Nucleophilic Defense Against PM Toxicity
Phase: NA
Conditions: Air Pollution Toxicity
Sponsor: University of Louisville

Nucleophilic Defense Against PM Toxicity (NEAT) Trial 
 
NCT03314987 
 
Protocol 5/14/2020 

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STUDY PROTOCOL:
Nucleophilic Defense against PM 
Toxicity (NEAT) Trial
PRINCIPAL INVESTIGATORS: 
Timothy O’Toole, PhD
Christina Lee Brown Envirome Institute
University of Louisville School of Medicine
Aruni Bhatnagar, PhD
Christina Lee Brown Envirome Institute
University of Louisville School of Medicine

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1.
PRIMARY INFORMATION ...................................................................................................................3
2.
STUDY PERSONNEL ..........................................................................................................................4
3.
STUDY COMMITTEES.........................................................................................................................6
4.
LIST OF ABBREVIATIONS ..................................................................................................................9
5.
EXECUTIVE SUMMARY ....................................................................................................................10
6.
STUDY OBJECTIVES ........................................................................................................................13
7.
BACKGROUND ..................................................................................................................................14
8.
INVESTIGATIONAL PLAN .................................................................................................................19
9.
STUDY ENROLLMENT AND WITHDRAWAL....................................................................................24
10.
STUDY VISITS ...............................................................................................................................29
11.
STUDY SUPPLEMENTS................................................................................................................32
12.
CLINICAL AND LABORATORY EVALUATIONS ...........................................................................37
13.
ADVERSE EVENT DEFINITIONS..................................................................................................41
14.
REPORTING BY INVESTIGATOR.................................................................................................43
15.
DATA AND SAFETY MONITORING BOARD.................................................................................46
16.
STATISTICAL PROCEDURES.......................................................................................................50
17.
TRIAL MANAGEMENT...................................................................................................................53
18.
REFERENCES ...............................................................................................................................57
19.
TABLES ..........................................................................................................................................61
20.
FIGURES........................................................................................................................................64
21.
APPENDIX A – SAMPLE PROCUREMENT AND PROCESSING.................................................71
22.
APPENDIX B – MEASUREMENT OF BLOOD BIOMARKERS......................................................75
23.
APPENDIX C – SUPPLEMENT MANUFACTURING AND QUALITY CONTROL PROTOCOL ....76
24.
APPENDIX D – PHYSICAL FUNCTION MEASUREMENTS .........................................................77

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1. PRIMARY INFORMATION
FULL TITLE: Nucleophilic Defense against PM Toxicity (NEAT) Trial
SHORT TITLE: Carnosine NEAT Trial
STUDY INSTITUTION(S): 
Christina Lee Brown Envirome Institute, University of Louisville, Louisville, KY 
University of Louisville School of Medicine, Division of Environmental Medicine, Louisville, KY
Clinical Trials Unit, University of Louisville, Louisville, KY
COLLABORATING INSTITUTION(S): 
Bellarmine University, Louisville, KY 
University of Colorado, Skaggs School of Pharmacy, Aurora, CO
STUDY SPONSOR: National Institutes of Health (5R01ES019217-07)
CLINICALTRIALS.GOV # NCT03314987
PRINCIPAL INVESTIGATOR: Timothy O’Toole, PhD – Christina Lee Brown Envirome Institute, 
University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 309, Louisville, KY 40202
Office: 502-852-5886
Tim.OToole@Louisville.Edu 
ETHICS BOARD REVIEW: 
University of Louisville Institutional Review Board (IRB) 
Approval # 17.1067
Approval Status: Current

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2. INVESTIGATORS & PERSONNEL
PRINCIPAL INVESTIGATORS
Timothy O’Toole, PhD
Co-Principal Investigator
Christina Lee Brown Envirome Institute, University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 309
Louisville, KY 40202
Office Phone: 502-852-5886
Tim.otoole@Louisville.Edu
Aruni Bhatnagar, PhD
Co-Principal Investigator
Director, Christina Lee Brown Envirome Institute, University of Louisville 
302 E. Muhammad Ali Blvd, Room 106
Louisville, KY 40202
Office Phone: 502-852-5873
Aruni@Louisville.Edu
CO-INVESTIGATORS
Alok Amraotkar, MD
Study Management, Primary Contact
Christina Lee Brown Envirome Institute 
University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 209
Louisville, KY 40202
Office Phone: 502-852-7559
Alok.Amraotkar@Louisville.Edu
Andrew DeFilippis, MD
Study Physician
Division of Cardiovascular Medicine,
University of Louisville School of Medicine
580 S. Preston Street, Room 404-A
Louisville, KY 40202
Office Phone: 502-852-2459
Andrew.DeFilippis@Louisville.Edu
Rachel Keith, PhD, ARNP
Data and Safety Monitoring Board Member

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Christina Lee Brown Envirome Institute, 
University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 207
Louisville, KY 40202
Office Phone: 502-852-4211
Rachel.Keith@Louisville.Edu
Shesh N. Rai, PhD
Study Biostatistician
Department of Biostatistics and Bioinformatics, University of Louisville 
Louisville, KY 40202
Office Phone: 502-852-4030
Shesh.Rai@Louisville.Edu
REGULATORY AND LOGISTICS SUPPORT
Clinical Trials Unit
University of Louisville 
501 East Broadway, Suite 210
Louisville, KY 40202
Office Phone: 502-852-7559
CTU@Louisville.Edu

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3. STUDY COMMITTEES 
Executive Committee (EC)
The EC will comprise of three components: study PI, study Medical and Safety Unit, and UofL 
Clinical Trials Unit. The EC will advise the study PI on various aspects of the study.
  
1.
Timothy O’Toole, PhD
Co-Principal Investigator
Christina Lee Brown Envirome Institute, University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 309
Louisville, KY 40202
502-852-5886
Tim.otoole@Louisville.Edu
2.
Andrew DeFilippis, MD
Co-Investigator, Study Physician
Division of Cardiovascular Medicine,
University of Louisville School of Medicine
580 S. Preston Street, Room 404-A
Louisville, KY 40202
Office Phone: 502-852-2459
Andrew.DeFilippis@Louisville.Edu
3.
Clinical Trials Unit
Regulatory and Logistics Support
University of Louisville 
501 East Broadway, Suite 210
Louisville, KY 40202
502-852-7559
CTU@Louisville.Edu
Data Safety Monitoring Board (DSMB)
The DSMB will comprise of three independent experts responsible for reviewing clinical trial 
data on an ongoing basis to ensure the safety of study participants and validity and integrity of 
the data. The DSMB will use a combination of, notifications, event specific reports, and 
scheduled cumulative trial reports to keep the Executive Committee (EC) and the Institutional 
Review Board (IRB) informed about real and potential safety issues.
1.
Matthew Cave, MD
Associate Professor of Medicine

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Division of Gastroenterology, Hepatology, and Nutrition,
University of Louisville School of Medicine
Louisville, KY 40202
Office Phone: 502-852-6991
Matt.Cave@Louisville.Edu
2.
Shahab Ghafghazi, MD
Assistant Professor of Medicine, 
University of Louisville School of Medicine
Office Phone: 502-852-7959
Shahab.Ghafghazi@Louisville.Edu
3.
Rachel Keith, PhD, ARNP
Assistant Professor of Medicine 
Christina Lee Brown Envirome Institute, 
University of Louisville School of Medicine
302 E. Muhammad Ali Blvd, Room 207
Louisville, KY 40202
Office Phone: 502-852-4211
Rachel.Keith@Louisville.Edu
4.
Shesh N. Rai, PhD
Professor of Bioinformatics and Biostatistics
Biostatistics and Bioinformatics Facility
Department of Biostatistics and Bioinformatics, University of Louisville 
Louisville, KY 40202
502-852-4030
Shesh.Rai@Louisville.Edu
Randomization Committee (RC)
The RC will be in possession of the randomization key so that accurate unblinding of the 
randomization assignment may be identified in event of a medical or other emergency. The 
randomization key will be held by four individuals: the pharmacist compounding the supplement 
(Dr. Rice), the study physician (Dr. DeFilippis), a member of the UofL Clinical Trials Unit, and 
the study biostatistician (Dr. Rai). 
1.
Peter Rice, PharmD, PhD
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado
12850 East Montview Blvd C238
Aurora, CO 80045
303-724-2613

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Peter.Rice@CUAnschutz.Edu
2.
Andrew DeFilippis, MD
Co-Investigator – Study Physician
Division of Cardiovascular Medicine,
University of Louisville School of Medicine
580 S. Preston Street, Room 404-A
Louisville, KY 40202
Office Phone: 502-852-2459
Andrew.DeFilippis@Louisville.Edu
3.
Clinical Trials Unit
University of Louisville 
501 East Broadway, Suite 210
Louisville, KY 40202
502-852-7559
CTU@Louisville.Edu
4.
Shesh N. Rai, PhD
Professor of Bioinformatics and Biostatistics
Biostatistics and Bioinformatics Facility
Department of Biostatistics and Bioinformatics, University of Louisville 
Louisville, KY 40202
502-852-4030
Shesh.Rai@Louisville.Edu

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4. LIST OF ABBREVIATIONS
AE
Adverse Event
EPC
Endothelial Progenitor Cells
CTU
Clinical Trials Unit, University of Louisville
DHHS
Department of Health and Human Services
DSHEA
Dietary Supplement Health and Education Act of 1994
DSMB
Data Safety Monitoring Board
EC
Executive Committee
FDA
Food and Drugs Administration
IRB
Institutional Review Board
MedDRA
Medical Dictionary for Regulatory Activities
NEAT
Nucleophilic Defense Against PM Toxicity
SAE
Serious Adverse Event
SAR
Suspected Adverse Reaction
SOC
System Organ Classification
SSAR
Serious Suspected Adverse Reaction
Supplement
Dietary Supplement Freely Available Over the Counter
UofL
University of Louisville
UPs
Unanticipated Problems
CVD
Cardiovascular Disease
WHO
World Health Organization
PMA
Platelet Monocyte Aggregates 
CAP
Concentration Air Particles
OC
Organic Carbons
PM2.5
Particulate Matter with diameter less than 2.5 micrometers
VOC
Volatile Organic Compounds

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5. EXECUTIVE SUMMARY
Study Design: This is a randomized, double-blind, placebo-controlled longitudinal clinical 
intervention study to assess the physiologic effects of dietary supplementation of carnosine in 
healthy human participants. Carnosine is a naturally synthesized peptide in the human body, 
and is also commercially available as a dietary supplement. The primary endpoint in this study 
are endothelial progenitor cells (EPCs) found in peripheral blood. Exposure to PM2.5 is known to 
decrease levels of EPCs, which represent angiogenesis. 
EPCs will be compared between carnosine and placebo groups at 6 weeks and also their 
change from baseline to 6 weeks to 12 weeks post initiation of intervention. The secondary 
endpoints are difference in EPCs at 12 weeks between carnosine versus placebo groups and 
difference in arterial stiffness, endothelial microparticles, and platelet monocyte aggregates 
between carnosine versus placebo groups at 6 weeks and 12 weeks.
Target Population: Eligible study candidates will be males or females from all ethnicities, 
between 22 and 65 years of age, living within 30 miles radius of Louisville, KY, willing to provide 
a urine sample, and willing to participate in the entire study if found eligible for the main study. 
Eligible candidates will be identified from an existing database of people interested in being 
contacted for research studies or the University of Louisville outpatient clinics. Study information 
will be advertised using flyers, social media, and other outreach methods to reach out to a 
broader population and increase broader representation. Study candidates must agree to the 
consent and will have no indication of concurrent illness. We will exclude individuals with active 
treatment for alcohol and drug abuse, or those using immunosuppressant agents or any other 
medical condition that might compromise the successful completion of the study. Subjects will 
be prescreened, and those with conditions known to effect peripheral blood cell counts and 
bone marrow function will be excluded from the study. These conditions included malignancies, 
organ transplant, renal replacement therapy, type 1 diabetes, untreated thyroid disease, 
anemia, acute infections, HIV infection, hepatitis, and unhealed wounds. Subjects on hormone 
replacement therapy or medications affecting bone marrow function or peripheral blood cell 
counts will also be excluded from the study as will those unwilling or unable to provide informed 
consent, pregnant or lactating women, prisoners and other vulnerable populations. Personal 
identifiers will be recorded and secured by clinical personnel. 
Eligible candidates will be identified via a screening visit during which the following data will be 
collected: primary contact and demographic information, and a urine sample. The urine sample 
will be used to assess levels of carnosine. Individuals with less than median level of carnosine 
will be invited to participate in the study. 
Selected Sample: A total of 240 participants will be recruited (120 receiving Carnosine dietary 
supplementation and 120 receiving placebo); males and females, all ethnicities, who live in 

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Louisville and surrounding Kentucky counties, with carnosine levels below the median value of 
the population. The participants should also agree to complete 4 study visits (1 screening, 1 
baseline assessment, and 2 follow-ups) and to comply with study intervention regimen. 
Accounting for approximately 40% attrition, the goal is to randomize a total of 400 participants – 
200 in the carnosine group and 200 in the placebo group. 
Enrollment Period: Study enrollment will be split into two consecutive years, with a goal of 
equal enrollment each year (60 carnosine supplementation and 60 placebo in 2020; and 60 
carnosine supplementation and 60 placebo in 2021). There will be a total of four study visits: 
Screening (Visit # 1), Baseline Assessment (Visit # 2), Follow-Up # 1 (Visit # 3), and Follow-Up 
# 2 (Visit # 4). Screening can occur throughout the year. Baseline assessment will occur from 
May through June. Follow-up # 1 will occur, at 6 weeks post initiation of study supplement, from 
June through August. Follow-up # 2 will occur, at 12 weeks post initiation of study supplement, 
from July through September. Study intervention (carnosine supplementation or placebo) will 
begin in May and continue through September. 
Rationale: Carnosine is a naturally occurring peptide that is also available commercially as a 
dietary supplement. Since carnosine has anti-oxidant properties and air pollution exposure 
induces a state of oxidative stress, the purpose of this study is to see if those taking carnosine 
as a dietary supplement are protected from air pollution-induced oxidative stress and adverse 
cardiovascular outcomes.
The protective effects of carnosine against PM2.5 toxicity are consistent with the biological role of 
this peptide and its physiochemical properties. Carnosine is a dipeptide (-alanyl-L-histidine), 
which is present in very high concentration in the skeletal muscle, brain and heart. The 
concentration of carnosine in human skeletal muscle exceeds 10mM, whereas its concentration 
in brain and heart is between 0.1 to 1mM.  High levels of carnosine have also been detected in 
monocytes and red blood cells. Because of its high buffering capacity as a zwitterion, carnosine 
is thought to prevent proton-induced inhibition of glycolysis, and sustain glycolytic activity under 
reduced oxygen concentrations. Hence, carnosine, or its precursor – -alanine, are widely used 
as performance enhancing supplements. In addition to maintaining intracellular pH, carnosine 
has antioxidant, anti-inflammatory and anti-glycating activities. Because of the high 
nucleophilicity of its histidyl ring, carnosine binds avidly to -unsaturated aldehydes such as 
acrolein11 and 4-hydroxy-trans-2-nonenal (HNE), which are major products of lipid peroxidation 
and important “second messengers” of ROS toxicity. Carnosine also reduces the formation of 
advance glycation end-products (AGEs) that contribute to the progression of several 
degenerative diseases including diabetes and atherosclerosis. Carnosine supplementation has 
been shown to prevent inflammation and has beneficial immunomodulatory effects, and our 
studies have shown that carnosine facilitates the removal of lipid peroxidation products and 
decreases atherosclerosis in mice as well.

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 We have identified multiple adverse outcomes of PM2.5 exposure in humans including 
endothelial damage, platelet activation, immune response dysfunction and a depletion of 
circulating EPCs which are involved in many physiological functions.  We have obtained similar 
results in animal models, which show that exposure to concentrated ambient particles depletes 
EPCs, and results in defective hematopoiesis. Mechanistic studies from our group and others 
suggests these diverse outcomes result from the induction of oxidative stress. Hence, carnosine 
supplementation represents a viable option for attenuating the oxidative and inflammatory 
effects of PM2.5 exposure.  Because of its anti-inflammatory, antioxidant, buffering, metal 
chelating roles,2 2 carnosine may be more effective than other dietary supplements tested to-
date. Moreover, our preliminary data show that carnosine levels are significant determinants of 
PM2.5 toxicity, and, in preclinical models, effective in attenuating the vascular effects of PM2.5.
Primary Endpoints (reflecting change from baseline to 6 weeks and 12 weeks and 
between groups)
1.
Difference in EPC-2 between Carnosine and Placebo Groups
2.
Difference in EPC-3 between Carnosine and Placebo Groups
3.
Difference in EPC-4 between Carnosine and Placebo Groups
4.
Difference in EPC-7 between Carnosine and Placebo Groups
5.
Difference in EPC-8 between Carnosine and Placebo Groups 
Secondary Endpoints (reflecting change from baseline to 6 weeks and 12 weeks and 
between groups)
1.
Difference in Arterial Stiffness between Carnosine and Placebo Groups 
2.
Difference in Endothelial Microparticles between Carnosine and Placebo Groups 
3.
Difference in Platelet Monocyte Aggregates between Carnosine and Placebo Groups 
Subgroup Analyses: First series of analyses will include an examination of the effects of 
carnosine supplementation on sub-groups:
1.
Age
2.
Sex
3.
Race
4.
Socioeconomic Status
5.
Diabetes
6.
Smoking
7.
Hypertension
Hypothesis (for Primary and Secondary Endpoints):
Carnosine supplementation blunts adverse effects of PM2.5 pollution on vasculature of healthy 
participants. 

