Sex-related Differences in Arterial Stiffness in Type 2 Diabetics: Role of Uric Acid
Phase: PHASE2
Conditions: Diabetes Mellitus, Type 2, Hyperuricemia
Sponsor: University of Missouri-Columbia

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BIOMEDICAL RESEARCH PROTOCOL UNIVERSITY OF MISSOURI 
 
Project Title: Sex-related differences in arterial stiffness in type 2 diabetics: role of uric acid 
IRB Number: 2012106 
Version Number: 18 
Version Date: March 25, 2021 
Principal Investigator: Camila Manrique Acevedo, MD 
Funding Source: NIDDK 
 
Clinical Trial Phase: Delete if NA. 
Study Drug/Study Device: Allopurinol 
 
Collaborating Investigators: 
Celso Velazquez, MD 
Jaume Padilla, PhD 
Elizabeth Parks, PhD 
 
Study purpose: The purpose of the present project is to determine the effects of a low fructose diet 
(isocaloric or hypocaloric) or allopurinol on arterial stiffness in patients with type 2 diabetes (T2D). 
 
This study is classified as a clinical trial and is registered on Clinicaltrials.gov #NCT03648996 
 
I. 
RESEARCH OBJECTIVES/BACKGROUND 
 Background and significance 
Cardiovascular disease (CVD) is one of the main complications of 
type 2 diabetes mellitus (T2D) 2, 3. One third of the US population over 
age 65 has diabetes 4, increasing the public health burden of 
diabetes-related CVD. Increased arterial stiffness is a potential 
mechanism behind the higher incidence of CVD in T2D 5. Aortic pulse 
wave velocity (PWV), a measure of arterial stiffness, is a marker and 
predictor of CVD risk and mortality 6. A 1 m/s increase in aortic PWV 
corresponds to a 15% increased risk for total CV events, CV mortality, 
and all-cause mortality, as adjusted for age, sex, and other risk 
factors 7. Stiffening of the vasculature is a hallmark of aging in men 
and women 8, 9 but its progression is accelerated by hypertension, 
obesity and T2D 5, 10-12. Insulin-resistant and T2D-post-menopausal 
women are at higher risk for stiffening than men 13-16 but the 
mechanisms behind this 
differential augmentation 
remain unknown. 
Elevated serum uric acid 
(SUA) levels and 
xanthine oxidase (XO) 
activation are linked to 
increased arterial 
stiffness in T2D women.  
3.0
3.5
4.0
CD-F
WD-F CD-M WD-M
PWV (mm/ms)
P <0.05
*
Figure 1. Female C57Bl6/J mice fed a 
Western diet (WD) are more likely to 
develop aortic stiffness. CD=control diet. 
*p<0.05 vs CD, n=7. 
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
WD
WD+Allo
MDA (mM)
Endothelial cell 3-NT 
(aortic  staining)
WD
WD + Allo
C
20
60
50
40
30
10
0
*
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
WDC
WD-Allo
Uric acid (mg/dL)
A
*
Stiffness  (kPA)
20
16
12
18
14
10
8
6
4
2
0
*
B
WD
WD + Allo
*
D
Figure 2. XO inhibition with allopurinol (allo; 0.125 mg/ml) reduced (A) uric acid, (B) vascular 
stiffness, (C) endothelial cell oxidative stress (3-NT) and (D) serum MDA in WD-fed female C57Bl6/J 
mice. n=4-8; *p<0.05 1  

Page 2 of 27 
SUA is a product of XO activation. Analysis of the NHANES database found an elevated SUA 
prevalence of 21.2% in men and 21.6% in women 17. Elevated SUA is associated with increased 
morbidity in patients with hypertension 18, 19, CVD and stroke 20, chronic kidney disease (CKD) and 
insulin resistance 21. In the Framingham Heart Study, SUA was shown to be independently 
associated with aortic stiffness 22, particularly in women, as also reported by others 23-30. In pre-
hypertensive subjects, elevated SUA was related to a higher risk of developing hypertension in 
women than in men 31. The presence of diabetes significantly increases the risk of arterial stiffness in 
women 32. Elevated uric acid (UA) and XO activation can result in chronic inflammation, endothelial 
dysfunction and increased oxidative stress, key events in the pathogenesis of stiffness 33, 34. Diet is 
the main driver of SUA in Western societies. Fructose supplementation elevates SUA in a dose-
dependent manner 35. Reducing SUA with allopurinol, an XO inhibitor, reduces arterial stiffness 36, 37 
but whether reducing SUA, by either drug or diet, improves arterial stiffness in T2D subjects is 
unknown. Importantly, the differential effects of treatment on T2D men and women have not been 
tested. The proposed study will bridge this knowledge gap. 
Distinct contributions of XO activity and SUA. High SUA levels can damage proteins and other 
macromolecules through mechanisms related to oxidative stress and inflammation as XO 
simultaneously generates both UA and reactive oxygen species 38. In the vasculature, elevated UA 
has been shown to increase expression of inflammatory molecules such as C-reactive protein (CRP) 
39, 40, as well as production of reactive oxygen species 41, 42 with parallel decreases in nitric oxide (NO) 
production 41, 43. Perez-Pozo et al. suggested that the observed benefits of allopurinol treatment on 
blood pressure may be the result of either reductions in the deleterious effects of SUA, or from 
reductions in systemic inflammation following XO inhibition 44. Consequently, in this proposal we will 
assess whether XO inhibition, and its direct effects on endothelial function, oxidative stress and 
inflammation, is superior to dietary UA reduction in improving arterial stiffness. 
Dietary fructose and UA. Stanhope et al. found a linear relationship between fructose 
supplementation and increasing SUA 35. A recent meta-analysis of the effects of fructose on SUA 45 
revealed that most studies were confounded by study designs comparing fructose with sucrose 
(fructose-containing disaccharide) and allowed for inadvertent weight loss in both groups. Such 
weight loss would drive down SUA in both arms of the study 46 negating the ability to independently 
test the impact of fructose on hyperuricemia. With regard to hyper-caloric feeding of fructose, the data 
are very clear: fructose supplementation was shown to increase SUA concentrations in over 10 
studies 45. None of the long-term studies assessed arterial stiffness as an outcome, nor were there a 
priori comparisons between T2D men and post-menopausal T2D women in the designs. The 
proposed study design will allow for an accurate assessment of the effect of dietary fructose reduction 
on SUA levels and arterial stiffness, without the 
confounding effect of weight loss. 
Endothelial dysfunction and arterial stiffness. 
Endothelial dysfunction plays a central role in the 
development and progression of CVD 47, and has 
been linked to the genesis of arterial stiffening 48-51. 
Arterial stiffness has also been associated with 
microvascular dysfunction 52 and target organ 
damage 53. A recent analysis of the Framingham 
cohort indicates that up to 13% of the relationship 
between measures of aortic stiffness and CVD 
events is mediated by impaired microvascular 
function 54. Unfortunately, studies of the effect of XO 
inhibition on endothelial function have produced 
conflicting results 55-58. A recent clinical trial in a 
B
0
2
4
6
8
10
12
Serum Uric Acid
(mg/dL)
BL Wt loss
BL Wt loss
p=0.130
p=0.464
Men
Women
p=0.05
Energy intake from sugars
% total calories
0%
5%
10%
15%
20%
25%
p=0.006
p=0.02
A
p=0.02
BL
Wt loss BL Wt loss
Figure 3. (A) Dietary sugar intake, higher in women at baseline, 
was significantly reduced after a sugar and weight reduction 
program. (B) Preliminary data in a small number of subjects 
indicate that although both sexes lost an equal amount of body 
weight (10%), SUA trended lower only in females (n=7 men; 9 
women). 

