Effect of Age and Fitness on Vascular Function and Oxidative Stress During Acute Inflammation
Phase: PHASE4
Conditions: Aging, Inflammation
Sponsor: University of Illinois at Chicago

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Effect of age and fitness on vascular function and oxidative stress during acute inflammation 
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Effect of age and fitness on vascular function and oxidative stress during acute 
inflammation 
 
 
NCT03889158 
 
 
 
Version: 6 
Date: 12/05/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
Page 
Table of Contents ................................................................................................................ 2 
List of Abbreviations ............................................................................................................ 3 
1.0 Project Summary/Abstract ...................................................................................... 4 
2.0 Background/Scientific Rationale ............................................................................ 5 
3.0 Objectives/Aims ........................................................................................................ 7 
4.0 Eligibility ..................................................................................................................... 8 
4.1 Inclusion Criteria ................................................................................................. 8 
4.2 Exclusion Criteria................................................................................................ 8 
4.3 Excluded or Vulnerable Populations ................................................................ 8 
5.0 Subject Enrollment ................................................................................................... 9 
6.0 Study Design and Procedures................................................................................. 9 
7.0 Expected Risks/Benefits ........................................................................................ 17 
8.0 Data Collection and Management Procedures .................................................... 19 
9.0 Data Analysis ........................................................................................................... 19 
10.0 Quality Control and Quality Assurance ............................................................... 19 
11.0 Data and Safety Monitoring ................................................................................... 19 
12.0 Statistical Considerations ...................................................................................... 20 
13.0 Regulatory Requirements ...................................................................................... 20 
13.1 
Informed Consent ........................................................................................ 20 
13.2 
Subject Confidentiality ................................................................................ 21 
13.3 
Unanticipated Problems .............................................................................. 21 
14.0 References ............................................................................................................... 22 
 

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LIST OF ABBREVIATIONS  
 
ACSM  
American College of Sports Medicine 
CV 
 
Cardiovascular 
FMD  
Flow-mediated dilation 
HR 
 
Heart rate 
IPL 
 
Integrative Physiology Lab 
 
 

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1.0 
Project Summary/Abstract 
Acute and chronic inflammation both increase cardiovascular disease risk, especially 
with aging, which may be due to vascular dysfunction. Aging and inflammation also lead 
to increased oxidative stress, which impairs vascular function. During acute 
inflammation, endothelial function is altered differently in younger and older adults with 
decreases in endothelial function in younger, but not older adults. However, 
cardiorespiratory fitness is cardio-protective, impacting inflammation, vascular function, 
and oxidative stress. During acute inflammation, moderately fit older adults exhibit 
similar responses to younger adults, suggesting preserved endothelial reactivity. 
However, whether the protective mechanism is oxidative stress has not been confirmed. 
Furthermore, it is undetermined whether the vascular dysfunction is further propagated 
down the arterial tree during acute inflammation to the microvasculature. We will 
investigate these questions through 2 specific aims: 1) To determine if age and fitness 
moderate the vascular response to acute inflammation; 2) To determine if antioxidant 
administration eliminates vascular dysfunction during acute inflammation.   
We will recruit 15 young low fit (18-35 years) and 30 older low or high fit (55-75 years) 
adults. An acute inflammatory bout will be induced by typhoid vaccination. The influence 
of oxidative stress will be assessed using an oral antioxidant. Baseline influences of the 
antioxidant on endothelial function will be assessed on Visit 1. Acute inflammation 
measures will be taken at baseline (Visit 2, prior to) and 24-hours after the vaccine (Visit 
3). The antioxidant will be administered again on Visit 3.    
We anticipate low fit older adults will have reduced endothelial responsiveness and 
microvascular reactivity compared to young and high fit older adults. Second, we 
anticipate the antioxidant will improve vascular function at baseline in low fit older 
adults. During acute inflammation, we anticipate improvement in vascular function to 
baseline levels in young and older high fit adults.  
The results from this study will help to elucidate if fitness is a protective and preventive 
measure to ameliorate the detrimental cardiovascular response to acute inflammation. 
Thus, this study may provide health professionals with a behavioral intervention to 
reduce cardiovascular disease burden in the rapidly growing aging population.  
 

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2.0 
Background/Scientific Rationale  
Acute inflammation increases CV risk [1–4] with a higher prevalence of older adults 
experiencing adverse CV events[3]. Aging is associated with immunosenescence[5,6], 
rendering older adults more susceptible to infection[7] and may contribute to the higher 
mortality and acute CV events following acute inflammation in the elderly[1,2,8]. Indeed, 
infectious disease hospitalization and the concomitant inflammatory insult leads to an 8-
fold increased risk of acute coronary syndromes in older adults within 15 days after 
admission[9].  
Increased CV risk with acute inflammation may also stem from detrimental changes in 
endothelial function[10–12] and arterial stiffness[13–15]. Indeed, our laboratory has 
shown that endothelial function during acute inflammation decreases in younger, but not 
in older, adults [16,17]. Moreover, acute inflammation results in concomitant increases 
in arterial stiffness, a predictor of CV events. These large artery impairments during 
acute inflammation may be further propagated down the arterial tree to the 
microvasculature, altering microvascular reactivity. During a severe acute inflammation 
model, peripheral near-infrared spectroscopy (NIRS) evaluation of microvascular 
reperfusion is altered earlier than other systemic variables[18]. NIRS may therefore 
serve as an early detector of altered peripheral perfusion during acute inflammation. As 
such, identifying means to restore vascular reactivity during acute inflammation to levels 
seen in young adults may help attenuate CV risk in older adults.  
Higher cardiorespiratory fitness and habitual physical activity are protective for many 
aspects of CV health[19,20] and appear to also be protective during acute inflammation. 
Recent data suggests voluntary running is protective against acute inflammation and 
the concomitant microvascular dysfunction in a mouse-model of sepsis[21]. 
Furthermore, population-based data suggests inadequate exercise (<1.07 MET h/day) 
is associated with a doubling of the risk of mortality 
during severe 
acute inflammation (sepsis), independent of other risk 
factors[22]. Additionally, during acute inflammation, our 
group has shown moderately fit older adults 1) maintain 
vascular reactivity similar to that of younger adults[23], 
which may help protect maintenance of blood pressure 
and 2) do not experience increases in arterial stiffness 
as seen in low fit older adults.[15] These data suggest 
exercise has beneficial effects in clinical manifestations 
of inflammation and has shown potential 
cardioprotective effects during acute, low-grade inflammation. 
Figure 1. 