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6. STUDY OBJECTIVES 
Primary Objective
The primary purpose of this study is to determine whether taking carnosine as a dietary 
supplement protects the vasculature from PM2.5 air pollution-induced oxidative stress in healthy 
human subjects. The primary endpoint is difference in EPCs between Carnosine versus placebo 
groups at 6 weeks and 12 weeks.
Secondary Objective
The secondary objective of this study is to determine whether taking carnosine as a dietary 
supplement is protective from PM2.5 air pollution-induced arterial dysfunction, endothelial 
damage, and pathological platelet activation in healthy human subjects. The secondary 
endpoints are difference in arterial stiffness, endothelial microparticles, and platelet monocyte 
aggregates between Carnosine versus placebo groups at 6 weeks and 12 weeks.
Hypothesis (for Primary and Secondary Objectives)
Carnosine supplementation blunts adverse effects of PM2.5 pollution on vasculature of healthy 
participants.
7. BACKGROUND 
Rationale
Exposure to particulate air pollution has been found to be robustly associated with increased 
risk of cardiovascular disease (CVD) and CVD mortality. Acute exposure to fine particulate 
matter (PM2.5) has been linked to adverse cardiovascular events such as myocardial infarction, 
stroke, cardiac arrhythmias, and sudden cardiac death, whereas chronic exposures are 
associated with an increased burden to CVD risk factors, insulin resistance and accelerated 
progression of atherosclerotic disease. However, the mechanisms by which PM2.5 induces 
cardiovascular injury remain unclear and the pathophysiological conditions and co-exposures 
that determine individual susceptibility to PM2.5 toxicity have not been identified. Although 
reductions in PM levels have been found to reduce CVD mortality, no effective individual 
interventions are available to mitigate the adverse health effects of PM2.5. To design and test 
such interventions, it is important to understand not only the mechanisms underlying PM 
toxicity, but also to identify pre-existing conditions and co-exposures that moderate the 
cardiovascular effects of PM, so that appropriate, mechanism-based interventions can be 
devised and targeted to those individuals who are susceptible to the harmful cardiovascular 
effects of PM2.5.
Our previous work has shown that in young, healthy adults, exposure to PM2.5 is associated with 
inflammation and subclinical vascular injury. In addition, we have found that exposure to PM2.5 

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decreases levels of EPCs that could potentially compromise vascular repair. We have obtained 
similar results in animal models, which show that exposure to concentrated ambient particles 
depletes EPCs, and results in defective hematopoiesis. In pilot experiments, we have found that 
PM2.5-induced defects in angiogenic cell function could be prevented by treating mice with the 
endogenous, naturally occurring dipeptide - carnosine, which quenches reactive oxygen species 
and removes toxic products of lipid peroxidation. We have also found that individuals with high 
CVD risk are more susceptible to the effects of PM2.5 than are low-risk individuals. Our studies 
also show that individuals with high levels of carnosine (a natural dipeptide) are relatively 
impervious to the adverse vascular effects of PM2.5. Building upon these observations, we now 
plan to identify individual risk factors that determine cardiovascular susceptibility to PM2.5, and to 
conduct a clinical trial to test the hypothesis that in the presence of CVD risk factors, exposure 
to PM2.5 induces vascular dysfunction and injury, and that these could be prevented by 
carnosine, which removes toxic products generated by PM2.5 exposure. To test this hypothesis, 
we will assess the impact of PM2.5 on vascular function in individuals with mild-to-moderate CVD 
risk, identify susceptibility factors and co-exposures that predispose individuals to PM toxicity, 
and determine whether the vascular effects of PM could be prevented by carnosine 
supplementation.
Extensive epidemiologic data show that exposure to particulate air pollution is associated with 
adverse health outcomes and excessive mortality.3-5  The World Health Organization (WHO) 
estimates that exposure to air pollution is linked to 6.5 million deaths (approximately 12% of 
total deaths) per year worldwide. The pervasiveness of the health impact of air pollution is 
underscored by data showing that 92% of the world’s population lives in areas where the levels 
of fine particulate matter (PM2.5) exceed the WHO recommended annual mean concentration 
limit of 10g/m3. Thus, exposure to air pollution is a major global health threat, and is likely to 
remain so in future. Assessments of the impact of PM2.5 and understanding of how PM2.5 affects 
health and how these effects could be mitigated are, therefore, priority research areas of high 
scientific and social significance.
Although exposure to PM2.5 increases the risk of several diseases, most (70-80%) of the 
excessive mortality associated with PM is attributable to CVD.6-8 Short-term elevations in PM2.5 
levels increase the incidence of myocardial infarction, arrhythmias, stroke, and CVD mortality. In 
some estimates, chronic exposure to 10 g/m3 PM2.5 is associated with an 8-18 % increase in 
CVD mortality.9 In humans, as well as animal models,10-12 chronic exposure to PM2.5 is 
associated with increased blood pressure, progressive atherosclerotic disease, and worsening 
heart failure.  Given the marked cardiovascular toxicity of PM2.5, the health burden of PM2.5 
could not be substantially decreased without diminishing its cardiovascular impact. Accordingly, 
our studies are focused on understanding the cardiovascular effects of PM2.5, and how they 
could be mitigated.  Our previous work has shown that exposure to PM2.5 decreases the levels 
of EPCs and that these changes are accompanied by an increase in the levels of circulating 
endothelial microparticles, immune cells, and anti-angiogenic cytokines13-16. These findings 
provide new insights into the nature of PM2.5 -induced cardiovascular injury, and identify new 

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targets and biomarkers of PM2.5 injury. Based on these findings, we now plan to elucidate the 
mechanistic link between PM2.5 exposure and cardiovascular injury. Elucidation of such 
mechanisms is important not only for a better understanding of PM2.5 toxicity, but also for 
developing mechanism-based interventions to mitigate the adverse effects of PM2.5.
Despite its high disease burden, no effective individual-level interventions are currently available 
to prevent the adverse cardiovascular effects of PM2.5.  Although the deleterious effects of PM2.5 
could be attenuated by decreasing its ambient levels, such reductions are difficult, even in 
developed countries, due to difficulties associated with reducing traffic, or changing industrial 
and agricultural practices. Such societal changes take many years. Meanwhile, millions across 
the world are exposed to unhealthy levels of PM2.5. Hence, effective interventions are urgently 
needed to lessen the adverse health effects of PM2.5. Such interventions, even if partly 
successful, could significantly reduce mortality worldwide. However, attempts to develop 
effective interventions are thwarted by a paucity of mechanistic understanding of PM2.5 toxicity, 
which is a pre-requisite for designing effective interventions that target key steps in the sequelae 
of PM2.5 injury.
In addition to being mechanism-based, such interventions must also target specific populations. 
It has long been suspected that not all individuals are equally sensitive to PM2.5. Indeed, some 
studies have reported that individuals of low socio-economic status, or those with diabetes or 
advanced age are particularly sensitive to PM2.5,17-19but others have reported contradictory 
results.20 Overall, the literature is sparse and inconsistent, and many studies are limited by the 
lack of information available on CVD risk factor burden, which might partly account for variability 
in susceptibility, which could also stem from co-exposures to other pollutants.20 Hence, we will 
identify susceptible states and moderating co-exposures that determine vulnerability to PM2.5, 
and delineate their contribution to cardiovascular harm associated with PM2.5. These findings 
would not only lead to a better understanding of the cardiovascular pathways affected by PM2.5, 
but also aid in targeting interventions to mitigate PM toxicity.  Hence, the results of our project to 
understand the mechanisms that contribute to the cardiovascular effects of PM, to identify and 
validate indices and biomarker of PM-induced vascular injury, and then to test whether targeted 
intervention to these processes could attenuate PM associated changes address key 
knowledge gaps in this area and move the field forward.  Additionally, findings of our project 
could also lead to the development of clinical guidelines for preventing PM2.5 toxicity in 
susceptible individuals, and evidence-based advisories to mitigate the effects of PM2.5 by 
avoiding co-exposure and protecting vulnerable populations.
  
Background Pre-Clinical 
In order to test whether carnosine supplementation will attenuate the effects of PM2.5; however, 
to obtain proof-of-principle data for this Aim, we employed a preclinical mouse model to 
determine whether carnosine supplementation would attenuate the effects of PM2.5. In these 
experiments, we found that mice inhaling concentrated air particles (CAPs) for 9 days show 

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defects in hematopoiesis, i.e., the bone marrow of mice exposed to CAPs was populated by 
fewer hematopoietic stem cells. 
Furthermore, EPCs isolated from the bone marrow of exposed mice exhibited an impaired 
ability to form tubes in vitro.   However, mice that were placed on carnosine-containing drinking 
water showed no significant defect in tube formation. To assess the physiological significance of 
these findings, we exposed mice to CAPs ± carnosine in drinking water. After 9 days of 
exposure, EPCs were isolated from these mice and injected into other mice subjected to hind 
limb ischemia.  EPCs from exposed mice fed carnosine were able to stimulate blood perfusion, 
while those on normal drinking water did not.  These observations, showing that carnosine 
attenuates the hematopoietic injury induced by PM2.5, and prevents EPC dysfunction in mice; 
strengthen the rationale for conducting an interventional clinical study. Thus, collectively, our 
preliminary studies support key concepts of the project, i.e., PM2.5 exposure in humans is 
associated with marked vascular injury that is moderated by pre-existing CVD risk and that 
carnosine levels and that carnosine supplementation blunts adverse effects of PM2.5.
Vascular homeostasis and endothelial health are thought to be maintained in part by a 
subpopulation of pro-angiogenic cells - the EPCs that reside in the bone marrow and circulate in 
the peripheral blood. 21 It has been shown that upon hypoxia or vascular injury, EPCs are 
mobilized from the bone marrow and home to the site of tissue damage. At these sites, EPCs 
contribute to angiogenesis either through terminal differentiation into mature endothelial cells or 
by paracrine stimulation of wound healing processes. 22
Background Clinical 
Exposure to PM2.5 has been found to be associated with an increase in blood pressure and 
vascular dysfunction.23 Our studies have shown that PM2.5 exposure is associated with changes 
in several biomarkers of vascular injury, including EPCs, endothelial microparticles, fibrinogen, 
platelet-monocyte aggregates as well as plasma cytokines and growth factors in a cohort of 
young individuals with low CVD risk.13  However, it remains unclear whether vascular function 
and biomarkers of vascular injury are similarly affected in our cohort of individuals with mild-to-
moderate CVD risk (Framingham Risk Score, >10). Extending our previous work with a young 
disease-free cohort to an older, at-risk cohort is essential to identify which specific indices of 
vascular function and which specific biomarkers of vascular injury are associated with PM2.5 
exposure in individuals with mild-to-moderate CVD risk, as our preliminary work shows that 
changes in vascular parameters vary with CVD risk. Moreover, this work will permit assessment 
of the relationship between PM2.5-induced changes in function and the underlying 
pathophysiological changes. We will further examine their interrelationships and contribution to 
vascular injury. 
However, not all individuals are equally sensitive to PM2.5. Previous studies have variably shown 
some individuals are more sensitive to PM2.5-associated mortality than are young healthy 

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individuals,18, 19, 24, 25 but the literature is sparse and conflicting.20 Moreover, it is unclear whether 
changes in PM2.5-associated vascular injury varies with individual differences in age, SES, sex, 
ethnicity, and pre-existing CVD risk. Our past studies have shown that PM2.5 exposure is 
associated with endothelial injury even in young, healthy individuals,13 which might be important 
factors contributing to progressive cardiovascular injury, hence it is important to distinguish 
between changes that contribute to the progression of PM2.5-induced injury in healthy 
individuals, and those that heighten PM2.5 sensitivity in individuals with high CVD risk. This 
distinction is important for appropriate targeting of interventions via carnosine supplementation.
In addition to individual level variables, co-exposure to other pollutants could also affect the 
relationship between PM2.5 exposure and vascular injury and dysfunction. There is sparse and 
conflicting evidence for an association between CVD and exposure to gaseous pollutants such 
as ozone, oxides of nitrogen, carbon monoxide, and organic carbon (OC).20 Identifying the role 
of these pollutants in epidemiological studies is difficult due to their strong correlation with each 
other and with PM.  Moreover, epidemiological studies continue to focus on measures of OC 
without speciation, because of the expense, as a result, data on the effects of volatile organic 
compounds (VOCs) are lacking.20  Evaluation of the role of VOCs is important, because even as 
the particle emission are reduced, there is increased risk of higher toxicity of remaining particles 
and an increase in gaseous copollutants,26 which are expected to increase with a changing 
climate and by 2050, and their impact may exceed that of particulate pollution.27   Moreover, 
animal data from our laboratories suggest that they cause significant cardiovascular injury.28-32  
Limited data from our group also link VOCs to CVD risk and cardiovascular injury in humans.33  
Hence, in addition to our focus on PM2.5, we will examine the contribution of gaseous pollutants, 
as they are likely to affect pathways (inflammation, oxidative stress, thrombosis) similar to PM2.5. 
The results of these investigations will not only provide a new understanding of the role of 
gaseous pollutants in inducing vascular injury, but also aid in assessing whether they heighten 
sensitivity to PM, which would be important in identifying individuals suitable for intervention via 
carnosine supplementation. 
The intervention of carnosine supplementation as blinded placebo-controlled clinical trials has 
been extensively published in the existing literature. 34-37 Daily dose of dietary carnosine 
supplementation in these studies is predominantly in the range of 2 grams to 4 grams per day. A 
daily dietary supplementation of 2 grams carnosine has been shown to be safe and efficacious 
in healthy human participants.34, 37 

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8. INVESTIGATIONAL PLAN
Research Questions
1.
Does dietary supplementation of carnosine during a period of high PM2.5 pollution 
mitigates effects on vascular indices?
2.
Does dietary supplementation of carnosine during a period of high PM2.5 pollution 
improve the arterial function?
3.
Does dietary supplementation of carnosine during a period of high PM2.5 pollution 
decrease endothelial damage?
4.
Does dietary supplementation of carnosine during a period of high PM2.5 pollution 
decrease platelet activation?
Study Design
This is a randomized, double-blind, placebo-controlled longitudinal clinical intervention study to 
assess whether dietary supplementation of a naturally occurring peptide in the body will mitigate 
the harmful effects of seasonal PM2.5 air pollution on indices of vascular biology. A total of 240 
participants will be enrolled and randomized in 1:1 ratio to receive either dietary carnosine or 
placebo (cornstarch).  Stratification factors are sex (birth male and female), age (22-44; 45-65) 
and carnosine level (very low, low).  A block (or size 6) stratified randomization scheme will be 
used. Accounting for a conservative attrition rate of 40% (failure to complete all study time-
points), a total of 400 participants will be randomized to receive either dietary carnosine 
supplementation or placebo (cornstarch). 
The mode of dietary supplementation will be oral capsules. Intervention of dietary carnosine 
supplementation or placebo will occur daily, for 12 consecutive weeks during a period of high 
PM2.5 pollution – May through September. Due to logistical limitations, enrollment may be split 
over multiple years, but will occur during the same monthly period of high PM2.5 pollution (May 
through September) in the study region (30 miles radius from Louisville Kentucky). The 12 
weeks supplementation period was selected based upon previous dietary carnosine 
supplementation trials as a reasonable and commonly used time frame to assess benefit and 
risk.34, 37  Based on existing literature, a measureable effect of carnosine on many indices is 
noticeable by 6 weeks of starting supplementation and can reach peak effect by 12 weeks. 34, 37   
The general study enrollment and investigational plan for Year # 1 (2020) and Year # 2 (2021) is 
illustrated and outlined in Figure 1 and Figure 2 respectively. Details regarding each individual 
study visit are illustrated and outlined in Figures 3-7. The study visits and details regarding each 
visit are further detailed in section 10 “Study Visits”.
The study PI and other investigators will be blinded to the allocation of participants to either 
carnosine or placebo. The participants will also be blinded to their allocation. The 
Randomization Committee (RC) will be in possession of the randomization key so that accurate 
unblinding of the randomization assignment may be identified in the event of a medical or other 