Page 3 of 27 
population of mostly male subjects with stage III 
CKD did not show a beneficial impact from 
allopurinol on endothelial function 59. Nevertheless, 
a recent meta-analysis, by Cicero et al., of ten 
published trials, (670 subjects; allopurinol doses 
from 300-900 mg/day and 2 weeks to 9 months 
duration) reported improvements in flow-mediated 
dilation (FMD) with allopurinol therapy 57. In the 
proposed study we will evaluate the impact of two 
SUA-reducing interventions on endothelial function 
of conduit arteries, assessed via FMD, and on 
microvascular function, assessed via insulin-
stimulated blood flow using Doppler and contrast-
enhanced ultrasound. The ultimate goal is to 
correlate these results with arterial stiffening.  
Preliminary data. In our previous studies in a rodent model of 
insulin resistance and obesity, a high fructose/high fat diet 
(Western diet (WD)) increased aortic stiffness only in females 
(Figure 1), supporting a sex-specific predisposition for the 
development of arterial stiffness in females. When WD-fed female 
mice were treated with allopurinol (0.125 mg/mL) (Figure 2), SUA 
(Figure 2A), aortic stiffness assessed by atomic force microscopy 
(AFM) (Figure 2B), aortic oxidative stress assessed by 3-
nitrotyrosine staining (3-NT) (Figure 2C), and systemic oxidative 
stress assessed by plasma malondialdehyde (MDA) (Figure 2D), 
were all markedly reduced 1. The proposed study will translate 
these findings from mice to humans, using similar methodologies, 
to determine the separate contributions of XO activity and SUA 
levels to arterial stiffness in men and women with T2D. Data from 
a weight loss study support the feasibility of our approach and the 
expertise of our research team. In men, ad libitum diets contained 9.3 ± 3.6% of energy as added 
sugars, significantly lower than the baseline intake in women (16.6 ± 8.1%, P<0.02). After a 6-month 
program designed to reduce dietary sugar intake and body weight, weight loss was similar in men (-
10 ± 3%) and women (-10 ± 2%), P=0.99, and systolic and diastolic pressure was lower in both (data 
not shown), but women reduced their sugar intake more than men (Figure 3A), highlighting the 
relevance of dietary sugar intake, particularly in women. Further, even in this relatively small sample 
size, SUA trended lower with weight loss in women but not in men (Figure 3B) exhibiting a significant 
time-by- sex interaction (P=0.006 by ANOVA). Overall, the data demonstrate that added sugar intake 
in rodents and humans is a modifiable factor that can be used to impact arterial stiffness. Sexual 
dimorphism in isocaloric substitution of carbohydrates has yet to be tested in a controlled manner, 
and the paucity of data in this field leaves a critical gap in our understanding of the role of diet in uric 
acid-associated arterial stiffness in T2D women at risk for CVD.  
In another recent fructose restriction/weight loss intervention in middle-aged adults, obese women 
showed a significant improvement in PWV, while men did not (Figure 4A). Relevant to this proposal, 
we found that PWV correlated with SUA in women after 4 weeks of weight loss (Figure 4B). TNFα 
and UA were the only variables that predicted PWV = 3.604 + (0.270*UA) + (0.132*TNFα), r=0.712, 
p=0.0025 following the weight loss in women, suggesting that SUA is a modifiable risk factor in the 
development of arterial stiffening in women. In another preliminary study, exposure to high glucose 
and high UA increased production of inflammatory markers in cultured human aortic endothelial cells 
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
1
PWV (m/sec)
Weight 
loss
BL
Weight 
loss
p=0.144
p=0.028
BL
Men
Women
y = 1.8172x - 4.8026
R² = 0.4979
4.00
5.00
6.00
7.00
8.00
9.00
10.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Uric acid mg/dL
PWV m/s
A
B
Figure 4. In women, (A) PWV improved significantly, and (B) was 
significantly correlated to SUA after 4 weeks on a fructose and 
weight reduction program (n=10 men; 10 women). 
0
0.5
1
1.5
2
2.5
3
VCAM-1
(fold expression)
0
0.5
1
1.5
2
2.5
3
3.5
MCP-1
(fold expression)
High glucose
-
+
-
+
*
*
High uric acid 
-
+
-
+
Figure 5. Increased inflammatory marker 
expression upon exposure of human aortic 
endothelial cells to high glucose (30mM) and 
high uric acid (0.1 mg/ml) cells. n=3; *p<0.05 
vs. control. 

Page 4 of 27 
(Figure 5). Data from our basic and human studies complement growing epidemiological evidence 
that hyperuricemia in women is related to increased arterial stiffness, putting women at a high risk for 
CVD. We propose that the sexual dimorphism in arterial stiffness associated with T2D is mediated, in 
part, by sex-dependent effects of UA and XO activation. 
 Summary of Study Objectives 
We will accomplish the following specific aims: 
Aim 1:  To determine if sex-related differences exist in the relationship between SUA levels and 
arterial stiffness (via cfPWV) in age-matched T2D men and women, and to what degree these 
differences are related to augmented oxidative stress, inflammation and endothelial dysfunction. 
Hypothesis: The relationship between arterial stiffness and elevations in SUA/augmented oxidative 
stress/endothelial dysfunction/increased inflammatory marker activity seen in T2D women will be 
weaker or absent in men. 
Aim 2:  To assess whether XO inhibition is superior to dietary-induced SUA reduction in ameliorating 
arterial stiffness in T2D subjects. Hypothesis: Although we expect XO inhibition to have the greatest 
impact, we posit that SUA reduction by either strategy will lead to more improvement in endothelial 
function and stiffness. 
As shown in Figure 6, this project has three parts and each subject will participate in only one part.  
These separate treatment arms are combined into one IRB protocol because they will test an 
integrated hypothesis regarding uric acid and arterial stiffness in a similar population with identical 
procedures. 
Part 1:  Diet treatment 
The overall goal of part 1 is to reduce fructose in the diets of T2D women and men at risk for future 
cardiovascular disease.  Dietary fructose will be replaced with starch to keep the research subjects' 
body weights stable.  Subjects will be treated for 6 months and measurements of arterial stiffness and 
endothelial function will be made before, during, and after treatment. 
 
Part 2:  Drug/placebo treatment 
 
The overall goal of part 2 is to lower serum uric acid concentration in T2D women and men.  The 
treatment will be allopurinol administration (or placebo), ramping up the dose as shown below to 
achieve a target dose of 300 mg/day. Measurements of arterial stiffness and endothelial function will 
be made before, during, and after. 
Figure 6. Duration and number of subjects enrolled in each of the four interventions. Interim visits occur at month 3 (12 weeks) for all 
arms of the study, and final visits occur at month 6 (24 weeks) for all arms of the study. 

Page 5 of 27 
Subjects that do not qualify based on blood biochemistry to participate in the above interventions will 
be offered to participate in a hypocaloric low fructose diet intervention. 
Part 3. Hypocaloric Low Fructose Diet Treatment 
The goal is to restrict fructose and calories in the diets of T2D women and men at risk for future 
cardiovascular disease to mediate weight loss. Dietary fructose will be reduced, but baseline fat and 
protein quantities will be held constant. A caloric restriction of 500 kilocalories will be prescribed to 
subjects. Subjects will be treated for 6 months and measurements of arterial stiffness and endothelial 
function will be made before, during, and after treatment.  
 
II. 
DRUGS 
A. Drug treatment, dose, route and regimen:  
Dr. Velázquez will oversee the drug 
and placebo treatments in conjunction 
with principal investigator (Dr. 
Manrique). They will use standardized 
clinical protocols to ramp the dosage 
of allopurinol to a target dose of 300 
mg. 60, 61. The medication will be given 
orally once a day. 
Dose escalation scheme:  The dose 
escalation scheme for allopurinol, 
Table 1, is supported by past work 60, 
62.  The doses of drug will be 
increased according to the scheme 
presented in Table 1 until a target 
dose of 300 mg is reached, while closely monitoring for any adverse effects. Safety visits will be 
scheduled every 2 weeks during months 1-2 (at weeks 2, 4,6, and 8) and every 4 weeks during 
months 3-6 (at weeks 12, 16, 20) to monitor blood chemistries. Subjects may have safety labs 
repeated at the discretion of the safety officer.  
B. Rationale for choosing the drug and dose: 
Allopurinol was chosen as it is used clinically for management of gout and hyperuricemia. As 
mentioned before the dose and the escalation scheme for allopurinol, below, is supported by past 
work and available literature. 60, 62  
C. Risks of not treating hyperuricemia in the placebo group: 
A 2013 Cochrane review emphasized the lack of sufficient evidence to support the use of urate-
lowering therapy to prevent future gout in hyperuricemic, asymptomatic patients 63.  A need for further 
clinical trials to test other benefits of lowering SUA was called for 64 which strongly supports the 
rationale for the current study.   
Given the limited duration of subject participation (6 months), the lack of known risk to the subject for 
not being treated is balanced by the significant knowledge gained from this study.  At the study's end, 
subjects in the placebo arm will be offered one, no-cost counseling sessions with the dietitian 
nutritionist, who will have analyzed their ad libitum food intake and body composition.  The dietitian 
nutritionist will make recommendations to these subjects for modifying lifestyle factors to reduce SUA 
and improve overall health. 
 
