Circadian Mechanisms of Cardiovascular Risk in Obesity
Conditions: Cardiovascular Risk Factor, Circadian Dysregulation, Obesity
Sponsor: Oregon Health and Science University

 
 
Body Weight, Sleep, and Heart Health 
ClinicalTrials.gov ID: NCT03388788 
 
 
Oregon Health & Science University  
IRB Protocol: 17489  
 

Version Date:  03.04.2024 
Page 1 of 3 
 
APPROVAL OF SUBMISSION 
November 5, 2025 
 
Dear Investigator: 
On 11/5/2025, the IRB reviewed the following submission: 
IRB ID: STUDY00017489 
MOD or CR 
ID: 
MODCR00033153 
Type of Review: Modification and Continuing Review 
Title of Study: Circadian Mechanisms of Cardiovascular Risk in 
Obesity 
Title of modification This is a continuing review 
Principal Investigator: Steven Shea 
Funding: Name: DHHS NIH Natl Ctr for Advancing Translational 
Sciences, PPQ #: 1010312; Name: Sleep Research 
Society Foundation, PPQ #: 1013221; Name: DHHS 
NIH Natl Heart, Lung, and Blood Inst, PPQ #: 1010921; 
Name: Ford Family Foundation, PPQ #: 1012078 
IND, IDE, or HDE: IND #138055; IND #138056; IND #138054 
Documents Reviewed: • Approval for 138055 
• Regadenoson packet insert 
• Ad_flyer 
• Approval for IND 138054 
• Screen_PET Scanner Pictures 
• REDCap Screening Survey 
• Screen_BDI-II 
• Memo_2018-03-21 
• driving-wavier 
• Ad_Craigslist_2021-03-24.pdf 
• Screen_PROMIS-sleep 
• Questionnaire_Stanford 
• Questionnaire_owl-lark 
• MOU-Emens-2017 
• CHOP_Ad_flyer-halfsheet 
• Screen_Primary care provider release form 
• SRS Career Development Award Application 
• IND # 138056 for S-(4-[3-(tert-butylamino)-2-

Version Date:  03.04.2024 
Page 2 of 3 
 
hydroxypropoxy]-1,3-dihydro-2H-benzimidazole-2-
[11C]-one or S-[11C]CGP12177 
• CHOP_hipaa-woa_2021-04-06.docx 
• Thosar_KL2 funded grant 
• reason for holding IND 
• Perflutren packet insert 
• DSMP 
• DSMB-Appendix-A 
• NHLBI-grant-submission 
• Questionnaire_Poms 
• IND # 138055 for [11C]-meta-hydroxyephedrine or 
[11C]mHED 
• Questionnaire_appetite 
• IND # 138054  [15O]water or Water O15 Injection 
• Screen_info-release 
• NIH NHLBI grant application 
• Screen_SDQ 
• Test_pvt 
• REDCap_Morning Sleep Diary 
• REDCap_Evening Sleep Diary 
• Questionnaire - MINI Interview screen 
• Participant-contact-card 
• sst-draft-20190913-response to ncats (REFERENCE 
ONLY) 
• Questionnaire_PANAS 
• Approval for 138056 
• Test_math 
• Ad_research-match 
• Screen_demographics 
• Questionnaire - MINI Interview 5.0.0 
• CHOP_visual analog scales 
• Questionnaire_exit 
• CHOP_COVID-questions_2020-06-29.xlsx 
• Ionizing-radiation-form 
• Sleep-diary 
• CHOP_protocol_2022-03-04.docx 
• Consent and Authorization-Main 3.24.21 
• Consent and Authorization-Screening 
• Phone Screen Script 
 
The IRB granted final approval on 11/5/2025.  The study is approved until 11/4/2026. 
Review Category:  Expedited Category # 8c 
Copies of all approved documents are available in the study's Final Documents (far right 
column under the documents tab) list in the eIRB.  Any additional documents that require 

Version Date:  03.04.2024 
Page 3 of 3 
 
an IRB signature (e.g., IIAs and IAAs) will be posted when signed.  If this applies to your 
study, you will receive a notification when these additional signed documents are 
available. 
Ongoing PI Responsibilities: 
• Six to ten weeks before the expiration date, submit a continuing review to request 
continuing approval. 
• Submit changes to the project for IRB approval prior to implementation. 
• Submit Reportable New Information per OHSU policy. 
• Submit a continuing review to close the study when the research is completed. 
Guidelines for Study Conduct 
In conducting this study, you are required to follow the guidelines in the document 
entitled, “Roles and Responsibilities in the Conduct of Research,” as well as all other 
applicable OHSU IRB Policies and Procedures. 
Requirements under HIPAA 
If your study involves the collection, use, or disclosure of Protected Health Information 
(PHI), you must comply with all applicable requirements under HIPAA. See the HIPAA 
and Research website and the Information Privacy and Security website for more 
information. 
IRB Compliance 
The OHSU IRB (FWA00000161; IRB00000471) complies with 45 CFR Part 46, 21 CFR 
Parts 50 and 56, and other federal and Oregon laws and regulations, as applicable, as well 
as ICH-GCP codes 3.1-3.4, which outline Responsibilities, Composition, Functions, and 
Operations, Procedures, and Records of the IRB. 
Sincerely, 
 
The OHSU IRB Office 
 

 
Page 1 of 24 
Revised: 11.DEC.2018 
IRB #17489 Protocol 
1. Protocol Title: Circadian mechanisms of cardiovascular risk in obesity 
2. Objectives 
Endogenous circadian rhythms of cardiovascular (CV) risk factors are rarely studied in obese humans. Thus, 
we aim to determine whether obese individuals have elevated morning CV risk because of normal CV 
rhythms on top of their existing increased CV risk (e.g., hypertension and increased sympathetic activation), 
or whether obesity disrupts CV rhythms and thereby furthers CV risk. Using an established multi-day 
circadian protocol in humans performed in dim light with behaviors scheduled evenly across the entire 
circadian cycle, our aims are to determine: (1) if obese individuals have normal or abnormal basal circadian 
CV rhythms, and (2) if obese individuals have normal or abnormal circadian rhythms in CV reactivity to 
stresses.  
 
Specifically, we aim: 
 
a. To test if the circadian amplitudes and phases of important obesity-related CV risk markers are 
affected by obesity. We hypothesize that there will be endogenous circadian rhythms in CV risk 
markers including important hemodynamic variables (blood pressure, heart rate, vascular 
endothelial function), plus circulating markers of prothrombotic state, oxidative stress and 
inflammation in both obese and lean individuals. We expect the mean levels of these markers to be 
impaired in obese individuals along with changes in circadian rhythm amplitude contributing to 
increased CV risk during the morning period of CV vulnerability.  
Sub aim a1: Determine in lean healthy humans if CMF, measured as coronary microvascular 
blood flux, has an endogenous circadian rhythm with lowest function in the morning. 
i. Sub aim a2: Test the hypothesis that people with obesity have impaired coronary 
microvascular blood flux compared to lean individuals, with the exaggerated impairment 
during the morning. 
b. To determine how obese individuals differ in their rhythms of CV responses to standardized stresses 
(i.e. change in posture and exercise) compared to lean controls, with special attention to the 
morning period of CV vulnerability. We hypothesize that impaired sympatho-vagal balance (2) and 
impaired vascular function (3) that are characteristic of obesity will lead to exaggerated CV 
responses to standardized stressors. For example, the morning increase in cortisol is higher in 
people with increased abdominal obesity (4) and the morning surge in blood pressure (BP) is higher 
with increasing central obesity (5) and increasing serum cholesterol levels (6). We anticipate 
replicating this finding, and here we will determine whether this is specific to the subjective morning 
as encoded by the internal circadian clock.  
c.  Although obesity increases efferent sympathetic activity, it is unknown how effector organs 
like the heart adapt to chronically increased sympathetic tone especially with respect to 
changes across the day. We will use short-lived positron emission tomography (PET) ligands 
to measure in the cardiomyocytes the balance between sympathetic pre-synaptic function 
(norepinephrine) and postsynaptic function (beta-adrenergic receptor density) across the 
circadian cycle in lean and obese individuals.  
 
Overall, these studies will help us answer whether CV rhythms predispose obese individuals to increased CV 
disease risk - particularly around the vulnerable morning period. The results will serve as a foundation for clinical 

 
Page 2 of 24 
Revised: 11.DEC.2018 
trials of appropriately timed dosing of medications targeting aspects of the CV system in obesity that increase 
effectiveness while decreasing side-effects. 
 