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The protective effect of fitness in older adults is potentially due to lower oxidative stress. 
Aging alone increases oxidative stress which reduces nitric oxide bioavailability, 
impairing baseline endothelial function (Figure 1). In older adults, habitual physical 
activity maintains endothelial function to similar levels as young healthy adults[24,25] 
and increases antioxidant defense by reducing NADPH oxidase and nuclear factor-𝜅B 
(NF𝜅B) activity[26]. Salsalate administration inhibits NF𝜅B, reducing oxidative stress, 
and improving endothelial function[27]. Salsalate administration improves endothelial 
function in older, sedentary adults but not their trained counterparts or younger 
adults[28]. This supports the role of fitness in reducing oxidative stress with aging.  
During acute inflammation in young adults, endothelium-independent vasodilation 
remains intact, indicating the change in endothelial function is likely due to reduced 
nitric oxide bioavailability and increased oxidative stress[11,12]. Increases in oxidative 
stress have been shown at 48 hours following a vaccination[10]. Additionally, ascorbic 
acid has partially resurrected forearm blood flow responses to acetylcholine during 
acute inflammation[11]. Since being physically active while aging maintains baseline 
levels of oxidative stress and endothelial function similar to that of young adults, 
changes in oxidative stress may explain the differing conduit endothelial responses 
between older low and high fit adults during acute inflammation. However, the 
mechanism of impairment has not been fully investigated. Antioxidant intake improves 
vascular function in older adults to similar levels of young adults, suggesting a role of 
oxidative stress[29,30]. Thus, ingestion of an antioxidant such as ascorbic acid may 
eliminate vascular dysfunction during acute inflammation.  
To understand the vascular response to acute inflammation, an integrative approach 
must be applied to identify both the mechanism of vascular dysfunction and a potential 
therapeutic target to abrogate acute inflammation-induced vascular dysfunction. This 
study addresses both gaps of knowledge by investigating the role of oxidative stress 
and by recruiting participants based on training levels to effectively examine the effect of 
high fitness.  
Relevance 
CV disease is the leading cause of death in the United States and particularly burdens 
older individuals[31]. Aerobic exercise is recommended by the United States Physical 
Activity Guidelines, American Heart Association, and American College of Sports 
Medicine (ACSM) to improve quality of life, reduce risk of heart disease, diabetes, 
stroke, and osteoporosis, and improve immune function. If fitness moderates the effects 
of acute inflammation on vascular function, our study will provide insight into how 
physical activity and exercise interventions could reduce the risk of CV events during 
acute inflammation.  

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Preliminary Studies 
Our laboratory has conducted a series of studies 
evaluating the effect of acute inflammation on 
cardiovascular function[15–17,32–34]. evaluated 
both cardiac and endothelial function during acute 
inflammation in younger and older 
adults[16,17,34]. These studies suggest age-
associated differences in endothelial function, 
cardiac function, and blood pressure that may be 
detrimental to older adults[16,33,34].  We have 
shown that fitness, an indicator of chronic 
exercise, may exert protective effects. In older 
adults, high fitness protected from acute 
inflammation-induced arterial stiffening[15]. 
Recently our lab has shown that high fit older adults also have a similar endothelial 
response to acute inflammation as younger adults. The high fit older adults exhibit a 
greater reduction in flow-mediated dilation than older low fit adults. Fitness level did not 
influence the response in young adults (Figure 2)[23]. Additionally, we have collected 
pilot data on microvascular reactivity in young adults (n=16), showing no changes in 
tissue saturation index from baseline to 24h during acute inflammation in peak 
hyperemic response (13.7±3.9% to 13.8±2.9%) or reperfusion slope (1.84±0.52%/s to 
1.83±0.49%/s).  
 
 
3.0 
Objectives/Aims 
 
Aim 1: To determine if fitness moderates the vascular response to acute 
inflammation with aging. We hypothesize that during acute inflammation A) high fit 
older adults will have similar endothelial and microvascular reactivity as young adults; 
B) high fit older adults will have greater endothelial and microvascular reactivity than low 
fit older adults.  
Aim 2: To determine if antioxidant administration eliminates vascular dysfunction 
during acute inflammation. We hypothesize A) at baseline, ascorbic acid will improve 
endothelial function only in low fit older adults; B) during acute inflammation, ascorbic 
acid will improve vascular function to baseline values in young and older high fit adults.  
 
