Dietary Nitrate Supplementation and Physiological Function in Older Adults
Phase: NA
Conditions: Aging
Sponsor: Darren P Casey

 
 
 
 
 
 
 
Dietary Nitrate Supplementation and Physiological Function in Older Adults   
 
NCT02593305 
 
July 1, 2015 
 
 

Blood pressure responsiveness in older adults following dietary nitrate 
supplementation. 
 
Principal Investigator: 
Darren P. Casey, Ph.D. 
 
 
Co-investigator(s):  
Kenichi Ueda, M.D. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Abstract 
 
1 in 3 adults in the U.S. have hypertension which is known to increase the risk of heart 
disease and stroke.  Blood pressure regulation is mediated by many factors including but 
not limited to metabolic factors, pressure/stretch sensing receptors located in the aorta 
and carotid arteries, endothelial cell function, and kidney function (via water and sodium 
absorption/excretion).  It has been shown that many of these regulatory mechanisms are 
altered and/or become dysfunctional with aging.  Specifically older adults commonly 
demonstrate a decreased sensitivity in pressure receptors and/or endothelial dysfunction.  
Additionally with aging, there is an increase in sympathetic nervous system activity and 
arterial stiffness which can contribute to higher blood pressure.  The important 
vasodilator, nitric oxide (NO), has been implicated as a key signaling molecule involved 
in several of the mechanisms in blood pressure regulation outlined above.  A decreased 
NO production or bioavailability is thought to contribute to several of the physiologic 
changes that come with aging.  Therefore, the overall aim of this study is to use a 
randomized, double-blind crossover, placebo controlled study to examine the effects of 
acute dietary nitrate supplementation (to boost bioavailable NO) on various parameters 
of blood pressure regulation and responsiveness in normal healthy older adults.  Our main 
hypothesis is that 4 weeks of dietary nitrate supplementation will decrease the blood 
pressure responses in older adults in response to a variety of physiological stressors.   
 
 
Research Plan 
 
I. Specific Aims: 
 
Specific Aim 1:  Determine whether carotid chemoreceptor sensitivity is altered with 
healthy aging.  We will measure heart rate, blood pressure, and ventilatory (minute 
ventilation) responses to acute hypoxia in young and older adults.  We hypothesize 
carotid body chemosensitivity to hypoxia is augmented with healthy aging.   
 
Specific Aim 2:  Determine whether increasing NO bioavailability via inorganic nitrate 
supplementation 1) attenuates carotid chemoreceptor sensitivity and 2) decreases blood 
pressure responsiveness in older adults.  We will measure heart rate, blood pressure, 
and ventilatory (minute ventilation) responses to various physiological stressors in older 
adults before and after 4 weeks of beet root juice supplementation.  We hypothesize 
carotid body chemosensitivity to hypoxia and blood pressure responsiveness to exercise 
and exposure to cold will be reduced following dietary nitrate supplementation via beet 
root juice.  In addition, we hypothesize improvements in each of these reflex responses 
will be associated with the change in circulating nitrate and nitrite levels. 
 
 
 
 
II. Background 
 

Older adults are known to exhibit increased sympathetic nervous system activity (SNA), 
which is predictive of morbidity and mortality.  This increase in SNA is related to elevations 
in blood pressure (BP) in older adults (17, 18, 19).  The exact mechanisms responsible 
for the increase in sympathetic activation with aging are not completely understood, but 
may be related to alterations in baroreceptor sensitivity (BRS) (9, 12, 35, 37).  Additionally, 
there is evidence to suggest carotid chemoreceptors in older adults are “overactive” and 
thus contribute to increases in sympathetic activation.  However, controversy exists 
regarding aging and chemoreceptor activity such that data regarding the effect of aging 
on carotid chemoreceptor activity is inconclusive.  Specifically, older adults have been 
shown to exhibit increased (7, 12, 30), decreased (13, 16, 25, 30, 40, 44, 46), or preserved 
(1, 42, 43, 48) carotid body chemoreceptor activity (as assessed by the heart rate and/or 
ventilatory response to acute hypoxic exposure).   
 
The Carotid Chemoreceptors:  The carotid chemoreceptors are primarily known for their 
oxygen sensing capabilities.  More recently, these peripheral chemoreceptors have been 
found to sense and respond to a variety of other stimuli (e.g. acidosis, hypotension, 
hyperthermia, etc).  In addition, the chemoreceptors have been identified as a potential 
therapeutic target for sympathetically mediated diseases (e.g. heart failure, hypertension, 
insulin resistance) such that removal of the carotid bodies can improve prognosis (38).  
For these reasons, the carotid chemoreceptors may be a viable therapeutic target for 
many of the comorbidities associated with normal aging. 
 