3. Background 
 
Obesity and cardiovascular risk. The prevalence of obesity in the US has increased dramatically over the last 25 
years such that now over a third of adults are obese, and this is projected to reach 50% by 2030 (7-9). Obesity 
increases the risk for cardiovascular (CV) disease, the leading cause of death in both males and females (10-12). 
Increased body fat, in particular central adiposity, contributes 
substantially to circulating levels of leptin, tumor necrosis factor-α 
(TNF-α), interleukin-6 (IL-6) [leading to CRP release from the liver], 
plasminogen activator inhibitor-1 (PAI-1), and angiotensinogen 
leading to a relatively pro-thrombotic state with chronic 
inflammation, increased sympathetic nervous system (SNS) 
activation, hypertension (HTN) and obstructive sleep apnea (OSA) 
– all notable CV risk factors (13-18). There are also adaptations in 
cardiac structure and function with increased adiposity even in the 
absence of HTN or other pathological states (19,20). To meet 
increased metabolic needs with obesity, blood volume and cardiac 
output increase, leading to heart chamber enlargement, left 
ventricular hypertrophy, reduced diastolic compliance, increased 
left ventricular filling pressure and in some cases, heart failure. 
Obesity related systemic HTN combined with other 
cardiometabolic risk factors leads to coronary heart disease (19). 
Ultimately, there is a fivefold increased risk of death from CV 
disease in obese individuals (21,22).  
 
Circadian pattern of adverse CV events. Overall, obesity increases 
the risk of adverse CV events including myocardial infarction (MI) 
(23,24), sudden cardiac death (SCD) (12,25,26), and stroke (27). 
These adverse events do not occur randomly across the day (28) but are clustered around a morning vulnerable 
period (29-31). This daily pattern of vulnerability likely emerges from three sources. First, CV responses to rapid 
changes in behavior during the morning—including waking from sleep, posture changes, and sudden physical 
activity—precipitate adverse events (17,18,32-34). Second, the time of day itself represents a time of 
vulnerability to CV events due to the function of the circadian clock that times 24 h rhythms in physiology and 
behavior (Clock-Risk Model) (35,36). The likeliest scenario, is a mixture of these, in which a behavior like exercise 
is harmless at most times of day, but may trigger an adverse event in this clock-defined window of vulnerability 
(Clock-Reactivity Model). Thus, we aim to determine how obesity affects: (1) circadian rhythms of CV function 
and (2) the time-of-day dependent reactivity of the CV system to typical physical stressors i.e., posture and 
exercise. 
 
Studying circadian rhythms in humans. To separate the endogenous circadian and behavioral influences on CV 
function in humans, our research team has applied ‘Forced Desynchrony’ protocols – as proposed in the current 
study. This separation is achieved by scheduling all behaviors evenly across all phases of the circadian cycle via 
numerous recurring days that have non-24 h periods while the participant remains in dim light to avoid circadian 
entrainment to light (37,38). Using this technique, we discovered in healthy lean individuals that certain CV risk 
factors surge around the vulnerable morning period for increased MI and CV deaths, including circulating 
cortisol (39), sympathetic activation, cardiac vagal withdrawal, heart rate (HR) (39), (40), platelet aggregability 
(41) and the pro-thrombotic plasminogen activator inhibitor-1 (PAI-1) (42). These circadian rhythms are 
presumably beneficial in healthy people as they prepare individuals for rapid physiological responses associated 
 
Fig. 1. The FD consists of a baseline night 
and day, then ten 5 h 20 m cycles of sleep 
(black) and wake (gray). Stressors are 
scheduled each wake period. Lights are dim 
(≤3 lux). Letters indicate PET scanning for 
cardiac 
sympathetic 
function. 
Each 
participant undergoes up to 3 scans across 
the entire FD, 16 h apart across the circadian 
cycle, in a randomized order (a1, a2 & a3, or 
b1, b2 & b3, or c1, c2 & c3, balanced among 
participants).  

 
Page 3 of 24 
Revised: 11.DEC.2018 
with anticipated rigorous daytime behaviors. But these same rhythms might be deleterious in obese individuals 
due to their increased CV risk. Despite the information on CV rhythms in healthy and generally lean subjects, 
how obesity alters the timing or amplitude of these rhythms is unknown. 
 
4. Study Design 
 
The goals of this study are to elucidate circadian rhythms in CV risk markers in obesity. To meet this goal, we will 
evaluate endogenous circadian rhythms, responses to behaviors and the interaction of behaviors and 
endogenous circadian rhythms in hemodynamic variables (BP, HR, vascular endothelial function) and primary 
physiological regulators, in obese and lean individuals. To study independent effects of the circadian clock, we 
will conduct a 5-day forced desynchrony protocol (FD, Fig 1) where all behaviors such as arousal from sleep, 
eating a meal, performing an exercise test, and sleep will occur evenly spaced across the entire circadian cycle 
by scheduling 10 recurring ‘day’ of 5 h 20 min. Repeated measurements of CV variables throughout the 10 
wake/sleep cycles allow for assessment of independent effects of circadian rhythms and scheduled behaviors. 
Due to repeated measures, we propose to measure selected clinically relevant CV variables that are reliable, 
relatively noninvasive, and independent. 
 
Baseline routine: To stabilize circadian rhythms, participants will maintain a consistent sleep-wake schedule for 
at least 1 week prior to admission to the laboratory. The bedtime will be determined by a participant’s habitual 
bedtime with 8-h time in bed. To ensure compliance to this schedule, each participant’s activity will be 
monitored by wrist Actigraphy (ActiGraph wGT3X-BT, ActiGraph, Pensacola, FL). Actigraphy will provide an 
estimate of daily activity levels and sleep characteristics in real world settings. Participants fill out a sleep diary 
every day, and call into a time-stamped voice-mailbox when going to bed and getting out of bed. If more than 
one deviation (>1 h) from the target times are detected per week, the subject is asked to repeat the screening 
schedule. To identify daily BP patterns, subjects will wear an ambulatory BP monitor (Spacelabs Healthcare, WA) 
for 48-h during this home routine.  
 
Acclimatization and baseline day: Participants report to the laboratory at Oregon Clinical & Translational 
Research Institute (OCTRI) on day 1 and are admitted to an individual room free of external time cues (e.g., 
clocks, radios, computers, visitors and sunlight). Participants maintain contact with study investigators. Room 
temperature is maintained at 21-22.2 °C and light intensity set at £3 lux during scheduled wakefulness and <0.2 
lux (darkness) during scheduled sleep opportunities. Participants will be instrumented for full polysomnography, 
and a Holter monitor throughout the study. Our laboratory suites have a porthole for 24 h blood sampling 
without disturbing sleep and an IV line will be inserted and kept patent by a heparin solution.  
 
Investigators/nurses are present in the lab or in a central control room 24h/day to monitor subject health, data 
acquisition, provide meals, collect biologic specimens, perform tests, and record sleep. A physician is always on 
call when a participant is in the laboratory. An extensive series of written protocols and checklists and team 
practices are used to ensure uniformity in execution of standard procedures.  
 
FD Protocol (Fig. 1): Designed by Kleitman (37), the FD protocol desynchronizes the sleep-wake cycles from the 
circadian pacemaker and is performed in dim light to ensure the circadian pacemaker ‘free-runs’ at its intrinsic 
rate of ~24.1 h (43). At ~6 PM on day 2 after having been awake for 10 h, a participant’s sleep-wake cycle is 
scheduled to a period of 5 h 20 min with a 2:1 ratio of wake: sleep-opportunity.  
 
Imaging: PET imaging of the heart (detailed below in section 6h) will occur up to 3 times for each subject across 
the FD protocol (Fig. 1).  The scans will be a minimum of 16 h apart (Fig. 1) and represent up to 3 different 
circadian phases.  
 
Cardiac ultrasound: Myocardial perfusion imaging (detailed in section 6i) may occur up to 3 times for each 

 
Page 4 of 24 
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participant across the FD protocol. 
 
5. Study Population 
a. Number of Subjects 
To determine the effects of obesity and specifically abdominal fat on our outcomes of interest, we 
will compare 2 groups of participants, ages 25-65: (1) healthy obese [30≤BMI<40 kg/m2] and (2) 
healthy lean controls [18.5<BMI<25 kg/m2]; n=14 in each group. To ensure some degree of stability 
and avoid recent changes due to significant recent weight gain, all obese participants must report 
the presence of obesity with abdominal adiposity for >5 years. Participants must also be free of 
medication use. Based on experience in these protocols, we estimate that we will medically screen 
120 participants in order to complete 28 participants.  
b. Inclusion and Exclusion Criteria 
In order to control for additional varation among obese individuals such as worse sleep at night or 
reduced daytime activity we will screen and match for insomnia, sleep apnea and overall habitual 
activity among our participants. Thus, we will exclude participants with moderate to severe 
obstructive sleep apnea based on an overnight sleep screen (Watch-PAT, Itamar Medical, Israel), we 
will exclude subjects with clinically significant insomnia (sleep efficiency less than desired, and 
residual daytime sleepiness based on the PROMIS Sleep Distrubance and Sleep-Related Impairment 
surveys).. The healthy control group will also be matched for age (+/- 5 years) and sex. 
 