 
 
 
 
Figure 2 

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4.0 
Eligibility 
 
4.1 Inclusion Criteria 
• Males and females willing to provide informed consent 
• 18-35 or 55-75 years of age 
• Non-smoker 
• No antioxidant/vitamin supplementation 
• No use of anti-inflammatory medication within last 2 weeks 
• Aerobically trained (defined as performing aerobic exercise on ≥4 
days/week, for ≥30 minutes, for at least the past 3 months AND a 
VO2max ≥75th age- and sex-specific percentile according to ACSM)  
 
OR 
 
Sedentary (defined as being involved in less than 30 minutes of 
moderately-intense physical activity per day, < 3 days/week AND a 
VO2max ≤ 50th age- and sex-specific percentile according to ACSM)  
 
 
4.2 Exclusion Criteria 
• Body mass index >35 kg/m2 
• Pregnancy, hormone replacement therapy, or peri-menopausal 
• Known cardiovascular (i.e. atherosclerosis, uncontrolled hypertension, 
stroke, myocardial infarction, etc.), inflammatory (i.e. Crohn’s disease, 
arthritis, etc.), or metabolic (i.e. Diabetes mellitus) disease 
• Current use of >2 blood pressure medications, change in vasoactive 
medication type or dose within past 2 months. 
• Regular use of medications to reduce inflammation (NSAIDS, aspirin, 
steroids, etc) 
• Bleeding disorders 
• Illness, other vaccination, or antioxidant use within 2 weeks prior to 
screening 
• Typhoid vaccination within previous 2 years or prior adverse reaction 
• VO2max in 51st – 74th age- and sex-specific percentile according to ACSM 
• Non-English speaking participants 
 
4.3 Excluded or Vulnerable Populations 
• Pregnant women, women on hormone replacement therapy, and peri-
menopausal women will be excluded due to the changes in hormonal 
concentrations, hemodynamics, and altered exercise capacity 
• In addition, we exclude non-English speakers as the researchers must 
ensure the subjects completely understand the purposes and risks 
associated with our study and we do not have any Spanish speaking 
research personnel.  

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5.0 
Subject Enrollment 
 
Subjects will be recruited from the UIC campus and Chicago community using an 
IRB approved recruitment flyer, internet recruitment ad (i.e. UIC website, Craig’s 
List, ResearchMatch, etc.), social media (i.e. Facebook, Twitter, etc.) and word of 
mouth including speaking engagements. Subjects will be asked to contact the 
IPL for further information about the study and subsequent screening for eligibility 
by either phone or e-mailing Elizabeth Schroeder.  
 
After the screening is complete, participants will be asked if they are still 
interested in the study. If interested, their age, gender, and group (sedentary or 
active) will be kept by Elizabeth Schroeder. Once an individual of the same 
gender, similar age (within 2 years) and opposite group has been identified, 
participants will be contacted again for scheduling of visits. We have not had 
issues with this type of recruitment in the past. 
 
If still interested, a packet will be sent (mail or email) to the potential subject with 
a copy of the informed consent and information regarding the first laboratory visit. 
All subjects will sign an institutionally approved informed consent prior to 
participation in the study during visit one.  
 
 
6.0 
Study Design and Procedures 
 
Study site 
All study procedures except the vaccination will be conducted in the Integrative 
Physiology Laboratory at the University of Illinois at Chicago located at 1640 W. 
Roosevelt Road Suite 158 in the Disability, Health and Social Policy building 
(DHSP). The typhoid vaccination will be administered by a registered nurse at 
the Clinical Research Center at UIC. 
 
Design 
A comparative, observational study design will be conducted with a group of 15 
low fit young adults, 15 low fit older adults, and 15 high fit older adults, for a total 
of 45 research participants.  
 
Subjects will participate in three study visits, with at least 72 hours between the 
first two visits, and exactly 24 hours between the last two visits. Menstruating 
females will be studied during the first 7 days of menses, or during the placebo 
phase if taking oral contraceptives. Participants will arrive to each visit having 
refrained from exercise, alcohol, and caffeine for 24h prior to each test day. 
Participants will be tested in the post-prandial (no food, only water) state (>10 
hours) for all visits. (Figure 1) 

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Figure 1. Study protocol schematic 
 
Visit 1. Following informed consent, participants will complete a health history, 
food recall, and physical activity questionnaire and undergo anthropometric 
(height, weight, waist and hip circumference) and body composition measures 
(DEXA scan). For women of childbearing age, the first visit will include a urine 
pregnancy test using a test stick. Baseline influence of oxidative stress on 
endothelial and microvascular function will be assessed following supine rest and 
resting hemodynamics. Measures will include: 1) endothelial function, 2) 
microvascular function, 3) arterial stiffness, and 4) circulating markers of 
oxidative stress. Participants will then orally ingest 2g of ascorbic acid. This dose 
produces a physiological level of ascorbate in plasma[35], has been used in 
previous studies to improve endothelial function[35–39], and reaches optimal 
absorption after 2 hours[40,41]. During this time, an assessment of general 
cognitive function will be completed using the Montreal Cognitive Assessment 
(MOCA) and Symbol Digits Modality Test (SDMT), and be familiarized with a 
working memory cognitive function task (NBACK) by completing the task four 
times throughout the two hour period. Following 2 hours, all measures will be 
repeated to determine the influence of the antioxidant. The visit will finish with a 
treadmill exercise test to evaluate cardiorespiratory fitness (VO2peak). 
Visit 2. At least 72 hours later, participants will arrive after an overnight fast (>8 h) 
and undergo supine baseline measures of: 1) endothelial function, 2) 
microvascular function, 3) arterial stiffness, 4) cerebrovascular function and 5) 
cognitive function, 6) heart function and 7) circulating markers of inflammation 

