Vitamin C Supplementation Intervention for Patients With Heart Failure-A Pilot Study
Phase: NA
Conditions: Heart Failure
Sponsor: University of North Carolina, Chapel Hill

 
Vitamin C Supplementation Intervention for Patients with Heart 
Failure—A Pilot Study 
 
 
NCT number NCT04036110 
Document Date 04/21/2020 
 

Vit C Study Protocol / 1 
Protocol 17-1809 –  
Protocol version: 4  
Version date: 04-21-2020 
 
Protocol Title: Vitamin C Supplementation Intervention for Patients with Heart Failure—A 
pilot study 
Principal Investigator:  Jia-Rong Wu, PhD, RN, FAHA   
Email Address: jiarongw@email.unc.edu 
Office Address: 4015 Carrington Hall, School of Nursing, Chapel Hill, NC 27599-7460 
Office Telephone Number: 919-966-8057 
Medical Monitor: Sarah Waters, Heart Failure Nurse Practitioner, Cardiology, UNC Hospital 
 
 
PI Signature: 
I confirm that I have read this protocol and understand it.    
Principal Investigator Name:   
Jia-Rong Wu  
 
 
 
 
Principal Investigator Signature:  
 
 
 
 
 
 
 
Date:   
 
 
 
4/21/2020 
 
 
 
 
 

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TABLE OF CONTENTS 
Table of Contents ................................................................................................................. 2 
 
Abbreviations and Definitions of Terms ................................................................................ 3 
1 BACKGROUND  AND RATIONALE ............................................................................... 4 
2 STUDY OBJECTIVES ................................................................................................ 4-5 
3 INVESTIGATIONAL PLAN ............................................................................................. 5 
4 STUDY INTERVENTION (DEVICE OR OTHER INTERVENTION) ............................ 5-6 
5 PROCEDURES AND RECRUITMENT STRATEGY ................................................... 6-7 
6 CONSENT PROCESS.................................................................................................... 7 
7 MEASUREMENTS AND STUDY EVALUATIONS ...................................................... 7-8 
8 SAFETY EVALUATION .............................................................................................. 8-9 
9    DATA MANAGEMENT AND STATISTICAL ANALYSIS ..........................................10-11 
10  APPENDIX ................................................................................................................... 12 
11 REFERENCES ........................................................................................................13-14 
 
 
 
 

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Abbreviations and Definitions of Terms  
 
Abbreviation 
Definition 
HF 
Heart failure 
HRQOL 
health related quality of life  
RCT 
Randomized controlled trial 
G6PD 
glucose-6-phosphate dehydrogenase deficiency 
UNCH 
University of North Carolina at Chapel Hill Hospital 
MLHFQ 
Minnesota Living with Heart Failure questionnaire 
MSAS-HF 
Memorial Symptom Assessment Scale—Heart Failure 
ELISA 
Enzyme-linked immunosorbent assay 
HIPAA 
Health Insurance Portability and Accountability Act 
EDTA 
Ethylenediaminetetraacetic acid 
BHT 
Butylated hydroxytoluene 
 
 
 

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1. BACKGROUND: 
Heart failure  
Although significant advancements in morbidity and mortality statistics have been 
established with medical treatment, HF remains a prevalent, morbid, and costly condition.1,2 
Currently, 6.5 million Americans have heart failure (HF),1,3,4 50% are readmitted within 6 months 
of hospital discharge for exacerbations of HF.5,6 Thus, hospitals are financially penalized for HF 
readmissions within 30 days based on the Readmissions Reduction Program initiated by the 
Centers for Medicare & Medicaid Services. Moreover, patients with HF continue to be 
symptomatic even after optimization of medical treatment. These symptoms are associated with 
frequent hospitalization, as well as decreased health related quality of life (HRQOL).7,8 
Therefore, HF symptom reduction is still the major target of treatment to reduce frequent re-
hospitalizations and improve HRQOL.7,8 
Vitamin C Supplementation  
 
Mean serum vitamin C level was significantly lower in patients with HF compared to 
those with other non-HF chronic illness.9-11  Up to 39% of patients with HF had vitamin C 
deficiency.10 Many studies have shown that higher vitamin C levels are associated with 
improved endothelial function.12-14  There was a significant positive correlation between cardiac 
function and serum vitamin C level.15-17  Furthermore, in a prospective study in HF, vitamin C 
deficiency predicted shorter cardiac event-free survival.11 
 