 
Diabetes. For participants who have a fasting blood glucose level of ≥ 100 mg/dL we will measure 
hemoglobin A1c to exclude for diabetes (HbA1c>6.5%).  
 
Hypertension. An upper cut off of 160/100 mmHg during an office BP measure will be used as an 
exclusion criterion as it would not be adviseable to delay initiation of therapy in patients with severe 
hypertension.  
 
Cardiovascular disease. Individuals actively treated or previously hospitalized for heart failure, 
arrhythmia or coronary artery disease, a history of coronary artery bypass grafts and coronary 
stents, and those that use a pacemaker or defibrillator will be excluded.  
 
Psychiatric/psychological suitability. Each participant will undergo a structured interview (Mini 
International Neuropsychiatric Interview) with study staff approved by a qualified OHSU physician. 
This physician will supervise the administration and scoring of a Beck Depression Inventory II (BDI-II) 
questionnaire for each potential participant. Individuals with evidence of psychopathology on the 
BDI-II, or in a structured clinical interview with the physician, will be excluded from study. 
Individuals will be excluded if they have a history of severe psychiatric illnesses or psychiatric 
disorders such as alcoholism, drug dependency, major depression, manic depressive illness, 
schizophrenic disorders, panic disorder, generalized anxiety disorder, post-traumatic stress disorder, 
agoraphobia, claustrophobia, paranoid personality disorder, schizoid personality disorder, 
schizotypal personality disorder, borderline personality disorder, and antisocial personality disorder. 
Finally, individuals who are unaware of specific psychiatric diagnoses and who have a history of 
having been treated with antidepressants, neuroleptic medications, or tranquilizers will be excluded. 
However, a personal history of limited prior counseling or psychotherapy (e.g., for adjustment 
reactions) will not necessarily be exclusionary. Participants must demonstrate a full understanding 
of the requirements and demands of the study. 
 
Drug/alcohol use. Volunteers must be drug-free (including caffeine, nicotine, alcohol and herbal 
medications) for the duration of the screening and study period, with no history of drug or alcohol 

 
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dependency. All participants must be current non-smokers, and are required to have a history of 
less than 5 ‘pack years’ of smoking. A comprehensive toxicological analysis of blood and urine for 
prescription medication, non-prescription medication, herbal remedies/medications and drugs of 
abuse will be carried out for verification of reported non-use during the initial screening and on the 
day of admission to the laboratory.  
 
Medication/drug use. Volunteers must not be taking any prescribed medications. Over the counter 
medications will need to be avoided throughout the at-home period and the in-lab study. As above, 
compliance is verified by comprehensive toxicological assessment before admission. Oral 
contraceptives are allowed, and women will continue to take these throughout the experiment. 
 
Prior shift work. For stability of endogenous circadian rhythmicity, volunteers must have no history 
of working irregular day and night hours, regular night work, or rotating shift work for the 6 months 
prior to the study. They must not have traveled across more than two time zones during the month 
prior to the study. 
 
All candidates who are excluded from study for medical and/or psychological reasons are so informed. In 
addition, they will be encouraged to inform their primary care physician of any elevated blood pressures, 
increased blood glucose levels or obstructive sleep apnea, and to potentially seek treatment.   
 
c. Vulnerable Populations 
No vulnerable populations will be studied. 
 
d. Setting 
All research is performed in the research facilities at Oregon Clinical and Translation Research 
Institute (OCTRI) in the Hatfield Research Center at OHSU. Participant screenings occur in the 
Outpatient Research Unit and in-lab stays occur on 10D. 
 
e. Recruitment Methods 
Using established methods, participants will be recruited from the general population via internet 
(ResearchMatch.com, OCTRI’s research Data Warehouse, advertisements, clinicaltrials.gov), flyers across the 
OHSU campus and at local organizations, as well as Epic Reporting Workbench and OCTRI RVR. Medical 
suitability will be established on the basis of phone screens, medical history, biochemical and toxicology 
screens of blood and urine, and a physical exam conducted by an OHSU physician. Written informed consent 
will be obtained from each volunteer before study participation. 
 
f. Consent Process 
There are two written consents and a verbal consent involved in this study. The verbal consent is to allow 
the researchers to screen for the exclusion criteria in a phone screen. At the time of the phone screen prior 
to collecting any data, we will ask potential participants for verbal consent to keep their answers to the 
phone screen questionnaire for purposes of future recruitment (STUDY00015966).  We will inform the 
potential participant that not giving consent will not affect his/her consideration for participation in the 
current study or their relationship with OHSU.  We will also state that should the participant wish to 
withdraw consent at any time, he/she could do so by contacting the repository guardian or study staff via 
phone, email or written correspondence and request to no longer be approached for research purposes by 
our group. Additionally, we will collect basic demographics, gender/sex, and COVID-19 screening 
information during the phone screen. 
 
Once deemed eligible by the phone screen, participants will receive a summarized explanation of the 
purpose, procedures, risks and discomforts involved in the proposed study in their first screening visit.  The 

 
Page 6 of 24 
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investigator will answer any questions in plain English. Participants will be informed that they will not be 
permitted any timepieces during their stay in the laboratory. Every attempt will be made to acquaint each 
prospective participant with all of the procedures involved in this study in order to minimize the possible 
effect of uncertainty about the experimental procedures.   
 
Before undergoing the initial screening tests, the Screening Consent form, summarizing the information 
given above, will be reviewed with the prospective participant. When the prospective participant is satisfied 
with his/her understanding of the study, and fully comprehends his/her freedom to withdraw at any time, 
the potential participant will be asked to sign the screening consent form if they wish to do so.  
 
If they volunteer, an informed consent (for the intervention) will be presented to them. Investigators will go 
over each aspect of the consent form in plain English and answer any questions the participants may have. 
When the participant is satisfied with his/her understanding of the study, and fully comprehends his/her 
freedom to withdraw at any time, the potential participant will be asked to sign the research consent form if 
they wish to do so.  
 
The investigators will take all necessary steps to answer questions raised by volunteers pertaining to the 
nature, purpose and risks of the study.  An investigator will be available at all times to answer questions the 
participant may have. By both verbal and written instruction, the participant will be explicitly informed of 
his/her freedom to withdraw consent and discontinue participation in the project at any time. 
6. Procedures Involved 
a. Screening: Screening will involve at least two visits. During the first visit, height, weight, waist 
circumference and blood pressure will be measured. BMI will be calculated. Participants will have a 
12-lead ECG. A blood draw will be collected for testing basic metabolic panel, lipid levels, plasma 
glucose and HbA1c and a urine sample will be collected for toxicology screening. Participants will fill 
out sleep and psychological questionnaires.  
 
All individuals who pass this initial screening will undergo up to two sessions of 48 hour ambulatory 
BP recording with BP measurements automatically taken every 20-30 minutes across 2 days and 
nights, and undergo a home screening test for Obstructive Sleep Apnea (Watch-PAT, Itamar Medical, 
Israel).   
 
The participants will be notified of significant findings from the blood and urine test results. If they 
remain eligible for participation after the toxicology and the screening questionnaires, they will be 
scheduled for a review of completed screening questionnaires and a physical exam with a study 
physician. The participants will come in on a mutually convenient day for this part of the screening. 
Once the physician determines study eligibility, participants will be asked if they want to continue 
and participate in the study. 
 
The week of the participant’s in-laboratory visit we may also ask them to complete a COVID-19 test 
and self-isolate for the days following the test and leading up to your in-laboratory study. 
 
b. Establishing ambulatory baseline: It is essential to ensure initial stability of circadian rhythms and 
sleep. Hence, participants will maintain a consistent sleep-wake schedule for at least 1 week prior to 
admission to the laboratory. The bedtime will be determined by the participants’ habitual bedtime 
with 8-h time in bed. To ensure compliance to this schedule, each participant’s activity will be 
monitored by wrist Actigraphy (ActiGraph, Pensacola, FL). Additionally, participants will call a time-
stamped voice-mailbox both when going to bed and getting out of bed. For the first and last two 
days of the acclimatization, the participants may be asked to wear an ambulatory BP monitor 
(Spacelabs Healthcare, WA) to get accustomed to the regular inflation-deflation cycles. Actigraphy 

 
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Revised: 11.DEC.2018 
and ambulatory BP measures will provide an estimate of daily activity levels and sleep 
characteristics in real world settings. Actigraphy is widely accepted as a noninvasive means to 
estimate sleep patterns under non-laboratory conditions (44). The estimated sleep from at least 1 
week of home recordings with timed sleep opportunities will also provide an estimate of sleep 
duration, which will complement the results from the polysomnographically scored sleep on the first 
night in the lab. 
 