Study window 
Visit 
number 
Daily 
dose 
How many 
days 
Day -30 
v0, screening  
 
Day 0 
v1, day 1 
 
 
Day 1-14 
v2, day 14 
100 mg 
daily 
X 14 days 
Day 15-28 
v3, day 28 
200 mg 
daily 
X 14 days 
Day 29-168 
v4, day 42 
300 mg 
daily 
X 133-147 
days 
Table 1

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D. Risks and safety information 
Side effects of allopurinol include hepatitis, rash and pruritus. Our team is aware of the existing 
recommendations by the American College of Rheumatology in regard to the use of anti-inflammatory 
agents when starting allopurinol in patients with acute gout 60, 65. As the present study will not include 
patients with diagnosis of gout and given that available studies done in subjects with asymptomatic 
hyperuricemia demonstrate that monotherapy with allopurinol is safe 66, 67, we plan to treat subjects 
our hyperuricemic subjects only with allopurinol. Dr. Velázquez and Dr. Manrique will closely monitor 
these subjects and might consider using a low-dose of naproxen in selected cases.  
 
III. 
RECRUITMENT PROCESS 
A. Recruitment Pool 
The study population includes male and female subjects with 
BMI (25.1-50 kg/m2) and age 40-75. We used the MU i2b2 
database to determine the recruiting pool for the project this 
September. From a total of 519,153 patients older than 40, 
after excluding for lack of T2D diagnoses (497,325) and 
adding the population estimates (overweight/obesity of 65% in 
Missouri), a potential recruitment pool of 14,188 patients 
remains.  Thus, there are sufficient patients (men and 
women) in our database to serve as study subjects  The 
number of subjects entered in each study allows for a 10% 
dropout rate. Our past dietary studies have run from 6 weeks 
to 6 months and dropout has been 1-10%. In our experience, 
high public interest in reducing dietary fructose intake 
suggests that recruiting subjects for this arm will not be 
difficult. 
B. Subjects will be recruited by a number of means: 
1. A chart review of clinic patients from the Divisions of 
Rheumatology, Endocrinology and the Department of 
Medicine who are between the ages of 40 and 75, and 
have been diagnosed with type 2 diabetes will be completed in Powerchart by study 
coordinator/staff based on the study inclusion/exclusion criteria. A letter, chart message, or phone 
call will be utilized to notifying them of their eligibility to participate in a research study. Study 
coordinator/staff will work in conjunction with healthcare providers that have a direct patient care 
relationship to recruit subjects from clinics. 
2. Potential subjects may also be identified via use of i2b2 screening. Study coordinator/staff will work 
in conjunction with healthcare providers that have a direct patient care relationship to recruit these 
subjects. Subjects may be informed of their eligibility via chart messaging, letter, or phone call. 
3. Subjects may also respond to the recruitment flyers or advertisements posted in MU Hospital 
clinics, MU Hospital television screens, MU Info, MU campus, newspapers, Facebook, or any place 
generally accessible to the public (i.e grocery stores, community centers, etc). For all recruitment, 
subjects will be given an email address and/or a phone number to contact. Subjects may also be 
given a link to complete a screening questionnaire via Qualtrics.  
Figure 7. Pathway for subjects to enter into research 
arms 

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IV. 
CONSENT PROCESS 
Four consent forms will be used:  
1)  A screening consent form (general consent) describing the isocaloric low fructose diet treatment, 
the allopurinol treatment, the hypocaloric low fructose diet treatment, and the control arm; 
2)  A treatment consent form that describes the isocaloric low fructose diet;   
3)  A treatment consent form that describes the allopurinol/placebo study; 
4) A treatment consent form that describes the hypocaloric low fructose diet   
A. Preliminary Screening 
Subjects who inquire about participation will answer screening questions, with their waiver of written 
consent.  These questions are for preliminary screening only and are not used as study data since 
the data represent self-report.  The questions include queries about health history/habits, age, sex, 
height, weight, tobacco use, medications/ supplements, over the counter medications use, illness, 
dietary habits, and chronic conditions.  The study design is described in general terms to subjects, 
with mention of factors most likely to impact subject interest in participating.  These factors can 
include limited food options and the requirement to remain weigh stable, drug treatment or no drug 
treatment.  If the subject is interested in being formally screened for participation, an appointment will 
be scheduled for a CRC visit for consenting, measurement of fasting blood chemistries, DEXA, 
vascular measurements, and an Oral Glucose Tolerance Test (OGTT). 
B. Formal clinical trial consenting 
Subjects who are eligible following the CRC screening visit will be formally consented for the specific 
arm of the study they are participating in (the isocaloric fructose restriction arm, the 
allopurinol/placebo treatment arm, or the hypocaloric fructose restriction arm of this project) before 
their baseline visit.  Formal consenting up to 1 hour and during that process, 1) the subject will be 
asked to describe, in their own words, what the baseline visit will be like, and 2) the subject (in either 
of the fructose restriction arms) is given a three-day food record and instructed on how to fill it out.  
They will be provided with the study details. Review of the consent form will occur with study 
personnel in a quiet, unhurried setting. Comprehension will be assessed by asking the subject to 
explain the study in their own words. Participants will have adequate opportunity to review the 
informed consent and to ask any questions they may have about the research protocol, 
compensation, risks, and benefits of taking part in the study. 
 
V. 
INCLUSION AND EXCLUSION CRITERIA 
Inclusion criteria: 
1) Men and women with BMI 25.1-50 kg/m2, who are 40-75 years of age at randomization.  
2) T2D diagnosed > 3 months ago. Patients with T2D will be classified based on physician diagnosis. 
3) No vulnerable populations (e.g., prisoners, pregnant, children) will be enrolled. 
Exclusion criteria for part 1 and part 2 of the study: 
1) Serum uric acid < 5.5 mg/dL 
2) Habitual diet containing low amount of sugars < 5% of total energy intake 
3) Recent CVD event (stroke, heart failure hospitalization, revascularization or acute coronary event 
in the last 12 months), abnormal thyroid tests or chronic liver disease, stage IV renal disease 
(GFR <30) and hyperparathyroidism. 
4) Use of azathioprine 
 
5) Active cancer 
6) Autoimmune diseases 

Page 8 of 27 
7) Excessive alcohol consumption (>14 drinks/week for men, >7 drinks/week for women) 
8) Current tobacco use 
9) Bodyweight change ≥10% within the last 6 months 
10) History of gout or uncontrolled hypertension 
11) A1C >10.0 
12) Pregnancy or lactation in women (or women not using contraceptives) 
Medications for glucose control (insulin, metformin, gliptins, GLP-1 agonists, sulfonylureas, sodium 
glucose co-transporter inhibitors), antihypertensives, hormone replacement therapy, antidepressants, 
anticoagulants, or statins, will be considered during recruitment, analyses (e.g., using covariate 
analysis) and interpretation. 
Exclusion criteria for hypocaloric low-fructose diet intervention (part 3): 
1) Habitual diet containing low amount of sugars < 5% of total energy intake 
2) Recent CVD event (stroke, heart failure hospitalization, revascularization or acute coronary event 
in the last 12 months), abnormal thyroid tests or chronic liver disease, stage IV renal disease 
(GFR <30) and hyperparathyroidism. 
3) Use of azathioprine 
 
4) Active cancer 
5) Autoimmune diseases 
6) Excessive alcohol consumption (>14 drinks/week for men, >7 drinks/week for women) 
7) Current tobacco use 
8) Bodyweight change ≥10% within the last 6 months 
9) History of gout or uncontrolled hypertension 
10) Pregnancy or lactation in women (or women not using contraceptives) 
Medications for glucose control (insulin, metformin, gliptins, GLP-1 agonists, sulfonylureas, sodium 
glucose co-transporter inhibitors), antihypertensives, hormone replacement therapy, antidepressants, 
anticoagulants, or statins, will be considered during recruitment, analyses (e.g., using covariate 
analysis) and interpretation. 
 
