The Effects of Orange Juice Compared With Sugar-sweetened Beverage on Risk Factors and Metabolic Processes Associated With the Development of Cardiovascular Disease and Type 2 Diabetes
Phase: NA
Conditions: Cardiovascular Risk Factor, Type2 Diabetes Mellitus, Insulin Sensitivity, Metabolic Syndrome
Sponsor: University of California, Davis

STUDY PROTOCOL: The effects of orange juice compared with sugar-sweetened beverage on risk factors 
and metabolic processes associated with the development of cardiovascular disease and type 2 diabetes  
APPROVAL DATE: 11-15-2022 
NCT number: 03527277
Principal Investigator: Kimber Stanhope 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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HRP-503-PTOTOCOL TEMPLATE-General 
 
 
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1) 
Protocol Title 
The effects of orange juice compared with sugar-sweetened beverage on risk factors 
and metabolic processes associated with the development of cardiovascular disease and 
type 2 diabetes  
 
PROTOCOL MODIFICATIONS TO ACCOMMODATE COVID-19 STUDY 
SAFETY RESTRICTIONS: Our original protocol includes inpatient metabolic 
studies that require multiple close interactions between participants and the research 
team. NIH has approved modifications of the inpatient protocol to an outpatient 
protocol in order to safely conduct this study while the pandemic continues. The 
modifications to the original inpatient procedures are described, plus other safety 
mitigations including the elimination of non-essential interactions between study staff 
and study participants. 
 
Protocol Version Date 11-09-2022 
2) 
Objectives 
Specific Aims: There is considerable epidemiological evidence that demonstrates associations 
between added sugar/sugar-sweetened beverage consumption and increased risk for or 
prevalence of chronic diseases such as cardiovascular disease (CVD), type 2 diabetes (T2D), 
metabolic syndrome, and gout. Especially concerning is recent evidence from National Health 
and Nutrition Examination Survey III that demonstrates that there is increased risk of CVD 
mortality with increased intake of added sugar across quintiles.  Even the US mean added sugar 
intake, 15% of daily calories, was associated with an 18% increase in risk of CVD mortality over 
15 years. The results from our recently completed study (1R01 HL09133) corroborate these 
findings. They demonstrate that supplementing the ad libitum diets of young adults with 
beverages containing 0, 10, 17.5 or 25% of daily energy requirement (Ereq) as high fructose corn 
syrup (HFCS) affects lipid/lipoprotein risk factors for CVD in a dose response manner. 
Specifically, levels of nonHDL-cholesterol(C), LDL-C, apolipoprotein B (apoB), and 
postprandial triglycerides (TG) increased linearly over a 2-week period with increasing doses of 
HFCS. Furthermore, even the participants consuming the 10% Ereq dose exhibited increased 
levels of these risk factors compared to baseline.  
 
These and similar results have helped to lead to reductions in soda consumption in this country, 
and new dietary guidelines and FDA food labeling requirements to promote reductions in added 
sugar consumption. However, there are gaps in knowledge about other sugar-containing foods 
that lead to public confusion concerning healthier options for soda, and impede further progress 
in implementing public health policies that will promote further reductions in soda consumption.  
One such food is naturally-sweetened fruit juice. The amount of sugar in fruit juice is 
comparable to the amount in soda. Because of this, a consumer seeking answers on the internet 
will find many articles in which experts state or suggest that the effects of consuming fruit juice 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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are as detrimental as or even worse than those of soda. However, in contrast to soda, fruit juice 
contains micronutrients and bioactives that may promote health. Therefore the consumer can also 
find numerous articles on the internet where the health benefits of fruit juice and these bioactives 
are extolled. There are a limited number of clinical dietary intervention studies that have directly 
compared the metabolic effects of consuming fruit juice and sugar-sweetened beverage, and their 
results are not conclusive.  
 
The objectives of this proposal are to address the gaps in knowledge regarding the metabolic 
effects of consuming orange juice, the most frequently consumed fruit juice in this country, 
compared to sugar-sweetened beverage. Thus, we will pursue the following Specific Aims: 
 
1. Specific Aim 1: To compare the weight-independent effects of consuming 25%Ereq 
as orange juice or sugar-sweetened beverages for 4 weeks on risk factors for CVD and 
other chronic disease in normal weight and overweight men and women. We hypothesize 
that blood concentrations of LDL-C, apoB and uric acid will be lower in subjects consuming 
orange juice than in subjects consuming sugar-sweetened beverage. 
 
2. Specific Aim 2: To mechanistically compare the weight-independent effects of 
consuming 25%Ereq as orange juice or sugar-sweetened beverages on metabolic processes 
associated with the development of CVD and T2D in normal weight and overweight men 
and women. We hypothesize that consumption of orange juice will result in less detrimental 
changes on hepatic de novo lipogenesis (DNL), liver lipid accumulation and insulin sensitivity 
than consumption of sugar-sweetened beverage. 
 
3. Specific Aim 3: To relate the changes assessed under Specific Aims 1 and 2 to the 
changes in the urinary levels of metabolites and catabolites of the main flavanones in 
orange juice, hesperetin and naringenin.  We hypothesize the increased urinary levels of the 
hesperetin and naringenin metabolites and catabolites in subjects consuming orange juice will 
negatively associate their changes in risk factors and metabolic processes associated with chronic 
disease.  
 
Weight-maintaining, low sugar diets will be provided during the 2-week baseline period, and 
matched diets that contain 25% energy as the carbohydrate in orange juice or sucrose-sweetened 
beverage will be provided during the 4-week intervention period. All experimental procedures 
for assessment of blood risk factors, DNL, insulin sensitivity, liver lipid and abdominal fat 
distribution are conducted during baseline and during the end of intervention. 
 
The results from this study, whether supportive or not of our hypotheses, will assist the consumer 
in making informed beverage choices and also aid the efforts to establish evidence-based public 
health policy aimed at slowing the epidemics of CVD and T2D.  
3) 
Background 
As shown in Table 1, orange juice, the most commonly-consumed fruit juice in this country24, 
can contribute significant amounts of sugar to the diet. Thus, it is possible that consumption of 
high amounts of orange juice will result in increases in blood levels of LDL-C, apoB, 
postprandial TG, and uric acid that are comparable to what we have observed in subjects 
consuming HFCS-sweetened beverage 7. However, while the micronutrient content of sugar-

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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sweetened sodas and beverages is essentially zero, fruit juices contain a wide variety of 
micronutrients and other bioactive components (e.g. polyphenols, flavonoids, anthocyanins, etc.), 
which could possibly modify the metabolic processes by which excess sugar consumption leads 
to increased risk factors for chronic disease. In support of this, there are populations studies that 
report that incidence/prevalence of  metabolic syndrome 25, 26, CVD 27  and T2D 28-30 and their 
risk factors 31-33 are associated with consumption of SSB, but not with consumption of 100% 
fruit juice. However, this lack of adverse association may be due to lower levels of fruit juice 
consumption than SSB consumption in most of these studies. It is also possible that the usual 
background diets of regular consumers of SSB are less healthy than regular consumers of 100% 
fruit juice. Furthermore, several studies report both fruit juice and SSB consumption are 
positively and comparably associated with metabolic syndrome 34, T2D 35, 36 and gout 37. 
 
 
 
The direct experimental data does not resolve the discrepancy in the epidemiological results. A 
meta-analysis of 19 randomized controlled trials through 2012 investigating the effects of 
various fruit juices, concentrated fruit juices and fruit juice powders from elderberry, bilberry, 
grapefruit, blueberry, cranberry, pomegranate and several types of grape, suggested that fruit 
juice had a borderline significant effect to reduce diastolic blood pressure, and did not affect total 
or LDL-C 38. The unaffected lipid levels could be viewed as a positive finding since the majority 
of the trials utilized control groups that received water intervention or no intervention. However, 
the dose of provided fruit juice ranged from 40-270 kcal/d, with an approximate mean of 165 
kcal/d 38. Possibly the amount of fruit juice consumed in the majority of the 19 studies 38 was too 
low to induce unfavorable effects in only 2-12 weeks. The low dose group from our recent study, 
who exhibited increased levels of LDL-C and apoB in 2 weeks, consumed 234 kcal/d as HFCS. 
A more recent systematic review of the evidence concerning the metabolic impact of 100% fruit 
juice consumption concluded evidence-based recommendations were not possible5.  
 