c. Acclimatization and baseline days: Participants will be admitted to the OCTRI research laboratory 
on experimental day 1. Saliva will be collected with Salivettes for melatonin and cortisol assays and 
genetic testing at regular intervals. Sleep and cardiac variables will be continuously recorded by 
polysomnography. A venous catheter will be placed where the tip of the catheter is peripherally in 
an arm vein, or in some cases closer to the armpit (a ‘midline’ catheter) using standard hospital 
procedures. Ultrasound imaging and local anesthetic may be used to assist in catheter placement. 
The catheter will be kept patent using a solution of sodium and heparin. BP will be measured 
throughout the in-lab stay (e.g., every 30 min during the wake periods with sphygmomanometry 
and continuously during sleep using a finger plethysmography device). Light intensity will be set at 
150 lux (normal indoor room light) during the baseline wake period and reduced to <1 lux (darkness) 
during sleep.  
d. Cardiac Computed tomography (CT) examination: Subjects enrolled in this study will undergo 
cardiac computed tomography scan for the detection and quantification of coronary artery calcium. 
The presence and amount of coronary artery calcium as quantified by Agatston score (CAC score) as 
a predictor of major adverse cardiovascular events. The CT examinations are designed to efficiently 
and accurately provide volumetric CT image data for measuring coronary artery calcium (CAC), an 
accurate measure of coronary atherosclerosis burden (45,46). The CT examination for CAC consists 
of scout images and one cardiac prospectively ECG-triggered series of the heart to measure CAC. On 
average, 15 minutes of participant time will be spent within the CT scan suite; this includes 
instructions, setup and imaging. Participants will have ECG electrodes attached for cardiac gating 
and be instructed to use a standardized breath hold technique. The CT examination will be 
performed on a 256-detector row CT scanner (Philips iCT, Philips Medical Imaging, Cleveland, OH). 
The scan will be performed in prospectively ECG-triggered mode at 75% of RR interval with the tube 
current of 120 kVp and tube potential of 100 mAs. 
 
e. Forced Desynchrony ( F D ) protocol: See above and Fig. 1.    
 
f. Wake episodes: Tests are identical on all wake periods, except for additional PET scanning on up to 
3 of the 10 cycles (Fig. 1 and detailed below in section 6h). We may also make cardiac ultrasound on 
up to 3 cycles. Participants are aroused from sleep using a standard auditory tone to minimize 
different arousal stimuli influences. Participants remain in bed in the supine posture for ~10 minutes 
(awake, eyes open, supervised by technical staff and EEG monitoring). During this ‘constant posture’ 
period, baseline waking hemodynamic, autonomic, endothelial and hemostatic measures are 
recorded (details of dependent measures below). Participants complete a test battery consisting of 
endothelial function assessment, a possible postural stress, cognitive challenges, PET imaging (on up 
to 3 of 10 wake periods) or cardiac ultrasound (on up to 3 of the remaining wake periods), mild 
exercise and an isocaloric meal containing 22% of the daily target intake. Participants are to finish 
each meal to ensure metabolic homeostasis. Participants will then have scheduled ‘free-time’ where 
they are able to move about the suite but are not permitted to lie down, nap, or exercise beyond 
light stretching. Activity will be monitored for compliance by closed circuit TV. 
 
g. Posture Test: Standing up after a period of lying down is a large physiological challenge requiring 
sympathetic activation to maintain perfusion pressure to the brain and avoid syncope.  If this test is 

 
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Revised: 11.DEC.2018 
performed, participants will be instrumented with EKG and an automated sphygmomanometer. 
Non-invasive BP recordings on a beat-by-beat basis (Nexfin, Netherlands) and may also be 
performed. The Nexfin finger cuffs will be placed on the non-dominant hand and held in place by an 
arm sling so that the BP cuffs will be at heart level. BP and HR will be assessed while participants 
stand up from a semi-recumbent posture and remain standing for ~5 min. A tilt table may also be 
used to transition the participant from supine to vertical posture at a controlled rate. For this 
procedure, participants will be strapped to a hydraulic tilt table with a weight-bearing footboard. 
Measurements will then be made under 3 conditions: (1) Baseline: Participants lie supine (0-
degrees), awake and resting for approximately 20-min (2) 60 degree tilt: within a 3 to 5 sec period 
the table will be tilted to a 60-degree head-up angle. The table will be held here for a further 15-
minutes (except in situations where the participant becomes uncomfortable, dizzy or faint). (3) 
Recovery: Within a 3 to 5 second period the table will be tilted to a 0-degree angle. The table will be 
held here for a further approximately 20-min. 
 
 
h. Proton Emission Tomography (PET): PET imaging of the heart will occur up to 3 times across the FD 
protocol (minimum 16 h between scans), with order randomized for each participant and beginning 
~40 min after waking (Fig.1 ). 
 
We will use O-15 water for blood flow, C-11 – meta-hydroxyephedrine for presynaptic 
norepinephrine transporter function and CGP 12177 for measure of beta adrenergic receptor 
density.  
 
After the endothelial and posture tests, participants are taken by wheelchair to the Nuclear 
Medicine Suite (in the same building but on a different floor) while wearing opaque goggles to 
prevent light exposure and contact with non-study personnel that could provide time cues. One in 
the scanner suite, the lights will be kept dim, and the participant will lie supine on the imaging table, 
movement may be restricted with foam or other form of padding, and the participant will be 
connected for heart rate, oxygen saturation,  and blood pressure monitoring. The IV from the 
circadian lab will already be in place and will be used for periodic blood sampling during the imaging 
period. A second IV will be put in place and used to inject the radiopharmaceuticals.  Once settled, 
an attenuation image will be acquired using a low dose CT scan. All images will be dynamic 
acquisition collected in “list mode”. Cardiac beta adrenergic receptor density via CGP12177 images: 
[11C]-CGP12177 (≤0.1 mCi/kg body weight up to 10 mCi of high specific activity), will be injected via 
IV over ~1 min. Image acquisition will begin one min before tracer injection and continue for ~20 
min after which time ≤0.1 mCi/kg up to 10 mCi of low specific activity [11C]-CGP12177 will be 
administered via IV over ~1 min and images acquired for up to an additional 40 min. Myocardial 
blood flow may be measured via [15O]-water (<0.5 mCi/kg body weight) up to 50 mCi injected via IV 
and images acquired for ~ 5 min. Coronary blood flow is calculated using the Bergmann model (47). 
The 15O water study will be performed between the norepinephrine and post-synaptic receptor 
studies. Cardiac presynaptic norepinephrine transporter function will be measured via infusion of 
[11C]-meta-hydroxyephedrine (mHED: ≤0.2 mCi/kg body weight up to 20 mCi) over ~30 sec with PET 
images acquired continuously for 40 min. Following completion of the images, IV lines will be 
disconnected (but not removed) and the participant will return to the circadian lab in a wheelchair 
while wearing opaque goggles. In wake periods without imaging, participants will remain in bed to 
match the duration of lying in the scanner. During that time there may be interactions with lab staff 
and completion of computerized tests that measure vigilance and neurocognitive performance and 
ensure wakefulness.  
 
i. Perfusion imaging: A commercially-produced FDA-approved microbubble contrast agent (Definity, 

 
Page 9 of 24 
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Bristol Myers Squibb) will be used. One vial of Definity (1.3 mL) will be diluted to a total volume of 
30 mL and infused at 1.5 ml/min intravenously. Myocardial contrast echocardiography will be 
performed with a phased-array transducer interfaced with an ultrasound system (iE33, Philips 
Ultrasound). A contrast-specific multipulse amplitude-modulation sequence will be performed at a 
centerline transmission frequency of 1.8 MHz, a mechanical index of 0.12, and a compression of 60. 
Images will be acquired in the apical 4-chamber, 2- chamber, and 3-chamber views. End-systolic 
images will be acquired for 10 seconds after a 5-frame destructive pulse sequence performed at a 
mechanical index of 1.0. Vasodilator stress will be applied using i.v. regadenoson and 
echocardiography will be repeated to measure the effect of hyperemia. 
 
j. 
Exercise Test: All team members have extensive experience in performing exercise testing under 
laboratory conditions. The power setting of the cycle ergometer will be tailored to induce 50% HRMAX 
for each individual during their initial exercise test and kept constant for subsequent tests to enable 
us to quantify changes across the circadian cycle. This workload is similar to that in our preliminary 
data and chosen to reflect the type of workload placed on the CV system during normal daily 
activities (e.g. walking). This level elicits characteristic hemodynamic responses without causing an 
anaerobic state or training effects (48,49). The procedure is: (i) sit quietly on the ergometer for 5-10 
min for baseline recordings; (ii) cycle for 15 min at 50% HRmax, and (iii) remain on the ergometer, 
stationary and resting for a 5-10 min recovery period. 
 
k. Energy Expenditure: We will measure energy expenditure via indirect calorimetry for assessment of 
EE and macronutrient oxidation (calculated using equations of (Jequier, 1987) during different 
portions of the inpatient protocol. O2 consumption and CO2 production are used to calculate 
metabolic rate and the oxidation of carbohydrate and fat (50). 
 