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emergency. The RC will consist of four individuals: the pharmacist compounding the 
supplement (Dr. Peter Rice), the study physician (Dr. Andrew DeFilippis), a member of the UofL 
Clinical Trials Unit, and the study biostatistician (Dr. Shesh Rai). Study supplements (carnosine 
or placebo) will be prepackaged into punch cards and marked with letters “A” or “B”. The 
contents of series “A” or “B” will be part of the blinded randomization and will be known only to 
the RC. The study supplements will be directly shipped, stored and handled by the UofL CTU.  
An initial screening (Visit # 1) of potential / interested participants will be conducted to identify 
eligible candidates based on eligibility criteria established a priori. Eligible candidates will be 
invited to a complete baseline assessment (Visit # 2). Randomization and allocation will occur 
between Visit # 1 and Visit # 2. Participants invited for Visit # 2 will already be randomized into 
one of the two study groups (carnosine or placebo). Participants from both study groups will be 
followed up at 6 weeks (Visit # 3) and 12 weeks (Visit # 4). Relevant and extensive data for 
primary and secondary endpoints will be collected at Visit # 2 through Visit # 4. The data 
collected throughout all visits (# 1 through # 4) may be used to generate preliminary data for 
future studies. 
Visit # 1 will focus on consenting the participant for the entire study, collect contact/demographic 
information, and collect a urine sample. Safety assessments will be conducted at Visit # 3 and 
Visit # 4. Participants may be contacted for up to 12 months after enrollment via 
phone/email/mail for safety, adverse events, and or general follow up questions.
Study Endpoints
Primary Endpoints (reflecting change from baseline to 6 weeks 
and 12 weeks and between groups)
1.
Difference in EPC-2 between Carnosine and Placebo Groups
2.
Difference in EPC-3 between Carnosine and Placebo Groups
3.
Difference in EPC-4 between Carnosine and Placebo Groups
4.
Difference in EPC-7 between Carnosine and Placebo Groups
5.
Difference in EPC-8 between Carnosine and Placebo Groups 
Secondary Endpoints (reflecting change from baseline to 6 
weeks and 12 weeks and between groups)
1.
Difference in Arterial Stiffness between Carnosine and Placebo Groups 
2.
Difference in Endothelial Microparticles between Carnosine and Placebo Groups 
3.
Difference in Platelet Monocyte Aggregates between Carnosine and Placebo Groups 

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Data Collection Tools
Blood Samples
A sample of ~3 table spoons (~45 ml) of blood will be obtained by venipuncture 
performed by a study staff trained in phlebotomy using standard procedures and 
precautions to insure the safety of the participants and phlebotomist. If any subject 
becomes ill, uncomfortable, or desires to withdraw, he/she may do so at any time. Blood 
sample collection protocol is detailed in Appendix A. Samples will be promptly 
transported to the Diabetes and Obesity Center’s research laboratory on the 4th floor of 
the Delia Baxter building (580 S. Preston Street) for processing, storage, and analysis. 
After processing, samples will be aliquoted in appropriately labeled vials and stored in (-
80C) freezer. Stored samples will contain study name, participant ID, date of sample 
acquisition, type of sample, and visit type. Samples will be stored in de-identified fashion 
and participant personal information or PHI will not be displayed/barcoded on any stored 
sample. Samples will be stored for future use, ancillary studies, and or preliminary data 
development. 
The measurement of blood biomarkers for primary and secondary endpoints from blood 
samples (EPCs, microparticles, and PMA) are further detailed in Appendix B.  
Urine Samples
Standard clean catch urine sample will be obtained from participants and stored at 4°C. 
Urine sample collection protocol is detailed in Appendix A. The samples will be 
transported on ice to the Diabetes and Obesity Center’s research laboratory on the 4th 
floor of the Delia Baxter building (580 S. Preston Street) for processing, storage and 
mass spectrometric analysis. After processing, samples will be aliquoted in appropriately 
labeled vials and stored in (-80C) freezer. Stored samples will contain study name, 
participant ID, date of sample acquisition, type of sample, and visit type. Samples will be 
stored in de-identified fashion and participant personal information or PHI will not be 
displayed/barcoded on any stored sample. Samples will be stored for future use, 
ancillary studies, and or preliminary data development. 
Arterial Stiffness Test (Vascular Function)
To assess CVD risk, we will acquire arterial stiffness measures (augmentation index; AI, 
augmentation pressure; AP, and pulse wave velocity; PWV), aortic systolic pressure and 
SEVR derived from pulse wave analysis using SphygmoCor, which provides highly 
reproducible values.38-40 The participant will be asked to provide the initials of their first 
and last name and date of birth. The device will also store the participant ID, date, and 
time of data acquisition. These parameters are necessary to calculate age and for data 
retrieval. Data will be stored on the device and other HIPAA compliant storage 
databases.

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Measures of arterial stiffness are positively and robustly associated with increased risk 
for CV events, 41 and are associated with atherosclerosis, hypertension, stroke, 
Alzheimer-type dementia, and kidney disease. They provide novel and clinically-relevant 
information beyond that provided by standard risk factors; have been increasingly 
incorporated into longitudinal cohort studies, 41 and are particularly sensitive to air 
pollution exposure. 42. 
SphygmoCor Xcel is a class-II FDA approved device used in clinical and research 
settings. SphygmoCor is a non-invasive, cuff-based tool used for obtaining brachial 
artery blood pressure and the central arterial pressure waveform with associated indices 
(augmentation index, augmentation pressure, and pulse wave velocity). A cuff will be 
applied on the upper arm in the standard position. The cuff will be partially inflated to 
record the brachial waveforms. These waveforms are detected by sensing changes in 
the pressure inside the cuff related to arterial pulsation. The ascending aortic waveform 
is subsequently derived using a validated mathematical transfer function.
The participant may be provided with their current blood pressure and heart rate, as 
measured by this FDA approved device.  
Questionnaires
Self-reported interview-style questionnaires are quick, convenient and reliable source of 
research data collection. This modality is used by many large NIH funded research 
studies like the Multi-Ethnic Study of Atherosclerosis (MESA), the National Health and 
Nutrition Examination Survey (NHANES), and many of our group’s other NIH funded 
studies – the Green Heart Project, Cardiovascular Injury in Tobacco, Louisville Healthy 
Heart Study, etc. We will utilize these existing questionnaires (internal and external) to 
compile a list of relevant questions which will help in high quality data acquisition. The 
questionnaires will collect data on medical history, medications, environmental pollution 
exposure, sleep, diet, exercise, tobacco, and women’s health. 
Data will be recorded in real-time in a REDCap database created specifically for this 
study. Since, these questionnaires will collect multiple personal information and PHI, 
only trained research personnel with relevant reason will be granted access. 
Physical Examination
Physical examination will be conducted to record the participant’s height, weight, and 
waist circumference. Blood pressure and heart rate measurements will be provided 
during the arterial stiffness measurement.
Physical Function Test
To assess the impact of carnosine supplementation on physical performance during 
times of variable PM2.5 levels, we will utilize three physical performance tests: handgrip 

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strength test, 2-minute step test (2MST), and bilateral calf-raise test. The protocol for 
these measurements are further detailed in Appendix E.  
Sample Size Computations and Assumptions
The primary outcome measures are EPC-2, EPC -3, EPC -4, EPC -7, and EPC -8. Also, 
there are many secondary outcome measures. Based on our pilot study, mean and SD 
(standard deviation) are reported below. 
EPC-2
EPC-3
EPC-4
EPC-7
EPC-8
Mean
0.675783
0.016016
0.050813
0.297092
0.631728
SD
0.702561
0.010805
0.056709
0.412414
0.691299
This is a longitudinal study and primary observations will be made at baseline, 6 weeks 
and 12 weeks follow-ups. Due to 5 primary outcome measures, we adjusted alpha=0.01 
(=.05/5). Due to multiple sub-studies, we plan to enroll a large evaluable cohort of size 
n=240. With a correlation of 0.5 on any two consecutive time-point measures, (this is a 
conservative estimate) and n1=120, n=120 at alpha= 0.01 and power=0.90, we can 
detect the effect size of 0.5 SD using a two sample t-test in a longitudinal setting. The 
detectable effects for each EPC is given below.
Power
Correlation
EPC-2
EPC-3
EPC-4
EPC-7
EPC-8
80%
0.50
0.351
0.005
0.028
0.206
0.346
90%
0.50
0.223
0.003
0.018
0.131
0.219
80%
0.80
0.197
0.003
0.016
0.116
0.194
90%
0.80
0.156
0.002
0.013
0.092
0.153
To adjust for attrition/drop out we have increased the sample size to 400.
Associations of PM2.5 exposures with measured outcomes will be estimated using mixed 
effects models. We will use interaction variables to detect whether the association 
between PM2.5 and outcomes is significantly different between with or without carnosine 
groups. We will perform a formal mediation analysis, which allows for interactions to 
determine the degree to which carnosine levels mediate the effect of PM2.5 on our 
outcomes. Plots of subject-specific differences over pollution concentrations along with 
regression plots will be generated.
Sample Size Recommendation
Sample size of 240 participants (120 carnosine supplementation, 120 placebo group) 
provides sufficient sample size for the primary endpoint as well as other secondary 
endpoints. 

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Due to the prolonged intervention period (12 weeks), two follow-up visits and the 
inconvenience of taking multiple oral capsules daily, we anticipate a 40% attrition from 
screening (Visit # 1) through 12-week follow-up (Visit # 4). Therefore, we have 
established a goal of stratified identification of a total of 400 participants (1:1) at 
screening (Visit # 1). We anticipate that with a conservative estimate of 60% retention, 
120 participants in each group (supplementation, placebo) will complete the entire study.  
Multiple Testing Issue
Type I error correction in the context of an early phase clinical trial that performs multiple 
tests can serve to protect the study interpretation against Type I error inflation. However, 
the use of corrections for multiple comparisons in “proof-of-concept-studies” is 
problematic. At this investigational level, tight control of the overall family-wise Type I 
error rate would increase the likelihood that the investigators would miss a finding of 
potential importance by attributing it to chance simply because the p-value is large. The 
study investigators have, in this protocol, attempted to balance the need to control the 
number of evaluations with the need to identify new effects by not incorporating a 
multiplicity correction. The primary outcomes measures will be declared significant for 
alpha <0.01. There will not be any adjustments for secondary outcome measures and 
will be declared significant at alpha <0.05. 
9. STUDY ENROLLMENT AND WITHDRAWAL
Introduction
Potential subjects will be males and females, all ethnicities, 22-65 years of age, living within or 
near the Louisville metropolitan area, with carnosine levels below the median value of the 
population.
Criteria to Invite for Screening Visit (Visit # 1)
To be considered eligible to participate in the screening visit of the study, the participant must 
meet the following criteria:
1.
Living within 30 miles radius of Louisville Metropolitan area (Kentucky counties: 
Jefferson, Bullitt, Spencer, Shelby, and Oldham; Indiana counties: Clark, Floyd).
2.
Age 22-65 at time of baseline assessment (Visit # 2)
3.
Healthy subjects
4.
Subjects with a diagnosis that puts them at high risk for acute or chronic disease, 
specifically but not limited to hypertension or diabetes, are eligible for the study if the 
condition is treated by a physician or provider and is controlled via medications and or 
lifestyle that are otherwise not excluded by the exclusion criteria.
5.
Consumes some type of meat/fish at least once a month during the past 3 months 

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6.
Agree to complete all four study visits (one screening, one baseline, two follow-up visits)
7.
Will be living in the Louisville Metropolitan area throughout the study period (May 
through September), with planned vacations of no more than 2 weeks away from study 
area.
8.
Agree to take supplement/placebo intervention during the study period and follow study 
intervention regimen
Main Study Inclusion Criteria (Visits # 2-4)
To be considered eligible to participate in this study, the participant must meet the following 
criteria:
1.
Living within 30 miles radius of Louisville Metropolitan area (Kentucky counties: 
Jefferson, Bullitt, Spencer, Shelby, and Oldham; Indiana counties: Clark, Floyd).
2.
Age 22-65 at time of baseline assessment (Visit # 2)
3.
Consumes some type of meat/fish at least once a month during the past 3 months 
4.
Carnosine level less than median (value of the population
5.
Agree to complete all four study visits (one screening, one baseline, two follow-up visits)
6.
Will be living in the Louisville Metropolitan area throughout the study period (May 
through September), with planned vacations of no more than 2 weeks away from study 
area.
7.
Agree to take supplement/placebo intervention during the study period and follow study 
intervention regimen
Exclusion Criteria (All Visits)
Participants will be excluded from the study if they have any of the following conditions:
1.
Consumed any carnosine supplement of more than 6 grams per a week in the past 4 
weeks (one month)
2.
Current / ongoing treatment for substance abuse 
3.
Currently undergoing treatment or have conditions which may cause participant to be 
immunosuppressed
4.
Diseases Affecting Peripheral Cell Count (i.e. Autoimmune Diseases – Hashimoto, 
Rheumatoid Arthritis, SLE, Rheumatoid Arthritis, Sjogren syndrome, Ankylosing 
Spondylitis, Takayasu arteritis, Kawasaki disease, Polyarteritis nodosa.) 
5.
Diseases Affecting Bone Marrow capacity
6.
Diagnosis of any active cancer
7.
Recent organ / kidney transplant or replacement (Active/Long-Term Medications)
8.
Type 1 Diabetes Mellitus 
9.
Untreated thyroid disease
10. Untreated anemia
11. Current acute infections (Influenza, fever, etc.)
12. HIV positive status
13. Active/current Hepatitis HepA, HepB or HepC or in past 6 months  
14. Pregnant / lactating or planning to  

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15. Prisoners / vulnerable populations
16. Other medical conditions that compromise completion of study 
17. Unwilling to provide consent
Special Conditions of Exclusion (Screening or Main Study)
Candidates with the following conditions or history may be excluded: 
1.
Local reactions: diarrhea, stomach upset, tingling sensation in extremities, rash or 
itchiness, dry mouth, changes in appetite, feelings of tiredness, or vivid dreams.
2.
Systemic reactions: fever, allergic reaction, anaphylaxis or any clinical untoward event 
that occurred with a known cause of taking dietary supplement.
3.
Abnormal laboratory results which are deemed clinically significant with a known 
cause of taking dietary supplement.
4.
Pregnancy and breast-feeding: Currently, there is not enough reliable information 
about the safety of taking carnosine supplement during pregnancy or breast-feeding. 
Hence, participants/candidates who are currently pregnant or breast-feeding or plan to 
be pregnant/breast-feeding during the study period will not be enrolled in the study.  
5.
Low blood pressure: Carnosine synthesized in the body has been found to decrease 
blood pressure via influencing the autonomic nervous system. 43 Hence, in theory, taking 
supplemental carnosine might lower blood pressure and or exacerbate lowering of blood 
pressure in people taking antihypertensive medications. Hence, all participants taking 
blood pressure lowering medication will be cautioned for this interaction and advised that 
in case of an episode of low blood pressure to discontinue study supplement and 
immediately seek medical attention from a licensed healthcare provider.
6.
Low blood sugar: Carnosine synthesized in the body has been found to decrease blood 
sugar via influencing the autonomic nervous system. 43 Hence, in theory, taking 
supplemental carnosine might lower blood sugar and or exacerbate lowering of blood 
sugar in people taking blood sugar lowering medications. Hence, all participants taking 
blood sugar lowering medication will be cautioned for this interaction and advised that in 
case of an episode of low blood sugar to discontinue study supplement and immediately 
seek medical attention from a licensed healthcare provider.
Prescreening (Prior to Consent)
Prescreening participants includes reviewing personal or protected health information for 
inclusion/exclusions prior to consent. Also, currently we have a database of about 1,000 
subjects who have previously participated or contacted us for participating in one of our 
research studies and expressed interest in participating in future research studies. Only 
candidates who have given permission to contact them for future studies will be prescreened for 
relevant eligibility criteria and contact information. The script for phone and email/letter 
communication is detailed in Appendix C.  