VI. 
NUMBER OF SUBJECTS 
Sample size calculations are based primarily on 
satisfying the requirements of Aim 2, i.e. to show 
that XO inhibition and fructose restriction reduce 
PWV, and that these interventions are more 
effective in women than in men.  Calculations 
are based on the paired t-test for pre- to post-
treatment change, and independent t-tests for 
comparisons between treatment arms.  In both 
cases, we assume an alpha of .05 and a two-
sided alternative.  The sample size calculations shown in Table 2 are derived from the literature as 
follows:  De Angelis et al have shown that in men, T2D does not increase PWV in age- and BMI-
matched men without T2D 32.  By contrast, in women, T2D significantly increases PWV compared to 
women without T2D and importantly, compared to men with T2D.  Any reduction in PWV represents a 
significant reduction in cardiovascular risk 7.  However, with 6 months of allopurinol treatment, we 
have chosen a sample size to be able to detect a reduction in PWV of 0.6 m/s, which Ng et al showed 
to be clinically meaningful 68.  Nine subjects per group (9 men and 9 women) completing the 
allopurinol arm will allow for 90% power to detect a change of 0.6 m/s and seven subjects/group will 
allow for 80% power to detect this change Table 2. With regard to fructose restriction, our preliminary 
Treatments 
Power 
Alpha 
Change 
PWV 
SE of ∆ 
in PWV 
# of 
subjects 
Allopurinol 
90% 
0.05 
0.6 m/s 
0.4 m/s 
9 
80% 
0.05 
0.6 m/s 
0.4 m/s 
7 
Low-fructose 
90% 
0.05 
0.5 m/s 
0.4 m/s 
13 
80% 
0.05 
0.5 m/s 
0.4 m/s 
10 
Table 2

Page 9 of 27 
data demonstrate a significant improvement in PWV only in the females.  We anticipate that the 
reduction in PWV with diet may be smaller than that with allopurinol.  We would need 13 
subjects/group to detect a 0.5 reduction in PWV with 90% power; 10/group would give 80% power.  
Further, as we do not expect changes in the PWV during 6 month of follow up in the placebo group 
and to accommodate variability in response and potential dropout, we will include 40 subjects in the 
low-fructose intervention, 40 subjects in the allopurinol cohort and 20 in the placebo group (half men 
and half women for all the cohorts). Further and based in our previous weight loss/dietary 
intervention69, we have chosen a sample size to be able to detect a reduction in PWV of 0.6 m/s of 20 
diabetic males and 20 diabetic females. 
 
VII. 
STUDY PROCEDURES/ DESIGN/TREATMENT PLAN 
Following screening, eligible male and female subjects will be assigned to one of the three treatment 
arms. In the medication arm of the study, they will be randomized to either the allopurinol group 
(n=40; 20 men and 20 women), or the placebo (n=20; 10 men and 10 women) for 6 months, with a 
final drug/placebo allocation ratio of 2:1 using a permuted block design with random variation of block 
size.  In the isocaloric low-fructose arm of the study, 20 women and 20 men will be recruited to enter 
a dietary treatment of fructose restriction for 6 months. In the hypocaloric low-fructose arm of the 
study, 20 women and 20 men will be recruited to enter a dietary treatment of calorie and fructose 
restriction for 6 months.  
 Screening/Initial Consenting Visit 
The first visit to the CRC will take approximately 6 hours and include a written consent to be 
screened.  The subject is instructed to hold their glucose lowering AM medications (subject will 
resume regular medications after blood has been drawn), and not to eat or drink anything besides 
water for at least 10 hours before this visit.  The subject is also told not to exercise the morning of this 
visit.  After signing the screening consent form, medical information is obtained by the study team, 
including: DOB, gender, ethnic/racial category, height, body weight (history of body weight gain or 
loss), waist circumference, vitals, and a medical history questionnaire. A fasting blood draw is taken 
to test for biochemistries.  Subjects will also undergo a DEXA scan and vascular measurements 
including carotid intima media thickness (cIMT), carotid femoral pulse wave velocity (cfPWV), 
Glycocheck, Near-Infrared Spectroscopy (NIRS), Brachial artery FMD, popliteal artery FMD testing, 
and OGTT with femoral artery blood flow (Figure 8). 
An introduction to the clinical trial will be done which includes abbreviated descriptions of the various 
treatment arms and the placebo arm (no treatment).  As soon as the blood biochemistry results are 
available, the subject is contacted, provided with a copy of the results. If eligible for the 
allopurinol/placebo arm or (isocaloric) low fructose diet intervention arm, study staff discuss with 
him/her whether they would like to be consented for enrollment in one of the present studies.  
Figure 8. Order of events at the Screening Visit occurring at the clinical research center (CRC). 

Page 10 of 27 
Subjects that are ineligible for both the allopurinol/placebo and (isocaloric) low fructose arms or do 
not want to pursue the 6-month intervention will be offered the opportunity to participate in the 
hypocaloric low fructose arm. Participants that have previously failed initial screening (were ineligible 
to participate in arm 1 or 2 of the study) will be offered the opportunity to re-consent to the study and 
have vascular measurements taken at an additional paid screening visit and to enroll in the 5-month 
long hypocaloric low fructose arm (arm 3). 
 Baseline/Formal Consenting Visit 
The subject will arrive to the CRC in the morning to perform further metabolic and vascular tests 
(Figure 9).  The subject is instructed to hold their glucose lowering AM medications (subject will 
resume regular medications after the visit has been completed), and not to eat or drink anything 
besides water for at least 10 hours before this visit.   The subject is also told not to exercise the 
morning of this visit.  The visit will take up to 8 hours and the subjects must be fasted.  Upon arrival, 
the participant meets with one of the study’s physicians to review and sign the study consent forms. 
The subject then voids and the study staff takes anthropometric measurements and repeats a DEXA 
scan at the discretion of the investigator. Subjects will then be placed supine in a quiet, climate-
controlled room (22–23 ° C) to rest before vascular measurements are repeated by study staff, 
including: cIMT, PWV, Glycocheck, FMD and NIRS. Vital signs are measured and an IV line is placed 
in each arm by the nurse in order to draw blood and infuse during a hyperinsulinemic-euglycemic 
clamp. The CRC vascular procedures will be repeated again at interim and final visits at 3 and 6 
months.  
 Interim visit 
An interim visit will include fasting blood draws. Study staff will assess blood pressure, heart rate, 
weight, and waist circumference as well as body composition (DEXA), cIMT, FMD, NIRS, 
Glycocheck, and cfPWV. This visit will take up to 4 hours and must occur in the fasting state. 
 Final visit 
A final appointment will repeat all baseline measurements including DEXA, cIMT, Glycocheck, 
cfPWV, FMD, NIRS and insulin clamp with CEU. The study staff will assess the body weight, waist 
circumference, heart rate, and blood pressure.  Blood will be collected at the beginning and 
throughout the study procedures at this visit. This visit will take up to 8 hours and must occur in the 
fasting state. Following the final visit, the treatment portions of each arm are over.  Subjects in Part 2 
will be offered a meeting with the dietitian nutritionist to discuss diet and exercise. Subjects may be 
asked to complete an exit survey upon completion of the study. Exit surveys will be administered via 
Qualtrics or in person depending upon subject preference. 
Figure 9. Order of events at the Baseline Visit occurring at the clinical research center (CRC). 