l. 
Sleep: Autonomic and hemodynamic (HR and BP), but not endothelial function, will be measured 
during sleep for comparison with standardized wake periods while in the same posture. Sleep 
episodes will be polysomnographically recorded. Electronic sensors (Emfit Bed Monitor) may also be 
placed under the bed to estimate sleep and wakefulness. Because changes in posture and activity 
can affect results, participants will be instructed not to get out of bed, even if they should awaken 
before the end of the scheduled sleep episode. If requested, a nurse or technician will bring the 
participant a urinal, bedpan, or commode during scheduled sleep time. The ambient light intensity 
in the suite will be <1 lux. Blood sampling will continue every ~2 h. Standard polysomnographic 
recordings during sleep will include EKG, electroencephalogram (EEG), electro-oculogram (EOG) and 
submental electromyograms (EMG), and arterial oxygen saturation via pulse oximetry measured on 
the non-dominant index finger. In addition, continuous beat-by-beat BP will be recorded. Sleep 
recordings will be scored by a registered polysomnographic technician in 30-sec epochs for sleep 
stage, arousals, and any periodic limb movements and respiratory events according to scoring 
criteria of the American Academy of Sleep (51).  
 
m. Circadian Phase: Analysis of dim light melatonin onset (DLMO) from saliva collected over the study 
duration yields an accurate estimate of circadian phase. With mathematical techniques that we 
have previously used (39,41,42,52) see statistical techniques) the phase, period and amplitude of 
the DLMO rhythm will be established. The phase and period information will be used as phase 
markers for assigning circadian phase to all other collected data. Saliva will be collected with 
Salivettes.  
 
 

 
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n. Genetics: Single nucleotide polymorphisms (SNPs) will be measured from saliva or blood to 
determine how polymorphisms within genes that regulate biological rhythms may vary in obese and 
lean participants. Additional consent will be obtained for this portion of the study. 
 
o. Primary dependent variables:  
i. BP: We may measure beat-to-beat BP (using a non-invasive device employing the volume-
clamp method with hydrostatic correction (53)). We have previously used this technique 
during recordings over extended in-lab stays (up to 12-days) with success during rest, 
posture test, cycle exercise and in sleep. Beat-to-beat BP will be recorded during: (i) each 
sleep episode; (ii) each constant posture period immediately following awakening; and (iii) 
throughout each posture test session. In addition to the finger-cuff BP, an automated 
calibrated sphygmomanometer will be used to record BP at intervals during the waking 
stresses for safety and cross-reference to the finger device. As a secondary variable, cardiac 
output will also be estimated using algorithms built into the beat-to-beat BP system. We 
may also be monitoring ambulatory BP (Spacelabs Healthcare, Washington, USA) every ~30 
min during wake and every ~15 min during sleep as is standard in most ambulatory BP 
assessments (54). 
ii. Heart Rate: For the duration of the study, 2 channels of EKG will be recorded (RA-V6) and 
stored. Software will be used for peak detection (R-wave detection and subsequent HRV 
analysis to estimate cardiac vagal tone (see below). 
iii. Sympathetic activity: Primary estimators of sympathetic output/effect include (1) venous 
epinephrine (adrenal cortex contribution), (2) venous norepinephrine (sympathetic nerve 
activity), (3) saliva cortisol (sympathetic potentiating hormone), and (4) venous aldosterone 
(end point of renin-angiotensin, sympathetic nerve activation). Endocannabinoid analysis 
from plasma may also be measured as a secondary marker of HPA-axis and sympathetic 
regulation.  
iv. Parasympathetic activity: We will use the high frequency power (HF) of the HRV power 
spectrum to estimate cardiac parasympathetic activity as modulated by respiratory sinus 
arrhythmia (55,56). The R-R intervals resulting from ectopic beats or beats >3 SD from the 
mean are deleted from datasets. Thereafter, R-R interval data for sequential 5-minute time 
segments will be used for HRV analysis in each condition (i.e. semi-recumbent vs. standing, 
resting vs. exercise, wakefulness vs. stable sleep stages) according to published criteria (57). 
Frequency domain measures are calculated by interpolating the R-R tachogram with a cubic 
spline and re-sampling. Power spectral density will be calculated using Welch’s technique (58) 
of FFT. Calculated time domain HRV measures will include mean R-R, SD of all normal-normal 
R-R intervals (SDNN), and percentage of R-R intervals differing by greater than 50 msec 
(pNN50). 
v. Oxidative stress: We will measure markers of lipid peroxidation from plasma. 
vi. Endothelial Function: We will measure endothelium-dependent flow mediated dilation 
(FMD) and endothelium-independent vasodilation starting ~20-min after each awakening in 
a constant posture following an overnight fast. Brachial artery FMD will be measured in the 
supine rested position using the standard guidelines and protocol (59). We have expertise in 
these technique, especially in longitudinal measures within an individual (60-62). 
vii. Inflammation: We will measure plasma TNFa because of its strong association with obesity 
related CV disease, effect on vascular function, and known circadian variation (34,63,64). 
viii. Coronary blood flow (using [15O]-water), cardiac presynaptic norepinephrine transporter 
function (reuptake; using [11C]-mHED), and beta-adrenergic receptor density (using [11C]-
CGP12177) and will be measured as described above in the procedures section. 

 
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ix. Anthropometric measures.  Dual energy X-ray absorptiometry (DEXA, Model Model GE 
Lunar iDXA) will be used to assess body fat percentage and fat free mass at during the study. 
This is currently considered the gold standard for body composition analysis. 
x. Mental Stress Test: Approximately 3 h after awakening participants start the mental stress 
test. Measurements will be made under 3 conditions: (1) Baseline: Participants sit in a semi-
recumbent posture (45°), awake and resting for ~20-min; (2) Mental Stress: for ~20 min. 
p. Discharge: Following the last FD waking period, participants will be given 9 hours of recovery sleep 
during their extended habitual sleep time. The additional hour beyond habitual sleep time will 
account for possible sleep deprivation during the protocol and account for the natural drift the 
participant may experience as a result of a free running circadian system (a result of constant dark 
or dim light). Upon awakening participants will have the opportunity to shower and eat a breakfast 
of their choosing. Following the meal, participants will complete the Stanford Sleepiness Scale and 
have a final nursing assessment. If an individual does not rate a score of 1 or 2 (Feeling active, wide 
awake OR Functioning at high levels), participants will be given the opportunity for additional rest, 
provided a cab ride home, or picked up by a family member/friend. We may also use a 10-minute 
psychomotor vigilance test (PTV) initially provided during the second screening visit. Failure to 
obtain within 10% of their initial score would be similar to having a blood alcohol score above the 
legal limit.  If the participant insists on leaving and driving themselves home, they must sign a waiver 
stating that upon research and nursing staff, and physician advice they chose to drive home and 
assume liability. Finally, upon departure participants will be provided a card that once more 
(originally in consent form) provides staff contact information. The card will also remind participants 
of possible residual side effects associated with the study. 
 
7. Data and Specimens 
a. Handling of Data and Specimens 
i. Electronic Data Handling:  While study is active, all PHI will be stored in a database housed 
on a secure OHSU server.  PHI will be accessible to only investigators, and research staff 
listed on the protocol. The PET images are reconstructed and saved on the tomograph data 
system until the data are converted and stored as de-identified DICOM files for further data 
analyses. At the end of the study, participants who gave verbal consent to have their 
information stored in a recruitment repository (STUDY00015966), PHI collected on the 
‘Phone Screen Questionnaire’ will be transferred to a password protected database on a 
network drive behind the OHSU firewall. For those who did not give verbal consent to have 
their information stored in a recruitment repository, PHI will be destroyed by removing all 
18 identifiers. No PHI will be stored or transferred via USB or other portable drives. We will 
obtain a waiver of the HIPPA authorization requirement for PHI collect at the time of the 
phone screen.  
 