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Consent
All participants enrolled in this clinical trial will be identified as males or females, age 22-65, 
living within 30 miles radius of Louisville metropolitan area, and with carnosine levels less than 
the median of local population. After being identified (from existing database, from clinics, flyers, 
or other routes), potential participants will be approached by one of the investigators or research 
coordinators. The information provided to the potential participant is included in the informed 
consent. The informed consent will provide information regarding PM2.5 pollution, prevalence of 
PM2.5 pollution in the study area, synthesis and natural role of carnosine in the body, natural and 
supplemental sources of carnosine, cornstarch (constituent of placebo), and will include 
information regarding possible risks of participation. 
Randomization
Eligible participants identified from Screening (Visit # 1) will be randomized in to supplement or 
placebo groups by 1:1 distribution. Randomization will be performed in accordance with written 
standard operating procedures by personnel that are not involved in the selection, screening, or 
enrollment of subjects and blinded as to study documentation. Randomization to supplement 
assignment will be conducted using a web access database created and maintained by the 
Randomization Unit. After the investigator or research coordinator verifies that screening testing 
is complete and the inclusion and exclusion criteria for the study have been satisfied, an un-
blinded member of the Randomization Unit will have secured access to the participant’s 
supplement assignment. The participant will be randomized a minimum of 2 days before the 
scheduled date of the baseline assessment (Visit # 2) to arrange for the timely availability of the 
study supplement. See Manual of Operations for details 
Stopping Early or Withdrawal from the Study
Monitoring rules and statistical boundaries for adverse outcomes are further detailed in Section 
15 “Data and Safety Monitoring Board”. Below is the outline for recusal or withdrawal of 
participant from the study due to a non-health / safety related reason. 
Participants that have consumed study supplement for at least 4 weeks during the duration of 
the study period, but missed taking the study supplement for no more than 1 week during the 
study period will still be followed if feasible, and included in intent to treat analysis. 
Participants that withdraw from the study before Follow-Up # 1 (Visit # 3) or do not complete 
Visit # 3 will be considered lost to follow-up. Existing data of these participants will be retained 
for further analysis.
Screen Failure
Candidates are considered screen failures when they meet one of the following criteria after 
signing consent: 
1.
Screening (Visit # 1) data reveals that the candidate is ineligible
OR 

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2.
Candidate withdraws and does not complete Baseline Assessment (Visit # 2)
Discontinued
Participants are considered discontinued when they meet one or more of the following criteria: 
1.
Participant withdraws consent after being randomized 
OR 
2.
Participant withdraws consent after completion of Baseline Assessment (Visit # 2)
OR 
3.
Participant fails to follow study supplement regimen 
OR
4.
Participant is unable to fulfill the inclusion/exclusion criteria during the study
OR
5.
Participant refuses/is unable to provide critical data at follow-up visits as deemed 
necessary by the investigator 
OR
6.
Participant is withdrawn after randomization by investigator for reasons that may 
compromise completion of the study
Medical or Health Concerns
Participants will be instructed that in an event of an acute health concern (related or unrelated to 
the study supplement), they should immediately discontinue the study supplement and seek 
medical attention from a licensed healthcare provider. Participants will be provided with a study 
identification card. This card will contain the following information: name of the study, name of 
study institution, a statement to the effect of “each capsule may contain 0.5 grams of L-
Carnosine or 0.5 grams of cornstarch”, method of contacting the study physician, and a method 
of contacting the study coordinator. 
Participants may be able to re-start supplement regimen and continue the randomization after 
detailed discussions with the study physician. The study physician may seek advisement from 
the DSMB on these matters.  
Compensation 
Participants will be given a University of Louisville branded prepaid VISA gift card for 
participating in the study. The following information will be collected: full name, address, and 
social security number. Social security number will not be saved. As required by the Internal 
Revenue Service, participants may need to report the payment as income on their taxes. If 
participants choose not to provide their social security number or other required information, 
they will not be paid for participating in the study. Participants can still participate in the study 
even if they don’t want to be paid.
Participants will be compensated for completing a clinical visit as per the following schedule:

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Visit Description
Amount
Screening (Visit # 1)
$25
Baseline Assessment (Visit # 2)
$50
Follow-Up # 1 (Visit # 3)
$75
Follow-Up # 2 (Visit # 4)
$100
4-12 Month Safety Check (Visit # 5)*
None
*Visit # 5 is not an actual physical visit, contact will be made via phone, email, or 
mailed paper survey
10.
STUDY VISITS 
Screening (Visit # 1)
The screening period extends from the date informed consent is signed until the day of 
randomization (Figure 3). The screening window will not exceed 180 days prior to 
randomization.
The following activities will be carried out at screening:
1.
Informed Consent (for entire study)
2.
Blood and or Urine sample collection
3.
Demographic data
4.
Contact information
5.
Prepaid $25 VISA gift card 
Participants may be excluded from the study if any of the above screening data collection is not 
completed or meets an exclusion criterion during the screening period. Data gathered from 
screening (Visit # 1) will be used to identify whether the candidate meets the enrollment criteria 
for the main study and whether they are eligible to be randomized. Eligible candidates will be 
invited to participate in the main study and subsequently randomized in a double blind fashion. 
Baseline Assessment (Visit # 2)
Baseline assessment will occur after eligible candidates have been identified via screening (Visit 
# 1) (Figure 4). Participants attending the baseline assessment (Visit # 2) will already be 
randomized into carnosine or placebo group. Participants will be provided with the study 
supplement during this visit and they will start their dietary supplement regimen (carnosine or 
placebo) immediately on the day of baseline assessment. The following activities will be carried 
out at Baseline Assessment:

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1.
Update Demographics and Contact Information
2.
Questionnaire data on Medical History, Medications, Exposure to Environmental 
Pollution, Sleep, Diet, Exercise, Tobacco, and Women’s Health
3.
Blood sample collection (~3 tablespoons or ~45ml)
4.
Urine sample collection (~2 tablespoons or ~30ml)
5.
Arterial stiffness test (SphygmoCor)
6.
Physical strength test (hand grip test, step test, calf raise test)
7.
Provide 8 weeks of study dietary supplement capsules (carnosine or placebo)
8.
Discuss schedule and related instructions related to taking the study dietary supplement
9.
Prepaid $50 VISA gift card  
Participants may be excluded from the study if any of the above data collection is not completed 
or meets an exclusion criterion during this period. 
Follow-Up # 1 (Visit # 3)
Follow-Up # 1 will occur ~6 weeks after initiation of study supplement at the baseline 
assessment (Visit # 2) (Figure 5). Participants attending follow-up # 1 (Visit # 3) will have 
already been taking the study supplement for at least 5 weeks and no more than 7 weeks (6 
weeks + 1 from Visit # 2). The following activities will be carried out at Follow-Up # 1:
1.
Update Demographics and Contact Information
2.
Update Questionnaire data on Medical History, Medications, and Women’s Health 
3.
Questionnaire data Exposure to Environmental Pollution, Sleep, Diet, Exercise, Tobacco
4.
Blood sample collection (~3 tablespoons or ~45ml)
5.
Urine sample collection (~2 tablespoons or ~30ml)
6.
Arterial stiffness test (SphygmoCor)
7.
Physical strength test (hand grip test, step test, calf raise test)
8.
Review the participant’s dietary supplement regimen and any instances of deviation in 
regimen (missed or over consumption).
9.
Provide 4 more weeks of study dietary supplement capsules (carnosine or placebo)
10. Discuss any side-effects which may have occurred or may arise from study supplement. 
Discuss any health changes during the study period. 
11. Prepaid $75 VISA gift card 
Participants may be excluded from the study if any of the above data collection is not completed 
or meets an exclusion criterion during this period.
Follow-Up # 2 (Visit # 4)
Follow-Up # 2 will occur ~12 weeks after initiation of study supplement at the baseline 
assessment (Visit # 2) (Figure 6). Participants attending follow-up # 2 (Visit # 4) will have 
already been taking the study supplement for at least 10 weeks and no more than 12 weeks (11 
weeks + 1 from Visit # 2). The following activities will be carried out at Follow-Up # 2:
1.
Update Demographics and Contact Information

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2.
Update Questionnaire data on Medical History, Medications, and Women’s Health 
3.
Questionnaire data Exposure to Environmental Pollution, Sleep, Diet, Exercise, Tobacco
4.
Blood sample collection (~3 tablespoons or ~45ml)
5.
Urine sample collection (~2 tablespoons or ~30ml)
6.
Arterial stiffness test (SphygmoCor)
7.
Physical strength test (hand grip test, step test, calf raise test)
8.
Review the participant’s dietary supplement regimen and any instances of deviation in 
regimen (missed or over consumption).
9.
Discuss any side-effects which may have occurred or may arise from study supplement. 
Discuss any health changes during the study period. 
10. Prepaid $100 VISA gift card 
Participants may be excluded from the study if any of the above data collection is not completed 
or meets an exclusion criterion during this period.
4 – 12 Month Safety Check (Visit # 5)
Although this interaction is termed as a “Visit”, it will not involve participants to 
physically be present. A follow-up email / phone call / mailed paper questionnaire will be 
conducted 4-12 months after the end of supplement intervention (Figure 7). This contact will 
focus on updating about any SAEs & medical / medication history changes since end of 
intervention:
1.
Update Demographics and Contact Information
2.
Questionnaire data on Medical / Medications History since end of intervention 
3.
Questionnaire data on adverse effects or SAEs since end of intervention.
4.
No monetary compensation for this follow-up / check. 
Participants will not be excluded from the study if any of the above data collection is not 
completed. Instead, they will be categorized as “lost to safety follow-up”. 
Safety Evaluations during Study Visits
Adverse events will be assessed during Visit # 3 and Visit # 4 and through the 12 months after 
participants have stopped taking study supplement. The following will be documented in the 
adverse event reporting system: 
1.
Local reactions: diarrhea, stomach upset, tingling sensation in extremities, rash or 
itchiness, dry mouth, changes in appetite, feelings of tiredness, or vivid dreams.
2.
Systemic reactions: fever, allergic reaction, anaphylaxis or any clinical untoward event 
that occurs within 30 days of beginning study supplement.
3.
Abnormal laboratory results which are deemed clinically significant.
4.
Pregnancy and breast-feeding: Currently, there is not enough reliable information 
about the safety of taking carnosine supplement during pregnancy or breast-feeding. 
Hence, participants/candidates who are currently pregnant or breast-feeding or plan to 
be pregnant/breast-feeding during the study period will be “discontinued” from the study. 

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5.
Low blood pressure: Carnosine synthesized in the body has been found to decrease 
blood pressure via influencing the autonomic nervous system. 43 Hence, in theory, taking 
supplemental carnosine might lower blood pressure and or exacerbate lowering of blood 
pressure in people taking antihypertensive medications. Hence, all participants taking 
blood pressure lowering medication will be cautioned for this interaction and advised that 
in case of an episode of low blood pressure to discontinue study supplement and 
immediately seek medical attention from a licensed healthcare provider.
6.
Low blood sugar: Carnosine synthesized in the body has been found to decrease blood 
sugar via influencing the autonomic nervous system. 43 Hence, in theory, taking 
supplemental carnosine might lower blood sugar and or exacerbate lowering of blood 
sugar in people taking blood sugar lowering medications. Hence, all participants taking 
blood sugar lowering medication will be cautioned for this interaction and advised that in 
case of an episode of low blood sugar to discontinue study supplement and immediately 
seek medical attention from a licensed healthcare provider.
Participants will be advised to continue / discontinue after a review by the study physician and 
the DSMB.
11.
STUDY SUPPLEMENTS
Eligible participants identified from Screening (Visit # 1) will be randomized into carnosine or 
placebo groups by 1:1 distribution.
Supplement Quality Control and Regulations 
As per the Office of Inspector General of the Department of Health and Human Services’ report 
on “Dietary Supplement Labels: Key Elements” – “The Dietary Supplement Health and 
Education Act of 1994 (DSHEA) 44 defines the term “dietary supplement” to mean a product 
(other than tobacco) intended to supplement the diet that bears or contains one or more of the 
following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a 
dietary substance for use by man to supplement the diet by increasing the total dietary intake, or 
a concentrate, metabolite, constituent, extract, or combination of any of the aforementioned 
ingredients”. 44, 45
The United States Food and Drug Administration (US FDA) guidelines specify that merchants 
selling dietary supplements in the United States are required to follow current good 
manufacturing practices and their manufacturing plants are required to be inspected by the FDA 
for “quality of the dietary supplement and to ensure that the dietary supplement is packaged and 
labeled as specified in the master manufacturing record”.45 Dietary supplements are not 
regulated by the FDA for their efficacy but will be inspected for quality, safety, and accuracy.44, 45 

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Both, Carnosine and placebo, supplements will be prepared by the University of Colorado, 
Skaggs School of Pharmacy and Pharmaceutical Sciences. Commercially available L-carnosine 
powder and cornstarch (placebo) will be packaged into gelatin coated capsules in laboratory 
facilities associated with the state licensed pharmacy of the University of Colorado, Skaggs 
School of Pharmacy and Pharmaceutical Sciences.
Supplement Capsule Preparation
Empty size 000 capsules will be purchased from www.CapsulCN.com and filled by trained 
individuals (technicians and Doctorate of Pharmacy students) under the supervision of a 
compounding pharmacist. Personnel involved in capsule preparation will be trained in but not 
limited to: practice and verification of capsule weight consistency. An appropriate amount of L-
carnosine will be weighed, placed on a capsule packing device, and evenly distributed to each 
capsule. Capsules will be then sealed, locked and “polished” to remove any residual powder. 
Capsules will be then checked for weight consistency. USP standards allow 10% variation; 
variation in capsules prepared by this pharmacy is under 5% and typically less than 2%.  
L-carnosine will be purchased in bulk from www.BulkSupplements.com. The FDA inspection 
registration number of www.BulkSupplements.com is 17938017996. Placebo supplements will 
be composed of cornstarch, and will match the composition routinely used in clinical 
interventional studies. Cornstarch placebo will be purchased from Medisca (Plattsburgh, NY). 
Further details about the materials, preparation, safety testing and other items related to 
supplement or placebo are detailed in Appendix D.
Supplement Assignment
The study supplements (carnosine and placebo) are each individually described below but shall 
thereafter be referred to throughout the protocol as “study supplement”. In the circumstance that 
the participant misses taking the supplement on for less than 3 consecutive and 4 total days in 
the 2 weeks leading to Follow-Up # 1 (Visit # 3), the participant will continue to be followed 
(intent to supplement analysis). 
Carnosine Supplement
Two hundred participants will receive Carnosine supplement. Carnosine supplement will be 
available in forms of oral capsules coated with bovine bone derived gelatin. Each capsule will 
contain 0.5 grams of Carnosine supplement. The participants of the Carnosine group will be 
required to take a total of 2 grams of daily carnosine supplement. Participants will be required to 
take Carnosine supplement twice per day: 1) Morning – 2 capsules, each containing 0.5 grams 
of Carnosine and 2) Evening – 2 capsules, each containing 0.5 grams of Carnosine. As per 
manufacturer instructions, the capsules can be taken with water or any other non-alcoholic 
drink. There is no pre-requisite of consuming a meal prior to taking the supplement. 

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The following activities and documentations will be performed and saved by the University of 
Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences for the Carnosine 
supplement: 
1.
Prepare, label, package, and deliver carnosine supplement and chain of custody form to 
courier as appropriate,
2.
Maintain Carnosine chain of custody forms, certificate of analysis forms, and 
accountability logs (so that clinical / recruitment staff does not have access). 
3.
Keep completed chain of custody forms, on site for study files and quality assurance 
purposes. 
  
Placebo Supplement
Placebo supplements will be composed of cornstarch, and will match the composition routinely 
used in clinical interventional studies. The placebo supplement will also be manufactured by the 
University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences. 
Two hundred participants will receive placebo supplement. Placebo supplement will be available 
in forms of oral capsules coated with bovine bone derived gelatin. Each capsule will contain 0.5 
grams of cornstarch. The participants of the placebo group will be required to take a total of 2 
grams of daily cornstarch. Participants will be required to take cornstarch twice per day: 1) 
Morning – 2 capsules, each containing 0.5 grams of cornstarch and 2) Evening – 2 capsules, 
each containing 0.5 grams of cornstarch. As per manufacturer instructions, the capsules can be 
taken with water or any other non-alcoholic drink. There is no pre-requisite of consuming a meal 
prior to taking the supplement. 
The following activities and documentations will be performed and saved by the University of 
Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences for the placebo 
(cornstarch) supplement: 
1.
Prepare, label, package, and deliver placebo (cornstarch) supplement and chain of 
custody form to courier as appropriate,
2.
Maintain placebo (cornstarch) chain of custody forms, certificate of analysis forms, and 
accountability logs (so that clinical / recruitment staff does not have access). 
3.
Keep completed chain of custody forms, on site for study files and quality assurance 
purposes. 
Randomization and Blinding
Blinding of the study personnel recruiting the participants and giving the pill-boxes will be 
facilitated by the fact that all study participants (Carnosine and placebo) will receive a dietary 
supplement. All study dietary supplements will be matched in color, consistency, and taste. The 
primary and secondary endpoints of the study will be determined by core laboratories whose 
personnel are blinded to supplementation assignment. In addition, the study recruitment and 
regulatory teams will take steps to ensure that endpoint and adverse event assessments are 
carried out in a blinded fashion.  