Background—Specific Aim 1: We propose to directly compare circulating levels LDL-C, 
apoB, uric acid and other risk factors in subjects consuming SSB or orange juice. Two published 
studies have compared the effects of orange juice with SSB on risk factors for chronic disease.  
In a 4-week trial, consumption of 500 ml/day (180 kcal)  orange juice lowered diastolic blood 
pressure, fasting plasma uric acid, and increased microvascular reactivity in middle-aged, 
overweight men 39 compared with the SSB control. Overweight, hypercholesterolemic men who 
consumed 250 ml (118 kcal) orange juice/day for 12 weeks tended (P=0.06) to have lower levels 
of TG compared with the men who consumed SSB 40. We have conducted a pilot study 
comparing the effects of consuming a much higher dose (25% Ereq) of orange juice or sucrose-
SB. Even at this high dose, orange juice consumption does not appear to have detrimental effects 
on LDL-C and apoB, and 24-hour uric acid concentrations compared with those following 
sucrose-SB consumption. Studies that investigated the effects of orange juice consumption 

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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without a SSB control also report positive effects. Women who consumed 500 ml/day (180 kcal) 
of orange juice for 3 months had improved LDL-C and HDL blood values compared with 
women who did not consume an intervention beverage 41.  Middle-aged, mildly hyperlipidemic 
adults who consumed 280 kcal/day of orange juice for 3 months exhibited decrease lipid 
peroxidation, increased total antioxidant status, and unaffected lipids 42. Men who consumed 750 
ml/day of orange juice for 2 months had decreased LDL-C concentrations and increased HDL 
function (measured by in vitro assay of transfer of radioactive lipid emulsion into the HDL 
fraction) 43. Consumption of 750 ml/day orange juice for 4 weeks increased HDL concentrations 
without affecting LDL and apoB in subjects with elevated cholesterol levels 44. Thus the 
available results specific to orange juice do suggest that it may have neutral or positive effects on 
risk factors and is a healthier beverage choice than SSB.  
 
Background—Specific Aim 2: We propose to directly compare metabolic processes by which 
excess sugar consumption leads to increased risk factors for chronic disease in subjects 
consuming SSB or orange juice. These include de novo lipogenesis, fatty acid oxidation, liver 
lipid accumulation, hepatic insulin resistance, and visceral fat accumulation. To the best of our 
knowledge none of these processes have been compared in subjects consuming SSB or orange 
juice.  
 
Background—Specific Aim 3: Our Specific Aim 3 investigation will be guided by 
Collaborator/Consultant Dr. Alan Crozier, a Thomson Reuters Highly Cited Researcher with 
more than 100 publications on the absorption, metabolism, disposition, excretion and health 
benefits of dietary flavonoids. We propose to directly compare the urinary levels of metabolites 
and catabolites of hesperetin and naringenin in subjects consuming SSB or orange juice. 
Hesperetin and naringenin are the main flavanones in orange juice and there is evidence from in 
vitro 66-68, animal 69-76, and human 27, 39, 77-79 studies to suggest that hesperetin and naringenin 
exert a wide range of beneficial actions that could possibly modify the metabolic processes by 
which excess sugar consumption leads to increased risk for chronic disease. However, 7 years 
ago Dr. Crozier pointed out the irrelevance of in vitro research that focused on the polyphenolic 
compounds as they are found in fruits and vegetables, because it is not these compounds that are 
transported around the human body in the circulatory system, or reach body tissues to elicit 
bioactive effects 80, 81. It is their metabolites, formed in the small intestine and hepatic cells, and 
also the low molecular weight catabolic products of the colonic microflora. It is these 
compounds that carry interest for drug-discovery and for dietary prevention of disease 80, 81. 
More recently, Dr. Crozier has published 1, 82, 83 and reviewed 84 the evidence that refutes the 
general belief that polyphenols are poorly bioavailable with only relatively small amounts of the 
ingested dose entering the systemic circulation, in the form of metabolites, prior to undergoing 
renal excretion. Instead, when metabolites and catabolites are included in the overall estimation 
of absorption, distribution, metabolism, and excretion, it is evident that many polyphenols, are 
highly bioavailable1. In the case of OJ, after acute intake, 0-24 h urinary excretion of hesperetin 
and naringenin metabolites; principally in the form of hesperetin-3-O-glucuronide and 
hesperetin-3-sulfate; was equivalent to 16% of intake1. Excretion of colon-derived phenolic 
catabolites was 88% of intake1, with 3-(3-hydroxy-4’-methoxyphenyl)hydracrylic acid being the 
main catabolite1 and a good biomarker of hesperetin intake 84.  This work also demonstrated that 
while plasma pharmacokinetic measurements provide a ‘snapshot’ of absorbed circulating 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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metabolites, 0-24 h urinary excretion of both metabolites, absorbed in the small intestine, and 
catabolites, of distal gastrointestinal origin that are products of bacterial processing, provide a 
more quantitative reflection of polyphenol absorption. We therefore propose to analyze 
hesperetin-3-O-glucuronide, hesperetin-3-sulfate and 3-(3-hydroxy-4’-
methoxyphenyl)hydracrylic acid; and also hesperetin-7-O-glucuronide, naringenin-4'-O-
glucuronide and naringen-7-O-glucuronide;  in urine collected over a 24-hour period at baseline 
and 4 week later during intervention when subjects consume orange juice or SSB with each 
meal.  
4) 
Inclusion and Exclusion Criteria 
Screening for eligibility: Subjects are first screened for eligibility via a 30-minute survey 
conducted over telephone or online (UC Davis Qualtrics) in which information about the study is 
provided. Qualifying respondents are asked to read the human consent form and then contacted 
to schedule a consenting appointment using Zoom, an online video communications application.  
During the consenting appointment, the study coordinator engages potential participants in a 
discussion about usual diet, social commitments, food-related activities and the difficulties that 
can be anticipated in adhering to the provided diet for approximately 6 weeks. The study 
coordinator discusses the consent form and all experimental procedures, the time commitment 
required by the study, and answers all questions. A list of all food ingredients served during the 
study is provided to determine acceptability, and potential subjects can request and arrange for 
pick-up of samples for taste testing if they wish. Interested potential participants will sign the 
consent form using DocuSign and a fasting blood draw appointment will be scheduled at either 
Ragle Human Nutrition Research Center on campus or at a UC Davis Health System affiliated 
clinic off campus if the former is unavailable due to scheduling confilicts. Clinical chemistry, 
CBC, and lipids are analyzed to determine eligibility. A follow up draw may be requested if 
necessary to determine eligibility. Female participants will be asked to provide the date of their 
last menstrual period and form of contraception, if applicable. Qualified respondents who are 
judged by the study coordinators as likely to be responsible and compliant are scheduled for 
study.  
 
Inclusion criteria: The first submission of this proposal to NIH limited inclusion to participants 
≤ 40 years of age with BMI ≤ 28 kgm2. The NIH reveiwers criticized this plan because it would 
preclude the ability to assess the effects of the 2 interventions across the metabolic spectrum of 
BMIs and detect differences in susceptibility between healthy and less healthy subjects. It was 
noted that inclusion in the statistical analysis of BMI and metabolic syndrome, liver fat and 
insulin sensitivity will allow us to gain insights on these variables in the response to both 
interventions. Therefore in the revised NIH application we expanded our inclusion criteria to 
include participants ≤ 50 years of age with BMI ≤ 35 kgm2. In order to ensure that we will be 
able to enroll obese subjects, who are likely to have plasma concentration of glucose, triglyceride 
or LDL-C that are out of the normal ranges, we also revised our exclusion criteria for these 
parameters to the values shown below. We will study men and pre-menopausal women (age 18-
50 y; BMI 20-35 kg/m2 with body weight > than 50 kg) with self-reported stable body weight 
during the prior six months. We will study 24 subjects per group, requiring a total of 48 subjects 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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to complete research procedures. We anticipate a completion rate of 70% therefore expect to 
enroll a total of 72 subjects.  
 
Exclusion criteria:   
• Fasting glucose >125 mg/dl 
• Evidence of liver disorder (AST or ALT >200% upper limit of normal range) 
• Evidence of kidney disorder (>2.0 mg/dl creatinine) 
• Evidence of thyroid disorder (out of normal range) 
• Systolic blood pressure consistently over 140 mmHg or diastolic blood pressure over 
90 mmHg 
• Triglycerides > 400 mg/dl 
• LDL-C > 160 mg/dl in combination with Chol:HDL > 4  
• Hemoglobin < 10 g/dL 
• Pregnant or lactating women 
• Current, prior (within 12 months), or anticipated use of any hypolipidemic or anti-
diabetic agents. 
• Use of thyroid, anti-hypertensive, anti-depressant, weight loss medications or any 
other medication which, in the opinion of the investigator, may confound study 
results 
• Use of tobacco 
• Use of marijuana 
• Strenuous exerciser (>3.5 hours/week at a level more vigorous than walking)  
• Surgery for weight loss 
• Diet exclusions: Food allergies, special dietary restrictions, routine consumption of 
less than 3 meals/day or dietary habits that may undermine compliance to dietary 
protocol, routine ingestion of more than 2 sugar-sweetened beverages or 1 alcoholic 
beverage/day, unwillingness to consume any food on study menu 
• Veins that are assessed by the R.N.s as being unsuitable for long-term infusions and 
multiple blood draws from a catheter.  
• Pre-existing claustrophobia or metal implants that preclude MRI 
• Any other condition that, in the opinion of the investigators, would put the subject at 
risk 
• A recent positive COVID-19 test  
 
We will exclude individuals from each of the following special populations: 
• Adults unable to consent 
• Individuals who are not yet adults (infants, children, teenagers < 18 years) 
• Pregnant women 
• Prisoners 
 
We will also exclude non-English speakers because study procedures can only be implemented 
with proficiency in English. At the current time we do not have staff that are proficient in any 
other language than English and we do not have the funds to hire extra staff with bi-lingual 

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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skills. This is also true for the staff at Touro University, where the 12-hour blood collection 
procedures are conducted. 
 