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and oxidative stress. The typhoid vaccination (Typhuim Vi, Sanofi Pasteur SA) 
will then be administered by a registered nurse. Acute inflammation is often 
induced in cardiovascular research by use of either the influenza or typhoid 
vaccination [10,11,13,23,33,34,42]. Vaccinations provide a safe and controlled 
inflammatory response, in which the typhoid vaccination has shown to increase 
hsCRP up to at least 32 hours post-vaccination[13].  
Visit 3. Exactly 24-hours following Visit 2, participants will return to the lab under 
the same conditions. Visit 2 will be repeated. Following baseline measures, 
participants will consume 2g of ascorbic acid, as completed in Visit 1. Following 2 
hours, measures of 1) endothelial function, 2) microvascular function, 3) arterial 
stiffness and 4) circulating markers of oxidative stress.will be repeated to 
determine the influence of the antioxidant.  
All procedures are being completed for research purposes. The table below 
shows the estimated time commitment for each study visit and procedure. 
  
Visit 1 
Visit 2 
Visit 3 
Informed Consent/ 
Questionnaires 
30 
  
  
Anthropometrics 
5 
  
  
DEXA 
10 
  
  
Blood Draw 
10 
10 
10 
Rest/Preparation 
10 
10 
10 
Vascular Assessment 
  
  
  
Blood pressure 
7 
7 
7 
FMD/NIRS 
13 
13 
13 
Arterial stiffness (PWV) 
10 
10 
10 
Brain blood flow (TCD) 
  
15 
15 
Carotid Flow 
5 
5 
Heart Function  
15 
15 
Cognitive: NBACK 
  
12 
  
Vaccination 
  
15 
  
Vit C 
120 
  
120 
  
NBACK 
Familiarization 
  
Cognitive: 
NBACK 
  
MOCA 
  
  
  
SDMT 
  
  
Blood Draw 
10 
  
10 
Vascular Assessment 
  
  
  
Blood pressure 
7 
  
7 
FMD/NIRS 
13 
  
13 

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Arterial stiffness (PWV) 
10 
  
10 
Maximal Exercise Test 
30 
  
  
TOTAL TIME (minutes) 
285 
112 
245 
Hours: 
4.75 
1.87 
4.08 
 
Measurements 
 
DEXA Scan 
We will determine percent body fat via a whole body scan by dual energy x-ray 
absorptiometry (GE, IDXA, Madison, Wi), which will be operated and calibrated 
using the manufacturer’s stated guidelines.  The duration of the scan will be less 
than 10 min and the subject will be exposed to a radiation dose of 0.3 mrem 
based upon the manufacturer’s specifications and calculations from Standford 
Dosiometry, LLC RADAR Medical Procedure Radiation Dose Calculator. This 
amount of research protocol radiation exposure is minimal given the estimated 
effective equivalent dose is below the 100 mrem per year, a limit set by the 
Nuclear Regulatory Commission for “general public” exposure.   
 
Venous blood samples 
A blood sample will be collected at baseline during each visit (3 times) and 
following Vit C consumption (2 times). During the first visit, each blood sample 
will be 15 mL (approximately 1 tbsp). On visit 2 and 3, the baseline blood draw 
will be 25 mL (approximately 5 tsp). The final blood draw on visit 3 will be 15 mL 
(approximately 1 tbsp). At all time points, we will measure C-reactive protein and 
interleukin-6 for inflammatory markers, oxidized LDL as a marker of oxidative 
stress, total antioxidant capacity and ascorbate for antioxidant capacity, and 
endothelial extracellular vesicles, in which additional analyses on inflammatory 
markers may be performed. The baseline blood draw of visit 2 and 3 will also 
assess the cholesterol panel (Total cholesterol, HDL cholesterol, non-HDL 
cholesterol, triglycerides, LDL cholesterol, TC/HDL ratio) and glucose. The 
additional 10 mL during the baseline blood draw on visit 2 and 3 will be used for 
analyses of peripheral blood mononuclear cells, which provide valuable 
information about immune function. Immediately after acquisition of venous 
blood, plasma or serum will be separated by centrifugation (3000g at 4°C for 15 
minutes), placed in aliquots, and stored at -80°C. 
 
 
Blood pressure 
Blood pressure will be measured in the arm using an automated microprocessor 
controlled ambulatory blood pressure monitor (Mobil-O-Graph 24 PWA, I.E.M, 
Stolberg Germany) in the supine position according to the guidelines of the 
American Heart Association. BP will be measured twice, one minute apart, and 
the average of the two values will use as resting BP. If the two BP measurements 

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differ by more than 5 mmHg, a third measurement will be taken. Cardiovascular 
variables such as derived ascending aortic BP waveform and central arterial 
indices (PWV and pulse wave analysis), stroke volume and cardiac output can 
be calculated from the pressure waveforms. 
 