At the initial phone-screen, all participants will be assigned a unique code that will be used 
to identify them on documents residing outside of the secure database.  Upon enrollment, 
participant study data will be recorded and stored in a password protected database and 
will be identified only by the unique code assigned at the initial phone screen. The database 
will reside on restricted and firewall protected OHSU network drive.  Only persons listed on 
the IRB approved protocol will be given access to the databases. 
 
ii. Paper Data Handling:  All paper files (e.g. signed consent forms, participant sleep and 
activity diaries) will be stored in locked filing cabinets in restricted access offices at OHSU.  
Whenever possible, original records will be retained as the source document. However, a 
photocopy is acceptable provided that it is a clear, legible and exact duplication of the 
original document.  Access to these files will be limited to study personnel listed on the IRB 

 
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approved protocol. The PET radiopharmaceutical manufacturing batch records are retained 
in the Center for Radiochemistry Research (CRR) under lock and key with access restricted to 
authorized CRR personnel, the PIs and to authorized manufacturing auditors. The batch 
records are retained as required by Federal and State laws for investigational drug 
manufacturing. 
 
iii. Specimen Handling:  Blood and urine samples will be identified only by the code assigned at 
the initial phone screen.  Processing and handling of samples will be done by the OCTRI 
nursing and core lab staff.  Samples will be stored and maintained by the OCTRI core lab -80 
degree freezers.  Access to study samples at OHSU will be limited to OCTRI core lab staff and 
study personnel listed on the IRB approved protocol. Coded biological samples may be sent 
to outside labs for analysis. 
 
 
b. Sharing of Results with Subjects 
With their consent, we will make known to the participant some of the information we have gathered 
from the physiological testing during screening and in the laboratory. There is a chance that the pre-
study screening or the various blood and urine samples taken during the study will reveal some medical 
abnormality. This information will be conveyed to the participant, together with a recommendation to 
discuss the results with a physician if appropriate. Specifically, since genetic information obtained does 
not have medical or treatment importance at this time it will not be shared with the participant. 
 
 
8. Data Analysis 
Analysis of circadian rhythmicity: Forced desynchrony data is composed of circadian effects (intrinsic 
period ~24 h), evoked effects (5 h 20 min behavioral cycles) and ‘noise’. The intrinsic period of the circadian 
pacemaker will be assessed with standard techniques as used for 20 years by the Principal Investigator 
(65,66). This intrinsic period, derived from melatonin, will be used to assign a circadian phase (from 0° to 
360°) to each minute of the study, with 0° corresponding to the minimum of the waveform fitted to the 
DLMO data from the FD portion of the study. Analysis then proceeds in two ways. (A) Binning: Data for each 
variable and each participant will be ‘binned’ according to circadian phase and an average value per bin will 
be calculated. Data will then be averaged across participants. Bin size will be determined by the nature of 
the data being analyzed; in most cases we have found that a bin size of 60° (~4 h) allows sufficient detail 
while at the same time provides data for each participant in each bin. (B) Cosinor analysis to estimate 
rhythm amplitude and phase (67): Using regression techniques, time series data, not necessarily evenly 
spaced, can be fit to the function. This can be done within-subject, so standard deviations of amplitude and 
phase can be estimated (68).  
 
Analysis of Cardiac Computed tomography (CT) examination: The images will be reconstructed with 2.5 
mm slice thickness. The gantry rotation time will be 280 ms resulting in temporal resolution of 140 ms. A 
dataset with a field-of-view focused on the heart for the purpose of the CAC quantification will be 
reconstructed. The reconstructed datasets will be transferred to Knight Cardiovascular Institute Imaging 
Core Laboratory. The coronary calcium CT scans will be analyzed on a dedicated workstation (Medis medical 

 
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imaging systems, Leiden, the Netherlands). A software application will be used for the semi-automated 
selection of CAC. The Agatston score (AS) will be calculated to quantify total CAC amount (69). 
 
Analysis of PET Imaging  Data: 
We will analyze regional radiotracer uptake in the left ventricle. The heart will be segmented into 5 mm 
thick slices with 8 sectors per slice (70). 
Norepinephrine re-uptake: Retention fraction: This is the sum of 11C activity (cpm/ml) per sector from 25-40 
min post-injection divided by the image-based blood pool activity integrated from 0-40 min and corrected 
for any metabolites.  Images will also be evaluated using a kinetic model on a sector-by-sector basis and the 
values, summarized in the legend of Fig. 2 are reported which also shows sample time activity curves from 
two sectors from a single heart-failure patient (9).  
Beta-receptor density: Will be analyzed by the graphical method of (71). Two injections of [11C]-CGP are 
required for the graphical analysis approach to determine regional LV beta adrenoceptor density. The 
Delforge model provides validated estimates of receptor density (B' max) on a sector by sector basis, 
expressed in terms of pmol/g (72).  
Analysis of myocardial perfusion imaging. Image analysis will be conducted offline. The immediate post-
destruction frame will be digitally subtracted from all subsequent frames. Time versus background 
subtracted intensity will be measured on the background-subtracted frames from regions-of-interest placed 
over the myocardium according to the American Society of Echocardiography’s 17-segment model. Data will 
be fit to the function y=A(1-e-βt) where y is the video intensity at time t; A is plateau video intensity, an index 
of capillary blood volume and β is the rate constant which provides a measure of average erythrocyte flux 
rate. Microvascular blood flow will be calculated by the product of A and β. Since flow reserve and β-reserve 
can be abnormal from increased resting values more so than reduced stress values, the definition of 
microvascular dysfunction will also require that complete refill of the microcirculation take >2 
postdestructive beats. These data will be further analyzed using mixed model analysis with β-reserve as the 
primary dependent variable and circadian time as the independent variable. 
9. Power / Sample Size Justification: To estimate sample size, we have preliminary data from a similar study 
population (4 obese, 6 lean), in the same age range, and under the same protocol as the current proposal. 
The variables will be compared across circadian phases, across stresses, and between obese and lean groups 
in terms of basal circadian rhythm amplitudes (Aim 1) and circadian rhythms of reactivity (Aim 2). The main 
outcomes of interest are: differences in systolic BP, HR or vascular endothelial function (FMD) between 
obese and lean individuals upon awakening; changes in these variables with behavioral stresses, and 
between the most vulnerable (~9AM) and less vulnerable other circadian phases (e.g., 12 h later). The 
clinically significant or meaningful differences that we aimed to detect between groups, between states and 
between circadian phases were 10 mmHg for systolic BP, 10 bpm for HR and an absolute 3 point different in 
% FMD (73-76). The sample size calculations revealed that for all 6 main comparisons, 14 participants per 
group was sufficient to provide at least 80% power to identify the assumed main effects. To account for 
multiple comparisons, even after adjusting for six outcomes of primary interest (adjusted level of 
significance = 0.00833 per comparison), use of 14 participants for each of obese and lean groups, we have 
80% power to detect effect sizes of at least 1.41 SD between groups. Thus, we will study 14 participants per 
group to adequately test our hypotheses. For our sub aims a1 and a2 related to circadian rhythmicity in 
coronary microvascular function between lean and obese individuals, in order to test the hypotheses with 

 
Page 14 of 24 
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80% statistical power, out of the total 28 participants, we will study 10 lean and 10 obese individuals. 
Privacy, Confidentiality, and Data Security 
Standard institutional practices will be followed as described in the OHSU Information Security and Research 
Data Resource Guide (http://ozone.ohsu.edu/cc/sec/isg/res_sec.pdf) to maintain the confidentiality and 
security of data collected in this study. Study staff will be trained with regard to these procedures.  The PI 
shall prepare and maintain complete and accurate study documentation in compliance with good clinical 
practice standards and applicable federal, state and local laws, rules and regulations. Study documentation 
shall be made available at the investigator’s site 
upon request for inspection, copying, review and 
audit at reasonable times by any regulatory 
agencies. The investigator agrees to promptly 
take any reasonable steps that are requested as a 
result of an audit to correct deficiencies in the 
study documentation. Confidentiality of data will 
be maintained. However, legal regulations 
regarding access to data by those other than the 
investigator and his/her representatives will be 
followed. All information will be entered into the 
study dataset by coded entry (de-identified data) 
only. The code linkage data will be kept in a 
locked file available only to the PI or his/her 
authorized designate unless requested by the 
appropriate regulatory authority 
 
10. Provisions to Monitor the Data to Ensure 
the Safety of Subjects 
a. Safety Monitoring: Dr. Steven A. Shea and 
Dr. Jeanne Link, the study mPIs, study 
physician(s), and an independent safety 
officer will monitor this study for all patient safety issues. A semi-annual evaluation will be conducted 
by the independent safety officer to identify any safety or other protocol-related concerns. A detailed 
Data Safety Monitoring Plan (DSMP) is included in the study materials. 
 
The data to be monitored for safety include hematocrit/hemoglobin levels and response to 
experimental events. Vitals, hemoglobin, computerized tests of mood, and post-sleep 
questionnaires are monitored daily by project leaders and RN staff. Attending physicians monitor 
these data while volunteers are participating in the inpatient portions of the study. Participants will 
be given a 24 h access number to reach Dr. Shea, the project leader, and the responsible 
recruiter/coordinator during inpatient and outpatient portions of the study. 
b. Protocol Issues: These studies will be conducted in a well-supervised hospital facility and participants 
will be under constant observation by skilled professionals highly qualified to perform the study 
procedures. A physician and nurse will be available at all times. Procedures will be terminated 
immediately if potentially serious side effects develop. In the event of adverse reactions, medical care 
will be available. PET imaging will be done under the supervision of a nuclear medicine physician who 
will perform each injection of the radiopharmaceutical and observe the subject for at least 15 minutes 
for any adverse reaction.  
c. Outcomes Monitoring: The project leader will review and report all data and safety issues after 
discussions with the principal investigator. The Center for Radiochemistry Research will have internal 
and external audits of their records. In consultation with the on-call physician associated with the 
 
Fig. 12. Model analysis of pre-synaptic NE function in LV sectors 
from a heart-failure patient. Points in black are the time activity 
curve points from the left atrial cavity. Green are metabolites in 
the blood. Blue are the activities in the LV tissue, and the red line 
is the model fit. The parameters are image spillover from the 
ventricular blood cavity (spillover), virtual volume of the nerve 
terminal (Vnt), volume for NE presynaptic vesicular storage 
(Gves) and transfer into the vesicle (PSves), and our main 
variable presynaptic reuptake rate (PSnt) (1).  