Effective and clear communication between the study staff and between the subjects is essential 
in order to properly implement the experimental and dietary protocols required for this study. 
Before confirming their willingness to participate in this study, the subjects must have a clear 
understanding of what is required of them in terms of time commitment, diet commitment, and 
also an understanding of procedures that they will undergo. For example, it is very important that 
potential subjects clearly understand that we need to know if they have metal implants in their 
bodies because certain metal implants can cause great harm to them during magnetic resonance 
imaging.  Potential subjects need to clearly understand that this study requires many blood 
samples and realize that if the insertions required for needle-stick collections or catheters makes 
them nervous or unhappy, this study is not for them.   
 
Throughout the study more than 30 text messages and/or emails get exchanged between 
subjects and the study coordinators regarding schedules, body weight, diet issues such as hunger 
or food that needs to be replaced. Those subjects who are most able to clearly communicate any 
problems they are having regarding schedules, diet, beverages, are the subjects who are most 
likely to consider participation in our study to be a rewarding experience.  
5) 
Study Timelines 
Timeline:  Our goal is to study 24 subjects (12/group) using the modified protocol and 24 
subjects (12/group) using the original protocol. Ten subjects have already completed the original 
protocol. Based on an expected retention rate of 70%, we will enroll 35 subjects during Years 4-
5 (spring 2021 to summer 2022) into the modified protocol. When we have achieved our sample 
size of 24 subjects completing the modified protocol, we will seek approval to resume conduct of 
the original protocol. If approval is not obtained due to the pandemic, we will enroll up to 24 
more subjects into the modified protocol to achieve a final sample size of 48 (10 on original 
protocol, 38 on modified protocol).   
 
Duration of individual subject’s participation: ~6 weeks (40-47 days) 
Duration of anticipated enrollment of all subjects: April 1, 2018-August 30, 2023 
Estimated date of investigator completion of primary analyses: January 31, 2024 
6) 
Study Endpoints 
Primary and secondary study endpoints: The primary endpoints are the changes in risk factors 
for metabolic disease which include blood lipid and lipoprotein levels.  The secondary endpoints 
are hepatic lipid content, the fractional rate of de novo lipogenesis,  and whole body insulin 
sensitivity.   
 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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Primary and secondary safety endpoints: The study intervention, which consists of 4-weeks 
consumption of 25%Ereq as orange juice or sucrose-sweetened beverages, is not associated with 
any short term safety risks.  
7) 
Procedures Involved 
Anyone coming onto campus (students, employees or visitors) is required to follow the 
university COVID-19 safety guidelines that are posted on the campus website: 
https://campusready.ucdavis.edu/, which may include filling out the UC Davis Daily Symptom 
Survey during each visit and completing COVID-19 testing. Additionally, participants will be 
required to wear a mask during all study interactions, have their temperature monitored and 
maintain a 6-foot distance when possible (e.g., except Registered Nurses performing blood 
draws). For transport by study staff in UCD vehicles to study procedure sites, participants will be 
provided and required to wear a KN95 or N95 mask, face shield and to sit 6 feet away from the 
driver and any other passenger. 
 
General study design: The study is designed as a parallel, 6-week study with a 2-week baseline 
period during which all subjects consume the provided low-sugar baseline diet and a 4-week 
intervention period during which the experimental beverages are consumed along with a low-
sugar diet.  
Baseline (Week 1-2):  All subjects will consume a provided, standardized, low sugar, energy-
balanced diet for approximately 9-13 days. All experimental procedures are conducted.  
Intervention (Week 3-6):  Subjects will be stratified by gender and normal 
weight/overweight status and randomized to one of the two groups listed below:   
1. Sugar-sweetened beverages (SSB): 25% of estimated Ereq/day as sucrose in water (11% 
weight/w/w), flavored with unsweetened Kool-Aid and divided into 3 servings/day. 
2. Orange Juice (OJ): 25% of estimated Ereq/day as the carbohydrate in OJ divided into 3 
servings/day. 
Subjects will consume three provided beverages/day along with provided, standardized, 
energy-balanced meals and snacks for approximately 28-33 days. All experimental procedures 
are repeated. 
 
Diets & weight monitoring: Approximately 2 weeks prior to the start of study, enrolled subjects 
will be scheduled at their convenience to report to Ragle HNRC. If Ragle HNRC is unavailable 
due to facility safety restrictions (due to the small dining and procedure rooms, the number of 
subjects that can be accommodated at this time is very limited) or schedules conflicts, the foyer 
at the entrance to Ragle HNRC will be used. Their height and body weight will be measured with 
a portable stadiometer and body weight scale.  They will be provided with a body weight scale 
and asked to provide (via text or email) a daily, minimally clad, fasted body weight for the next 2 
weeks and throughout the study. The purpose is to improve the accuracy of the initial estimation 
of Ereq and to facilitate the adjustment of energy intake required to maintain stable body weight. 
Also at this visit, female participants will be asked to take a urine pregnancy test. 
 
Study diet is prepared by the study staff in the kitchen of the Ragle HNRC on the UC Davis 
campus. Subjects are restricted to eating only the study food provided during the entire study. 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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The initial daily Ereq of each subject will be calculated by the Mifflin equation with an 
adjustment of 1.5 for activity 108. During the 2-week baseline period, all subjects consume an 
energy-balanced, low sugar diet that is 50% Ereq carbohydrate, 35% fat and 15% protein. During 
the 4-week beverage intervention, the subjects receive diets that are matched as closely as 
possible to the baseline diet with the exception of the substitution of 25% Ereq as the 
carbohydrate in sucrose-sweetened beverage or orange juice for 25% Ereq complex carbohydrate.  
Subjects will return all uneaten food for calculation of daily intake. Diet energy will be adjusted 
as required to maintain stable body weight. If energy adjustment of the meals consistently fails to 
prevent weight gain or weight loss the subject will not be studied further.  
 
Food is served/provided as 3 meals and a snack, with 23% of the energy provided as breakfast, 
32.5% as lunch, 37% as dinner and 7.5% as snack. Meals and snacks mainly consist of a 
heterogeneous mix of ingredients (i.e. stir-fry or casserole rather than separate servings of meat, 
grain and vegetable) formulated to the standardized specifications. This ensures that a subject 
who does not eat an entire meal is still consuming the required macronutrient distribution. The 
intervention meals contain 25% more fiber than the baseline meals in order that the fiber content 
of both the overall baseline and intervention (which includes the beverages) diets is 12.5 g/1000 
kcals. This is mainly achieved by a greater proportion of unrefined grains (e.g. whole wheat 
pasta) in the intervention meals. The overall fat content of the baseline and intervention diets is 
<10% Ereq saturated fat, and ~12% and ~13% Ereq polyunsaturated and mono-unsaturated fat, 
respectively.   
 
Beverages: We will utilize unfortified 100% orange juice that is pasteurized, ready-to-serve, and 
is not reconstituted from concentrate. We will utilize a top-selling brand and type that is widely 
available. The SSB will be made with sucrose from beet sugar at a weight/weight concentration 
to ensure that subjects with identical energy requirements will receive the identical volume of 
beverage, whether it be orange juice or SSB.  Samples of each beverage batch are retained and 
frozen for future quality control and nutrient/bioactive analyses. Subjects are provided with 25% 
of daily Ereq as the total carbohydrate in their assigned beverage (carbohydrate in orange juice is 
approximately 90% sugar and 10% oligosaccharides), divided into 3 daily servings to be 
consumed with meals (25% at breakfast, 35% at lunch, 40% at dinner). Subjects are required to 
consume all the beverage provided. The beverages will contain a biomarker (riboflavin) that will 
be assayed fluorometrically in urine collected 2 times/week during meal pick-up appointments 
(See Compliance monitoring section below).  
 