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Should unblinding occur, the Executive Committee consisting of the study PI, the study 
physician, and UofL CTU will be informed about the circumstances and they will discuss the 
circumstances related to the unblinding and further course of action. Any instance of unblinding 
will be documented in the study database and will be reported to the IRB as required.
Study Supplement Requirements
Study Supplement Packaging and Labeling
Study supplement packaging and labeling will meet all local and federal requirements. 
Study supplement (carnosine and placebo) label includes the product number, letter “A” 
or “B”, volume, expiration date and time, storage conditions, and contact name and 
address. All supplements will be labeled for investigational use only. 
Supplement capsules will be pre-sorted into monthly (28 day) pill boxes by the unblinded 
laboratory personnel at the University of Colorado, Skaggs School of Pharmacy and 
Pharmaceutical Sciences. Pre-sorted pill boxes will be created for a total of 12 weeks 
per participant. The University of Colorado, Skaggs School of Pharmacy and 
Pharmaceutical Sciences personnel will label each box clearly as letter “A” or letter “B”, 
indicating whether the box contains Carnosine or placebo supplement. The study 
personnel at the University of Louisville involved in recruitment, processing, analysis, or 
any other direct participant contact will be blinded to whether “A” boxes contain 
Carnosine or placebo, and similarly whether “B” boxes contain Carnosine or placebo. 
The University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences 
personnel will ensure that there is absolute consistency in the contents of each series of 
labeled boxes. The University of Colorado Skaggs School of Pharmacy and 
Pharmaceutical Sciences will ensure that the contents of each box are not disclosed to 
anyone involved with the study (staff, investigator, or participant) and will directly and 
securely send the randomization key to the Randomization Committee (study physician 
and UofL CTU).
Study Supplement Storage Requirements
L-Carnosine is tested on arrival to establish its purity and retested about every 6-12 
months. L-Carnosine receives a three-year dating each time it passes re-assay. Product 
is stored at controlled room temperature. A sample of packed capsules will be retained, 
which will be re-assayed about every six months. Potency and stability testing services 
will be provided using Nuclear Magnetic Resonance and Liquid Chromatography/Mass 
Spectrometry in the Medicinal Chemistry Core Facility of the University of Colorado, 
Skaggs School of Pharmacy and Pharmaceutical Sciences.  
United States Pharmacopeia (USP) <795> allows 180 day dating for compounded 
capsules, with extension of dating with ongoing stability studies. Initial potency and purity 
are established using Nuclear Magnetic Resonance and Liquid Chromatography/Mass 

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Spectrometry by Michael F. Wempe, PhD in the University of Colorado Skaggs School 
of Pharmacy, Medicinal Chemistry Core Facility. L-Carnosine (bulk powder) has up to a 
three-year dating from the last date of analysis. Dr. Wempe will perform ongoing stability 
studies to ensure that the doses we prepared remain in date throughout the study.  
Earlier stability studies support a dating of more than three years for our capsules.
Study supplement must be stored at room temperature from the time of manufacture 
until the time of giving to the study participant. The participant will be informed of these 
requirements and instructed to strictly follow them. 
Study Supplement Accountability
The study supplements will be stored in the UofL CTU, housed at Healthcare Outpatient 
Care Center (401 E. Chestnut Street, Suite 410, Louisville, KY, 40202). Upon receipt of 
study supplement from manufacturer, UofL CTU will open the shipment and verify 
shipment inventory, temperature, and condition of the study supplement by completing 
and signing the chain of custody form and forwarding the document to the unblinded 
manufacturing personnel. The UofL CTU personnel will also compare blinding letter (“A” 
or “B”). Records shall be kept of all study supplies received, disposition, and any study 
supplements not used or left over. Study supplement accountability logs contain 
unblinded information and should be maintained by the Clinical Trials Unit staff only.
The supplements accountability logs will record use of placebo as well.
Under no circumstances will any investigator(s) allow study supplement to be used for 
any purpose other than that specified by the protocol. The study supplement are only to 
be used as a dietary supplement only for participants of this study and for their specific 
study group (Carnosine or placebo) only.
Study Supplement Handling and Disposal
Destruction of unused study supplements will proceed as directed by manufacturer or designee, 
with appropriate documentation completed and retained by site personnel. All records involving 
receipt, disposition, administration, return and/or destruction of the study supplement will be 
made available by the PI (or designee) to the Sponsor or designee upon request. 
Taking Pre-Existing Medications
Participants can remain on their current / pre-existing medications that they were taking prior to 
enrollment. Data on medications added or discontinued during the course of the study will be 
recorded throughout the study to the 4-12 month safety check.  

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Other Supplements 
Participants may not consume any other dietary protein supplements (e.g. Whey protein drinks, 
Collagen powder, etc.) during the study period. Since Carnosine is an amino acid that serves as 
a protein building block for muscles, simultaneous consumption of protein supplement has the 
potential to interfere with the effects of Carnosine on muscles and body physiology.   
12.
CLINICAL AND LABORATORY EVALUATIONS
Schedule of Procedures
Study Activities
Screening
(Visit # 1)
Screening
(Visit # 2)
Screening
(Visit # 3)
Screening
(Visit # 4)
4-12 
Month 
Safety 
Check
Informed Consent
X
Contact & Demographics
X
X
X
X
Medical History
X
X
X
X
Medications History
X
X
X
X
Physical Exam
X
X
X
Blood Samples
X
X
X
Urine Samples
X
X
X
X
Arterial Stiffness Exam
X
X
X
Physical Function Exam
X
X
X
Environmental Pollution 
Exposure Questionnaire
X
X
X
Sleep Questionnaire
X
X
X
Diet & Exercise Questionnaire
X
X
X
Supplement Distribution
X
X
Adverse Events Check
X
X
X
Checkout (Prepaid Visa)
X
X
X
X

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Procedure Details
Contact and Demographics
A detailed account of the participant’s contact and demographic information will be 
collected at screening (Visit # 1). This information will be critical while implementing the 
screening criteria and selecting eligible candidates for randomization. This information is 
also required for NIH progress report reporting. It will be administered by a research 
team member at every clinical study visit (Visit # 1 through # 4).
Participant Medical History and Physical Exam
A complete participant history including medical, alcohol, tobacco, and medication 
review will be conducted at baseline assessment (Visit # 2). 
A complete physical exam including vitals, height, and weight will be completed at 
baseline assessment (Visit # 2). Similar physical exams will be conducted at each 
additional clinic visit (Visit # 3 and Visit # 4) during the study.
Blood and Urine Samples
A fresh catch urine sample will be collected at screening (Visit # 1). Urine sample 
collection details are in Section 8 “Investigational Plan” and Appendix A. This is 
necessary to identify the candidate’s body carnosine level and to implement the 
inclusion criteria. This information is critical in screening and selecting eligible 
candidates for randomization. A fresh catch urine sample will be collected by a research 
team member at every clinical study visit (Visit # 1 through # 4).  
Fresh stick blood samples will be collected at baseline assessment (Visit # 2). Blood 
sample collection details are in Section 8 “Investigational Plan” and Appendix A. Fresh 
stick blood samples will be collected at each additional clinical study visit from baseline 
onwards (Visit # 2 through Visit # 4). 
Arterial Stiffness Test
SphygmoCor will be used to measure indices of arterial stiffness (augmentation index; 
AI, augmentation pressure; AP, pulse wave velocity; PWV, aortic systolic pressure, and 
SEVR).
SphygmoCor is a non-invasive, cuff-based tool used for obtaining brachial artery blood 
pressure and the central arterial pressure waveform with associated indices 
(augmentation index, augmentation pressure, and pulse wave velocity). A cuff will be 
applied on the upper arm in the standard position. The cuff will be partially inflated to 
record the brachial waveforms. These waveforms are detected by sensing changes in 
the pressure inside the cuff related to arterial pulsation. The ascending aortic waveform 
is subsequently derived using a validated mathematical transfer function.

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Arterial stiffness test will be administered by a research team member at baseline 
assessment (Visit # 2) and then at each additional clinical study visit from baseline 
onwards (Visit # 2 through Visit # 4). Arterial stiffness testing (via SphygmoCor) is 
detailed in Section 8 “Investigational Plan”.
Physical Function Test
To assess the impact of carnosine supplementation on physical performance during 
times of variable PM2.5 levels, we will utilize three physical performance tests: handgrip 
strength test, 2-minute step test (2MST), and bilateral calf-raise test.
Physical function test will be administered by a research team member at baseline 
assessment (Visit # 2) and then at each additional clinical study visit from baseline 
onwards (Visit # 2 through Visit # 4). Testing protocol is detailed in Section 8 
“Investigational Plan” and Appendix E.
Environmental Pollution Exposure Questionnaire
This questionnaire will assess participant’s exposure to environmental pollution. It will be 
administered by a research team member at baseline assessment (Visit # 2), Follow-Up 
# 1 (Visit # 3) and Follow-Up # 2 (Visit # 4). 
Sleep Questionnaire
This questionnaire will assess participant’s sleep habits during the study period. It will be 
administered by a research team member at baseline assessment (Visit # 2), Follow-Up 
# 1 (Visit # 3) and Follow-Up # 2 (Visit # 4). 
Diet and Exercise Questionnaire
This questionnaire will assess participant’s dietary and exercise habits and their possible 
impacts on study supplement intake and processing throughout the study period. It will 
be administered by a research team member at baseline assessment (Visit # 2), Follow-
Up # 1 (Visit # 3) and Follow-Up # 2 (Visit # 4). 
Storage, Genetic Testing, and Future Use of Biospecimens
Blood and urine biospecimens collected during the study (detailed in Section 8 “Investigational 
Plan” and Appendix A) will be processed to be stored for long period. Majority samples will be 
processed, aliquoted, labeled, cataloged and immediately stored in -80C freezers in the 
Envirome Institute’s research laboratories located on the 2nd or 3rd floors of the Cardiovascular 
Innovation Institute (302 E. Muhammad Ali Blvd, Louisville, KY 40202). 
Samples will be used to test for current hypothesis / endpoints and or generation of preliminary 
data for future studies. Samples may be used to genetic testing. These collected samples will 
be used for research purposes only. Samples will be stored without personal identifying 

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information, and will be shared with approved researchers who will conduct studies to improve 
the scientific knowledge including but not limited to environmental pollution and cardiovascular 
diseases. Samples will be retained for as long as possible. 
Follow-Up Evaluations
Follow-Up Windows
The timeline for follow-up will begin on the day of baseline assessment (Visit # 2). The 
time windows for each of the subsequent follow-up visits will be as follows:
1.
Follow-Up # 1 (Visit # 3) will be 6 weeks (6 weeks + 1 week post initiation of 
study supplement)
2.
Follow-Up # 2 (Visit # 4) will be 12 weeks (11 weeks + 1 week post initiation of 
study supplement) 
3.
The 12 month safety check (via phone / email / mailed paper survey) will occur 
between 4 months and 12 months post completion of study supplement.  
Scheduling and Lost to Follow-Up
Randomized participants will be followed for efficacy for 12 weeks and for safety up to 
one year (12 months). Participants will be considered lost to follow-up after 3 
consecutive failed telephone/email contacts AND one certified letter returned to the site. 
Contact attempts will be documented in the participant’s study chart.
13.
ADVERSE EVENTS AND REPORTING
The definitions for adverse and related events for this study have been adapted from 21 CFR 
312.32(a) and FDA guidance for industry and investigators.46 The definitions are as follows:
Adverse Events (AEs)
An adverse event (AE) is defined as any untoward medical occurrence associated with the use 
of a supplement in humans, whether or not considered supplement related. 
An AE (also referred to as an adverse experience) can be any unfavorable and unintended sign 
(e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use 
of a supplement, and does not imply any judgment about causality. An adverse event can arise 
with any use of the supplement (e.g., off-label use, use in combination with another 
drug/supplement) and with any route of administration, formulation, or dose, including an 
overdose.
Suspected Adverse Reaction (SARs)
Suspected adverse reaction (SAR) is defined as any adverse event for which there is a 
reasonable possibility that the supplement caused the adverse event. For the purposes of safety 

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reporting, ‘reasonable possibility’ means there is evidence to suggest a causal relationship 
between the supplement and the adverse event. A suspected adverse reaction implies a lesser 
degree of certainty about causality than adverse reaction, which means any adverse event 
caused by a supplement.
Within the reporting requirement under 21 CFR 312.32(c)(1)(i), the FDA provides a clear 
illustration of ‘reasonable possibility’ by providing the following examples of types of evidence 
that would suggest a causal relationship between a supplement and an adverse event:

A single occurrence of an event that is uncommon and known to be strongly associated 
with supplement exposure (e.g., angioedema, hepatic injury, Stevens-Johnson 
Syndrome)

One or more occurrences of an event that is not commonly associated with supplement 
exposure, but is otherwise uncommon in the population exposed to the drug (e.g., 
tendon rupture)

An aggregate analysis of specific events observed in a clinical trial (such as known 
consequences of the underlying disease or condition under investigation or other events 
that commonly occur in the study population independent of supplementation therapy) 
that indicates those events occur more frequently in the supplement group than in a 
concurrent or historical control group
Suspected adverse reactions are the subset of all adverse events for which there is a 
reasonable possibility that the supplement caused the event. Inherent in this definition, and in 
the requirement to report suspected adverse reactions, is the need for the sponsor to evaluate 
the available evidence and make a judgment about the likelihood that the supplement actually 
caused the AE. 
Serious Adverse Events (SAEs) or Serious Suspected Adverse 
Reaction (SSARs)
A serious adverse event (SAE) or serious suspected adverse reaction (SSAR) is defined as an 
AE/SAR which, in the view of the Investigator or Sponsor, results in: 1) Death; 2) a life-
threatening event (i.e. an event in which the subject was at risk of death at the time of the event; 
it does not refer to an event which hypothetically might have caused death if it were more 
severe); 3) inpatient hospitalization of > 24 hours or prolongation of existing hospitalization; 4) a 
significant disability/incapacity; or 5) a congenital anomaly/birth defect. Other important medical 
events may be considered SAEs/SSARs if, in the opinion of the Investigator(s), they jeopardize 
the participant or require intervention to prevent one of the other outcomes listed above.
Role of Abnormal Test Findings in Classifying an Event
The tests conducted in this study, for the primary or secondary endpoints, are for research 
purposes only and unless specified otherwise, do not have neither a clinically appreciable 
significance nor serve any diagnostic/treatment purpose. 

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However, if a clinical grade test result is associated with accompanying symptoms, and/or the 
test result requires additional diagnostic testing or medical/surgical intervention, and/or the test 
result is considered to be an AE/SAR by the Investigator or Sponsor it should be reported as an 
adverse event. 
NOTE: Merely repeating an abnormal test, in the absence of any of the above conditions, does 
not constitute an AE/SAR. Any abnormal test result that is determined to be an error does not 
require reporting as an AE/SAR.
Role of Hospitalizations in Classifying an Event
AE/SARs associated with hospitalization or prolongation of hospitalization is considered serious. 
Admission also includes transfer within the hospital to an acute/intensive care unit (e.g., from 
the cardiac wing to the medical floor for an infection, or from the medical division to the 
neurologic unit for a stroke).
Hospitalization does not include rehabilitation facilities, hospice facilities, respite care (i.e., 
caregiver relief), skilled nursing facilities or homes, routine emergency room admissions, or 
same day surgeries (as outpatient/same day/ambulatory procedures). Hospitalization or 
prolongation of hospitalization in the absence of a precipitating, clinical AE/SAR is not in itself 
an SAE/SSAR.
Unanticipated Problems (UPs)
An UP is an incident, experience, or outcome that specifically causes increased risk to the study 
or to its participants which may be of medical or non-medical etiology, and meets the following 
criteria:
1.
Unexpected (in terms of nature, severity, or frequency), given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the characteristics 
of the subject population being studied
2.
Definitely, probably, or possibly related to participation in the research (i.e., there is a 
reasonable possibility that the incident, experience, or outcome may have been caused 
by the procedures or materials involved in the research); and
3.
Suggests that the research places participants or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously known 
or recognized
4.
All UP reporting will follow the same guidelines as noted above for SAE/SSAR reporting, 
and must include a corrective action plan/measures to prevent recurrence.
Reporting by Principal Investigator