 
Page 15 of 24 
Revised: 11.DEC.2018 
study and with the PIs, Steven A. Shea, or Jeanne Link, all abnormal values will be handled in one of 
two ways: 
1. If minor, the test (e.g. blood pressure) is repeated. If still abnormal, the participant is either 
withdrawn or not enrolled and a letter to the participant’s personal physician is sent if the 
participant requests. 
2. If major, the participant is either withdrawn or not enrolled and, with the participant’s 
permission, a letter will be sent to the participant’s physician detailing the abnormality. If 
the participant does not have a physician, we will endeavor to help the participant find a 
physician. A yearly progress report will be submitted to the Institutional Review Board 
(IRB).  
d. Reportable New Information Guidelines: Reportable New Information (RNI) will be reported to the 
IRB within 5 business days of discovery. RNI not meeting IRB requirements for prompt reporting will 
be submitted with the Continuing Review. Adverse events or unanticipated problems will be 
reported to the funder, National Heart Lung and Blood Insitute (NHLBI), according to current 
guidelines (https://www.nhlbi.nih.gov/grants-and-training/policies-and-guidelines/nhlbi-adverse-
event-and-unanticipated-problem-reporting-policy). Full details can be found in the Data Safety 
Monitoring Plan. 
 
11. Risks and Benefits 
a. Risks to Participants 
i. Blood sampling 
1. There may be some discomfort or bruising on initial insertion of the catheter into a 
vein, but wearing the catheter should not be painful. 
2. There may be some soreness of fingers secondary to an occasional finger prick. 
3. Occasionally, mild discomfort may occur from the tube in the vein. If this happens, it 
can be repositioned or removed, asking the participant's permission before any 
subsequent reinsertion.  
4. To help keep the venipuncture site clean, we may ask permission to shave the 
forearm hair of the participant prior to insertion of the IV. 
5. There is a rare possibility of developing a small blood clot, inflammation, or local 
infection around the vein where the catheter is inserted, or in rare cases a 
generalized infection spread through the bloodstream as a result of the IV catheter.  
6. Occasionally, there is a black and blue mark at the site of the IV insertion, which may 
last a couple of weeks; and, rarely, a small scar may remain permanently at the 
venipuncture site. 
7. There is the possibility that the participant may faint during or after the IV insertion 
procedure. Therefore, we ensure that the catheter insertion is made when the 
participant is supine. 
8. A member of the IV team who has been trained in midline catheter placement will 
place the midline catheter.  
9. Ultrasound imaging and local anesthetic may be used to aid catheter placement. 
10. There may be a minor skin rash or reaction to the sterile tape (contact dermatitis) 
used to hold the catheter in position. Hypoallergenic tape will be used as necessary. 
11. There may be some side effects from the use of heparin, such as bleeding, heparin-
induced thrombocytopenia, and/or allergy. 
12. The amount of blood drawn should not significantly alter blood volume, although 
there may be a small decrease in the hematocrit. We will only recruit participants 
with normal levels of hemoglobin, hematocrit, and ferritin. The participants’ 
hemoglobin and red blood cell concentration will be carefully monitored throughout 
the study as needed, and blood drawing, with the exception of the blood drawn 

 
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during the 3 PET scanning procedures (1mL each scan, total of 3mL), will be 
discontinued if at any time the hemoglobin and/or red blood cell concentration 
should fall below established guidelines in addition to falling more than 1.3 gm/dL 
below their hemoglobin levels at admission. In this case, upon discharge, 
participants will be given information on their hemoglobin levels and will be 
encouraged to see their primary care provider. In addition, participants are 
encouraged to eat iron rich food before and after the study. 
ii. Polysomnography: The tape and special paste used to attach the EEG electrodes may cause 
some minor discomfort, skin irritation, and the glue used to hold electrodes to the scalp may 
leave a flaky residue for several days. The adhesive ECG pads may cause some skin irritation; 
the participant will be instructed to ask for a change in their placement if this occurs. To 
prep skin for ECG electrodes, participant skin may need to be shaved which may cause some 
minor discomfort and skin irritation. 
iii. Flow mediated dilation (FMD): The blood pressure cuff on the forearm is inflated to 
suprasystolic pressure. This may cause numbness below the site of inflation, and slight pain 
at the cuff site. In our experience, this discomfort does not last for more than 5 minutes 
after deflation of the cuff. Standard guidelines will be followed to measure FMD in a safe 
manner. The gel used for the ultrasound imaging may cause some skin irritation and 
redness. In case participants are allergic to one particular gel, a different gel will be used. 
The gel will be wiped off after the measurement.  
iv. In-lab Protocol 
1. The participant may undergo some weight loss over the course of the study. This is 
typically due to a loss of fluid as a result of differences in the sodium content 
between the laboratory diet and the participants’ regular diet. However, it is also 
possible that participants may experience a modest loss in actual body mass. 
2. The participant’s mood, appetite and weight will be monitored. 
3. Participants will be visited by RN staff at least once per day while in the OCTRI to 
evaluate overall physical and mental health. The participant’s vital signs will be 
checked at least once per day. This will include pulse rate, respiratory rate, systolic 
and diastolic blood pressure. 
4. The participant may become sleepy during some segments of the study. The 
participant will be asked to remain awake during the entirety of their scheduled wake 
times. Should the participant feel that he/she is unable to remain awake, he/she is 
free to withdraw his/her consent to participate in this experiment and then go to 
sleep. 
5. The light level in the room is dim (~3 lux) for the “awake” portion of the experiment. 
Though it may be difficult for participants to read and stay awake, this has not been 
a problem.  
6. Volunteer participants are studied in the OCTRI research laboratories at OHSU. In the 
unlikely event that emergency treatment is required during the participant's stay in 
the research facility, this treatment will be provided. 
7. During the in-laboratory part of the study, if the resting SBP ≥180 mmHg or the DBP 
≥110 mmHg on three consecutive measurements taken at least 5 minutes apart 
during rest, the physician will be called to decide on continuing testing.  
8. In adherence with OHSU’s policies, we will have participants wear personal protective 
equipment and follow other relevant regulations when appropriate. 
 
v. Posture Test: The participant may experience some presyncopal symptoms (lightheadedness, 
nausea) when they move from the semi-recumbent to standing position. If we encounter any 
of these symptoms during the test battery, we will ask the participant to lie down until these 

 
Page 17 of 24 
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symptoms go away. Typically, such symptoms disappear within a few minutes following lying 
down. Blood pressure, EKG and heart rate are monitored as part of the test, and any unusual 
physiological responses will be reported immediately to the physician on call and appropriate 
action instigated. 
vi. PET Imaging: Activation or blockade of the cardiac adrenergic nervous system:  In terms of 
sympathetic activation or blockade: 1) The tracers, injected in the patients at 1000-fold higher 
doses than planned, could block either the pre-synaptic norepinephrine reuptake system or 
post-synaptic beta-adrenergic receptor system creating a potential for increased ventricular 
arrhythmias, hypotension or hypertension, bradycardia or sinus tachycardia. These risks are 
exceedingly small. For the 60 healthy control volunteers and 14 patients with serious heart 
disease that Dr. Link has studied with [11C]-mHED, there have been no symptoms reported 
and no notable changes in heart rate or blood pressure have been observed. PET studies of 
[11C]-mHED in diverse patient populations have not reported hemodynamic changes, except 
for a ~ 5 mmHg increase in BP over a 40 minute study period likely due to IV fluid 
administration (77-81). 
 
The potential side effects of beta receptor-blockade (decreased heart rate and blood 
pressure) were not observed in the control patients in the study by Merlet et al (82) using 
[11C]-CGP12177. In the congestive heart failure patients in our prior study, heart rate 
decreased 15% without change in blood pressure. Two patients had transient and mild 
dyspnea. Delforge et al reported no change in heart rate or blood pressure in a series of 5 
dogs given 11C-CGP12177 at a dose approximately twice that used in the human studies. Qing 
et al (83) did not report hemodynamic effects when using [11C]-CGP12177 in patients with 
asthma. Thus, we do not anticipate adverse effects; nevertheless, heart rate, oxygen 
saturation, and blood pressure will be monitored throughout the studies. For all studies, a 
physician will be present in the Nuclear Medicine suite for each injection and 15 minutes 
following each injection. A trained technician will monitor throughout the imaging period in 
order to anticipate any rhythm abnormalities or hemodynamic changes, and a physician will 
be on premises to treat these conditions appropriately. 
 