Beverage and meal pick-up/return: At appointments scheduled at their convenience, subjects 
will report to Ragle HNRC two times/week to pick up a 3- or 4-day supply of 
meals/snacks/beverages, and to return uneaten food and packaging. If Ragle HNRC is 
unavailable due to facility safety restrictions or schedules conflicts, the foyer at the entrance to 
Ragle HNRC will be used. Temperature will be measured using a contactless thermometer. 
Subject will be given a urine collection cup and and asked to provide a urine sample using the 
restroom in the foyer area. All exchanges of meal & beverage carrying cases and urine cup & 
urine sample will be conducted with both subject and study staff member wearing masks, face 
shields and maintaining a 6-foot distance at all time.  Thus there will be a designated cart or area 
in the foyer upon which all carrying bag/supplies/samples will be set for dispensation or for turn-
in. The cart or area will also have a supply of sterile wipes and disposable gloves so all items can 

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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be decontaminated prior to being picked up. Subjects who fail to pick up a new supply of 
meals/beverages (or make arrangements for it to be delivered to their homes) in time to allow for 
uninterrupted consumption as required by the protocol, will be dismissed from the study for non-
compliance. Subject will need to fill out study diet report forms, physical activity questionnaires 
and food surveys. These forms will be made available to complete electronically and are used to 
assess diet palatability and quantity, activity level changes and to describe diet deviations. These 
reports will be reviewed by the Study Coordinator and communication through text, email or 
Zoom call will be used to discuss issues or request clarifications. 
 
Compliance monitoring: The sweetened beverages will contain a biomarker (riboflavin). The 
biomarker will be assayed in urine collected 2 times/week during meal pick-up appointments. 
The subjects will be informed that they are being monitored for compliance, but not provided 
specifics as to the method. We have successfully used this method of enhancing compliance in 
three previous studies7, 14, 15.  
 
To identify subjects who consume or consumed non-study foods that contained HFCS or cane 
sugar or consume alcoholic beverages during the study period, we will measure biomarkers for 
added sugar and alcohol intake. We will measure the delta 13C value of blood drops collected at 
baseline and at the end of 4 weeks of intervention. The Delta 13C content in the blood drop will 
be measured at the UCD Stable Isotope Facility. Delta 13C is a novel validated biomarker of 
added sugar intake because most plants have a low natural abundance of delta 13C, while that of 
corn and sugar cane is conspicuously higher 110, 111. The foods with the highest amounts of delta 
13C are pure corn products, and beverages, hard candies and desserts sweetened with HFCS or 
cane sugar. Sugar from sugar beets has a low natural abundance of delta 13C, therefore beet sugar 
will be used to sweeten the sucrose-sweetened beverages provided as part of the intervention.  
Delta 13C in blood is an indicator of added HFCS and sugar cane sucrose consumption over the 
prior 2 weeks 3. Because all the meals and snacks provided during the 6-week study have a very 
low HFCS and sugar cane content, and all subjects are consuming the same foods, all compliant 
subjects will exhibit low and comparable levels of blood delta 13C at both the end of the 2nd week 
and 6th week of study. Data from subjects with delta 13C blood values that are more than 2 
standard deviations above the mean for all subjects will not be included in the statistical 
analyses.  
 
To identify subjects who routinely consume alcohol we will assay urinary ethyl glucuronide 
(immunoassay, Thermo Scientific, Fremont, CA) in the urine samples collected at each meal 
pick-up visit. Detection of ethyl glucuronide in urine indicates recent alcohol consumption 112 
and is considered to be a specific, highly sensitive, and reliable marker of recent alcohol intake 
within the previous 5 days 4. Urinary ethyl glucuronide concentrations above the 500 ng/ml will 
be considered positive for alcohol intake. Urine will be monitored for alcohol intake upon 
collection, and subjects with positive values will be considered for dismissal. The subjects will 
be informed that they are being monitored for compliance to the requirement that they not 
consume non-study food, but will not be provided specifics as to the method. 
 
Experimental procedures: Procedures are performed as shown in the study schedule below 
(Table 2). This schedule is approximate and the 6-week length is approximate, as changes may 

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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be made to accommodate subject availability and availability of the study facilities, equipment & 
personnel. Outpatient procedures will occur at the Ellison Building, University of California 
Davis Medical Center (UCDMC), and at Touro Metabolic Research Center (TMRC) in Vallejo, 
CA. Transportation from Davis to UCDMC and TMRC and will be offered by study using UCD 
Fleet Service vehicles. Subjects will be provided and required to wear a KN95 or N95 mask and 
face shield while in the vehicle and maintain a 6-foot distance from the driver and any other 
passenger. Participants who wish to provide their own transportation to the facilities will be 
reimbursed at the UCD mileage reimbursement rate. For subjects who provide their own 
transportation, a study staff commucation plan that will include a wake-up call if needed will be 
set up to ensure their arrival at TMRC or the Ellison Building on schedule.  

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metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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Template Revision: June 15, 2017 
 
  
Week
Procedure
Location 
Time
Details
Compensation
-2
Scale Pick-up
Ragle HNRC
TBD by subject
2 weeks prior to study start; at-home body weight scale 
pick-up visit
$10
-2 - 6
Fasting BW
Home
pre-breakfast
Text, email or phone in fasting BW daily
$65
0
Study Overview
Home
TBD by subject
Zoom meeting for study/schedule review & questions
$10
0
1st Meal Pick-up
Ragle HNRC
TBD by subject
1st meal pick-up visit 
$10
1 - 2
Diet
Home/Ragle 
HNRC
TBD by subject
Daily consumption of provided baseline study meals 
Pick-up/dropoff for study meals; urine collection:  2 
times/week
$40
1
MRI #1
UCDMC 
Radiology
7:00
Hepatic & abdominal magnetic resonance imaging
$50
1
Stool Collection #1
Home
TBD by subject
Stool collection
$20
2
24-h UC #1
Home
TBD by subject
24 hour urine collection
$30
2
Pre-TMRC Evening
Home
20:00, 23:00
Consumption of acetate tracer dose
$40
7:00
Transport to TMRC, Consumption of acetate tracer dose
8:00
Check-in, vitals, anthropometrics, catheter insertion
8:45
Start blood collection every 30 or 60 minutes
9:00 - 12:00
Oral glucose tolerance test
12:00
Lunch, Consumption of acetate tracer dose
13:00 - 15:00
Study questionnaires, DEXA scan
16:00
Dinner, Consumption of acetate tracer dose
20:00
Final blood draw, remove catheter, check-out
3-6
Diet
Home/Ragle 
HNRC
TBD by subject
Consumption of provided intervention study meals & 
beverages
Pick-up/dropoff for study meals/beverages; urine 
collection:  2 times/week
$140
3
24-h UC #2
Home
TBD by subject
24 hour urine collection
$40
5
Stool Collection #2
Home
TBD by subject
Stool collection
$20
6
24-h UC #3
Home
TBD by subject
24 hour urine collection
$50
6
MRI #2
UCDMC 
Radiology
7:00
Hepatic & abdominal magnetic resonance imaging
$50
6
Pre-TMRC Evening
Home
20:00, 23:00
Consumption acetate tracer dose
$45
7:00
Transport to TMRC; Consumption of acetate tracer dose
8:00
Check-in, vitals, anthropometrics, catheter insertion
8:45
Start blood collection every 30 or 60 minutes
9:00 - 12:00
Oral glucose tolerance test
12:00
Lunch, Consumption of acetate tracer dose
13:00 - 15:00
Study questionnaires, DEXA scan
16:00
Dinner, Consumption of acetate tracer dose
20:00
Final blood draw, remove catheter, check-out
TOTAL
$970
Table 2: Study schedule with procedures, timing and compensation  
Baseline ~ 2 weeks
Energy-balanced baseline diet: carbohydrate (mainly starch--50%Ereq), fat (35%Ereq),  protein 
(15%Ereq)
Procedure days are approximate and dependent on clinic and subject availability (baseline visits will occur in the first 2 weeks of the study and 
intervention visits will occur between the 5th and 6th week of the study)
Ragle HNRC: Ragle Human Nutrition Research Center at UCD campus, Department of Nutrition; UCDMC: University of California Davis 
Medical Center; TMRC: Touro (University) Metabolic Research Center; Ereq: energy requirement; BW: Body weight
$150
End of Study
Intervention ~ 4 weeks
2
TMRC Visit #1
TMRC
6
TMRC Visit #2
TMRC
Energy-balanced intervention diet: Orange juice or SSB (25%Ereq), carbohydrate (mainly starch--
25%Ereq), fat (35%Ereq),  protein (15%Ereq)
$200

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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Template Revision: June 15, 2017 
Procedures at UCD or UCDMC:  
 
Urinary flavanone metabolites and catabolites: During baseline and two times during 
intervention study subjects will collect all urine over a 24-hour period.  The urine collection 
period will begin with the first collection upon waking in the morning (no later than 9:00) and 
end at approximately 9:00 the next day. Subjects will be provided with pre-labeled bottles 
(enough to ensure all urine can be collected) containing a preservative solution (30 ml of 10% 
phosphoric acid) in a designated ice chest with enough cold packs to maintain refrigeration 
temperatures. Bottles will be transported to our laboratory in the designated ice chest (by either 
the subject or study staff). All collections are pooled by study staff, weighed and multiple 
aliquots are saved for metabolic analyses. 
 