Page 11 of 27 
 Special Consideration in case of unforeseeable hardships such as public health 
emergencies or weather-related events 
In the event that subjects are not able to complete study visits for the reasons described above, the 
following remedial steps will be taken: 
1. Safety visits #4, 6 and 7 at weeks 8, 16, and 20 (respectively) can be completed via a phone 
interview during which the subject will answer the safety questionnaire (no changes in 
compensation). 
2. In the case that the subject cannot complete safety visit #3 at week 6, every effort will be made to 
complete this visit within 7 days of the originally scheduled date. In the case that the visit cannot 
be completed, the subject will be instructed to discontinue the study medication and his/her 
participation in the study will be completed. Dr. Velazquez, who oversees the allopurinol/placebo 
treatment with the Principle Investigator will be informed. 
3. In the case that safety visit #4 at week 8 cannot be completed, and providing the subject has 
completed at least 2 in-person visits since the medication was started, study medication will be 
mailed by investigational pharmacy for the subject to continue treatment. Dr. Velazquez will be 
informed.  
4. In the case that the subject cannot complete dietary counseling in person, counseling visits may 
be completed via phone or comparable, secure tele-counseling set-up. 
5. In the case that subjects are unable to complete their interim visits at the originally scheduled 
date, their visits may be scheduled +/-14 days of the originally scheduled date. If the interim visit 
cannot be completed onsite, safety formulary will be completed via phone. 
6. In the case that subjects are unable to complete their final visits at the originally scheduled date, 
their visits may be scheduled +/-14 days of the originally scheduled date provided that the subject 
maintains the assigned intervention for the duration of the study.  
7. In the case that the Clinical Research Center is unavailable or at the discretion of the primary 
investigator, safety visits may be completed at MUPAW.  
8. In the event of the clinical research center not being available or if it is considered by the study 
physician that performing studies there can increase risk of exposure to infectious agents or 
related hazards, the following alternative sites will be made available for screening, safety and 
study visits:  
• 
Medical school room NW404  
• 
University hospital clinic rooms 
These locations are not carpeted and we do not anticipate an increased risk for the subjects given 
that medical supervision and nursing staff assistance will be unchanged.  
 Description of Study Procedures 
Metabolic testing: As described in the background section, hyperuricemia is characterized by 
increased inflammation and insulin resistance.  We will monitor changes in inflammation and 
oxidative stress markers through fasting blood sample measurement of serum concentrations of XO 
activity, TNFα, IL6, CRP, PAI-1, MCP-1, E-selectin, MDA, 3-NT, TBARS, oxidized LDL and 8-iso 
PGF2α. In addition, this blood sample will also be used for measurement of fructose, blood lipids, 
glucose, insulin, creatinine, estradiol, estrone, testosterone and sex-hormone binding globulin, uric 
acid, blood lipids, and estrogen, the latter used as a covariate. 70  
DEXA scan: to assess body composition with dual-energy X-ray absorptiometry.  

Page 12 of 27 
Arterial Stiffness and Blood Pressure: The SphygmoCor XCEL device will be used to assess blood 
pressure and aortic pulse wave velocity, a marker of arterial stiffness. A blood pressure cuff will be 
wrapped around the upper leg. It will periodically inflate to ~200 mmHg for less than 60 seconds. A 
pressure sensor (tonometer) will be placed over the skin of the neck region to obtain the pressure 
wave form in the carotid artery.  The total time for this procedure is 15 min. 
Brachial and popliteal artery flow-mediated dilation (FMD): Arterial measurements will be 
performed by imaging the brachial and the popliteal artery longitudinally using high-resolution duplex 
ultrasonography. Arterial vasodilatory responses to hyperemia (i.e., FMD) will be examined by 
inflating a cuff, distal to brachial and the popliteal artery, up to 250 mmHg for 5 minutes. Before, 
during and after rapid release of the cuff, brachial and popliteal artery blood flow velocity and 
diameter will be continuously measured. In addition, during the FMD measurements NIRS probes will 
be placed on the tibialis anterior muscle and the forearm; the probe will be secured via an elastic 
tensor bandage which will be loosely wrapped around the site. These probes will stay attached to the 
participant for the duration of testing. 
Carotid artery intima-medial thickness (cIMT): Wall thickness and diameter of the common carotid 
artery will be measured using high-resolution vascular ultrasound with a 7.5-12.0 MHz linear array 
transducer and longitudinal 2D mode (Logiq P5; GE Medical Systems, Milwaukee, WI). The left 
common carotid artery will be measured 2 cm proximal to the bulbus and 2 to 3 cm distal to the 
bifurcation. For this vascular evaluation, we will measure artery diameter and wall thickness for a brief 
period of time. 
OGTT with leg blood flow: After an overnight fast, a catheter will be inserted into an antecubital vein 
for sampling of venous blood.  Blood will be collected every 15-30 minutes over the next 120 minutes 
after consuming the glucose beverage (75 grams of dextrose).   Moreover, before and during the 
OGTT, we will be using an ultrasound to measure femoral artery blood flow. Throughout the 
assessment for leg blood flow, a NIRS probe will be placed on the belly of the tibialis anterior muscle 
in the same conditions previously described. A pneumatic cuff will be placed below the knee. The cuff 
will be inflated at 20 mmHg above diastolic blood pressure for 60 s to partially occlude blood flow at 
the time of the NIRS measurement. The total volume of blood drawn per oral glucose tolerance test 
will be less than 40mL. 
Insulin clamp: Coupled with measures of leg blood flow, will be performed following standard 
procedures at the University Hospital CRC. Subjects will be instrumented for measures of heart rate 
(using standard lead II ECG), arterial blood pressure (using automated sphygmomanometry and 
beat-to-beat via Finometer), and leg blood flow (using duplex Doppler ultrasound and contrast-
enhanced ultrasound, CEU). After a minimum of 20 min supine rest, baseline cardiovascular 
measurements will be collected, blood samples obtained and the hyperinsulinemic-euglycemic clamp 
will start. Briefly, insulin diluted in 0.9% saline with 5 ml of the subject's blood is infused with a 10 min 
priming dose followed by a constant infusion at 40 mU•m−2•min−1, a dose used to mimic 
postprandial insulin concentrations. Glucose is maintained at fasting values throughout via a variable 
20% dextrose infusion. Leg blood flow, arterial blood pressure, and heart rate will be collected for 5-
min periods at 15-min intervals throughout the 120-min clamp allowing the magnitude as well as the 
temporal profile of these variables to be determined. For microvascular blood flow using CEU, 
measurements will be performed before and during the first 60 min of insulin stimulation. Briefly, 
assessment of microvascular perfusion utilizing CEU involves ultrasound imaging of the leg skeletal 
muscle and abdomen during the administration of one vial (1.3mL) of an ultrasound contrast agent 
diluted in 30mL of 0.9% saline that will be given using a handheld syringe pump through the IV at a 
rate of 2mL/min.  This agent, called Definity, is composed of tiny microbubbles smaller than the size 
of a red blood cell.  These bubbles stay inside the blood vessels and go where the red blood cells go.  
This contrast agent allows us to evaluate microvascular blood flow.  Definity has been FDA approved 

Page 13 of 27 
for use in humans during ultrasound of the heart cavity and has been shown to be safe. The total 
volume of blood drawn per insulin clamp will be less than 140mL. 
GlycoCheck: The GlycoCheck video microscope instrument will be placed under the subject’s 
tongue for a brief period (5 min approximately) to measure capillaries, blood vessel density, red blood 
cell concentration, flow rate, and red blood cell penetration of the glycocalyx lining. The GlycoCheck 
instrument, computer system, and software will analyze the measurements and produce a 
Microvascular Health Score (MVHS). GlycoCheck is an FDA Class 1 Medical Device. 
 Details of treatment visits for the isocaloric low-fructose diet (part 1) 
 Figure 10 shows the appointments associated with pick up of study food, inpatient visits, dietary 
counseling, and the follow-up visit.  Subjects will consume a six-month diet with the goal of reducing 
dietary fructose intake to <5% of energy and keeping their weight stable.  Prescribed caloric intake 
will be 
based 
on 
baseline total energy expenditure per day. For the first two weeks, all foods will be provided by the 
study (made at the MU Nutrition Center for Health, MUNCH) for the subject to consume and he/she is 
instructed to not eat any other foods.  Beverages that may be consumed can include water, non-
calorie containing sodas, and regular coffee or tea without nondairy creamer, which may be 
sweetened with non-caloric sweeteners. A maximum of 2 oz of cream per day may be added to the 
coffee or tea.  Regular sodas and fruit juices that are high in fructose are to be avoided. Habitual 
alcohol consumption may be continued and subjects will be asked to avoid alcoholic drinks containing 
fructose and other sugars (e.g., margaritas, fruit-containing drinks).  Milk and milk products may be 
consumed given their low-fructose content.  Within week 2, the subject will attend a grocery store tour 
with study staff to identify high-fructose foods to avoid.  After two weeks, the subject will begin to 
prepare, cook, and eat his/her own foods while following the diet with the assistance of the dietitian 
nutritionist and study staff. 
The subject will meet one-on-one with the dietitian nutritionist who will counsel him/her on how to eat 
a diet that matches the study criteria.  Subjects’ weight, waist circumference and vitals will also be 
measured at nutrition counseling visits. Throughout the entire study, the subject will be in contact with 
the dietitian nutritionist by phone or email to have their questions answered and receive social 
support to maintain adherence.  At three months, the subject returns for an interim visit where PWV, 
 