For central blood pressure, radial artery pressure waveforms will be attained in 
the supine position from a 10-second epoch using applanation tonometry and a 
high-fidelity strain gauge transducer (Millar Instruments, Houston, TX) [43]. A 
central aortic pressure waveform is reconstructed from the aforementioned radial 
artery pressure waveform using a generalized validated transfer function 
(SphygmoCor, AtCor Medical, Sydney, Australia). From this reconstructed 
waveform, aortic pressures, end-systolic pressure, and sub-endocardial viability 
index (a ratio of diastolic to systolic pressure-time intervals) will be obtained [43]. 
Only recordings receiving an in-device quality index > 80% will be included in 
analysis [43].This index is derived from a weighted algorithm that examines 
average pulse height (average height of the pulse waveforms measured), pulse 
height variation (amount of variation in the height of the pulse waveforms 
measured), and the diastolic variation (an index of baseline pressure consistency 
during the measurement attained from the variation in the diastolic inflection point 
of the pulse waveform).  Previous work shows that reproducibility of measures 
attained from this technique is high [44]. 
 
 
Pulse wave velocity (PWV) 
Approximately 20-sec of pressure waveforms will be collected at the brachial, 
common carotid, and femoral arteries using a high-fidelity strain-gauge 
transducer. PWV will be calculated from the distances between measurement 
points and the measured time delay between proximal (carotid) and distal 
(femoral) waveforms (NIHem, Cardiovascular Engineering Inc.). The brachial 
waveforms will not be used for PWV calculations, but are necessary for 
calibrating carotid blood pressure (described below). 
 
Carotid Blood Pressure 
Carotid artery pressure waveforms obtained during the measurement of cf PWV 
will be ensemble averaged to construct a carotid pressure waveform. This carotid 
waveform is then calibrated to brachial waveforms and brachial mean and 
diastolic pressure. The resulting carotid systolic pressure is necessary for 
calibrating carotid stiffness measures (described below).   
 
 
Flow-Mediated Dilation (FMD) 
Brachial artery vasodilator function will be noninvasively measured through 
assessment of brachial artery dilatation using ultrasonography (Hitachi Aloka, 
Alpha 7 Japan). The brachial artery will be imaged in longitudinal section, 5–10 
cm proximal to placement of a blood pressure cuff, just below the antecubital 
fossa, using a high frequency (5–13 MHz) linear array probe. Endothelium-

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dependent, flow-mediated dilatation (FMD) of the brachial artery will be 
measured at baseline and again for 5 minutes following ischemic stimulus 
(inflation of a blood pressure cuff around the forearm to 250 mmHg for 5 
minutes).  After cuff deflation, resultant reactive hyperemia is calculated as the 
flow change from baseline and expressed as a percentage change in blood flow 
[45]. Analysis of the FMD will be carried out using a semi-automated edge 
detection software system. This system requires an ECG and will be measured 
by placing 3 electrodes on the torso of the subject. Arterial diameter response will 
be calculated as percentage change in brachial artery diameter from baseline 
and controlled for the shear stimulus created by the hyperemic flow. The 
computerized image analysis system with edge-detection software significantly 
attenuates observer error over manual methods and allows the detection of 
changes in endothelial function with substantially fewer subjects. This method 
conforms to the guidelines set out for the ultrasound measurement of 
endothelium-dependent FMD of the brachial artery [45]. 
 
Near-Infrared Spectroscopy (NIRS) 
Tissue oxygenation saturation will be measured non-invasively by near-infrared 
spectroscopy (OxiplexTS, ISS Inc., Champaign, IL) on the forearm during the 
flow mediated dilation. The small NIRS sensor will be held in place with a flexible 
elastic band.    
 
Transcranial Doppler  
Cerebral blood flow velocity will be measured for 7 minutes in a resting state in 
the middle cerebral artery (MCA) using two 2-MHz transcranial Doppler 
ultrasound probe (TOCM Neurovision, MultiGon Industries, INC. Elmsford NY) 
fitted with a specific cushioned headpiece to hold the probe in place. The Doppler 
probe will be placed at the temporal window on the side of the head to insonate 
the vessels. The blood velocity wave form will be recorded continuously at 500 
Hz and stored off-line for subsequent analysis of the systolic, diastolic and mean 
blood flow velocities. Brain blood velocity is influenced by carbon dioxide, thus, 
we will monitor end tidal CO2 throughout this testing period. A 5-minute average 
at the end of the 7 minute period will be used for analyses. 
 
Carotid Ultrasonography 
 
Carotid stiffness: 
Carotid stiffness is a common clinical marker and independent risk factor for 
future vascular events. Carotid stiffness will be assessed using high fidelity 
ultrasound (Hitachi Aloka Alpha 7, Japan) and high frequency (7-13 MHz) linear 
probe. Carotid artery distension will be assessed using wall-tracking and 
simultaneous measurement of blood velocity. Stiffness measures are generally 
calculated by adjusting carotid artery compliance for changes in carotid blood 
pressure. Measures of carotid stiffness will include carotid B-stiffness, Elastic 
modulus, and PWV-B using the following equations:  
B-stiffness = (log P1/P0)/(D1-D0/D0);   
Elastic modulus = (P1/P0)/((D1-D0)/D0) 

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PWV-B = (√((B-stiffness) x P0)/2ρ) 
where P1 and P0 are the highest (systolic) and lowest (diastolic) carotid pressures 
and D1 and D0 are the maximum (systolic) and minimum (diastolic) diameters. 
 