Baroreflex and Chemoreflex Interactions:  There are known interactions between the 
baroreflex and chemoreflex, such that activation of one will blunt responsiveness of the 
other.  For example, when the baroreceptors are stimulated by intravenous phenylephrine 
(resulting in an increase in blood pressure), the heart rate response to hypoxia is 
attenuated (49).  Similarly, when the carotid chemoreceptors are stimulated with hypoxia, 
baroreflex sensitivity may be reduced (8).  Taken together, not only may altered 
chemoreceptor sensitivity with aging contribute to chronic sympathoexcitation, it may also 
explain, in part, why baroreflex sensitivity is reduced in this population.  Thus, by 
attenuating chemoreceptor activation with a nitric oxide (NO) donor, improvements in 
baroreflex sensitivity may be observed. 
 
Nitric Oxide Bioavailability and Aging:  As humans age, there is a decrease in nitric 
oxide (NO) bioavailability due to endothelial cell dysfunction which causes less enzymatic 
production of NO through L-arginine-NOS pathway.  Inhibition of endothelium-dependent 
relaxation due to decreased NO bioavailability is the most prominent feature of endothelial 
dysfunction. Loss of NO bioavailability occurs early in human vascular disease and is a 
contributing factor to abnormal vasomotion, altered blood flow, and ischemic symptoms 
(36).  Older adults are known to exhibit decreased bioavailability of the important 
vasodilator, nitric oxide.  Interestingly, NO has been shown to have an inhibitory effect on 
carotid body activity (14, 21, 51, 52). Consistent with this, nitric oxide donors can inhibit 
sensory discharge in the carotid sinus nerve (21, 38) and depress the ventilatory 
response to acute hypoxia (14).  Additionally, in animal models, injection of NO alters 
baroreceptor activity (34).   It has also been shown that risk of hypertension is decreased 
when baseline levels of BRS is increased (9).  Taken together, decreased nitric oxide 

bioavailability with aging may contribute to increased carotid chemoreceptor activity and 
altered baroreceptor sensitivity.  
 
NO Bioavailability: NOS vs. nitrate-nitrite pathways. It is widely known that the L-
arginine-NOS system significantly contributes to the overall production of NO.  Until 
recently the inorganic anions nitrite (NO2) and nitrate (NO3) were considered inert end 
products of NO metabolism. However, accumulating evidence suggests that NO2 and 
NO3 metabolism occurs in the blood and various tissues to form NO (4, 31, 32).  Thus, 
nitrite reduction (i.e. nitrate-nitrite-NO pathway) represents an alternative and 
differentially regulated system for NO generation that operates in parallel to the classic L-
arginine-NOS pathway (33).  Therefore, increasing plasma NO2 and NO3 levels in older 
adults may enhance NO bioavailability and subsequently improve blood pressure 
responsiveness to physiological stressors that have previously been demonstrated to be 
altered with aging and/or involve NO mediated mechanisms.   
 
Benefits of dietary nitrate supplementation:  In recent years data have accumulated 
to suggest that dietary nitrate supplementation can have physiological and therapeutic 
effects.  In healthy young humans acute oral nitrate supplementation improves exercise 
efficiency and tolerance (2, 3, 27, 28).  Dietary nitrite also reverses age-associated 
vascular endothelial dysfunction and large artery stiffness in mice via restoration of NO 
bioavailability and reductions in oxidative stress and inflammation (47).  Furthermore, a 
number of studies in animals have reported a protective effect of low dose nitrite against 
ischemia-reperfusion injuries to various organs (10, 15, 22, 26, 54). Interestingly, acute 
ingestion of dietary nitrate prevents endothelial dysfunction induced by an acute ischemic 
insult in the forearm of healthy humans (53).  Acute (hours) and short term (1-3 days) 
dietary inorganic nitrate consumption has also been shown to have favorable effects on 
isolated measurements of resting blood pressure (BP) in healthy volunteers (23, 28, 29, 
53). Additionally, chronic dietary nitrate supplementation can have a sustained effect on 
BP in rats (41).  To our knowledge the medium term effects (4 weeks) of dietary nitrate 
supplementation on BP have not been examined in healthy adults.  Moreover, the longer 
term effects (weeks to months) of dietary nitrate on vascular function and/or arterial 
stiffness in humans are unknown. 
 