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For all events (AE/SAR and SAE/SSAR), monitoring and reporting to the PI or EC begins at the 
time that the participant provides informed consent, which is obtained prior to the participant’s 
participation in the study, i.e., prior to undergoing any study related procedure and/or receiving 
study supplement, through and including 30 calendar days after the subject completes the 
study. Events should be recorded on the Adverse Event electronic case report form (eCRF). Do 
not delay the initial reporting of an event in order to obtain resolution or follow up information.
For all events, the PI must pursue and obtain adequate information both to determine the 
severity and causality of the event. For events with a causal relationship to the study 
supplement, follow-up by the Investigator is required until the event or its sequelae resolve or 
stabilize at a level acceptable to the PI, who concurs with that assessment.
In the rare event that the PI does not become aware of the occurrence of a SAE/SSAR 
immediately (i.e., if an outpatient study subject initially seeks treatment elsewhere), the PI  is to 
report the event within 24 hours after learning of it and document the time of his/her first 
awareness of the event.
Severity Assessment
The Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, for detailed 
descriptions of Severity Grades has been used in this study. The CTCAE schema is classified 
by body system and event using the Medical Dictionary for Regulatory Activities (MedDRA) 
System Organ Classification (SOC) hierarchy and provides descriptions of events that qualify 
under each severity rating. The CTCAE v5.0 schema was accessed from 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5.0.xlsx 
and 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_R
eference_5x7.pdf. 
The following table contains general descriptions of Adverse Event Severity Grades.
Please note: Grade 1 (Mild) AE/SARs are not entered in the electronic CRF.
CTCAE Severity Grading Scale
Severity Grade
Description
1
Mild. Asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention is not indicated.
2
Moderate. Minimal, local, or non-invasive intervention indicated or limiting 
activities of daily living (i.e. preparing meals, shopping for groceries/clothes, 
managing money, using telephone, etc.)
3
Severe or medically significant but not immediately life-threatening. 
Hospitalization or prolongation of hospitalization indicated OR disabling OR 

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limiting self-care (e.g. bathing, dressing, feeding self, using toilet, taking 
medications, etc.)
4
Life-threatening consequences; urgent intervention indicated.
5
Death. Death related to adverse event.
Notice that severity and seriousness are different concepts. For example, a headache may be 
severe (interferes significantly with subject's usual function) but would not be classified as 
serious unless it met one of the criteria for SAE/SSARs.
Causality Assessment
The study nomenclature for assessing the causal relationship between the study supplement 
and an event is listed in the following table.
Adverse Event/Suspected Adverse Reaction Relationship to Study 
Supplement
Unrelated
No temporal association to study supplement
An alternate etiology has been established.
Unlikely
Clinical events that are likely to be caused by participant’s clinical state, 
environment or administration of other therapies or exposure to toxins
Possibly Related
Reasonable temporal relationship to study supplement.
Connection to study product cannot be ruled out
Probably Related
There is a reasonable temporal association with the study supplement.
There is a high degree of certainty that the event is related to the study 
supplement.
Definitely Related
There is a direct temporal relationship to the study supplement.
The event follows a known pattern of response to the study supplement.
The PI chooses the category that overall best describes the relationship between the event and 
the study supplement and records the evaluation on the Adverse Event eCRF. Note: If the PI is 
unable to identify whether or not the study supplement caused the event, then the event will be 
handled as “possibly related to investigational supplement” for reporting purposes.
14.
DATA AND SAFETY MONITORING BOARD
The Data and Safety Monitoring Board (DSMB) will comprise of four experts responsible for 
reviewing clinical trial data on an ongoing basis to ensure the safety of study participants and 
validity and integrity of the data. Members will be independent, with no vested interest in the 
specific outcomes of dietary supplementation. The DSMB will review evidence of adverse 
events and interim outcomes to recommend whether the trial should be continued, altered, or 

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terminated. The DSMB will have access to “unmasked” data during the course of the trial, 
meaning that DSMB members know which subjects are in which supplement group.
Study Review
The DSMB will be responsible for reviewing study documentation (e.g., study protocol, the 
informed consent and other participant handouts, etc.) and ensuring that it has adequate 
information to assess the safety of the study participants and the efficacy of the intervention 
during the study period. The DSMB will ensure that the data and safety monitoring plan is 
sufficient, given the complexity of the study and participants.
Study Evaluation
The DSMB will evaluate participant safety data throughout the duration of the trial; evaluate the 
efficacy of the study intervention at intervals specified in the DSMB charter (described below); 
and independently provide recommendations to the study Executive committee and the IRB to 
either continue, amend or terminate the clinical trial based on this information. The presence of 
early unanticipated supplementation results, side effects, or adverse events are all reasons that 
a DSMB might recommend termination of the clinical trial early due to safety or efficacy matters.
 
Study Monitoring
The DSMB will be responsible for monitoring the performance of clinical recruitments (e.g., 
protocol violations, improper participant enrollment criteria, slow accrual rate, low participation 
rate, failure of randomization, inadequate supplementation adherence, inadequate follow-up 
rate, severely compromised validity). The DSMB will independently make recommendations to 
the EC for improving the performance of the study or terminating the study if it determines the 
study would be unable to provide useful data, regardless of modifications. A summary of each 
board meeting and the board recommendation(s) will be provided to the EC and the IRB.
The DSMB will use a combination of, notifications, event specific reports, and scheduled 
cumulative trial reports to keep the EC and the IRB informed about real and potential safety 
issues.
Notifications 
Notifications are comprised of an email to the EC and DSMB with available information on the 
date and nature of the event, the study physician’s evaluation of the severity, expectedness, 
and relatedness to study supplement; and the assessment of the event given the information 
known at the time of the initial reporting.
Event Specific Reports
Event specific reports are formal written reports providing the details of the event (including 
circumstances surrounding the event, laboratory testing, concomitant medications, and any 
formal diagnoses made via medical intervention). These reports include a full assessment by 

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the study physician of the severity, expectedness, and relatedness to study supplement as well 
as any available status update on the participants.
Scheduled Cumulative Trial Reports
Scheduled cumulative trial reports will be prepared semi-annually by the study PI. These will be 
used by the DSMB to assess recruitment, subject safety, and continued trial feasibility. These 
reports include total numbers of AE/SARs and SAE/SSARs experienced in the overall trial. The 
information provided includes both new events reported since the last DSMB meeting and 
cumulative events reported during the life of the trial.
CTU Reporting Requirements to DSMB and IRB
Once the event has been reported to the PI or the study physician, it will be reported to the 
Executive Committee within 48 hours. The UofL CTU (part of the EC) will use the Medical 
Dictionary for Regulatory Activities (MedDRA) System Organ Classes (SOC) to classify all 
AEs/SARs (including SAEs/SSARs assessed by PI or study physician). Additional supporting 
documentation may be requested from the PI and study team to enable the UofL CTU to 
accurately assess the event for reporting.
Reportable Events:
All unexpected events, all events related to study product, and all events with a severity grade 
of 3 or higher are reported by the UofL to the DSMB and the IRB.
Reporting Timeframe:
For events which are unexpected AND associated with study supplement, the CTU will notify 
the IRB and DSMB within 72 hours of learning of the event; with an event specific report filed 
no later than 7 days post notification.
For all other reportable events, the CTU will notify the IRB and DSMB no later than 15 days of 
learning of the event; with an event specific report filed no later than 30 days post notification.
Cumulative trial reports (which include all events) will be generated for review by the CTU and 
DSMB at semi-annual DSMB meetings.
Reporting Requirements to the FDA
Carnosine is a freely available over the counter dietary supplement in the market. This is not an 
experimental drug or an untested product. Commercially available Carnosine (or beta-alanine) 
is well known in the market and it is generally recognized as “safe when used in accordance 
with good manufacturing practice”. The goal of this study is to provide dietary supplementation 
of a naturally occurring amino acid inside the body and to study whether dietary 
supplementation can enhance normally occurring physiological/biochemical processes in the 
body. 

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As per the Office of Inspector General of the Department of Health and Human Services’ report 
on “Dietary Supplement Labels: Key Elements” – “The Dietary Supplement Health and 
Education Act of 1994 (DSHEA) 44 defines the term “dietary supplement” to mean a product 
(other than tobacco) intended to supplement the diet that bears or contains one or more of the 
following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a 
dietary substance for use by man to supplement the diet by increasing the total dietary intake, or 
a concentrate, metabolite, constituent, extract, or combination of any of the aforementioned 
ingredients”. 47 As per the DSHEA, dietary supplements are not regulated by the FDA other than 
for their good manufacturing practices. 44
Hence, the study (EC or DSMB) has no obligations to report any findings to the FDA. The study 
will however take every precaution to ensure that the supplement providers (bulk supplier and 
pharmacist) are operating in facilities at par with good manufacturing practices and are in 
possession of appropriate certifications and licensure. These documents can be found in 
Appendix D.  
Guidelines for Canceling Study Supplement Administration
The events listed below will follow the same reporting criteria for SAE/SSARs as it relates to the 
PI as well as the CTU and are to be considered sufficient to halt the study procedure. If subject 
develops any of the following conditions within 48 hours of planned supplement consumption, 
study supplement delivery will be halted in that particular participant:
1.
Diarrhea / Vomiting / Stomach Upset
2.
Sudden drop in blood pressure
3.
Sudden drop in blood sugar
4.
Hemodynamically unstable
Holding Rules
The expected number of SAE/SSARs in this trial is anticipated to be too small to support formal 
statistical stopping rules. However, the following criteria are provided as guidelines under which 
this clinical trial may be put on hold pending a detailed investigation by the DSMB. The 
assessment of the event shall include the relationship to the trial procedures as unrelated or 
possibly related. If possibly related, an indication of causality to a specific aspect of the study 
supplement / procedures will be determined: (e.g. study supplement consumption related, study 
supplement safety related, study supplement manufacturing related).
Holding Criteria
The DSMB will recommend putting the trial on hold as soon as it is notified of one or more of 
holding criteria listed below. The study holding criteria have been created under the guidelines 
of current literature data and the 21 CFR code of the FDA regulatory information. The study 
holding criteria:

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1.
20% or more cases of out of the total enrollment with Diarrhea / Vomiting / Stomach 
Upset
2.
20% or more cases of out of the total enrollment with Sudden drop in blood pressure
3.
20% or more cases of out of the total enrollment with Sudden drop in blood sugar
4.
20% or more cases of out of the total enrollment with Hemodynamically unstable events 
5.
20% or more cases of out of the total enrollment with Hospitalizations
6.
Or any other reason specified by the DSMB, study EC, bulk supplier or manufacturing 
pharmacist
As soon as any of the above is identified, the EC will inform the IRB and the DSMB. In addition, 
the DSMB will monitor the distribution of all SAE/SSARs, and the PI will be responsive to all 
DSMB concerns regarding SAE/SSARs that are not part of holding criteria listed above.
In addition, the DSMB may also recommend stopping the trial for the performance-related 
issues that would prevent the study from meeting its scientific objectives, such as:

Failure to recruit and enroll participants

Inability to meet protocol requirements for delivery of study supplements at specified 
times

Inability to prepare study supplement which meets quality specifications
15.
STATISTICAL PROCEDURES
Randomization
Randomization to Carnosine assignment will be conducted using a statistical software by one of 
the study statisticians. The randomization key detailing which participant is in which group 
(Carnosine or placebo) will be maintained by the Randomization Committee (RC) consisting of 
the study physician (Andrew DeFilippis, MD) and a member of the UofL CTU. After the 
investigator or research coordinator verifies that screening testing is complete and the inclusion 
and exclusion criteria for the study have been satisfied, an un-blinded member of the 
Randomization Committee will have secured access to the participant’s supplement 
assignment. 
Participants will be randomized to the Carnosine or placebo group, using a block size of 6. The 
stratification factors are sex, age (two group) and carnosine level (two group). When a 
participant is identified from the screening process (data from Visit # 1), the clinical recruitment 
team will be informed whether the participant will be receiving supplements from pill-box labeled 
“A” or “B. The clinical recruitment team will assign the participant a study ID from a pre-defined 
series, which will have no bearings on supplement randomization process.  

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As detailed in Section 11 “Study Supplement”, the University of Colorado Skaggs School of 
Pharmacy and Pharmaceutical Sciences will ship 28-day pill-boxes (total 3 per participant) pre-
filled with either Carnosine or placebo. These boxes will also be pre-labeled with large sized 
visible letters “A” or “B”. The entire study team, excluding the Randomization Committee, will be 
blinded to whether “A” or “B” series contains Carnosine versus placebo. The University of 
Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences personnel will ensure that 
there is absolute consistency in the contents of each series of labeled boxes. The University of 
Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences will directly and securely 
transmit the randomization key and other relevant information to the Randomization Committee 
(study physician and UofL CTU). 
Participants of both the groups (“A” or “B”) will undergo the exact same procedures of data 
collection as detailed in section 10 “Study Visits”. The CTU will monitor participant recruitment 
by providing reports to the study co-PI and DSMB as appropriate during the recruitment phase. 
Updated reports will be maintained on an Internet site accessible to all units of the study. The 
recruitment reports will provide data on recruitment of women and minorities (African-
Americans, Hispanics, and Asians). Goals for recruitment will be set and will be reviewed by the 
PI.
Statistical Analyses
Biostatisticians at the Envirome Institute, with the assistance of bioinformatics experts, have 
adapted or developed a number of statistical programs for analyzing study data. Data are 
analyzed for both data monitoring purposes, as described above, and for the purpose of 
detecting effects of the supplement. The Envirome Institute uses standard statistical packages 
such as SAS, S-PLUS, R and Stata to perform statistical analyses. 
Participants receiving supplementation will be presented.  Participants receiving 
supplementation who are found not to have fully met the eligibility criteria will also be presented.  
On-study protocol violations will also be presented. Participants who do not complete the 
required observations will be listed and evaluated separately as necessary.  Reasons for study 
discontinuation and date of withdrawal from study will be presented.
Baseline Analyses
Although the stratified random assignment of participants to the various supplements should 
ensure comparability with respect to known and unknown variables, imbalance may occur by 
chance. Descriptive statistics for baseline characteristics known or suspected to be associated 
with outcomes will be prepared for the various supplementation groups. The variables 
considered in such a description can be categorized as: 1) demographic characteristics; 2) 
medical history; 3) physical examination; and 4) laboratory data. Exact testing for categorical 
variables and Student t testing for continuous variables will be used to evaluate the differences 
in baseline variables between supplement groups.

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Outcome Analyses
Primary and Secondary Endpoints
There are 5 primary endpoints: difference in EPCs (EPC-2, EPC-3, EPC-4, EPC-7, and 
EPC-8) between Carnosine and placebo groups at 6 weeks and 12 weeks. These 
endpoints will be evaluated at the alpha=0.01. There are four secondary endpoints: 
difference in EPCs between Carnosine and placebo groups at 12 weeks, difference in 
EPCs between Carnosine and placebo groups at 6 weeks, difference in arterial stiffness 
between carnosine and placebo groups at 6 and 12 weeks, difference in endothelial 
microparticles between carnosine and placebo groups at 6 and 12 weeks, and difference 
in platelet monocyte aggregates between carnosine and placebo groups at 6 and 12 
weeks. 
Continuous variables will be presented by summary statistics (such as mean, median, 
standard error, range, 95% CI, correlations) and the categorical variables by frequency 
distributions (frequency counts, percentages and 95% CI).  We will also calculate 
correlations along with confidence intervals. To identify significant differences due to 
intervention, we will compare the differences using a two sample t-test. We will use 
regression (logistic or linear) to model and test the effect of other covariates such as age 
and race.  We also will compare outcome measures using GLM and MIXED model 
procedures to account of repeat observations. General statistical analyses will be 
performed with the SAS statistical software package (SAS Institute Inc., Cary, NC).
Statistical Monitoring Rule
Safety monitoring of the accumulated outcomes data is designed to ensure the continuing 
safety of the currently enrolled participants and participants not yet enrolled.  This is achieved 
by stopping the trial early to reduce the number of participants exposed to a harmful or 
ineffective supplementation. 
Monitoring Non-efficacious Supplementation (Futility)
We do not plan any interim analyses for futility due to longer follow-up (1 year). 
Monitoring Toxic Supplementation (Safety)
The cumulative number of grade 3 or 4 toxic events will be monitored after each person 
is enrolled 48. If the cumulative number of toxic events produces enough evidence to 
conclude that the true toxicity rate is greater than or equal to 33% (Pt0 = 0.33) then the 
trial will be stopped early for safety reasons.  If the cumulative number of toxic events 
after person is treated is greater than or equal to the associated boundary value bi then 
the combination supplementation is rejected for safety considerations (monitoring table 
with these boundaries will be generated using Ray and Rai, 2011)48. With this rule, there 
is only a 5% chance of stopping the trial early for lack of safety if the true toxicity rate is 
less than 33%. Continual assessment of the toxic events ensures we do not expose an 
undue number of participants to a harmful supplementation.    