Carotid Blood Flow:  
Carotid blood flow will be measured on the neck using the same ultrasound 
probe described for carotid stiffness. Carotid artery diameter and blood velocity 
will be measured with the probe placed on the on the skin over the carotid artery 
with 60 degree insonation angle. Common carotid blood flow will be assessed 
approximately 1-2 cm proximal to the carotid bifurcation. Internal carotid blood 
flow will be measured just distal to the carotid bifurcation to obtain measures of 
carotid blood flow that feed directly into the middle cerebral arteries.  
 
Cognitive Function 
Montreal Cognitive Assessment (MOCA): 
The MOCA is a tool that can be used to systematically and thoroughly assess 
global cognitive status. It assesses different cognitive domains: attention and 
concentration, executive functions, memory, language, visuoconstructional skills, 
conceptual thinking, calculations, and orientation. The maximum score is 30, in 
which a score of 26 or greater is considered normal. The MOCA takes only 8-12 
minutes to administer and is therefore practical to use repeatedly and routinely.  
Symbol Digits Modality Test (SDMT):  
The SDMT is a global test of cognitive impairment and is associated with 
selective attention and working memory. This test involves a visual substitution 
task, whereby the individual has 90 seconds to orally pair specific numbers with 
given geometric figures as quickly as possible. The test score is the number of 
correctly paired shapes/numbers.  
 
N-back: 
The N-back is a working memory task that lasts approximately 8 minutes. 
Participants will be presented with a series of digits one at a time. They will be 
instructed to press the ‘right’ response button if the number presented to them 
matches the digit that was presented 2 numbers previously. Participants will be 
instructed to hit the ‘left’ response button for every digit presented to them that 
does not match the number 2 digits prior. This test is widely used in cognitive 
psychology as a measure working memory and has been used previously 
following acute perturbations. 
 
Heart function (cardiac echocardiography)  
Cardiac output (Q), stroke volume (SV), and end systolic volume (ESV) will be 
assessed at rest using two-dimensional echocardiography via a Hitachi Aloka 
Alpha 7 system (Tokyo, Japan). With subjects in the left lateral position, 
measurements will be obtained using the four-chamber apical view. The interior 
of the left ventricle will be traced manually during both end systole and end 

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diastole. Volumes will be measured using Simpson’s rule. Stroke volume will be 
calculated by subtracting EDV from ESV. Q will be calculated as HR multiplied by 
SV. Three beats will be measured and the average will be used in the analyses. 
Ejection fraction (EF) will be calculated from the ventricular volumes and 
expressed as a percentage of ESV to EDV.  Mitral valve velocities will be 
obtained from the apical 4 chamber view, whereby E, A, and E/A will be 
measured.  The slope of the inflow will also be determined.  Tissue Doppler will 
also be performed to obtain E' with E/E' calculated.  Lastly, speckle tracking will 
be performed using Aloka’s tissue tracking software.   
 
Maximal Oxygen Consumption (VO2peak) 
Cardiorespiratory fitness (VO2peak) will be measured using an incremental 
exercise test on a motorized treadmill following a Modified Balke protocol while 
analyzing expired gases (TrueOne, Parvo Medics, Sandy, UT) to determine 
maximal oxygen consumption. Due to differing fitness levels by design, 
participants will choose a comfortable walking/jogging pace to perform a 5-
minute warm-up at 0% incline. The work rate will increase by 2.5% grade every 2 
minutes up to 12.5%, after which speed will increase 0.5 mph/minute with 
constant grade until exhaustion. VO2peak (ml∙kg-1∙min-1 ) will be based on 
highest recorded 20-second VO2 value when two of three criteria are satisfied: 1) 
respiratory exchange ratio ≥1.10; 2) peak heart rate within 10 bpm of age-
predicted maximum; or 3) peak rating of perceived exertion ≥17. Heart rate, 
blood pressure, and oxygen uptake will be monitored throughout the exercise 
and recovery. 
 
For Specimen Collection Studies  
Blood samples (15 ml each; 30 ml Visit 1, 15 ml Visit 2, 30 ml Visit 3) will be 
collected from participants following standard procedures. All study personnel 
collecting blood samples have been trained by UIC’s CCTS/CRC staff. The 
samples will be analyzed for C-reactive protein and interleukin-6 for inflammatory 
markers, oxidized LDL as a marker of oxidative stress, and total antioxidant 
capacity and ascorbate for antioxidant capacity (in which additional analyses on 
inflammatory markers may be performed) following the specified ELISA 
procedures. Samples will be stored separate from the study data and be de-
identified. The samples will be kept indefinitely for future analysis if specific 
consent is provided by participants. If consent is not provided, samples will be 
properly destroyed following study completion.  
 
Studies involving use of product  
Participants will receive a Typhoid Vi Polysaccharide Vaccine (Typhim Vi, Sanofi 
Pasteur SA). It will be administered intramuscularly at the deltoid (upper arm) of 
the non-dominant arm in a dose of 0.5 mL by a registered nurse. The product will 
be purchased from the manufacturer through UIC Investigational Pharmacy. The 
vaccination will be stored at 35° to 46° F in a temperature controlled refrigerator 

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in the Integrative Physiology Lab in the DHSP Building in a locked room in a 
controlled access suite. On-study personnel will have access. A log will be kept 
with date and subject number for each dose of the vaccine used. Adverse events 
will be immediately reported to the principal investigator, who will complete an 
Adverse Event Report Form. If it is deemed a severe adverse event, it will be 
reported to the IRB within 24 to 48 hours. Based on data from clinical trials with 
over 4,000 subjects, adverse reactions may consist of: local tenderness, pain, 
induration or erythema at the injection site; or, a systemic response of malaise, 
headache, myalgia, nausea, diarrhea, feverish, or vomiting. No severe or 
unusual side effects were observed and most adverse experiences were 
generally limited to 48 hours.    
 