Taken together, augmented carotid chemoreceptor sensitivity is known to increase 
sympathetic nervous system activity and contributes to pathological conditions.  In this 
way, the carotid chemoreceptors have been recently sought as a potential therapeutic 
target for sympathoexcitatory conditions.  Controversy exists regarding the effect of aging 
on carotid chemoreceptor activity and the potential mechanisms involved.  Given nitric 
oxide is known to attenuate chemoreflex activity and older adults often exhibit reduced 
nitric oxide bioavailability, it is reasonable to propose carotid chemoreceptor activity is 
increased with aging and can be reduced with treatment of a NO boosting agent (beetroot 
juice).  Furthermore, given known interactions between the chemoreceptors and 
baroreceptors, we propose dietary nitrate supplementation (via beet root juice) in older 
adults will also result in an improvement in baroreflex sensitivity that is correlated with 
reductions in chemoreflex sensitivity.  With these ideas in mind, we seek to systematically 

examine carotid body chemosensitivity in older adults and the potential mechanisms 
behind these observations. 
 
In this proposal, we aim to examine the effects of 4 weeks of dietary nitrate 
supplementation on peripheral chemoreceptor sensitivity, spontaneous baroreflex 
sensitivity, and blood pressure responsiveness to a variety of physiological 
stressors in older healthy adults.  We will also examine the effect of dietary nitrate 
supplementation on measures of central artery stiffness and aortic blood pressure 
in older adults. 
 
 
 
III. Research Design and methods 
 
Subjects: 
A total of 25 normal healthy subjects will be recruited for this study.  We will recruit 15 
older (60-85 years of age) and 10 young (18-35 years of age) adults.  The young subjects 
will serve as a control study group and will not participate in the intervention portion (i.e. 
dietary nitrate supplementation) of the study.  Exclusion criteria include hypertension, 
hyperlipidemia, smoking, heart disease, diabetes, body mass index (BMI) ≥ 30 kg/m2, 
autonomic disorders and other conditions or medications that might normally alter blood 
pressure responses. They will not be taking any medications except for oral 
contraceptives (in young women).  Subjects who engage in rigorous physical exercise 
programs (greater than 45 minutes in duration and exceeding 3 times per week) will be 
excluded.  Subjects that have an aversion to beets will also be excluded.  Subjects will 
be pre-screened by telephone and a brief health history will be assessed.  Young women 
will be studied in the early follicular phase of their menstrual cycle (or the low- hormone 
phase of oral contraceptive use), to minimize any effects of reproductive hormone status, 
which may make interpretation of results difficult.  All females of child bearing potential 
will have a negative pregnancy test before starting the study.  Recruitment will consist of 
a University-wide email to students, faculty, staff, and retired faculty.   
 
Experimental Procedures: 
 
1.  Subject Monitoring. Heart rate will be monitored by 3-lead ECG. Beat-to-beat arterial 
pressure will be measured using a Nexfin device placed on the middle finger.  Respiratory 
rate and tidal volumes will be measured by turbine (flow-volume turbine, Vacumetrics).  
End-tidal CO2 and inspired O2 will be measured by capnography.    
2.  Venipuncture and Blood Draw.   Single venous blood samples (~15-20 mL) from an 
antecubital vein using aseptic technique will be obtained on each study day for 
determination of nitric oxide metabolites (nitrate and nitrite) and cGMP.  Fasting glucose 
and lipid concentrations will be determined from the blood sample obtained on the first 
study day. 
3. Applanation Tonometry. High-fidelity radial artery pressure waveforms will be 
recorded by applanation tonometry of the radial pulse in the right wrist using a “pencil 
type” micromanometer (Millar Instruments, Houston, Texas).  The radial blood pressure 