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A second sub-analysis to be executed is the effect of Carnosine supplementation in 
participants with cardiovascular disease compared to the effect of Carnosine 
supplementation in participants without any cardiovascular disease.
Subgroup Evaluations
The effect of subgroup stratum on the relationship between carnosine supplementation and the 
endpoints (both primary and secondary) will be assessed. If a carnosine supplementation effect 
is demonstrated, it is not likely to behave identically among all important subgroups. The 
subgroups of interest are age, sex, race, socioeconomic status, diabetes, smoking, 
hypertension, and cardiovascular disease risk scores. 
These additional analyses can sometimes be helpful in identifying extreme differences in the 
effects of supplementation among subgroups, although the literature wisely warrants that 
caution be used in interpreting subgroup analyses.
Additional Analyses
We will use the 6 week and 12-week EPC levels to assess the change of EPCs from baseline to 
6 weeks and 12 weeks, assessing if this trajectory is related to carnosine supplementation using 
mixed model analysis of variance. 
We will evaluate the relationship among the indices of the primary endpoint – EPCs. 
Specifically, we will assess the relationship between the differences in EPCs, adjusting for 
important baseline covariates. 
Multiple Comparisons
In this study, no adjustments have been made for multiple comparisons. Measures of effect will 
be effect size, the standard error of the effect size, the 95% confidence interval for the effect 
size and the p-value. P-values will be interpreted at nominal 0.05 levels.
16.
TRIAL MANAGEMENT
Institutional Review Board (IRB) AND Informed Consent
This protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB or ethics committee responsible for oversight of the study. A 
signed consent form will be obtained from the participant. The consent form will describe the 
purpose of the study, the procedures to be followed, and the risks and benefits of participation. 
A copy of the consent form will be given to the subject and this fact will be documented in the 
participant’s medical record if recruited from a clinic.

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Informed Consent Process
Potential participants will be approached by one of the study investigators or research 
coordinators. Information regarding study participation will be provided to the potential 
participant. The informed consent includes descriptions of all study related procedures, all 
possible risks to participant, and the time commitment involved with participating. All consent 
forms will have IRB approval. Individuals who agree to participate will receive a copy of the 
signed informed consent. The research staff member obtaining consent will document the 
informed process in the participant’s chart if recruited from a clinic.
Risks Associated with Manufacturing and Consuming of Dietary 
Supplement 
Manufacturing Dietary Supplement 
Supplement manufacturing is done under strict sterile conditions; however, there is a rare 
chance that the capsules could become contaminated while being processed. Quality control 
checks will be done on the manufacturing process, and if the tests reveal break in protocol or 
contamination, the participant will be notified and instructed on whether or not he/she should be 
treated with antibiotics. The participant will be diligently assessed for adverse reactions or 
events at each follow-up visit (Visit # 3 and Visit # 4). If the participant notes a fever, he/she will 
be requested to notify the investigator/study team immediately.
Consuming Dietary Supplement
Possible risks of Carnosine supplementation include: tingling of upper / lower extremities, drop 
in blood pressure, nausea, vomiting, stomach upset. 
Participants who are taking antihypertension medications at the time of the study may be 
susceptible to a temporary sudden drop in blood pressure; during which time the participant 
may be at an increased risk of stroke and heart attack. The participant will be advised to inform 
the research team immediately of any symptoms of dizziness, light-headedness, chest pain, 
chest tightening, nausea, blurred vision, slurred speech, facial drooping, decrease sensations 
anywhere on your body, or weakness or a decrease in strength of the arms or legs. The 
participant will be closely monitored if started back on the study supplement regimen. The study 
physician will conduct a detailed assessment and provide its recommendations to the PI. 
Adequacy of Protection Against Risks
The precautionary measures mentioned in the last two sections will minimize the risk associated 
with taking study dietary supplement. Overall the study procedures are low risk and in previous 
similar trials there was no complications related to Carnosine or placebo supplementation in 
healthy participants.34, 37

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Potential Benefits of the Proposed Research to the Participants and 
Others
The potential benefits of this research for participants include reduced oxidative stress from 
PM2.5 pollution which will have therapeutic effects such as decrease in CVD progression and 
ultimately decreased CVD mortality. This project will also provide mechanistic insight into 
Carnosine supplementation which will be useful for finding new treatments for other diseases.
Risk Benefit Analysis
The supplementation of dietary Carnosine offers a new option to participants with existing CVD 
of different intensities. The goal of this supplementation is to improve ameliorate the progressive 
nature of CVD by facilitating the reduction of oxidative stress on vasculature. Having highly 
trained experts oversee the supplementation with close study monitoring substantially reduces 
the likelihood of AEs. The potential risks to the participants remain reasonably low in relation to 
the possible benefit of improving their CVD risk factors during high pollution season, without the 
involvement of prescription medications or invasive diagnostic / treatment procedures. 
Importance of Knowledge to be Gained
The knowledge to be gained from this clinical trial is significant in that 1) participants with high 
exposure to PM2.5 environmental pollution and related CVD risk factors may benefit from 
receiving a freely available over the counter dietary supplement that heretofore has 
demonstrated few risks to the participant. 
The study has been designed to address critical limitations in the previous published studies 
which have not looked at the effect of Carnosine supplementation on mitigating CVD risk 
factors. The risks to the participants are reasonable in relation to the knowledge gained from 
this study since this supplementation therapy may potentially reduce the widely prevalent and 
debilitating effects of PM2.5 pollution. 
Data Safety Monitoring Board (DSMB)
The Data and Safety Monitoring Plan has been outlined in Section 16 above. 
Clinical Recruitment
The PI and other involved investigators will oversee that the clinical research team and 
investigators are trained on but not limited to the following topics: 
1.
Maintaining IRB required trainings and certifications (e.g. CITI program, etc.)
2.
Obtaining informed consent 
3.
High quality data collection
4.
Good clinical practice 
5.
HIPAA privacy
6.
Institutional compliance
7.
Understanding the nature of the protocol or investigational plan.

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8.
The study PI understands and accepts his or her obligation to obtain IRB review and 
approval of a clinical investigation before the investigation may be initiated and to ensure 
continuing review of the study by the IRB in accordance with 21 CFR Part 56, and to 
keep the sponsor informed of such IRB approval and subsequent IRB actions 
concerning the study.
9.
Access to an adequate number of suitable potential candidates / participants to conduct 
the investigation.
10. Adequate facilities for supplement preparation and conducting the clinical investigation.
11. Sufficient time to carry out the responsibilities of best practices of clinical research
12. The PI understands periodic / random audits by the IRB may occur
Database
The Envirome Institute will maintain a study database in a web-accessible electronic format. 
Detailed documentation of study variables will be prepared and available to study Investigators, 
and where necessary, to external scientists. Appropriate confidentiality and security of these 
files will be maintained at all times.
Security
Several levels of security will be implemented to protect the confidentiality of the data. All 
authorized users will be provided a unique name/password and will be given access as 
identified by the Principal Investigator. Data will be stored on a REDCap database which is 
HIPAA compliant database routinely utilized by clinical scientists nationwide to store sensitive 
research data. 
 
Data Quality
All data will have to pass through range and logical checks in addition to intra- and inter-form 
checks for consistency. The sequence of events will be enforced by allowing subordinate forms 
to become accessible only after its primary form has been submitted. If a response to a question 
on a form requires ancillary forms to be completed, the user will receive reminder messages 
within the application to complete the proper form. Regular reports on the REDCap data entry 
and completeness will be generated. Data quality or management questions or concerns will be 
immediately addressed.  
Dissemination
The overall usefulness of scientific research depends not only on the importance of the findings, 
but also on its eventual reach and effect on population health. Therefore, research projects must 
integrate ways to promote the eventual diffusion of the results into their research plans. The 
Envirome Institute will work with professional associations like the NIH, NIEHS, American Heart 
Association for a number of initiatives including cardiovascular diseases and environmental 
pollution. The Envirome Institute will use multiple general dissemination methods that will be 

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tailored for the target audiences (e.g. manuscripts, presentations, television, multimedia, social 
media).
17.
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Miller MR. The role of oxidative stress in the cardiovascular actions of particulate air 
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Agarwal A, Wickramasinghe N, Haberzettl P, Sithu SD, Shah J, O'Toole TE, Rai SN, Bhatnagar 
A and Srivastava S. Biomarkers of Chronic Acrolein Inhalation Exposure in Mice: Implications 
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Ismahil MA, Hamid T, Haberzettl P, Gu Y, Chandrasekar B, Srivastava S, Bhatnagar A 
and Prabhu SD. Chronic oral exposure to the aldehyde pollutant acrolein induces dilated 
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Conklin DJ, Prough RA, Juvan P, Rezen T, Rozman D, Haberzettl P, Srivastava S and 
Bhatnagar A. Acrolein-induced dyslipidemia and acute-phase response are independent of 
HMG-CoA reductase. Mol Nutr Food Res. 2011;55:1411-22.
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Wheat LA, Haberzettl P, Hellmann J, Baba SP, Bertke M, Lee J, McCracken J, O'Toole 
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Vladykovskaya E, Liu S, Srivastava S and Bhatnagar A. Acrolein consumption induces systemic 
dyslipidemia and lipoprotein modification. Toxicol Appl Pharmacol. 2010;243:1-12.
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Chugh A, Ramos KS, Srivastava S, Higdon D, Tollerud DJ, DeFilippis A, Becher C, Wyatt B, 
McCracken J, Abplanalp W, Rai SN, Ciszewski T, Xie Z, Yeager R, Prabhu SD and Bhatnagar 
A. Acrolein exposure is associated with increased cardiovascular disease risk. J Am Heart 
Assoc. 2014;3.
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Valkovic L, Kurdiova T, Garzon D, Barbaresi S, Teede HJ, Derave W, Krssak M, Aldini G, 
Ukropec J and Ukropcova B. Effects of carnosine supplementation on glucose metabolism: Pilot 
clinical trial. Obesity (Silver Spring). 2016;24:1027-34.
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Derave W, Ozdemir MS, Harris RC, Pottier A, Reyngoudt H, Koppo K, Wise JA and 
Achten E. beta-Alanine supplementation augments muscle carnosine content and attenuates 
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Harris RC, Tallon MJ, Dunnett M, Boobis L, Coakley J, Kim HJ, Fallowfield JL, Hill CA, 
Sale C and Wise JA. The absorption of orally supplied beta-alanine and its effect on muscle 
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Regazzoni L, de Courten B, Garzon D, Altomare A, Marinello C, Jakubova M, Vallova S, 
Krumpolec P, Carini M, Ukropec J, Ukropcova B and Aldini G. A carnosine intervention study in 
overweight human volunteers: bioavailability and reactive carbonyl species sequestering effect. 
Sci Rep. 2016;6:27224.
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Weber T and American Heart Association Council on H. Recommendations for Improving and 
Standardizing Vascular Research on Arterial Stiffness: A Scientific Statement From the 
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D'Anca A, Signorelli SS and Castellino P. A systematic review of arterial stiffness, wave 
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of L-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by 
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calf muscle endurance in patients with chronic venous insufficiency. Clin Rehabil. 2005;19:339-
44.

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18.
TABLES
TABLE 1: Screening and Enrollment Timeline of NEAT Clinical Trial for Year # 1 (2020)
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Potential Candidates Identification
Clinical Screening (Visit # 1)
Select Eligible Candidates from Visit # 1
Supplement Intervention Period (Total 12 weeks per Participant)
Baseline Assessment (Visit 
#2) Start Supplement
Follow-Up # 1 at 6 weeks (Visit # 3)
Follow-Up # 2 at 12 weeks (Visit # 4)
TABLE 2: Screening and Enrollment Timeline of NEAT Clinical Trial for Year # 2 (2021)
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Potential Candidates Identification
Clinical Screening (Visit # 1)
Select Eligible Candidates from Visit # 1
Supplement Intervention Period (Total 12 weeks per Participant)
Baseline Assessment (Visit 
#2) Start Supplement
Follow-Up # 1 at 6 weeks (Visit # 3)
Follow-Up # 2 at 12 weeks (Visit # 4)

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Table 3: Schedule of Procedures At Each Visit / Encounter
Study Activities
Visit # 1
(Screening)
Visit # 2
(Baseline 
Assessment)
Visit # 3
(Follow-Up # 1)
Visit # 4
(Follow-Up # 2)
4-12 Month 
Safety Check
Informed Consent
X
Contact & Demographics
X
X
X
X
Medical History
X
X
X
X
Sleep / Dietary / Social / 
Exposure Questionnaires
X
X
X
Blood & Urine Samples
X (Urine only)
X
X
X
Physical Exam
X
X
X
Arterial Stiffness Exam
X
X
X
Physical Function Exam
X
X
X
Supplement Distribution
X
X
Checkout (Payment) 
X ($25)
X ($50)
X ($75)
X ($100)
Adverse Events Check
X
X
X

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Table 4: Study Visit Components’ Time Requirements
Study Activities
Visit # 1
(Screening)
Visit # 2
(Baseline 
Assessment)
Visit # 3
(Follow-Up # 1)
Visit # 4
(Follow-Up # 2)
4-12 Month 
Safety Check
Informed Consent
30 mins
Contact & Demographics
5 mins
Medical History
2 mins
Sleep / Dietary / Social / 
Exposure Questionnaires
30 mins
15 mins
15 mins
Blood & Urine Samples
10 min (Urine 
only)
15 mins
15 mins
15 mins
Physical Exam
10 mins
5 mins
5 mins
Arterial Stiffness Exam
20 mins
20 mins
20 mins
Physical Function Exam
20 mins
20 mins
20 mins
Supplement Distribution
10 mins
10 mins
Checkout  
10 mins
10 mins
10 mins
10 mins
Adverse Events Check
5 mins
5 mins
2 mins
Estimated Visit Time
60 mins
120 mins
100 mins
100 mins
5 mins

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19.
FIGURES
Figure 1: NEAT Study Flow-Chart for Year # 1 (2020)

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Figure 2: NEAT Study Flow-Chart for Year # 2 (2021)

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Figure 3: Individual Visit Flow-Chart – Screening (Visit # 1)

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Figure 4: Individual Visit Flow-Chart – Baseline Assessment (Visit # 2)

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Figure 5: Individual Visit Flow-Chart – Follow-Up # 1 (Visit # 3)

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Figure 6: Individual Visit Flow-Chart – Follow-Up # 1 (Visit # 4)

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Figure 7: Individual Visit Flow-Chart – 4-12 Month Safety Check (Visit # 5)

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20.
APPENDIX A – SAMPLE PROCUREMENT AND 
PROCESSING
Blood Sample Collection 
Every effort must be made to make venipuncture as easy and painless as possible for 
participants. The technician must remain calm and project an attitude of competence even when 
faced with the most nervous or inquiring participant. The best way to achieve this is for the 
technician to be thoroughly knowledgeable about all aspects of the procedures. This study 
involves the collection of a total of ~45 ml of blood. Any participant who is concerned about the 
volume of blood should be reassured that the total amount of blood drawn is about 1.5 ounces, 
although it may look like more. 
Blood Collection Tray and Tubes Preparation
Prior to venipuncture the tray is prepared for each participant. Each tray holds the Vacutainer 
tubes used in blood collection labeled with the subject identifier code (given by the research 
coordinator), date, type of blood being collected (serum whole blood, EDTA plasma, sodium 
citrate plasma, etc.) and the study visit (Visit # 1, 2, 3, 4). 
Blood Collection Procedure 
General Guidelines
Venipuncture should be performed using standard sterile technique in the sitting position. 
Handle all specimens as potentially infectious for laboratory workers. OSHA rules mandate that 
technicians must always wear disposable protective gloves when collecting and processing 
specimens. All needles and sharps must be discarded into puncture resistant containers. Avoid 
formation of potentially infectious aerosols when removing the rubber stoppers from Vacutainer 
tubes. In addition to wearing protective gloves, hold a piece of gauze over the stopper while 
slowly removing it from the tube. Place all used Vacutainer tubes and blood-contaminated 
products in biohazard bags for proper disposal. Use 0.5% sodium hypochlorite (household 
bleach diluted 1: 1 0) to clean up any spills of blood, plasma, or serum. 
For Extremely Apprehensive Participants: Do not under any circumstances force the 
participant to have blood drawn. It may help to explain to the subject that the blood drawing is 
designed to be nearly painless as possible. It may also be helpful to have the participant relax in 
the blood drawing chair just so the phlebotomist can check the veins in the participant's arms, 
without actually drawing blood. If the participant is very anxious, he/she may lie down during the 
blood draw. Give the participant enough time to feel comfortable after the blood collection, as 
well. In many cases the most memorable part of the experience for participants will be the 
contact with the technicians who draw the blood and their general attitude and competence. 