Figure 10 

Page 14 of 27 
FMD and DEXA will be repeated, while at six months, the subject returns for the final visit which 
includes vascular measurements and an insulin clamp.   
Beverages: The subject may consume non-sugar containing beverages including water, non-calorie 
containing sodas, and regular coffee or tea without nondairy creamer, which may be sweetened with 
non-caloric sweeteners. A maximum of 2 ounces of cream per day may be added to the coffee or tea.  
Regular sodas and fruit juices that are high in fructose are to be avoided. Habitual alcohol 
consumption may be continued and subjects will be asked to avoid alcoholic drinks containing 
fructose and other sugars (e.g., margaritas, fruit-containing drinks).  Milk and milk products may be 
consumed given their low-fructose content.   
Activity: The subject should not consciously change his/her physical activity while in the study.  At 
the conclusion of the study, subjects may meet with study staff to receive advice on how to increase 
their activity. 
 Details of treatment visits for the allopurinol/placebo arm (part 2) 
Figure 11 shows the appointments associated with the inpatient visits, drug treatment, and the final 
visit for this arm.  After completion of the baseline visit (procedures described above), subjects 
participating in the allopurinol treatment/placebo arm will begin with an initial dose of drug of 100 
mg/d p.o. daily for 2 wks.  The dose is then slowly increased over the next 4 weeks to achieve a 
target dose of 300 mg/d).  Dose escalation will be led by Dr. Celso Velázquez, Professor of Clinical 
Medicine and Director of the Division of Rheumatology, using his established algorithm, and Dr. 
Manrique, Internist and Endocrinologist. Once 300 mg/d dose has been reached, the subject stays on 
this dose; and is seen for the interim visit (3 months), at which time all procedures are repeated 
except insulin clamp-CEU and OGTT.  After this, drug treatment continues for another 3 months and 
the subject returns for the final visit at 6 months.  The DEXA, cIMT/cfPWV, Brachial and Popliteal 
FMD, and insulin clamp procedures are repeated at this time. The dose of allopurinol will be taken the 
morning of the final visit. 
 Details of treatment visits for the hypocaloric low-fructose diet (part 3) 
Figure 12 shows the appointments associated with pick up of study food, inpatient visits, dietary 
counseling, and the follow-up visit.  Subjects will consume a six-month diet with the goal of reducing 
Figure 11 

Page 15 of 27 
dietary fructose intake to <5% of energy and reducing total daily caloric intake from baseline total 
energy expenditure by 500 kilocalories per day to promote weight loss.  For the first two weeks, all 
foods will be provided by the study (made at the MU Nutrition Center for Health, MUNCH) for the 
subject to consume and he/she is instructed to not eat any other foods.  Within week 2, the subject 
will attend a grocery store tour with study staff to identify high-fructose and added sugar foods to 
avoid.  After two weeks, the subject will begin to prepare, cook, and eat his/her own foods while 
following the diet with the assistance of the dietitian nutritionist and study staff. 
The subject will meet one-on-one with the dietitian nutritionist who will counsel him/her on how to eat 
a diet that matches the study criteria.  Subjects’ weight, waist circumference and vitals will also be 
measured at nutrition counseling visits. Throughout the entire study, the subject will be in contact with 
the dietitian nutritionist by phone or email to have their questions answered and receive social 
support to maintain adherence.  At 3 months, the subject returns for an interim visit where PWV, FMD 
and DEXA will be repeated, while at 6 months, the subject returns for the final visit which includes 
vascular measurements and an insulin clamp.   
Beverages: The subject may consume non-sugar containing beverages including water, non-calorie 
containing sodas, and regular coffee or tea without nondairy creamer, which may be sweetened with 
non-caloric sweeteners. A maximum of 2 ounces of cream per day may be added to the coffee or tea.  
Regular sodas and fruit juices that are high in fructose and/or added sugar are to be eliminated during 
the dietary intervention. Habitual alcohol consumption may be continued and subjects will be asked to 
avoid alcoholic drinks containing fructose and/or added sugars (e.g., margaritas, fruit-containing 
drinks).  Milk and milk products may be consumed given their low-fructose content.   
Activity: The subject should not consciously change his/her physical activity while in the study.  At 
the conclusion of the study, subjects may meet with study staff to receive advice on how to increase 
their activity. 
 
Figure 12 

Page 16 of 27 
 Sources of research material  
Sources will include the subject’s medical history, physical exam, screening laboratory tests (CMP, 
lipid panel, uric acid, insulin, A1c), dual-energy X-ray absorptiometry (DEXA, total and regional fat 
mass and fat-free mass), blood tests, aortic PWV, cIMT, brachial and popliteal artery flow-mediated 
dilation (FMD), OGTT with femoral artery blood flow results, CEU-insulin clamp results, and food 
intake.  Demographic data (plus other related data: emergency contact person, pregnancy status), 
blood pressure, and anthropometrics will be measured (height, weight, waist circumference).  Each 
subject in parts 1 or 3 (diet interventions) of the study, will donate up to 410 ml of blood (screening 
through final visit) which will occur over a period of up to 6 months.  For Part 2 drug treatment, each 
subject will donate up to 470mls of whole blood (screening through final visit) which will occur over a 
period of 6 months. 
 Blinding and subject safety  
All of Dr. Manrique’ s laboratory staff will be blinded as they perform biochemical assays on serum 
from the drug/placebo study, the isocaloric fructose-restriction study, or the hypocaloric fructose-
restriction study.  Dr. Padilla and the assistant performing vascular measurements will be blinded. For 
the drug study, safety officer cardiologist Brian Bostick, MD, rheumatologist Dr. Celso Velázquez and 
Dr. Camila Manrique will review screening values to determine subject eligibility.  Once the subject is 
randomized, the subjects themselves, investigators, and all CRC staff working with the subjects will 
be blinded to treatment group (allopurinol or placebo).  During the fructose-restriction studies, the 
CRC staff and the study dietitian nutritionist will not be blinded because subjects serve as their own 
control and all subjects will be changing their diets (cannot blind to this treatment).  No adverse 
effects of fructose restriction are anticipated but should subjects exhibit changes in their health status 
across any system related or unrelated to the dietary treatment, Dr. Bostick or Velázquez will be 
consulted for advice.  
 
VIII. 
Potential Risks/Adverse Events 
A. Potential Risks to Human Subjects 
The following are the potential risks: 
Phlebotomy:  The total blood withdrawn will not exceed 550 mL 
in any 8 week period. Hematocrit is measured during screening 
and before the interim and final visits to check that it is within safe 
limits (Table 3 and Table 4). All subjects will be advised to refrain 
from donating blood during public blood drives, during their 
participation in this project.   
These activities are associated with a small risk of phlebitis, 
bruising and minor pain.  Antiseptic technique will be used by 
nurses who are experts in phlebotomy and catheter placement. If 
phlebitis occurs, it will be treated conservatively with heat and 
when appropriate, with antibiotics. 
 
Time point 
Volume of whole 
blood (mls) 
Screening 
60 
Baseline visit 
145 
SUA concentration 
10 
SUA concentration 
10 
SUA concentration 
10 
Interim visit 
35 
SUA concentration 
10 
SUA concentration 
10 
SUA concentration 
10 
Final metabolic test 
170 
Total           470 
Table 3. Blood volume taken over the study in 
the allopurinol/placebo arm 