Participants will also receive 2g Vitamin C on the first and third visit. It will be 
administered orally. Supplementation will be ingested in front of laboratory 
personnel. This supplementation does not exceed daily recommendation and has 
been previously shown to acutely improve endothelial function[35–39]. The 
product will be purchased and stored in the IPL in the DHSP Building. On-study 
personnel will have access. A log will be kept with the date and subject number 
for each dose given. Adverse events will be immediately reported to the principal 
investigator, who will complete an Adverse Event Report Form. If it is deemed a 
severe adverse event, it will be reported to the IRB within 24 to 48 hours. There 
are no known risks associated with the administration of Vitamin C. The dosage 
of antioxidant vitamins are the same as those sold over the counter and safe. No 
side effects have been reported with this dose. 
 
7.0 
Expected Risks/Benefits 
1. Typhoid vaccine: Mild reactions to the typhoid vaccine may occur, such as 
fever, headache, redness/swelling at the site of injection, nausea, or muscle pain. 
You may also faint or an allergic reaction may occur. The risk of serious harm or 
death is extremely small and serious problems from the typhoid vaccine are very 
rare.  Participants with a history of adverse reactions to the typhoid vaccine, will 
be removed from the study. 
2. Health information: Risk of loss of privacy or confidentiality of health 
information. To minimize the risk, all data will be stored coded. 
3. Questionnaires: Participants may feel uncomfortable providing personal 
information in the questionnaires. 
4. Blood pressure: Blood pressure will be measured using a cuff around the arm. 
Subjects may feel some discomfort when the cuff is inflated on the arm or may 
feel some tingling in the fingers. 
5. ECG (Heart rate): Skin redness may appear where the electrodes are placed on 
the skin for the ECG. 
6. Pulse wave analysis and Pulse wave velocity: No known risks. 
7. Ultrasound: No known risks. All efforts will be made to maintain patient’s 
modesty. The gel used during the ultrasound may feel cool and sticky and is 
hypoallergenic (not likely to cause an allergic reaction) and washes off easily.   

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8. Fasting: May cause mild discomfort and hunger pains. If participants develop 
any further symptoms due to fasting, we have snacks and drinks (water and 
juice) available. Participants will not have to perform the treadmill test in the 
fasted state, reducing risk.  
9. Flow-mediated dilation: Subjects may feel uncomfortable (i.e. the arm may “fall 
asleep”) when the blood pressure cuff is inflated for 5 minutes during flow-
mediated dilation but this will subside when the cuff is released.  
10. Treadmill test (VO2max): The treadmill test may result in muscle soreness, 
feeling tired, or out of breath. The soreness should go away in a few days.  
Participants may also feel dizzy, faint, trip, fall, or sprain an ankle during the 
testing.  Spotters will be used during the test.  A mouthpiece will be worn to 
measure breathing so participants may feel uncomfortable. Participants may feel 
warm during the test. Temperature will be kept at a comfortable temperature 
between 72-75 degrees Fahrenheit. Additional fans will be provided, as needed, 
to keep the participant cool and comfortable.  Participants will be instructed that 
they can stop the test at any time. Other rare, but serious, risks associated with a 
peak exercise test include: rapid or irregular heart rhythms, chest pain, heart 
attack, and very rarely death. All individuals assisting with the treadmill test are 
certified in cardiopulmonary resuscitation (CPR) and automated external 
defibrillator (AED) use. An AED is readily available, as well as a landline phone 
to call emergency services.  
11. Blood draw: Subjects may experience mild pain, bleeding or bruising at the site 
of the blood draw. They may also faint or get a mild infection at the site of the 
draw. 
12. Near-infrared spectroscopy: No known risks.  
13. Transcranial Doppler: Participants may find the headpiece for the brain blood 
flow measurements uncomfortable and may develop a headache from wearing 
the head piece over a prolonged period of time (>45 min). The headpiece will be 
adjusted so that it is as comfortable as possible and the short time frame for wear 
(<15min) should minimize likelihood of a headache developing.  
14. Cognitive Function Tests: No known risks.  
15. DEXA: The DEXA scan involves radiation exposure: The duration of the scan will 
be less than 10 minutes. You will be exposed to a radiation dose of less than or 
equal to 0.3 mrem based upon manufacturer’s specifications and pre-determined 
calculations. This amount of radiation exposure is minimal given the estimated 
effective equivalent dose is below 100 mrem per year, a limit set by the Nuclear 
Regulatory Commission for “general public” exposure. 
16. Vitamin C: There are no known risks associated with the administration of the 
Vitamin C, as the dosage does not exceed the upper limit for dietary 
consumption. Additionally, any excess will be excreted out. No side effects have 
been reported with these doses. 
 
Expected Benefits: 
 
There will be no direct benefit to the subjects for participating in this research. 
 