The pooled urine samples from baseline and from the two intervention collections will be 
assayed by the NIH West Coast Metabolomics Center at UC Davis for hesperetin-3'-sulfate, 
hesperetin-3'-O-glucuronide, 3-(3'-hydroxy-4’-methoxyphenyl)hydracrylic acid, hesperetin-7-O-
glucuronide, naringenin-4'-O-glucuronide and naringen-7-O-glucuronide using liquid 
chromatography-high resolution mass spectrometry 83. Standards for each compound will be 
obtained from Toronto Research Chemicals (Toronto, Canada). All outcomes will be adjusted for 
urinary creatinine concentration, which will be measured on a PolyChem Chemistry Analyzer. 
 
Abdominal and hepatic fat/fasting blood sample: A fasting Magnetic Resonance Imaging 
(MRI) for hepatic lipid content and quantification of visceral and subcutaneous fat will be 
performed at the beginning and at the end of the study at the UCDMC Department of Radiology. 
These appointments are usually scheduled at 7:00 and study staff provide transport in university 
vehicles to subjects. Due to the pandemic, some subjects may prefer to drive themselves, and 
these subjects will be reimbursed at the UCD mileage reimbursement rate. Female participants 
will have a urine pregnancy test prior to the MRI. Dr. John McGahan, Department of Radiology 
UCD Medical Center, will supervise the pre- and post- quantification of images for intra-/extra-
abdominal fat. Image post-processing, tissue segmentation, and analysis of liver fat will be 
performed by Post-doctoral fellow Dr. Desiree Sigala using a commercial semi-automated 
software tool (SliceOmatic; Tomovision, Inc.). 
 
Microbiome - Fecal collection: Each subject will receive instructions and a sample collection 
kit containing ice packs, labeled sample vials, a fecal collection kit, zip-lock bags, and a sealable 
secondary container (freezer box). Feces will be collected in a disposable fecal collection vessel 
that attaches to the toilet. Subjects will be instructed to label sample vials with date and times of 
sample production. Subjects will transfer approximately 10mL of sample into the sample vials 
using the scoop that is attached to the sample vial lid. The lid is replaced on sample vial and the 
vial is placed in the ziplock bag and then in the secondary container and stored in the subject’s 
freezer until subject is ready to transport sample back to UC Davis. Upon arrival at UC Davis, 
labeled sample will be placed in a -80°C freezer until processed for future analyses.  Subjects 
will be asked to collect one sample during baseline and one sample during the intervention 
period. 
 
 

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metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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Template Revision: June 15, 2017 
Procedures at TMRC: These tests will be conducted during a 13-hour period at TMRC, which 
will occur during the baseline period and be repeated 4 weeks later during the intervention 
period. 
 
Isotope consumption for assessment of DNL (Figure 1): We are replacing our original 26-
hour isotope infusion protocol with an oral bolus protocol. Each subject will be provided with 
two doses of an oral stable-isotope tracer (1-13C acetate) in water that will be consumed at 20:00 
and 23:00 the night before the outpatient visit to the TMRC. Subject will be picked up at 6:30 -
7:00 by study staff for transport to TMRC.  Due to the pandemic, some subjects may prefer to 
drive themselves to TMRC, and these subjects will be reimbursed at the UCD milage 
reimbursement rate. A third dose of the acetate tracer will be consumed at 7:00. Additional doses 
of acetate tracer will be provided and consumed at the TMRC at 9:00, and with meals at 12:00 
and 16:00. Each oral dose of the acetate tracer will consist of 1-2 grams 1-13C acetate in 
approximately 4 ounces of water.  
 
Blood pressure: Blood pressure will be measured at check-in, before dinner, and prior to 
check-out. Weight, height, waist and hip will be measured. 
 
Blood collection (Figure 1): A catheter will be inserted at 8:15-8:30 and blood sampling will 
start at 8:45 and continue until 20:00 at the timepoints indicated on Figure 1 and Table 2. 
 
Urine Collection: A urine sample will be collected before 9:00 and after consumption of lunch 
and dinner.   
 
Oral glucose tolerance test (OGTT) for assessment of insulin sensitivity (Figure 1): We are 
replacing our original hyperinsulinemic euglycemic clamp protocol with a 3-hour oral glucose 
tolerance test. Following a second fasting blood draw at 9:00, subjects will consume 75 grams of 
glucose in 300 ml of water followed with blood draws at 9:30, 10:00, 10:30, 11:00, 11:30, 12:00. 
 
Lunch: Lunch will be provided following the 12:00 blood draw. 
 
Dual energy X-ray absorptiometry (DEXA): Total body fat will be determined by DEXA. 
In the event that the DEXA scanner at TMRC cannot be used, the DEXA scanner at CCRC 
will be used. Each subject’s baseline and intervention scans will be conducted using the same 
scanner. 
 
Questionnaires: Questionnaires about eating behaviors, food preferences and personality traits 
will be administered. 
 
Dinner: Dinner will be provided following the 16:00 blood draw. 
 
Check-out: Following the final blood draw at 20:00, subjects will be transported home. 
 
 
 

PROTOCOL TITLE:   
The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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HRP-503-PTOTOCOL TEMPLATE-General 
 
 
Template Revision: June 15, 2017 
 
 
 
 
Analyses: %Fractional DNL:  %Fractional DNL (fasting, during OGTT, during and following 
meal feeding) will be assessed in the sample collected from 8:45 – 20:00. 
 
OGTT: Glucose and insulin will be measured in the samples collected at 9:00, 9:30, 10:00, 
10:30, 11:00, 11:30 and 12:00. The 3-hour area under the curve (AUC) for glucose and insulin 
and the Matsuda insulin sensitivity index will be calculated. 
 
Fasting and postprandial blood profiles: Fasting blood samples will be collected at 8:45 and 
9:00. Pre-dinner and postprandial blood samples will be collected hourly until 20:00. These 
samples will be used to analyze hormones and metabolites that include insulin, leptin, lipids, 
lipoproteins, glucose, lactate, liver enzymes, free fatty acids and uric acid. 
 
The timing and volume of the blood samples to be collected during TMRC visit 1 & 2 are shown 
in Table 3.   
 
 
 

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metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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Table 3: Timing and volume of blood samples collected for procedures  
Scheduled time  
of draw 
TMRC visit 1 
(baseline)  
blood draws (cc) 
TMRC visit 2 
(intervention)  
blood draws (cc) 
EXIT study  
blood draw (cc) 
8:00 
  
  
6 
8:45 
27 
27 
  
9:00 
27 
27 
  
9:30 
13 
13 
  
10:00 
13 
13 
  
1030 
13 
13 
  
11:00 
13 
13 
  
11:30 
13 
13 
  
12:00 
17 
17 
  
13:00 
13 
13 
  
14:00 
13 
13 
  
15:00 
13 
13 
  
16:00 
13 
13 
  
16:30 
8 
8 
  
17:00 
13 
13 
  
17:30 
4 
4 
  
18:00 
13 
13 
  
19:00 
23 
23 
  
20:00 
23 
23 
  
Total 
272 
272 
6 
Total blood volume (cc) for study 
550 
 
 
Questionnaires: Subjects will fill out questionnaire about physical activity, eating behaviors, 
food preferences and personality traits during the study.  Some of these questionnaires will be 
filled out during the TMRC visits, and some will be provided and returned electronically. These 
questionnaires are a consolidation of the eleven tests described below: 
 
Reward-Based Eating Drive (RED). The RED taps conditioned hypereating by assessing 
adaptive (flexible) and maladaptive (rigid) dietary restraint, as well as three aspects of drive to 
eat (loss of control, lack of satiety and preoccupation with food). Items are answered on a Likert 
scale from 1 (not at all like me) to 5 (very much like me). The title of the RED questionnaire 
provided to the subjects is “Eating thought & experiences”. 
 
General State and Trait Food Craving Questionnaires (reduced). The G-FCQ-T assesses general 
trait and state food cravings, and was created from the FCQ-T and FCQ-S, which assesses 
specific food craving. The 15-item G-FCQ-T is a reliable measure of a general ‘desire for food’ 
or ‘desire to eat’ that has evidenced good internal consistency (α = .94). The G-FCQ-T comprises 
four factors: (1) preoccupation with food (i.e., obsessive thought about food and eating), (2) loss 
of control (i.e., difficulty regulating eating behavior when exposed to food cues), (3) positive 
outcome expectancy (i.e., believing eating to be positively reinforcing), and (4) emotional 

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
Page 17 of 27 
 
HRP-503-PTOTOCOL TEMPLATE-General 
 
 
Template Revision: June 15, 2017 
craving (i.e., tending to crave food when experiencing negative emotion). Internal consistencies 
for the entire G-FCQ-T (α = .94) and its subscales (αs = .72 - .87) are good. The title of the G-
FCQ-T questionnaire provided to the subjects is “General food cravings”. 
 