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Procedure 
Before applying the tourniquet, screw the Luer adapter into the plastic Vacutainer tube guide. 
Insert the butterfly tubing onto the adapter. With jacket or sweater removed, have the participant 
sit upright with the sleeves rolled up to expose the antecubital fossa. 
Wrap the tourniquet around the arm 3 to 4 inches (7.5 to 10.0 cm) above the venipuncture site. 
If the participant has a skin problem, put the tourniquet over the participant's shirt or use a piece 
of gauze or paper tissue so as not to pinch the skin. The tourniquet should be on the arm for the 
shortest time possible. Never leave the tourniquet on for longer than two (2) minutes. To do so 
may result in hemo-concentration or a variation in blood test values. If a tourniquet must be 
applied for preliminary vein selection, and it remains of the arm for longer than two minutes, it 
should be released and reapplied after a wait of two minutes. 
Venipuncture:
1.
Identify the vein, and then cleanse the venipuncture site. 
2.
Remove the alcohol prep from its sterile package. 
3.
Cleanse the vein site with the alcohol prep using a circular motion from the center to the 
periphery. 
4.
Allow the area to dry to prevent possible hemolysis of the specimen and a burning 
sensation to the subject when the venipuncture is performed. 
5.
If venipuncture becomes difficult, the vein may need to be touched again with your hand. 
If this happens, cleanse the site again with alcohol. 
After successful venipuncture: 
1.
Place tubes into the Vacutainer holder. 
2.
Grasp the participant's arm firmly, using your thumb to draw the skin taut. This anchors 
the vein. The thumb should be 1 or 2 inches below the venipuncture site. 
3.
With the needle bevel upward, enter the vein in a smooth continuous motion. 
4.
Make sure the participant's arm is in a flat or downward position while maintaining the 
tube below the site when the needle is in the vein. It may be helpful to have the 
participant make a fist with the opposite hand and place it under the elbow for support. 
5.
Grasp the flange of the needle holder and push the tube forward until the butt end of the 
needle punctures the stopper, exposing the full lumen of the needle. The tube should 
begin filling with blood. 
6.
Remove the tourniquet after tube #1 fills. 
7.
Keep a constant, slight forward pressure (in the direction of the adapter) on the end of 
the tube. This prevents release of the shutoff valve and stopping of blood flow. 
8.
Fill each Vacutainer tube as completely as possible; i.e. until the vacuum is exhausted 
and blood flow ceases. If a Vacutainer tube fills only partially, remove the tube and 
attach another without removing the needle from the vein. 
9.
When the blood flow into the collection tube ceases, remove the tube from the holder. 
The shutoff value covers the point, stopping blood flow until the next tube is inserted (if 
necessary). 

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The tubes should be gently inverted 6-8 times immediately following removal of the tube from 
the adapter. 
If a blood sample is not forthcoming, the following manipulations may be helpful. 
1.
Tum needle slightly or lift the holder in an effort to move the bevel away from the wall of 
the vein. 
2.
Move needle slightly in hope of entering vein. Do not probe. If not successful, release 
tourniquet and remove needle. A second attempt can be made on either arm. 
3.
Loosen the tourniquet. It may have been applied too tightly, thereby stopping the blood 
flow. Reapply the tourniquet loosely. If the tourniquet is a Velcro type, quickly release 
and press back together. Be sure, however, that the tourniquet remains on for no longer 
than two minutes at a time. 
At the conclusion of the draw 
1.
To remove the needle, lightly place clean gauze over venipuncture site. 
2.
Remove the needle quickly and immediately apply pressure to the site with a gauze pad. 
3.
Discard needle and its cap into needle box (DO NOT ATTEMPT TO RECAP NEEDLES!) 
4.
Have the participant hold the gauze pad firmly for one to two minutes to prevent a 
hematoma. 
Bandaging the arm under normal conditions: 
1.
Slip the gauze pad down over the site
2.
Continue mild pressure. 
3.
Apply an adhesive or gauze bandage over the venipuncture site 
4.
Make sure that blood flow has stopped. 
Bandaging the arm if the participant continues to bleed: 
1.
Apply pressure to the site with a gauze pad. 
2.
Keep the arm elevated until the bleeding stops. 
3.
Wrap a gauze bandage tightly around the arm over the pad. 
4.
Tell the participant to leave the bandage on for at least 15 minutes. 
Blood Collection Precautions when a subject feels faint or looks faint following the blood 
draw: 
1.
Have the person remain in the chair. If necessary, have him/her lie on the floor with their 
legs elevated. Use of a transfer belt may be indicated in this situation. 
2.
Take an ampule of smelling salts, crush it, and wave it under the person's nose for a few 
seconds. 
3.
Provide the person with a basin if he/she feels nauseous. 
4.
Have the person stay seated or lying down until the color returns and he/she feels better. 
5.
Have someone stay with the person to prevent them from falling and falling and injuring 
themselves if they should faint. 

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6.
Place a cold wet cloth on the back of the person's neck or on their forehead. 
7.
Once the episode has passed, some fruit juice may be given to the participant in order to 
counteract any possible hypoglycemia due to fasting. 
8.
If the person continues to feel sick, take a blood pressure and pulse reading. Contact a 
medical staff member, who will advise you on further action. 
Blood Tube Labeling and Processing 
For the study, a total of approximately 45mL blood will be drawn into different tubes with or 
without internal coating of anticoagulants. Biomedical analysis of genetics will be performed only 
if the subject has consented to the genetic sub-study. It is important that the technician know 
more than just the arrangement of the blood collection tubes and the sequence of the tube 
collection. He/she should also be familiar with the purpose of each tube, the type of 
anticoagulant in-each tube, and possible sources of error in the handling of each tube. 
Each tube should be labeled with the subject’s ID code (given by the study coordinator), study 
ID, the date, tube type, and time point (visit # 1, 2, 3, 4) 
After blood draw, all labeled tubes should be kept in a cooler until further processing and 
storage. 
Urine Collection 
Standard clean catch urine sample will be obtained in sterile cups from participants. The 
samples will be transported on ice to the Diabetes and Obesity Center’s research laboratory on 
the 4th floor of the Delia Baxter building for storage and mass spectrometric analysis. Urine 
samples not being processed immediately maybe placed in a biospecimens only refrigerator. 

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21.
APPENDIX B – MEASUREMENT OF BLOOD 
BIOMARKERS 
Endothelial Progenitor Cells (EPCs) 
Eight ml of venous blood collected in a CPT Mononuclear Cell preparation tube (Becton 
Dickinson) will be processed within 3 hours of collection. Mononuclear cells will be separated by 
centrifugation at 1700g for 30 minutes. The buffy coat layer will be collected and washed twice 
with PBS+1%BSA. Cells will be pelleted by centrifugation at 400g for 5min and incubated with 
Fc Block for 10 minutes (Miltenyi Biotec). 50ul aliquots will be then incubated with FITC anti-
CD41a (BD Biosciences), FITC-CD235a (eBioscience), FITC-CD16 (eBioscience), 
QDot655anti-CD14 (Invitrogen), PE-CF594 anti-CD34 (BD Biosciences), APC anti- AC133 
(Miltenyi Biotec), V450 anti-CD31 (BD Biosciences), V500 anti-CD45 (BD Biosciences), In 
addition, we will include a discriminator of cell viability (Live Dead Green; Becton Dickinson). An 
unstained cell aliquot will be used as a control. After a 30 min incubation, the cells will be 
washed, resuspended in PBS+1%BSA, and 50ul of volumetric counting beads (Accucount 
Particles; Spherotech) added. The samples will be then run on an LSR II flow cytometer (Becton 
Dickinson) set to acquire 20,000 beads. Collected events will be analyzed using FlowJo 
software. Initial gating will be selected a lymphocyte population that was negative for CD235a, 
CD41a, CD16, CD14 and the viability marker. Fifteen distinct EPC subgroups will be then 
identified by the variable expression of CD31, CD45, CD34, and AC133 (S1 Table). Positive 
boundaries for gating will be accomplished with the unstained sample. EPCs numbers will be 
quantified and normalized to volume of blood using bead counts.
Microparticles and Platelet-Monocyte Aggregates (PMA) 
For the analysis of microparticles and immune cell populations, 8 ml of blood will be collected in 
sodium citrate-containing cell preparation tubes (CPT Vacutainer; Becton Dickinson). These 
tubes will be centrifuged at 1700xg for 30 min at room temperature and shipped overnight to the 
University of Louisville for analysis. Upon arrival, the tubes will be centrifuged again and the 
upper layer containing mononuclear cells and plasma was collected. This material will be diluted 
with an equal volume of PBS and centrifuged at 500xg for 10 min. Aliquots of the supernatant 
will be used for analysis of microparticles while the cell pellet will be washed once with PBS and 
centrifuged. The final cell pellet will be resuspended in a volume of 300 µl PBS+2% FCS, 150 µl 
of which will be used for analysis of immune cell populations.
For analysis of platelet-monocyte aggregates, 3ml of blood will be collected in an acid-citrate 
dextrose tube (ACD Vacutainer; Becton-Dickinson), and then 1ml aliquots will be diluted with 
3ml PBS and fixed with 1.3ml of 4% paraformaldehyde for 30 min on ice.  Red blood cells will 
be lysed by addition of 24 ml of water, the samples will be centrifuged at 400xg for 10min and 
the cell pellet will be resuspended in 1 ml Tyrode’s buffer and shipped to University of Louisville 
as above.

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22.
APPENDIX C – PRESCREENING COMMUNICATION 
SCRIPTS
Phone Communication Script 
Hi _____________ (insert name), how are you today? My name is __________ 
(insert name) from the Department of Medicine at the University of Louisville. 
The reason I am calling is to invite you to take part in a research study being 
conducted by Dr. Tim O’Toole. You are being invited because you are between 22-
65 years of age and live in or around Louisville. 
The purpose of this study is to see whether taking an over the counter carnosine 
supplement can help protect you from harmful effects of PM pollution, which is 
significantly high in the Louisville area. Carnosine is a naturally occurring chemical 
in our body and is easily available as an over the counter supplement, just like a 
protein powder! Our laboratory studies have shown that taking supplemental 
carnosine is protects against particulate matter pollution.
Your participation in the study would be completely voluntary.
There will be total 4 visits for the study. The first visit will tell us whether you qualify 
for the study. First visit will last about 45 minutes. If you qualify, we will invite you to 
participate in the main study, during which you will be required to take a daily 
dietary supplement for 3 months. You will be given either carnosine or placebo. 
Placebo is plain cornstarch and harmless. 
There will be 3 study visits during these 3 months. All visits will be at the University 
of Louisville Physicians Outpatient Center (401 E. Chestnut Street 4th floor suite 
460, Louisville, KY). Each visit will take about 1.5 to 2 hours and we will collect 
some blood, urine, and other data about your health and personal history. 
We will not do any drug testing on your samples. You will not need to answer any 
questions that you are uncomfortable with. If you choose to participate, as a token 
of appreciation for completing the study you will receive a prepaid Gift Card at each 
visit as per following: 
$25 for Visit # 1, $50 for Visit # 2, $75 for Visit # 3, and $100 for Visit # 4. 
Do you have any questions about the study?
Is this something you would be interested in doing?
(If YES, proceed. If NO, thank them for their time and discontinue the call)
If you decide to participate in the study, what would be a good date and time for 
you to come for a screening visit? ______ (Note down date/time for screening 

Carnosine NEAT Trial Protocol 
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visit), so that we can schedule that appointment now and we will be sending you 
directions for the study visit and where to come for the visit. Would you prefer to 
get these by mail or email? Can I verify your contact information one more time? 
____________________(Insert contact information). 
Thank you for your time and we look forward to seeing you on ________ at 
________am/pm.
Email or Letter Communication Script 
Dear [Mr. / Ms. LAST NAME],
I am writing to tell you about the Nucleophilic Defense Against PM Toxicity (NEAT) 
study being conducted by the University Of Louisville Department Of Medicine.
We are contacting individuals who have expressed an interest in participating in 
research studies and live in or around Louisville. Based on your current physical 
address and age, we would like to invite you to learn more about the study and see 
if you would be interested in participating.
The purpose of this study is to see if taking a carnosine supplement can help 
protect against cardiovascular injury caused by exposure to naturally occurring 
particulate matter (PM) pollution in the Louisville area. Carnosine is a naturally 
occurring chemical in our body and is easily available as an over the counter 
supplement (just like a protein powder!).
We want to learn whether taking an over the counter carnosine supplement can 
help protect you from harmful effects of PM pollution, which is significantly high in 
the Louisville area. 
Your participation in the study would be completely voluntary.
A screening visit will be conducted to check whether you qualify for the main study. 
The screening visit will last about 45 minutes and will get some urine sample and 
basic information about you. If you qualify, we will invite you to come back for a 
visit on a later date.
In the main study, you will be required to take a daily dietary supplement of 
carnosine or placebo (looks like the supplement but is not) for a total of 3 months. 
We will see you three times during the main study period. All visits will be at the 
University of Louisville Physicians Outpatient Center (401 E. Chestnut Street 4th 
floor suite 460, Louisville, KY). Each visit will take about 1.5 to 2 hours. At each 
visit we will get some blood and urine samples, measure arterial stiffness using a 
blood pressure-like cuff, perform tests of physical strength and ask you questions 
about demographics, health history, and personal habits.
We will not do any drug testing on your samples. You will not need to answer any 

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questions that you are uncomfortable with. If you choose to participate, as a token 
of appreciation for completing the study you will receive a prepaid Gift Card at each 
visit as per following: 
$25 for Visit # 1, $50 for Visit # 2, $75 for Visit # 3, and $100 for Visit # 4. 
Your participation in this study is voluntary.
If you are interested in learning more about this study we would be excited to speak 
with you and give you more details if necessary, please call us at 502-852-7559 or 
email us back at Envirome@Louisville.edu.
Thank you for your time and consideration. We look forward to hearing from you. 
Sincerely,
Timothy O’Toole, PhD
Assistant Professor, University of Louisville, Department of Medicine 

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23.
APPENDIX D – SUPPLEMENT MANUFACTURING AND 
QUALITY CONTROL PROTOCOL
L-Carnosine Powder Certificate of Analysis 
The certificate of analysis for the L-Carnosine powder from 
www.bulksupplements.com is attached. It highlights that the dietary supplement 
conforms the regulatory standards. 
Supplement Compounding and Quality Control Standard Operating 
Protocol
The capsule sources, compounding methods, quality control measures and other 
standard operating protocol are detailed in the attached agreement letter from the 
University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences.

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24.
APPENDIX E – PHYSICAL FUNCTION MEASUREMENTS 
Handgrip Strength Test  
The first is the handgrip strength test, which assesses upper extremity strength and is reported 
to be a reliable indicator of a variety of impaired cardiopulmonary conditions. 49, 50 The handgrip 
strength static test will be assessed as described 51 using a Jamar Technologies Plus+ Digital 
Hand Dynamometer (JLW Instruments, Chicago, IL, USA). Subjects will be seated in an upright 
position with their shoulder adducted and elbow flexed at 90°, with the forearm allowed to rest 
lightly on the arm of the chair or on the subject’s thigh. For each subject, a maximal voluntary 
contraction will be determined as the highest of three initial contractions (INITIAL MAX). 52 After 
a one-minute rest period subjects will be instructed to squeeze the dynamometer in a static 
contraction at a level of 50% of their maximal voluntary contraction for as long as possible, and 
the duration recorded (DURATION). 53 The level of static contraction will be monitored by the 
assessor who provides verbal feedback to ensure that a level of 50% maximal voluntary 
contraction is maintained. Subjects will then perform an additional post-fatigue maximal 
contraction within 10 seconds of completion of the 50% maximal static contraction (FINAL 
MAX). Handgrip testing will be performed on both the dominant and non-dominant hands for 
each subject. In addition to (INITIAL MAX), (FINAL MAX) and (DURATION), additional 
calculated indices will be: a) work performed (WORK = DURATION x 50% INITIAL MAX); and 
b) Strength Decrement Index (SDI = 100 x (INITIAL MAX + FINAL MAX) ⁄ INITIAL MAX).
Two Minute Step Test (2MST)
The 2MST is an assessment of aerobic endurance and can be predictive of morbidity and 
mobility in older adults or those with CVD. Higher scores on the 2MST indicate increased 
functionality, quality of life, and decreased incidence of chronic health problems. 54 To perform 
the test, participants are asked to stand next to a wall and march in place with the intent of 
raising the knees to a marked target on the wall mid-way, representing a height between the 
lateral condyle and anterior superior iliac spine of the participant. The test administrator counts 
the number of times the right knee reaches the marked target over a 2-minute period.  If an 
individual cannot march for the full 2-minutes, they are permitted to take a standing or seated 
rest break and resume within the 2 minutes. 55
Bilateral Calf-Raise Test
This is a muscle strength and endurance test that has been used to evaluate the functional 
performance of older adults and those with CVD.56-59 Initially, participants will stand barefoot and 
in a bipedal position facing a wall and will be supported with their dominant hand on the wall and 
elbow in semi-flexion to maintain balance. The participant will perform plantar flexion with 
maximum range for all repetitions. The first flexion is executed using the full range of motion, up 
to the point of support on the metatarsophalangeal joints. At full flexion, the examiner marks the 
maximum height reached by the participant’s head with an adjustable height instrument. This 
will ensure that the individual performs plantar flexion using full range of motion in every 

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repetition. A verbal command will be given to start the test and the participant will perform the 
maximum number of plantar flexions possible, as fast as possible until the point of voluntary 
fatigue. Measurements include the number of plantar flexions performed during the bilateral 
calf-raise, total time in seconds, and repetition rate (repetitions per second). These outcomes 
can then be normalized for differences in participant physical characteristics such as BMI, 
height, weight and age.