 

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8.0 
Data Collection and Management Procedures 
Subject identification will be coded by numerical values. The subject number will 
be linked to a master key with subject identifiers.  The master key, informed 
consent, health history questionnaires, and documents that provide subject 
identifiers will be stored separately in a locked cabinet located in the IPL, which is 
only accessible by key. Only study personnel will have access to this information.  
During data analyses, shall any abnormalities be discovered regarding the 
echocardiogram procedure, a letter will be sent to participants suggesting the 
participant contact their physician for follow-up. 
 
Upon completion of the study, the master key will be destroyed. Subject 
demographics, such as height, weight, resting HR, and resting BP, will be 
recorded directly onto a coded data collection form. Data from each subject will 
be compiled, coded, and saved onto a computer hard drive that is only 
accessible to study personnel. Electronic data will be stored indefinitely following 
completion of the study. If the subject agrees to allow us to contact them for 
future research studies, we will store (by paper and electronically) their name, 
race, age, sex, height, weight, phone number and email address indefinitely 
following completion of the study in a separate document.  
 
9.0 
Data Analysis 
Statistical data analysis will be conducted using IBM SPSS statistical analysis 
software. This study is a hypothesis generating study, designed to test the 
anticipated relationship of fitness with aging on vascular function and oxidative 
stress during an acute bout of inflammation, in generally healthy individuals. This 
study will provide preliminary data to conduct power analyses for future studies.       
 
 
10.0 Quality Control and Quality Assurance 
All raw data will be checked by Elizabeth Schroeder, as it is collected and all 
procedures of the protocol will be performed by trained personnel.  Biweekly 
examination of the analyzed data will be performed by Dr. Fernhall to ensure 
high quality analysis is being performed.   
 
11.0 Data and Safety Monitoring 
In the event of an accident or injury occurring during the subject’s visit, only 
emergency care (such as CPR and dialing 911) would be performed by study 
personnel until emergency medical personnel arrive. No other medical care will 
be provided. Minor cases, such as subject distress, will be communicated 
confidentially between the subject and the study personnel. The subject is free to 
withdraw from the study at any time without affecting future care at UIC. This will 
be addressed in the informed consent documentation and will be verbally 
reiterated to the subject on the orientation visit. The informed consent and study 
personnel will express to the subject the methods by which subject confidentiality 
will be maintained.    
 

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12.0 Statistical Considerations 
The data will initially be examined for normality violations, entry errors, and 
missing values. Entry errors will be corrected by double-checking with original 
data files. Non-normally distributed data will have an appropriate transformation 
applied to achieve normality (i.e. log transformation, square root, etc.).  
  
All analyses will be adjusted for age, sex, and body mass index, which are all 
factors known to influence hemodynamic parameters. Additionally, due to 
multiple tests, a correction will be applied for multiple comparisons (α divided by 
the number of tests). Significance level will be set at α=0.05, with a Bonferroni 
correction used to correct for multiple comparisons.  
 
Aim 1: The effects of acute inflammation on endothelial and microvascular 
function will be tested with a two-way group (3) by time (2) repeated measures 
ANOVA (V2 and V3 baseline). We expect older high fit adults to have similar 
vascular reactivity as young adults, i.e. decreased FMD and NIRS reperfusion, 
suggesting that fitness maintains vascular reactivity during an inflammatory insult 
with age. If no difference in the response to acute inflammation is seen between 
low and high fit older adults (not expected), this would indicate fitness is not 
protective with age during acute inflammation. 
 
Aim 2: The effects of ascorbic acid ingestion will be tested with a two-way group 
(3) by time (2) repeated measures ANOVA to determine if the antioxidant caused 
changes at Visit 1 (baseline) or Visit 3 (acute inflammation). A two-way group (3) 
and time (3) repeated measures (V2 baseline, V3 before and after Vit C) ANOVA 
will determine if the antioxidant attenuated endothelial function during acute 
inflammation. At baseline, we expect ascorbic acid to improve vascular function, 
i.e. increase FMD and NIRS reperfusion, in older low fit adults, with no changes 
seen in young or high fit older adults. During acute inflammation, we expect 
ascorbic acid to restore vascular function to baseline in young and older high fit 
adults. If ascorbic acid does not alter vascular function during acute 
inflammation, this would indicate another mechanism other than oxidative stress 
is contributing to vascular dysfunction. 
 
13.0 Regulatory Requirements 
13.1 Informed Consent  
Oral consent will be obtained by phone or email during the screening 
process. All subjects will receive an informed consent document to read 
and sign upon arrival to the IPL during the first visit. The informed consent 
will describe the protocol of the study, the intended risks, and the intended 
benefits of study participation. After reviewing the information in the 
informed consent, individuals may decline to participate. For those who 
are willing to participate in the study, they will sign the informed consent 
document and continue with the visit. The original signed informed 

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consent document will be stored in a locked cabinet that will be accessible 
to IPL members and a copy will be provided to the subject.  
13.2 Subject Confidentiality  
Subject identity will be numerically coded. All data recorded for that 
subject will be labeled and stored under that numeric code. All computers 
used to record data are password protected.  
13.3 Unanticipated Problems 
When an unanticipated problem is identified, the staff member identifying 
the adverse event will immediately report it to the principal investigator. 
The principal investigator will then contact the participant and an Adverse 
Event Report Form will be completed.  If the principal investigator feels 
that a serious adverse event has occurred, a report of the serious adverse 
event will be forwarded to the UIC IRB within 24-48 hours of occurrence. If 
the event is not classified as a serious adverse event, it will be reported to 
the UIC IRB upon the next renewal.      

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