Snacking On Sweets. We will assess participants’ tendency to snack on sweet foods and drinks 
using two items created for this purpose: “How many servings a day did you eat of desserts or 
other sweets (cookies, ice cream, candy, cake, or other sweet foods)?” and “How many servings 
a day did you drink of regular (not diet) soda, or other sweet drinks (sweet tea, any juices, 
blended coffees, milkshakes)?” Items are rated on a scale from 0 (0 servings) to 5+ (5 or more 
servings). The title of the SOS questionnaire provided to the subjects is “Snacking”. 
 
Salty and Sweet. Participants will be asked if they tend to prefer salty foods or sweet foods on 7-
point Likert scale, ranging from preferring sweet foods much more / somewhat more / a bit more 
than salty foods, to equal preferences for sweet and salty foods, to preferring salty foods much 
more / somewhat more / a bit more than sweet foods. Participants will be asked to endorse this 
question (1) in general, over the course of their lives (Time 1 only), and (2) over the prior 3 days.  
 
Subjective Opiate Withdrawal Scale. The SOWS is designed to tap the experience of 
withdrawing from a substance, and items tap common motoric, autonomic, gastrointestinal, 
musculoskeletal, and psychiatric symptoms of opiate withdrawal. We will add 11 items tapping 
typical naltrexone-related responses (e.g., nausea, headache) and food (e.g., “I have trouble 
getting food off of my mind”). We will modify one existing SOWS item (replacing word ‘using’ 
with word ‘eating’). Items are answered on a scale from 0 (not at all) to 4 (extremely). The title 
of the SOWS questionnaire provided to the subjects is “In this moment”. 
 
Stress Eating. To tap stress eating, we will ask participants to respond to three general questions 
related to whether and how often they eat in response to stress, and how they feel when they do. 
The items are answered on 4-6 point Likert scales that grade the frequency of the behavior or the 
emotional response to the behavior.  
 
Palatable Eating Motives Scale (PEMS) The PEMS is a 19-item questionnaire that aims to gather 
information on what motivates the participant to consume food. Subjects will respond to 
questions ranging from 1 (Almost never/never) to 5 (Almost always/always). The questionnaire 
will be provided to subjects as the “Palatable Eating Motives Questionnaire”. 
 
Control of Eating Questionnaire (CoEQ): The CoEQ asks 21 questions related to appetite, 
hunger and cravings over the last 7 days. Subject responses regarding frequency or strength of 
emotional responses are marked on a continuous scale anchored by “not at all” and “extremely”. 
The title of the CoEQ provided to the subjects is “Food feelings during the last 7 days”. 
 
Food Security: The Food Security questionnaire will be administered at the beginning of the 
study and identifies subjects who have experienced food insecurity. All 6 questions include the 
option to respond “Don’t know/Prefer not to answer”. The title of the Food Security 
questionnaire provided to the subjects is “Budget and Food”. 

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metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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8) 
Data and/or Specimen Management and Confidentiality 
Data analysis plan: Janet Peerson, Senior Statistician (USDA, Western Human Nutrition 
Research Center), will assist with statistical analyses. All fasting and postprandial outcomes will 
be assessed for conformance to the normal distribution and transformed if appropriate. The 
dependent variable will be calculated as the change obtained at 4-wk intervention compared with 
baseline. All outcomes, excepting those related to insulin sensitivity, will be analyzed in a 3-
factor (beverage, gender, original or modified protocol) analysis of covariance (SAS 9.4) with 
adjustment for BMI and outcome at baseline 7. Other subject characteristics or baseline outcomes 
will be included as continuous or ranked covariables (e.g. metabolic syndrome risk factors, 
visceral fat, liver fat, insulin sensitivity) and retained if they improve the sensitivity of the model. 
Tukey’s posttest will be used to identify significant differences between beverage x gender 
groups. Significant changes compared to baseline will be identified as adjusted means 
significantly different from zero. Insulin sensitivity outcomes will mainly be analyzed separately 
by protocol (clamp or OGTT).  
 
Multivariable linear regression analyses will be used to evaluate the association between 
cardiometabolic risk markers and creatinine-adjusted urinary concentrations of the flavanone 
metabolites and catabolites, individually, and in hesperetin- and naringenin-specific groups. 
 
Sample Size: The proposed sample size of 24 subjects/group will detect an effect size 
(difference between group means/pooled standard deviation) of 0.83 at P<0.05, 80% power in a 
2-group comparison, two-sided test. The effect sizes for the 2-week comparison of 25% Ereq 
orange juice and sucrose-SB on LDL-C, apoB and the 24h uric acid were 0.89, 0.84 and 1.02 
respectively. Thus we expect 24 subjects/group to be sufficient to detect differences in these 
primary outcomes between the groups consuming orange juice and SSB.  
 
Confidentiality and data management: All research staff requiring access to subject data and 
specimens will be trained on HIPAA and confidentiality by completing Privacy and Security 
Training courses offered by the UCDHS Compliance Department. All enrolled subjects will be 
assigned an identification number upon start of study. Documentation of subject ID number and 
subject name will be stored in each subject’s patient medical chart, which is maintained by the 
TMRC or CCRC research and medical staff, and in each subject’s hard copy file, which is 
maintained by the Study Coordinator. During the study, each subject’s patient medical chart will 
be stored in a locked file cabinet that is located in a locked room in restricted area at the TMRC 
or CCRC. The TMRC or CCRC nursing staff has access to these files. When a subject has 
completed study, copies of the medical chart are placed in the hard copy subject files. When the 
5-years study is completed, copies of the CCRC patient medical charts from all subjects will be 
uploaded into UC Davis Health System Electronic Medical Records (EMR) and hard copy 
patient medical charts will be destroyed. During study, the hard copy subject files are transported 
to the various study sites by the Study Coordinator in a locked file case. The Study Coordinator 
and UCD research team have access to this locked file case. Upon completion of study, each 
subject’s hard copy file will be stored in a locked file cabinet in Dr. Stanhope’s locked office in 
VM3B, 1089 Veterinary Medicine Drive, on the UCD campus. The Study Coordinators, Dr. 
Stanhope and Dr. Sigala have access to these files. These files will be de-identified within 5 
years of publication.  

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The effects of orange juice compared with sugar-sweetened beverage on risk factors and 
metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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HRP-503-PTOTOCOL TEMPLATE-General 
 
 
Template Revision: June 15, 2017 
 
Quality control and specimen management: Samples needing transport will be done so in 
collection containers and a secondary hard-walled container such as an ice chest. Containers will 
be labeled as required, i.e. plasma samples will have a dry ice label and UN3373 Category B 
label on the outside. Study specimens will be stored in -80ºC freezers in the laboratories of Drs. 
Stanhope and Jean-Marc Schwarz. All specimens and data sent to and analyzed by other 
collaborators will only be identified by subject ID number. The subject ID number will also be 
used for all the analyses conducted in the laboratories of Dr. Stanhope and Dr. Schwarz and all 
collaborators. All electronic databases will be identified only by ID number and will be stored on 
secured servers. Therefore all laboratory staff will be blinded to subject identity and intervention 
during all sample processing and analyses. The only exception is when subject identity 
information is essential to resolve a safety concern (i.e. human pathogens).  
 
All assays are run with in-house quality control samples to allow calculation of the inter- and 
intra-assay coefficients of variation. All procedures are scheduled such that they are repeated in 
the time frame relative to the last meal at both baseline and intervention. Identical meals are 
served before and on procedure days at baseline and intervention and any deviations from the 
baseline trials are replicated. Samples of each beverage batch are retained and frozen for future 
quality control and nutrient/bioactive analyses.  
9) 
 Data and/or Specimen Banking 
Blood, feces and urine will be banked for future use in -80ºC freezers in the laboratory of Dr. 
Stanhope/Havel. These specimens will be stored for as long as they are viable (for up to 20 
years). Dr. Stanhope’s study staff will maintain records of the storage location of all specimens 
and have access to them. Dr. Schwarz’s study staff will have access to the blood samples 
collected for the purpose of measuring glucose production, insulin sensitivity, and DNL and lipid 
kinetics. Numerous other outcomes (metabolites, enzymes, hormones) will be measured from the 
blood samples, and the data will be stored on a secured server in excel files that are identified 
only by subject number. 
 
Riboflavin levels and ethyl glucuronide will be measured in the urine samples to monitor dietary 
compliance. Potential future analyses of urine samples could include indices of inflammation or 
oxidative stress.  
 
The following resource sharing plan will be utilized.  
 