Page 17 of 27 
Adherence and serum and urine biochemistries: The SUA response to drug treatment and the pill 
counts will be used to assess adherence to the drug/placebo arms.  Some plasma will be frozen for 
analysis of potential biomarkers of cardiac and arterial (CA) stiffness.  Estimated glomerular filtration 
rate (GFR) is calculated via the Modification of Diet in Renal Disease Study equation, GFR = 175 × 
standardized serum creatinine −1.154 × age−0.203 × 1.212 [if black] × 0.742 [if female], where GFR 
is expressed as mL/min/1.73 m2 of BSA and creatinine is 
expressed in mg/dL 71. 
Indirect calorimetry: Fasting energy expenditure and substrate 
oxidation are measured at the beginning of the metabolic test.  
Calorimetry carries no risk, however, this test may cause 
discomfort in those who are fearful of confined spaces or 
claustrophobic.  Before the procedure is performed, subjects are 
allowed to become familiarized with the hood used to collect 
expired gas. 
Aortic pulse wave velocity (cfPWV): This procedure poses not 
risks. 
Carotid artery intima-media thickness (cIMT): This procedure 
poses no risks. 
Brachial artery and popliteal flow-mediated dilation (FMD) with NIRS: This is a measurement of 
endothelial function.  There are no risks associated with this procedure. 
Oral glucose tolerance test (OGTT): At screening, subjects will have an oral glucose tolerance test 
performed. This might cause blood sugar levels to be elevated following the completion of the test. 
Hyperinsulinemic-euglycemic clamp: For premenopausal female subjects, a urine pregnancy test 
is administered before the clamp. The potential risks during the clamp include mild nausea or light-
headedness, and mild to moderately high or low blood glucose levels. However, blood glucose will be 
continually monitored during the infusion of insulin and dextrose will be available if needed to 
counteract hypoglycemia. Also, drinks and snacks will be available for the subjects at the completion 
of testing should they have low blood sugar or feel nauseous. Subjects are monitored for a minimum 
of 1 hour post clamp testing. This procedure is routinely performed at the University Hospital CRC by 
Dr. Manrique and Dr. Padilla. Dr. Manrique, board-certified endocrinologist, will be present during the 
procedures. 
GlycoCheck: This procedure poses no risks 
Near-Infrared Spectroscopy (NIRS): This procedure poses no risks. 
Skeletal muscle (calf) perfusion assessment using Perflutren (Definity): A potential side effect 
from the perflutren ultrasound contrast agents is temporary back pain, joint pain, headache, shortness 
of breath, or flushing. These symptoms occur in about one in 200 subjects and are mild in intensity in 
90% of those who experience this reaction. If this happens infusion of the agent will be stopped. The 
back and joint pain will go away in a few minutes. A serious allergic reaction to ultrasound contrast 
agents is unlikely (1 in 10,000), but possible. Symptoms of an allergic reaction include: Rash, itching, 
swelling, severe dizziness, chest pain and trouble breathing. We are using this contrast agent to 
measure microvascular perfusion in skeletal muscle using a dose approved by the FDA.  
DEXA and anthropometrics: Radiation exposure during DEXA is equivalent to about 2% of the 
average radiation dose from all sources (natural background radiation, consumer appliances, radon 
gas, medical tests, etc.) that a person in the United States receives each year.  Subjects who 
participated in any other research study or medical procedure involving significant ionizing radiation 
exposure (e.g., multiple chest x-rays) in the past 12 months will be excluded.  For premenopausal 
 
Time point 
Volume of whole 
blood (mls) 
Screening 
60 
Baseline visit 
145 
Interim visit 
35 
Final metabolic test 
170 
Total 
410 
Table 4. Blood volume taken over the study in 
the isocaloric low fructose arm and hypocaloric 
low fructose arm. 

Page 18 of 27 
female subjects, a urine pregnancy test is administered before the DEXA.  Body weight is measured 
to the nearest 0.1 kg and height to the nearest 0.1 cm.   
Food Intake and dietary control: We do not anticipate that fructose restriction will pose risk to the 
subjects.  
Xanthine oxidase inhibition with allopurinol: Dr. Celso Velázquez is an expert in the drug 
treatment of gout and he will use standardized clinical protocols to ramp the dosage of allopurinol 60, 
61. Risks: Side effects of allopurinol hepatitis, rash and pruritus. Our team is aware of the existing 
recommendations by the American College of Rheumatology in regards to the use of anti-
inflammatory agents when starting allopurinol in patients with acute gout 60, 65. As the present study 
will not include patients with diagnosis of gout and given that available studies done in subjects with 
asymptomatic hyperuricemia demonstrate that monotherapy with allopurinol is safe 66, 67, we plan to 
treat subjects our hyperuricemic subjects only with allopurinol. Dr. Velazquez will closely monitor 
these subjects and might consider using a low-dose of naproxen in selected cases.  
Risks of not treating hyperuricemia in the placebo group: A 2013 Cochrane review emphasized 
the lack of sufficient evidence to support the use of urate-lowering therapy to prevent future gout in 
hyperuricemic, asymptomatic patients 63.  A need for further clinical trials to test other benefits of 
lowering SUA was called for 64 which strongly supports the rationale for the current study.  Given the 
limited duration of subject participation (6 month), the lack of known risk to the subject for not being 
treated is balanced by the significant knowledge gained from this study.  At the study's end, subjects 
in the allopurinol/placebo arm will be offered one, no-cost counseling session with the dietitian 
nutritionist, who will have analyzed their ad libitum food intake and body composition.  The dietitian 
nutritionist will make recommendations to these subjects for modifying lifestyle factors to reduce SUA 
and improve overall health. 
Study exit survey: surveys will be optional for subjects that complete study treatment. This poses no 
risks. 
B. Protection Against Risks 
Risks to loss of confidentiality are reduced by assigning all subjects a data identifier code.  Hard 
copies of data are stored in a locked office, and only the PI and study coordinator have access to the 
locked files.  Individual names or initials are not used in any discussions or publications of the data.  
We have assembled a research team which includes scientists, physicians, and clinician-scientists 
with significant experience in human research and metabolic diseases to help anticipate and reduce 
the risks to subjects.  The specific protocol to minimize risk associated with each procedure is 
described below. 
Psychological stress: The psychological stress from participation in this study is minimal.  However, 
some of the questions about food intake and physical activity may make the subjects feel 
uncomfortable.  Subjects will be told they may skip any portion of a questionnaire if they feel 
uncomfortable about answering the questions. 
Fructose restriction: Dr. Manrique will be assisted by a dietitian nutritionist to manage the dietary 
portion of the treatment.  The software, NDSR is used to design diets which are balanced and provide 
the RDAs for all nutrients and micronutrients.  Subjects may find it challenging to comply to the 
dietary restriction, however, the assembled team has performed human nutritional research studies 
for the past 20 years and, as described in the grant, the diets will be individualized as much as 
possible, to meet the subject's personal preferences. 
Blood collection and IV lines: Aseptic technique is used to collect blood using butterfly needles 
and, if necessary, through an IV.  Only the amount of blood necessary for analyses is withdrawn and 

Page 19 of 27 
the staff members performing venipuncture are nurses and trained phlebotomists.  Risk of bleeding is 
reduced by applying pressure at the site of puncture.  Bruising is treated with ice.  Fainting is 
prevented by drawing blood in the semi-recumbent position. 
Indirect calorimetry: To reduce the feeling of claustrophobia, during the screening visit, the subject 
is allowed to become familiarized with the hood used to collect expired gas. 
Arterial stiffness, carotid intima-medial thickness, flow mediated dilation, and blood pressure: 
When assessing carotid-femoral PWV, the blood pressure cuff will squeeze the arm and leg tightly; 
however, any discomfort will be alleviated as soon as the pressure in the cuff is released. 
DEXA: Risks include a small radiation for the DEXA, equivalent to about 2% of the average radiation 
dose from all sources that a person in the U.S. receives each year.  Subjects who have participated in 
other research studies or medical procedures involving significant ionizing radiation exposure in the 
past 2 months will be excluded.  A urine pregnancy test will be performed before the DEXA and 
insulin clamps for premenopausal women. 
There are no alternative methods that will allow us to test our proposed hypotheses.  
C. Plan for Reporting Study Deviations:  
Any minor problem/deviation will be summarized and reported to the IRB within 5 working days of 
awareness, including any event that does not: 1) increase the risk to the subject, 2) decrease the 
benefit to the subject, or 3) significantly affect integrity of the research data.  Any major 
problem/deviation will be summarized and reported to the IRB within 5 working days of awareness, 
including any event that: 1) increases the risk to the subject, or 2) significantly affects integrity of the 
research data.   
D. Stopping Rules 
We will stop an individual study in the event of an unanticipated serious adverse event.  If 4 or more 
subjects experience adverse events requiring termination of the study, the study will be stopped and 
the events will be discussed with the IRB to determine whether it is appropriate to continue and/or 
determine appropriate modifications to the protocol to avoid further adverse events.  All adverse 
events will be submitted for review according to current protocols. The pharmacological treatment will 
be discontinued in case of severe elevation of liver enzymes, evidence of hypersensitivity to the 
medication or at the discretion of the safety office Dr. Bostick. Follow up blood work and clinic visits 
will be scheduled at the discretion of the safety officer in coordination with the investigators. 
E. Breach of Confidentiality 
 Subject confidentiality will be rigorously maintained.  The data collected as part of this study will be 
for research only.  It will be de-identified after collection.  Confidentiality of data will be assured by 
coding of unique subject identities and that coding will be known only to the research team, including 
the use of secure files, locked in an office, and a unique subject coding system.  The original study 
data will be kept in locked in an office (hard copy) or entered into a secure computer database 
password protected under a secure server space allocated for use by only the study team 
(electronic).  Furthermore, data analysis will be appropriately blinded and any individual data 
presented in manuscripts will also be presented in an anonymous nature.  No identifying information 
will be disclosed.  Confirming with University of Missouri policy, all research records will be retained 
for a period of 7-years following completion of the study. 
All protocols and techniques to be used will be approved by the Institutional Review Board (IRB) prior 
to initiation of any studies.  Each subject will give written informed consent after all questions have 
been answered by a study team member.  The consent form will also include a statement 
guaranteeing confidentiality.  Adverse event reports and annual summaries will not include subject-