The subject could develop claustrophobia from the imaging apparatus and discomfort from 
lying on the imaging table for up to two hours. Only one subject out of over 100 such studies 
that we have performed has asked to terminate the study early because of discomfort.  That 
subject was a young healthy control.  
 
vii. Radiation exposure: Radiation dose carries an associated risk to the patient. The estimated 
effective radiation dose from a CAC scan is approximately 1.5 mSv (as compared to annual 
natural background radiation of 3-4 mSv). The effective equivalent (EDE) for one PET + CT 
imaging session is calculated to be 7.4 mSv for a male and about 7.0 mSv for a female 
assuming 70 kg per male or female.  The radiation dose for 3 imaging sessions, the entire 
protocol, is 22 mSv for a male and 21 mSv for a female. The critical organ targeted in this study 
is the kidney with a dose of 17 mSv to male or female of 70 kg weight for one imaging session, 
the dose to the critical organ is 51 mSv for three imaging sessions. For the population in this 
study which includes both lean and obese individuals the increased risk of death from their 
radiation exposure is minimal. There are no methods to reduce the radiation exposure. To 
minimize risk to the patient, the injected doses will be kept as low as reasonably achievable 
and to still obtain reasonable counting statistics. 
viii. Myocardial perfusion imaging: Lipid-stabilized octafluoropropane microbubbles (Definity, 
Lantheus Imaging) will be used for perfusion imaging. Safety of this agent has been well-
established, including post-marketing registry studies involving >1 million doses 

 
Page 18 of 24 
Revised: 11.DEC.2018 
administered, and multicenter trials of hundreds of thousands of patients. The safety studies 
have demonstrated that serious pseudoanaphylactic reaction or any other major 
cardiopulmonary adverse event is exceedingly rare (1 in 8,000 to 10,000) and much lower 
than any other cardiovascular imaging contrast agent that has been used in humans. Dose of 
Definity will be within the limits approved by the US FDA for left ventricular opacification. 
Although non-serious adverse events (AEs) are very common with coronary vasodilators such 
as regadenoson that are widely used for pharmacologic stress testing in cardiovascular 
medicine, most of these AEs are non-harmful side effects of A1-receptor agonism such as 
flushing, headache, dyspnea and chest pain, and resolve within minutes of discontinuation of 
adenosine. If participants experience any discomfort, then we will immediately reverse the 
effect of regadenoson with an antidote, Aminophylline. Additionally, to reduce risk, each 
subject will be closely supervised for the duration of the study including evaluation of vital 
signs, monitoring of symptoms, and ECG. The procedures will be conducted on a hospital floor 
fully-equipped with emergency response kit for dealing with any anaphylactic, arrhythmic, or 
hypotensive event. 
ix. Exercise Test: In a population of individuals with no history of cardiovascular disease, the risk 
for complications during mild bicycle exercise at a level that raises the heart rate to 
approximately 50% of maximal heart rate for 15 minutes is negligible. Even though 
participants with HTN are at risk for cardiovascular disease, the exercise intensity used in our 
protocol is less than moderate intensity and simulates physical activity during activities of 
daily living. If the BP rises to ≥220 mmHg SBP or ≥110 mmHg DBP associated with the initiation 
of the exercise test, the exercise test will be terminated and the physician will be contacted. 
The participant will be monitored continuously until further orders from the on-call physician. 
x. Energy Expenditure: Measurement of energy expenditure can cause mild discomfort. Some 
participants may also find the mouthpiece or hood to be slightly claustrophobic. Participants 
will be shown the metabolic mouthpiece or hood and provided the opportunity to test the 
mouthpiece or lay down under the hood at the consent if they express concern with the 
procedure. Sometimes, the metabolic cart recording needs to be delayed by a few minutes 
and most participants have no concerns with the equipment. 
xi. Blood Pressure Tests: The supra-systolic cuff pressure used during measurement of blood 
pressure may be uncomfortable for a brief period (30 seconds), but this is not dangerous. The 
finger cuff device can also be mildly uncomfortable but will be removed if it becomes painful. 
xii. Anthropometric measures: As part of this study we will perform 1 DEXA scan of the 
participant’s whole body.  DEXA is a way of looking inside the body by using X-rays.  X-rays are 
a type of radiation. The natural environment has some radiation in it.  This DEXA will give 
participants about the same amount of radiation that they would get from the environment 
in 2 days. 
xiii. Sleep Disturbances: At the end of the experiment, the participant may find that they are no 
longer going to sleep and waking up at the same time that they ordinarily did before the study. 
In fact, it may take them several days to readjust to the regular routine. This is very similar to 
jet lag. Some commonly reported symptoms include upset stomach and/or digestive 
disorders, insomnia, irritability, and/or excessive daytime sleepiness.  These symptoms may 
last for 1-2 weeks, although most people report readjustment after only a few days. 
Participants may not sleep as well in the laboratory as they do when at home. While 
dangerous accidents due to sleep loss are unlikely in the controlled environment of the 
laboratory, we have less control when participants leave the laboratory. Thus, we will ensure 
that the participant fully appreciates this risk and they will be given the opportunity to have 
recovery sleep after completion of the protocol before leaving the laboratory. If needed 
transportation will be arranged so that participants can return home safely. 
xiv. Electrical risks: All amplifiers connected to the transducers and electrodes have been 

 
Page 19 of 24 
Revised: 11.DEC.2018 
designed and constructed to clinical safety standards, and are tested and approved by the 
Bioengineering Group at the Oregon Clinical & Translational Research Institute. There is 
always a possibility of electric shock or burn but this risk is extremely remote and is further 
minimized by isolation of amplifiers from ground, and routine mandatory inspection for 
leakage currents. Amplifiers designed for safety in human studies will be used.  
xv. Genetic Testing: The genetic information examined in this study does not have medical or 
treatment importance at this time. Therefore, risks to the participant are currently 
unforeseeable. We will not place information about participation in the genetic study or the 
results of the genetic study tests in the participant’s medical record. There is a small risk of 
loss of confidentiality. If the results were to be accidentally released, it might be possible that 
the information we will gather about could become available to an insurer or an employer, or 
a relative, or someone else outside the study. Even though there are certain genetic 
discrimination and confidentiality protections in both Oregon law and federal law, there is 
still a small chance that harm could occur if this information. 
xvi. Emfit Bed Monitor: The Emfit bed monitor will send de-identified data directly to the study 
sponsor. While every effort will be made to ensure that the transmitted data is safe and 
secure, there is a small risk that if the device were lost or stolen, that certain sleep 
measurement data relating to the participant could be compromised.   
b. Potential Benefits to Participants 
Although there will be no direct physical benefit resulting from participation in this study, we will make 
known to the participant some of the information we have gathered from the physical exam, 
physiological testing during screening and in the laboratory. There is a chance that the pre-study 
screening or the various blood and urine samples taken during the study will reveal some medical 
abnormality. This information will be conveyed to the participant, together with a recommendation of a 
local clinic or physician from whom to seek treatment. 
 
Participant remuneration: Monetary compensation for participation in the study will be based on the 
following criteria.  
 
Table 1: Participant Remuneration 
Screening Phase 
Travel 
Based on mileage/public transit 
Toxicology screening 
$25 
Psychological screening 
$25 
At home screening 
$25/week (up to 3 weeks) 
Activity monitor return 
$25 
COVID-19 Testing and Wait time for drive-thru  
$25/hr up to $50 
Screening possible max 
$200 + travel 
In-Laboratory Phase 
Per day stay 
$240 
x 5 (days) 
= $1,200 
CT Scan 
25 
X 1 time 
$25 
Myocardial perfusion imaging 
$50 
X 3 times 
$150 
Completion bonus 
$350 
 
= $350 
 
 
laboratory total 
= $1,550 

 
Page 20 of 24 
Revised: 11.DEC.2018 
Screening + In-laboratory Phase 
= $1,925 +travel 
 
 
Drugs or Devices  
Throughout the in-lab portion of the study, heparin may be infused continuously to maintain patency. If 
used, heparin use is the same as the FDA approved indication, population, dose, and route of 
administration. The PET radiopharmaceuticals will be manufactured and the administered dose will be done 
under an approved IND for these radiopharmaceuticals, [11C]-mHED, [11C]-CGP12177 and [15O]-water as 
described in this protocol.  For practical reasons, the [15O]-water test may not be performed at each PET 
session. Perflutren and regadenoson will be used for myocardial perfusion imaging. The study will not 
proceed until the protocol, consent form, data safety monitoring plan and IND are approved. 
 
 
 
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