(BP) and waveforms will be calibrated from the systolic and diastolic brachial artery BP 
(cuff). A validated, generalized transfer function will be used to generate the 
corresponding aortic pressure waveform (SphygmoCor system, AtCor Medical, Sydney, 
Australia). Additionally, ECG gated arterial waveforms will be obtained from the carotid 
and femoral arteries. Pulse wave velocity (PWV; index of arterial stiffness) will be 
determined using the Sphygmocor PVX system.  The time (t) between the feet of recorded 
pressure waves will be determined as the mean of 10 consecutive cardiac cycles. PWV 
is calculated by the system software from the distance (D; meters) between measurement 
points and the measured time delay (t): PWV = D/Δt (m/s). 
4. Spontaneous Baroreflex Test. Heart rate will be measured via ECG and matched to 
beat-to-beat arterial pressure recording over a 10-minute resting period.  Respiratory rate 
and tidal volume will be measured during this time to determine the influence of 
respiratory rate and volume on heart rate.  Computer software will be used to determine 
all sequences of three or more successive heart beats in which there are concordant 
increases or decreases in systolic BP and RR interval. A linear regression will be applied 
to each of the sequences, and an average regression slope calculated for the sequences 
detected during each recording period. This slope represents the cardiac baroreflex 
sensitivity (in milliseconds per millimeter of mercury) by the spontaneous baroreflex 
method (SBR slope).  
5.  Hypoxic ventilatory response. The sensitivity of the carotid body chemoreceptors 
will be assessed using a hypoxic ventilatory response test (completed in duplicate, 
separated by a minimum of 15 minutes of quiet normoxic breathing).  End-tidal CO2 levels 
will be clamped at eupneic values throughout hypoxia by using a partial rebreathe system.  
Hypoxia will be achieved by titrating the level of inspired oxygen using an anesthesia gas 
blender [2 gas reservoirs (21% oxygen, 10% oxygen)]  to achieve an arterial oxygen 
saturation of ~80% SpO2 (lasting ~2-6 minutes).  
This will be followed by 2 minutes of normoxic 
recovery.  Ventilation, expired gases, and arterial 
oxygen saturation (finger pulse oximetry) will be 
continuously measured.  Chemosensitivity will be 
assessed as the slope of the linear regression line for 
minute ventilation (L/min), heart rate (beat/min), or 
blood pressure (mmHg) versus arterial oxygen 
saturation (%) (Figure 1).  
 
Figure 1. Regression line used to determine 
chemosensitivity. 
 
6. Metaboreflex test.  Subjects will be studied while 
in the supine position under standardized conditions.  
Maximal voluntary contraction (MVC) of the nondominant hand will be measured by 
having subjects squeeze a handgrip device at maximal effort three times; the average 
value will be used as the MVC. The MVC will be used to calculate a relative work rate of 
30% MVC for the experimental protocol. Subjects will then rest quietly for 10 min after 
which an isometric handgrip test will be performed for 2 min.  During the last 3 s of the 
isometric handgrip test, an occlusion cuff placed on the exercising arm above the elbow 

will be rapidly inflated to suprasystolic pressure (240 mmHg) and remain inflated for 2 min 
and 15 s (post exercise ischemia, PEI). The additional 15 s of post exercise ischemia is 
incorporated to account for the potential effects of the initial decrease in BP that occurs 
when exercise is immediately stopped.   
8. Rhythmic Forearm Exercise. Rhythmic forearm exercise will be conducted with a 
handgrip dynamometer at a light (~4 kg) and moderate (~8 kg) level of exercise (4 minutes 
each), as previously described (6). In addition, this technique has been adapted to 
facilitate beat-to-beat measurements of brachial artery blood flow via Doppler ultrasound. 
9. Measurement of Forearm Blood Flow by Doppler Ultrasound. Forearm blood flow 
(FBF) will be determined from the simultaneous measures of brachial artery diameter and 
mean blood velocity (MBV).  The echo-Doppler image of the brachial artery will be 
obtained in real time using a hand-held 12 MHz linear probe, operating in B-mode.  The 
probe will be positioned over the brachial artery, approximately 9 cm proximal to the 
medial epicondyle.  Brachial artery blood flow is the product of mean blood velocity and 
artery cross sectional area.  Forearm vascular conductance (FVC) will be calculated using 
blood flow and mean arterial pressure 
9. Cold Pressor Test. After 10 minutes of quiet rest, a 2-minute baseline period will be 
recorded, followed by immersion of the hand in ice water (0-4 degrees C) for 2 minutes. 
Heart rate and blood pressure will be averaged over each minute of cold immersion and 
recovery; these values will be compared to the average values during the pre-stress 
period. 
10.  Plasma Nitrate/Nitrite and cGMP. Plasma nitrate and nitrite levels will be 
determined from blood samples using a Sievers chemiluminescence NO analyzer (NOA 
280i; Sievers Instruments, Boulder, CO). [NO2−] will be determined by addition of plasma 
samples to potassium iodide in acetic acid at room temperature in a gas-sealed purging 
vessel.  To minimize the influence of diet on plasma levels of nitrate/nitrite and 
measurements of physiologic function, all subjects will be asked to follow a 3 day low 
nitrate diet prior to each study day.  cGMP will be determined using an enzyme 
immunoassay (Cayman Chemical). 
11. Dietary Nitrate Supplementation. At study entry, the 15 older subjects will be 
randomized to receive either a beetroot powder containing nitrate (active) or a beetroot 
powder without nitrate (placebo) (Neogenis Sport, Austin TX) for 4 weeks.  After visit 2 
and the subsequent 4-week washout period, all subjects will be crossed over to receive 
either the active or placebo powder (opposite of the one they received at study entry) for 
another 4 weeks (Figure. 2). 
12. Data Analysis and Statistics.   
Sample-size/ Statistical Power:  The primary aim of this investigation is to assess the 
potential efficacy of dietary nitrate supplementation for improving physiologic function on 
blood pressure within older individuals.  With this aim in mind, we have designed a 
randomized, double-blind crossover, placebo-controlled study to assess potential efficacy 
and provide preliminary data for designing a larger trial in the future. Thus the primary 
endpoint is the change from baseline to end of treatment in blood pressure responses to 
various physiological stressors in older individuals. 
Data Analysis:  The primary variable of interest for this investigation is blood pressure 
responses to physiological stressors which will be measured at baseline and at the end 
of each respective 4 week treatment (beetroot juice with nitrate vs. placebo) and analyzed 