Page 20 of 27 
identifiable material.  No information will be given to anyone without permission from the subject.  
Electronic communication with study team members will involve only coded, unidentifiable 
information.  Any unanticipated breach of confidentiality will be summarized and reported to the IRB 
within 5 working days of awareness 
 
IX. 
ANTICIPATED BENEFITS 
There may be no benefit to the subjects in this study.  These data will aid in the understanding of how 
the characteristics of the T2D and the level of SUA may increase the risk of CVD in women and men.  
From the screening and baseline tests, all subjects will gain health information about themselves.  At 
the end of the study, results collected will be shared with the subject in face-to-face meetings.  At the 
study's end, subjects in the placebo arm will be offered a no-cost counseling sessions with the 
dietitian, who will have analyzed their ad libitum food intake, physical activity, and body composition.  
The dietitian will make recommendations to these subjects for modifying lifestyle factors to reduce 
SUA and improve overall health. 
In summary, the risks of the treatments are minimal, as witnessed by millions of Americans who are 
treated with similar drugs each year.  The health testing offers some risk, which includes the blood 
draws.  Overall, the benefits to the subjects may be substantial, and the new information on 
characteristics that increase cardiovascular risk in T2D women will benefit the society.  There are 
risks associated with the study, but the experience and medical expertise of the research team should 
keep these to a minimum, and our track record with these types of studies indicates that this diligence 
has been effective. 
 
X. 
COMPENSATION 
We have completed multiple clinical studies involving 
human subjects with adherence rates of greater than 95% 
and subject retention rates of greater than 85% 72-74.  We 
utilize a validated retention strategy published by Jeffrey et 
al 75 that has been successful.  With respect to subject 
honoraria, as shown in Table 5, subjects will be paid $70 
for a screening test, $80 for a baseline test, $50/month for 
research participation (taking drug or placebo, or 
complying with the diet) (6 months = $300), $150 for 
interim study tests, and $300 for the final.  Any additional 
unscheduled safety visits will be compensated $25. Thus, a total compensation of $900 will be 
provided as study events are completed not including any additional visits. Subjects that reside more 
than 25 miles away from the University Hospital CRC will receive an additional compensation of $7 
per 25 miles traveled per visit.  
 
XI. 
COSTS 
 
You will not be charged for any procedures that are part of this research study.  The costs of the 
study will be covered by a NIDDK R21 grant. 
 
XII. 
DATA SAFETY AND MONITORING PLAN 
Overall framework for safety monitoring and what information will be monitored.  
Visit 
Compensation 
Screening test 
$  70 
Baseline test 
$  80 
$50/month x 6 mo 
$ 300 
Interim test 
$ 150 
Final test 
$ 300 
Table 5. Subject compensation for all arms of the 
study. 

Page 21 of 27 
A. Plan for data management:   
A password-protected database will be used to manage all study data.  To ensure confidentiality only 
subject ID numbers will be entered into the database.  Signed informed consent forms are kept in a 
locked office.  Participants will not be individually identified in any publication. Participants’ right to 
privacy will be protected. 
B. Plan for safety monitoring:  
Although risks from the dietary intervention protocol in our proposal are minimal, the drug treatment 
could pose risk to participants.  The data and safety monitoring plan (DSMP) for this trial focuses on 
close monitoring by the PI and the safety officer.  
Frequency of monitoring, including any plans for interim analysis and stopping rules.  Monitoring will 
include enrollment, attrition, efficacy end-points, and adverse events. In addition to monitoring by the 
PI, study coordinator, and safety officer monitoring, the MU IRB monitors all aspects of the project on 
an annual basis. The DSMC conducts its reviews on a semiannual basis. The frequency of the 
structured data review for this study can be summarized in the following table: 
 
 
 
      
 
 
 
 
 
 
 
C. Process for Managing and Reporting Adverse Events, Serious Adverse Events and 
Unanticipated Problems:  
Events that meet the three IRB criteria of being: unexpected, related or possibly related to research 
participation, and suggesting that research places subjects or others at a greater risk of harm than 
previously known or recognized will be reported to the IRB. Both serious and non-serious adverse 
events will be reported to the MU School of Medicine Data and Safety Monitoring Committee 
(DSMC). For reporting to the DSMC, adverse events will be categorized and classified according to 
Common Terminology Criteria for Adverse Events Scale (CTCAE v3.0). Safety reports will be sent to 
the safety officer. The PI will be responsible for assembling the data and producing these reports, as 
well as assuring that all parties obtain copies of these reports. 
D. Qualifications and responsibilities of the Safety Officer:  
The safety officer for this trial will be Brian Bostick, MD/PhD. Dr. Bostick is an Assistant Professor 
in the Division of Cardiology.  In addition to practicing medicine, he is a clinician scientist.  As 
Safety Officer, Dr. Bostick will review eligibility criteria with Dr. Manrique, and will be unblinded as to 
treatment assignment. He will review all reports sent by the study coordinator to determine whether 
Data type 
Frequency of review 
Subject accrual (adherence to protocol regarding 
demographics, inclusion/exclusion) 
At the end of each recruitment 
Adverse event rates (injuries)  
semi-annually 
Compliance to treatment  
semi-annually 
Out of range laboratory data 
semi-annually 
Stopping rules report regarding statistical power implications of 
dropouts and missing data 
semi-annually 
IRB review 
annually 
DSMC review  
semi-annually 
Table 6 

Page 22 of 27 
there is any corrective action, trigger of an ad hoc review, or stopping rule violation that should be 
communicated to the MU IRB and the DSMC. In addition, the safety officer may comment on whether 
the study PI needs to report any specific out-of-range laboratory data. 
E. MU Data and Safety Monitoring Committee (DSMC):  
The MU School of Medicine DSMC was formed by the MU Institute for Clinical and Translational 
Science and activated as an advisory committee within the MU School of Medicine.  Its structure and 
processes are consistent with NIH, NIDDK, and U.S. Food and Drug Administration guidelines. The 
DSMC helps investigators, and MU research compliance oversight mechanisms protect human-
research participants and ensure the integrity and scientific validity of research data.  The DSMC 
provides education for investigators, research teams, and faculty regarding data and safety 
monitoring; reviews proposed Data and Safety Monitoring Plans; establishes Data and Safety 
Monitoring Boards; conducts independent, interim reviews of study safety and progress; and makes 
recommendations concerning the continuation of studies, including recommendations regarding the 
modification, suspension, or termination of a study.  The DSMC has 13 voting members representing: 
medicine, nursing, health professions, pharmacology, ethics, biostatistics, behavioral science, 
information technology, social science, and research participant advocacy. There are also seven non-
voting members: compliance officers for the health science and campus institutional review boards, 
compliance officer for the campus conflict of interest committee, managers of the clinical trials offices 
at University Hospital and Ellis Fischel Cancer Center, director of compliance for the school of 
medicine, and the executive secretary of the DSMC. Leadership of the DSMC is provided by the 
chair, Greg Flaker, MD, Brent Parker Professor of Medicine (Cardiology); Vice Chair. David Fleming, 
MD, Professor of Clinical Medicine and Director, MU Center for Health Ethics; and the Executive 
Secretary. Don Reynolds, JD, Director, Office for Responsible Research. Administrative Coordination 
is provided by the School of Medicine Office of Compliance and Quality. 
 
XIII. 
MULTIPLE SITES 
Not applicable 
 
 

Page 23 of 27 
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