The final dataset will primarily include data collected specifically for the study, but it will also 
contain some demographic information and historical data regarding family history of diabetes, 
obesity, and cardiovascular disease. Upon publication of the primary manuscript, we will 
consider requests to make the study-related data and relevant demographic information (such as 
ethnicity) available to other investigators who submit an outline of the rationale for their request. 
This request will include the following elements: 
 
1. 
Hypotheses and aims  

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metabolic processes associated with the development of cardiovascular disease and type 2 
diabetes    
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2. 
Assurance that the data will be stored only on a secured computer 
3. 
Assurance that access will be limited to those who are named in the proposal 
4. 
Commitment to destroy or return the data after analyses are completed 
5. 
Guarantee that the grant and the primary investigators will be acknowledged in any  
publications arising from analysis of these data.  
 
The final dataset, which will not include subject names, will be further stripped of any other 
unique identifiers and prepared in accordance with all HIPAA regulations prior to release for 
sharing.  No unique subject characteristics will be included in any reports generated by use of the 
data. This plan is specific to data as it is expected that there will not be sufficient blood 
specimens for sharing.   
10) Provisions to Monitor the Data to Ensure the Safety of Subjects 
The Study Physician (Dr. Valentina Medici), the TMRC nurses, Dr. Kimber Stanhope, Dr. Jay 
Shubrook of Touro University, and the Study Coordinators will monitor the performance and 
safety of each procedure. All adverse events (e.g. bruises, diarrhea) which are reported by 
subjects or which occur at TMRC, Ragle HNRC or CCRC are noted in the patient medical chart. 
Upon presentation to Touro Facilities the UC Davis team will provide a paper medical chart with 
important screening and inclusion and exclusion documentation. This will be used during the 
TMRC study procedures and will include description of all TMRC procedures and results. A 
copy of this chart will be housed on the TMRC and a copy will be provided to the UC Davis 
team. All adverse events reported to the Study Coordinator during meal pick-up appointments or 
at other times are noted in the subject file. Any unanticipated adverse event is reported to Dr. 
Stanhope. Dr. Stanhope will discuss this event with Dr. Medici or Dr. Shubrook  if she deems it a 
concern. It will be reported to IRB if Dr. Medici or Dr. Shubrook or Dr. Stanhope deem it a 
concern. All serious adverse events will be reported immediately to the IRBs and reviewed in the 
yearly report to the IRBs.   
 
The safety of the intervention (drinking 25%Ereq as sucrose-sweetened beverages for 4 weeks) 
will be monitored by the Study Coordinators. A fasting blood draw will be scheduled at either 
Ragle HNRC on campus or at a UC Davis Health System affiliated clinic off campus if the 
former is unavailable during the final week of study and a clinical chemistry, lipid and blood 
profile will be performed. These ‘end of study results’ will be compared with screening results 
by the Study Coordinator. The Study Coordinator will have the Study Physician review any 
values that have changed negatively to levels not in the normal range compared to pre-study 
values, and the Study Physician will determine the appropriate action (e.g. no action, 
communication with subject, follow-up blood draw, diet counseling, referral to primary care 
physician). 
11) Withdrawal of Subjects 
For all subjects, diet energy will be adjusted upward or downward as required to maintain stable 
body weight. If energy adjustment consistently fails to prevent weight loss or gain, it will be 

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assumed that the subject is non-compliant with regards to consuming all or only the food 
provided, and the subject will not be further studied. 
 
Subjects will also be withdrawn if there is failure to pick-up study beverages/meals or make 
other arrangements that will allow for uninterrupted consumption of study diet, failure to 
consume study meals or study beverages, or if there is evidence that non-study foods are being 
consumed. 
12) Risks to Subjects 
Pre-existing hyperlipidemia: During recruitment/screening, it may be determined that a 
potential subject qualifies for study, but has TG or LDL-C levels higher than the normal range 
(TG > 150 and < 400 mg/dl; LDL-C >130 and < 160mg/dl). Subjects identified with existing 
hypertriglyceridemia will be informed of the potential risk that consuming sugar-sweetened 
beverages during the study period will further increase their blood levels of TG or LDL-C. They 
will be informed they have the option to not participate in the study and advised to discuss their 
clinical report and the decision to participate in this study with their primary care physician.  
 
Hyperlipidemia: Consuming sucrose-sweetened beverages for 4 weeks at 25% of energy 
requirements is likely to increase risk factors (TG, LDL-cholesterol, apolipoprotein B) for 
cardiovascular disease in hyperlipidemic and normolipidemic subjects. It is expected that any 
changes should be fully reversible when the subjects consume a healthy, low fructose diet. 
Therefore, at conclusion of study, the subjects will receive information about following a low 
saturated fat, low fructose diet. If analysis of fasting lipids at end of study indicates unfavorable 
changes to levels out of the normal range, the subject will be asked to return for a follow-up 
fasting blood draw. If lipids are still elevated, Dr. Medici will recommend further counseling 
with a Registered Dietitian and referral to primary care physician for treatment. 
 
Diarrhea and gastrointestinal distress: Consuming 3 servings of sugar-sweetened beverage or 
orange juice/day can cause diarrhea, stomach aches, bloating, heartburn or acid reflux due to 
fructose malabsorption or the acid content of orange juice. It is also possible that the changes in 
the subject’s normal dietary routine will cause gastrointestinal distress. Imodium is 
recommended to subjects who experience diarrhea. GasX or Pepto Bismol is recommended to 
subjects who experience gastrointestinal distress. 
 
Other diet-related effects: Subjects are restricted to only the food provided. They will not be 
allowed to eat or drink anything outside of the study diet. The subjects are likely to eventually 
find this diet to be restrictive and boring.  
 
The riboflavin biomarker in the sugar beverages can cause urine to darken or appear to have a 
greenish tint.  
 
Blood collection: There is potential for reduced hematocrit due to blood collection procedures. 
Screened participants with low levels of hemoglobin are not enrolled into the study. Screenees 
with below normal hematocrit/hemoglobin are informed that they may have their levels retested 

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following iron supplementation or increased meat consumption. The amount of blood collected 
during the study will be within generally accepted guidelines for drawing blood from research 
subjects with   body weight > 50kg (550 ml/8 weeks: Body weight > 50 kg is an inclusion 
criterion).  
(https://irb.duhs.duke.edu/sites/irb.duhs.duke.edu/files/Blood_Collect_Policy_Statement_12-13-
2012.pdf). As shown in Table 3, we will collect 550 ml from each subject throughout the 
duration of the study. 
 
There is a chance of bruising, or very rarely, an infection at the site where blood will be taken by 
needle stick or catheter. Proper aseptic blood collection techniques will be used to minimize 
these risks. There is a small risk that subject may experience a local skin irritation at the site that 
the catheter was inserted due to sensitivity to the standard dressing that is used to hold the 
catheter in place. A paper tape dressing will be used instead if a reaction occurs. 
 
There is a chance that insertion of the catheter may not be successful on the first attempt and 
multiple attempts may be necessary. There is also the chance that a working catheter can stop 
working later during the 12-hour blood collection period and the nurse will need to insert a new 
catheter. The subject may request that prior to doing so, that the nurse review other options. If 
most of the essential blood samples have been collected, collecting the remaining essential 
samples using a needle and syringe may be an option. The subject’s other option will be to end 
their participation from the study. To prevent clotting of a catheter the nurses will flush the line 
with saline after each blood collection, and some subjects find this uncomfortable.  
 
There is a moderate risk that subjects will become bored and tired during the 12-hour blood 
collection period at the TMRC where activity and mobility is limited. There is a moderate risk 
that subject will become irritable, fatigued, anxious, or nauseated. We recommend subjects bring 
from home movies and books and any other activities that will help them to pass the time more 
pleasantly. We inform the subjects that there are some people whose personalities are not suited 
to the limited mobility and activity required during the long blood collection procedures. We ask 
the subjects to please carefully consider their ability to undergo these all day and night blood 
collection procedures.  
 
Stable isotope infusion: There are no risks associated with the consumption of the stable isotope 
acetate tracer. 
 
Oral glucose tolerance test:  Some subjects may experience mild hypoglycemia during the last 
hour of the OGTT. If a subject complains of dizziness, shakiness, sweating, fast heartbeat or 
inability to concentrate, a small blood sample will be drawn for testing on the glucometer. If the 
blood glucose concentration is above 55 mg/dl, a second sample will be tested 10 minutes later. 
If the blood glucose concentration is still above 55 mg/dl, the OGTT will proceed unless the 
subject states that they wish to be provided a snack & lunch immediately. If the first blood 
glucose concentration is below 55 mg/dl, a second blood sample will be drawn and tested 
immediately. If  blood glucose concentration is still below 55 mg/dl, 25 grams of glucose tablets 
will be provided, followed by snack and lunch. The blood glucose will be rechecked 15 minutes 
after the glucose tablets/snack/lunch are provided to ensure it has returned to normal range.  

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Radiological (DEXA): Subjects will be exposed to a very small amount of x-ray radiation (0.04 
mrad) during DEXA procedure. There are no known risks associated with radiation exposure 
levels this low. There may be some side effects with repeated exposure to radiation, including an 
increased risk of cancer. However, exposure to doses several thousand times greater than that 
obtained from this study would be required before an increased risk of cancer could be detected. 
Thus, the radiation exposure derived from this procedure represents a negligible health risk. 
 