as change from baseline. For the primary analysis, the change in pressure response from 
baseline to end of treatment will be compared between treatment groups (beetroot juice 
with nitrate vs. placebo) using a two-way repeated measures analysis of variance 
(ANOVA).  Exploratory analyses will be performed to assess whether improvements in BP 
are positively correlated with changes in indices of arterial stiffness and whether 
improvements in blood pressure responses are associated with changes in vascular 
function.   
 
PROTOCOL - To address Specific Aims 
 
Rationale and hypothesis: 
As humans age, nitric oxide bioavailability decreases and there is an elevation in resting 
sympathetic activity (17, 19).  This increase contributes to increased arterial stiffness and 
blood pressure during rest and exercise.  Blood pressure responsiveness is also 
increased due to many factors including a decrease in the sensitivity of pressure 
receptors located in the carotid arteries and aorta.  Recent evidence has shown that acute 
and short term (days) dietary nitrate supplementation is potentially effective in increasing 
NO bioavailability (5, 23, 27) and has favorable effects on measures of peripheral blood 
pressure at rest (23, 24). We will examine whether longer durations of dietary nitrate 
supplementation (4 weeks) have favorable effects on blood pressure responsiveness to 
a variety of physiological stressors in older adults. 
 
Approach:  
 
15 healthy older subjects will be studied.  All older adults will undergo 4 separate days of 
testing as well as the placebo/supplementation portion of the study (Figure 2). We will 
also recruit 10 young adults to serve as a control group to compare baseline (prior to any 
intervention) data between age groups. The young group will only participate in one day 
of testing and will not undergo any of the placebo/supplementation portions of this study. 
Each group on their respective testing days will be subjected to a battery of tests that 
include a blood draw, spontaneous baroreflex testing, two separate consecutive hypoxic 
ventilatory response tests, handgrip exercise (both isometric and rhythmic),and a cold 
pressor test.   The older group after undergoing testing on visits 1 and 3 will then be given 
their placebo/supplementation beetroot packets to be consumed once per day for 4 
weeks.  On each study day, heart rate will be monitored by 3-lead ECG and beat-to-beat 
arterial pressure will be measured using a Nexfin device on the middle finger during each 
test.  Arterial O2 saturation levels will be monitored by pulse oximetry throughout the 
hypoxic ventilatory tests.  A single venous blood draw will be performed at the start of 
each study day (Figure 3) for the determination of plasma nitrate and nitrite levels. 
 
