC) have the authority to stop or suspend the study or require 
modifications. 
 
2. The risks associated with the current study are deemed greater than minimal for the following 
reasons: 
 
1. We do not view the risks associated with the use of stable isotopes as minimal risks. 
 
Although we have assessed the proposed study as one of greater than minimal risk, the potential exists 
for anticipated and/or unanticipated adverse events, serious or otherwise, to occur since it is not possible 
to predict with certainty the absolute risk in any given individual or in advance of first-hand experience 
with the proposed study methods. Therefore, we provide a plan for monitoring the data and safety of the 
proposed study as follows: 
 
3. Attribution of Adverse Events: 
Adverse events will be monitored for each subject participating in the study and attributed to the study 
procedures / design by the principal investigator (Nicola Santoro) according to the following categories: 
 
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s). 
b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s). 
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s). 
d.) Unlikely: Adverse event is likely not to be related to the investigational procedures(s)/agent(s). 
e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s). 
 
4. Plan for Grading Adverse Events: 
 
The following scale will be used in grading the severity of adverse events noted during the study: 
 
1. Mild adverse event 
2. Moderate adverse event 
3. Severe 

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5. Plan for Determining Seriousness of Adverse Events: 
 
Serious Adverse Events: 
In addition to grading the adverse event, the PI will determine whether the adverse event meets the 
criteria for a Serious Adverse Event (SAE). An adverse event is considered serious if it results in any of 
the following outcomes: 
 
1. Death; 
2. A life-threatening experience in-patient hospitalization or prolongation of existing 
hospitalization; 
3. A persistent or significant disability or incapacity; 
4. A congenital anomaly or birth defect; OR 
5. Any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the other 
outcomes listed in this definition. 
 
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse Event. 
Similarly, an adverse event may be graded as moderate but still meet the criteria for an SAE. It is 
important for the PI to consider the grade of the event as well as its “seriousness” when determining 
whether reporting to the IRB is necessary. 
 
6. Plan for reporting UPIRSOs (including Adverse Events) to the IRB 
 
The principal investigator will report the following types of events to the IRB: 
Any incident, experience or outcome that meets ALL 3 of the following criteria: 
 
1. Is unexpected (in terms of nature, specificity, severity, or frequency) given (a) the research 
procedures described in the protocol-related documents, such as the IRB-approved protocol 
and informed consent document and (b) the characteristics of the subject population being 
studied; AND 
2. Is related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); AND 
3. Suggests that the research places subjects or others at greater risk of harm (including 
physical, psychological, economic, legal, or social harm) than was previously known or 
recognized. 
Unanticipated Problems Involving Risks to Subjects or Others (UPIRSOs) may be medical or 
nonmedical in nature, and include – but are not limited to – serious, unexpected, and related adverse 
events and unanticipated adverse device effects. Please note that adverse events are reportable to 
the IRB as UPIRSOs only if they meet all 3 criteria listed above. 
 
These UPIRSOs/SAEs will be reported to the IRB in accordance with IRB Policy 710, using the 
appropriate forms found on the website. All related events involving risk but not meeting the prompt 

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reporting requirements described in IRB Policy 710 should be reported to the IRB in summary form at 
the time of continuing review. If appropriate, such summary may be a simple brief statement that events 
have occurred at the expected frequency and level of severity as previously documented. In lieu of a 
summary of external events, a current DSMB report can be submitted for research studies that are 
subject to oversight by a DSMB (or other monitoring entity that is monitoring the study on behalf of an 
industry sponsor). 
The DSMB is composed by: 
1) 
Stuart A Weinzimer, MD. Professor of Pediatrics at Yale University. Dr. Weinzimer is an 
expert of type 1 diabetes and has participated to several clinical trials studying the efficacy of closed 
loop system in children with type 1 diabetes. 
2) 
Eda Cengiz, MD. Associate Professor of Pediatrics at Yale University. Dr. Cengiz has a 
longstanding expertise in the pharmacokinetic properties of insulin analogues in type 1 diabetes. The 
PI will report to DSMB every 6 months by conference call and every year by in person meeting. 
Unscheduled meetings will be arranged in case any events should occur. The PI will provide the data 
obtained from the study and the potential adverse events reported by the patients. The DSMB will 
review the data and provide report the findings to the PI by email or in person meeting. 
The DSMB will be responsible for reviewing comprehensive, cumulative, unblinded safety reports, and 
employing stopping rules if there is evidence of differential effects in either risk or benefit. 
 
7. Plan for reporting adverse events to co-investigators on the study, as appropriate the protocol’s 
research monitor(s), e.g., industrial sponsor, Yale Cancer Center Data and Safety Monitoring 
Committee (DSMC), Protocol Review Committee (PRC), DSMBs, study sponsors, funding and 
regulatory agencies, and regulatory and decision-making bodies. 
 
For the current study, the following individuals, funding, and/or regulatory agencies will be notified: 
 
 
□ All Co-Investigators listed on the protocol. 
□ National Institutes of Health 
□ Data Safety Monitoring Board (DSMB) 
The principal investigator (Nicola Santoro) will conduct a review of all adverse events upon completion 
of every study subject. The research participant will also be given a study safety questionnaire at the end 
of the study that asks about the degree to which they experienced any side effects. The principal 
investigator will evaluate the frequency and severity of the adverse events and determine if 
modifications to the protocol or consent form are required. 
 
Please note: For any study that may be considered high risk, the IRB will be more focused on the safety 
requirements for the study and a DSMB will likely be required. 

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For more guidance on Adverse Event reporting and DSMPs, see IRB Policy 710 Reporting 
Unanticipated Problems Involving Risks to Subjects or Others, including Adverse Events