Magnetic resonance imaging (MRI): There is a small risk of that subject will experience anxiety 
or feelings of claustrophobia during the MRI scan. The radiology technician is in constant 
communication with the subject and will end the test if subject experiences undue anxiety or 
claustrophobia. Subjects with pre-existing claustrophobia or metal implants that preclude MRI of 
the abdominal region will not be enrolled. The MRI scanner makes a loud banging noise that some 
subjects may find disturbing. Subjects are provided with ear plugs to protect again the loud banging 
noise. Because the risks to a fetus from MRI are unknown, pregnant participants will not be 
allowed to participate. Urine pregnancy tests will be conducted before each MRI. 
13) Potential Benefits to Subjects 
There are no direct benefits to the subjects for participating in the research.  
14) Multi-Site Research: N/A 
15) Community-Based Participatory Research: N/A 
16) Sharing of Results with Subjects 
The results that the will be shared with the subjects include the anthropometric results (body 
weight, waist and hip measurement) and the body composition results generated by DEXA. 
Upon completion of their participation in the study, subjects will be provided with copies of their 
clinical chemistry, lipid and blood reports, which were generated on the blood samples collected 
during screening and during the final week of study. Other study results will not be shared with 
subjects. In general they will not be available until the end of the 5-year study.  
17) Prior Approvals 
NIH, the funding agency, will be sent a copy of the IRB-approved Protocol and Consent form for 
this study, when it is available.  
 
Dr. Schwarz will obtain approval to conduct this research from the Touro University, California 
Committee on Human Research. 
 
The Human Radiation Use Research Application (Form 35) has approved conduct of the DEXA 
procedures. 
 

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The UC Davis BUA, #R2217B, for the laboratories of Drs. Havel and Stanhope was approved on 
2/25/19 and is good through 2/28/22. 
18) Provisions to Protect the Privacy Interests of Subjects 
Following the 30-minute screening survey, most of the interactions with the potential and 
enrolled subjects that involve providing personal information are handled by the Study 
Coordinators, research and medical team. We anticipate that our experienced study coordinators 
and medical team will be able to make the subjects feel at ease with the research situation with 
honest, open answers about any concerns that the subjects may have. The only information we 
are unable to share with the subjects will be concerning the metabolic effects we anticipate that 
we will observe in subjects consuming sucrose. They will be able to share that the amount of 
sugar in the sugar-sweetened beverages represents an amount that is consumed daily by at least 
13% of the US.    
As required by NIH, this study will be issued the Certificate of Confidentiality. We will 
ensure that all research personnel and collaborators listed understand the responsibilities 
for protecting the confidentiality of the participants’ information and will comply with 
the prohibitions on disclosure. No other collaborator receiving specimens or data will 
receive identifiable information linking to the participants. Sources of information 
accessed by the research team include the data collected by the team, including test and 
scan results, and data generated from subject participation. Subject EMR records will be 
accessed only for the purpose to assigning them to the study for procedure scheduling and 
billing purposes. All files pertaining to personal and medical information will be 
safeguarded using locked filing cases and cabinets, locked offices, password-protected 
computers and encrypted software.  
19) Compensation for Research-Related Injury 
We will provide this information to the subjects concerning research-related injury:  
 
Subjects will be informed that it is important that they promptly tell the Researchers if they 
believe that they have been injured because of taking part in this study. If subjects are injured as 
a result of being in this study, the University of California will provide necessary medical 
treatment. The costs of the treatment may be covered by University or by the Principal 
Investigator’s Research Grant. The University does not normally provide any other form of 
compensation for injury. The subject does not lose any legal rights by signing the consent form. 
20) Economic Burden to Subjects 
There is no cost to the subject for participating in the study, or for the provision of food and 
beverage. There will likely be costs for the subjects for transportation to Ragle HNRC twice 
weekly for meal/beverage pickup/drop-off (approximately 12 times total). Subjects will be 
offered transportation from Davis to UCDMC and Touro University by study staff in university 
vehicles. In the case of staff shortages, subjects will be offered transportation from Davis to 

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UCDMC and Touro University via rideshare service, such as Uber or Lyft, that the study will 
pay for or reimburse. Subjects may prefer to drive themselves due to COVID risk. These subjects 
will be compensated at the UC mileage reimbursement rate for 44 miles to and from UCDMC 
and for 100 miles to and from TMRC. Additionally, subjects will be compensated $970 upon 
completion of the study. This amount may be greater if scheduling adjustments result in a subject 
remaining on the standardized diet for longer than 47 days. Beyond 47 days, subjects will receive 
$10/day for each additional day that they consume the study diet and an additional $15 for any 
extra meal pick-up visits. Subjects who do not complete the study will receive prorated 
compensation as shown in Table 2. Payment forms for each subject will be processed when 
participation in the study has ended.  
 
21) Drugs or Devices: N/A 
If the research involves drugs or device, describe your plans to store, handle, and 
administer those drugs or devices so that they will be used only on subjects and 
be used only by authorized investigators. 
☐ I confirm that all investigational drugs will be received by the Investigational Drug 
Service (IDS).  The IDS will store, handle, and administer those drugs so that they will 
be used only on subjects and be used only by authorized investigators. 
 
☐ I confirm that all investigational devices will be labelled in accordance with FDA 
regulations and stored and dispensed in such a manner that they will be used only on 
subjects and be used only by authorized investigators. 
 
22) ClinicalTrials.gov Registration 
 
FDAAA 801 establishes penalties for Responsible Parties who fail to comply 
withClinicalTrials.gov registration or results submission requirements. Penalties include 
civil monetary penalties and, for federally funded studies, the withholding of grant 
funds. 
 
For additional information on registration and results submissions requirements click 
here.  
If registration is necessary the following are required: 
 
• Registration and, in some instances, submission of results at Clinicaltrials.gov 
• Specific clinicaltrials.gov language in the consent form for this research. The 
language can be found in HRP 502 Template Consent under the heading “What 
happens to the information collected for the research?” 
 

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Complete each section below. If you finish a section indicating the research must be 
registered on Clinicaltrials.gov is required you do not have to complete the remaining 
sections.  
 
Section 1:  NIH Funded Studies 
If yes to BOTH, the study must be registered on Clinicaltrials.gov. 
Yes  
 
This study is funded by the NIH. (If this study is not funded by NIH, go to Section 2.) 
 
One or more human subjects will be prospectively assigned to one or more interventions 
(which may include placebo or other control) to evaluate the effects of those 
interventions on health-related biomedical or behavioral outcomes. 
 
Section 2:  Studies subject to FDA jurisdiction 
If yes to ANY the study must be registered on Clinicaltrials.gov. 
Yes 
 
 
This is a prospective clinical study of health outcomes in human subjects that compares 
an intervention with an FDA-regulated device against a control. This is not a small 
clinical trial to determine the feasibility of a device, or a clinical trial to test prototype 
devices where the primary outcome measure relates to feasibility and not to health 
outcomes. 
 
This is a pediatric postmarket surveillance of a device as required under section 522 of 
the Federal Food, Drug, and Cosmetic Act. 
 
This is a controlled clinical investigation, other than a phase I clinical investigation, of 
a drug subject to section 505 of the Federal Food, Drug, and Cosmetic Act or to section 
351 of the Public Health Service Act. 
To view a flowchart describing applicable clinical trials subject to FDA jurisdiction click here. 
 
Section 3: Publishing the results 
If yes to BOTH the study must be registered on Clinicaltrials.gov. 
Yes  
 
This study prospectively assigns people or a group of people to an intervention, with or 
without concurrent comparison or control groups, to study the cause-and-effect 
relationship between a health-related intervention and a health outcome. 
 
The PI has access to and control over all the data from the clinical trial and has the right 
to publish the results of the trial and plans to publish the results in a journal that follows 
the ICMJE recommendations.  
This requirement includes studies of behavioral interventions. 
 
Section 4: Registration on Clinicaltrials.gov is not required 
Yes  

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I have read sections 1-3 above and registration on clinicaltrials.gov is not required for 
this research.  
23) Criteria for 10 Year Approval  
If yes to all items below this research may qualify for a 10-year approval period. 
 
Yes  
 
This research involves no more than minimal risk. 
 
This research does not receive any federal or state government funding or funding 
from a private funder who requires annual review per contract. 
 
This research is not subject to FDA jurisdiction.  
 
This research does not include prisoners as participants. 
 
This research is not subject to SCRO oversight. 
 
This research is not subject to oversight by the Research Advisory Panel of 
California (RAP of C). 
 
This research does not involve identifiable information held by the State of 
California Department or Agency 
 
No personnel involved in the design, conduct, or reporting of this research have a 
new unreported related financial interest (RFI) in this study.