 
 
 
 
 
 
 
 
Consent  
Experimental day 
1 
Baseline measurements.  Assign 
to placebo/Beetroot supplement 
Experimental Day 
2 
Post beetroot/placebo 
measurements.  
Experimental Day 
3 
Experimental Day 
4 
Post beetroot/placebo 
measurements. 
= 4 week period on supplement 
or placebo 
= 4 week washout period 
Baseline measurements.  Assign 
to placebo/Beetroot supplement 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Expected results, Interpretation, and Follow-up studies:  We predict that following 4 
weeks of nitrate supplementation compared to placebo, older adults will demonstrate a 
decrease in blood pressure responsiveness to each of the physiological stressors 
employed in the lab.  Additionally, we hypothesize that dietary nitrate supplementation 
will have a beneficial effect on central artery stiffness.  If our hypotheses are correct, it 
would suggest a beneficial therapeutic effect of nitrate supplementation in older adults. 
 
General Issues: All techniques in this proposal have been successfully used by Dr. 
Casey for many years and are not expected to present difficulties.  Additionally, Dr. 
Kenichi Ueda (Co-investigator) has extensive experience with venous blood draws in 
humans.   
  
 
 
 
 
 
Human Subjects Research 
 
Recruitment of Subjects 
The subjects will be recruited from the faculty, staff, and students of the University of Iowa 
and from persons in Iowa City, Iowa and surrounding communities.  Subjects will be 
eligible based on their health history questionnaire.   
= Applanation Tonometry 
Figure 3.  Experimental  testing on each study day 
Spontaneous 
baroreflex 
Hypoxic 
ventilatory 
response 
Metaboreflex 
Cold Pressor 
Test 
= 15 minute rest period 
= Blood Draw 
Hypoxic 
ventilatory 
response 
Rhythmic 
forearm exercise 
(4kg, 8kg)  

 
Potential Risks. 
Risks from participation in these studies are not considered minimal. 
1.  Venipuncture.  Risks of venipuncture in the arm include bruise or clot formation and 
infection. Additionally, risks associated with a needle stick and blood draws may include 
feeling light headed or nauseous and/or fainting. 
2. Hypoxic ventilatory response tests: The hypoxic ventilatory response will be 
measured before and after beet root juice supplementation. There are theoretical risks 
related to prolonged exposure to hypoxia, but acute exposures as planned in this proposal 
should not pose any risk to the volunteers.  Pulse oximetry, blood pressure, capnography, 
and heart rate monitoring will be used to ensure adequate gas exchange, circulatory 
parameters, and patient safety when arterial oxygen is altered. We will closely monitor 
subject arterial oxygen saturation to keep saturation at 70% or higher during the testing.  
We will have hyperoxia available in the unlikely case saturations fall below 70%.  
3. Consumption of Neogenis BeetElite Powder. There is no risk associated with 
consumption of beetroot juice.  However, pink/red urine or stools has been reported. 
4. Applanation tonometry. There is no risk associated with arterial tonometry 
measurements. 
5. Forearm exercise.  Isometric and/or rhythmic handgrip exercise can lead to minor 
fatigue in the forearm and soreness for a day or two.  There are no long term risks 
associated with hand gripping exercise. 
6. Cold Pressor Test. The cold pressor test may cause some discomfort, but the subjects 
will be informed that they can discontinue the trial in case of unusual or extreme 
discomfort.  This discomfort lasts only as long as the hand remains immersed in the ice 
water.  
 
 
Protection. 
Each subject will give written informed consent after all questions have been answered 
by an investigator or study coordinator.  Confidentiality of data will be assured by coding 
of subject identities, and that coding will be known only to the investigators.  No identified 
individual data will be presented or published, rather group mean data will serve as the 
basis for comparisons.  Participation in these studies is safe, with minimal short-term 
(days) complications and minimal anticipated long-term risks.  Procedures used in this 
study are performed regularly in the laboratory or clinical setting by the investigators.   
 
Inclusion of Women and Minorities. 
The proposed studies will include healthy young (18-35 yr) and older (50-85 yr) men and 
women of all races and ethnic backgrounds recruited from the faculty, staff, and students 
of the University of Iowa and from persons in Iowa City, Iowa and surrounding 
communities.  This is a therapeutic protocol.  We will strive to enroll an equal number of 
males and females and recruit minority participants at least to the extent representative 
of the participation of minority populations in Iowa City and Johnson County.   
 
Inclusion of Children. 

Children between the ages 18-21 will be studied.  We believe these individuals have 
physiologically mature blood pressure regulating systems.  Additionally, children in this 
age group can give informed consent for invasive procedures.  We will exclude children 
less than 18 years of age because they are unable to give consent. 
 
 
 
 
 

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