In two intervention studies in patients with HF,11,12 investigators found that vitamin C 
supplementation increased serum vitamin C level18 and also improved endothelial function.13,17 
Other interventional studies also provided evidence that vitamin C supplementation not only 
improved cardiac function,4,12,19 but also reduced oxidative stress in patients with HF.13,20 
However, most of these trials tested effects of high dose vitamin C supplementation via intra-
venous or intra-arterial administration.  Only two trials with very small sample size (n=10) tested 
oral, 1 month vitamin C supplementation and found vitamin C supplementation increased serum 
vitamin C level and also improved endothelial function, although neither study controlled for 
confounders such as age, race, sex, BMI or social status.13,17 
Preliminary Study 
The PI has conducted a preliminary study that serves as the foundation for this feasibility 
pilot proposal.  This was an observational study in which we collected dietary vitamin C intake 
from a 4-day food diary, symptom burden using the Memorial Symptom Assessment Scale—
Heart Failure, and HRQOL using the Minnesota Living with Heart Failure questionnaire at 
baseline, and prospectively followed cardiac hospitalization and death for up to 4 years in 264 
patients with HF. In this study, 41% of HF patients had vitamin C deficiency. Patients with 
vitamin C deficiency had higher symptom burden (p =.05), worse HRQOL (p <.001) and were 
about twice as likely to have a cardiac event compared to patients with adequate vitamin C 
intake (p = .015). This study demonstrated the high prevalence and worse consequences of 
vitamin C deficiency in patients with HF.   
 
2. STUDY OBJECTIVE 
The purpose of this study is to test a low-cost, simple vitamin C supplementation 
intervention, that is, comparing placebo to 500mg/day vitamin C and 1gram/day vitamin C daily 
to assess feasibility and acceptability of vitamin C supplementation and effects on serum 
vitamin C level, cardiac function, HF symptoms, and HRQOL. Our specific aim 1 is to test the 
effect of a vitamin C supplementation intervention on the primary outcome (serum vitamin C 
level) from baseline to 3-month follow-up among 3 groups (0.5 g, 1 g vitamin C, and placebo). 

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Specific aim 2 is test the effect of a vitamin C supplementation intervention on the secondary 
outcomes (HF symptom burden, health related quality of life (HRQOL), cardiac function, and 
oxidative stress) from baseline to 3-month follow-up among 3 groups.  Specific aim 3 is to 
collect data to assess feasibility (recruitment, retention, attrition rates). 
 
3. INVESTIGATIONAL PLAN  
Design. This project is a feasibility pilot study to test the effects of vitamin C supplementation, 
and to obtain initial estimates of effect size to support a future RCT. The proposed vitamin C 
supplementation intervention study is a three-group, single blind, placebo controlled prospective 
design (control n=14, Vit C 500mg n=14, Vit C 1000mg n=14).  The most commonly tested dose 
of vitamin C in current trials on vitamin C supplementation is 1 g/day.18  The primary endpoint to 
evaluate the effect of vitamin C supplementation is serum vitamin C level. The secondary 
outcomes include HF symptom burden, HRQOL, cardiac function, and oxidative stress. The 
variables would be measured in person at baseline and at 3 months include: HF symptom 
burden, HRQOL, PROMIS fatigue, depression and sleep disturbance, serum vitamin C level, 
biomarker of cardiac function (i.e., cardiac output using impedance cardiograph) and oxidative 
stress (8-iso-PGF2a isoprostane, see below in detail), as well as demographic, clinical, and 
other variables that can affect study outcomes (e.g., age, gender, race/ethnicity, body mass 
index, serum creatinine) and medications. HF symptom burden and HRQOL will also be 
measured at Month 1 and 2 by phone. We will also collect data to assess feasibility 
(recruitment, retention, attrition rates). 
Sample and Setting.  A total of 42 participants (14 per group) will be randomly assigned to 
either the control or the intervention group. Patients with HF will be recruited at the University of 
North Carolina Hospital internal medicine/cardiology outpatient clinics.  
Inclusion/Exclusion Criteria. Inclusion criteria include patients (a) have a diagnosis of chronic 
HF. The diagnosis and etiology of chronic HF will be confirmed by a HF cardiologist using 
established criteria,6,21,22 (b) have undergone evaluation of HF and optimization of medical 
therapy, (c) are not taking any vitamin C supplementation at all or are willing to stop taking their 
current vitamin C supplementation, (d) able to read and speak English, (e) have >1 month from 
any inpatient hospitalization, (e) treating cardiology healthcare provider indicates the individual 
is a good candidate for this study that involves 3 months of vitamin C supplement of 500 
mg/day, 1g/day, or placebo, (f) Per treating healthcare provider's assessment, patient’s 
projected lifespan is greater than or equal to 6 months.   
Exclusion criteria include: (a) stage D HF or New York Heart Association (NYHA) functional 
class IV, (b) history of renal stones or renal disease (serum creatinine >1.5), (c) history of 
glucose-6-phosphate dehydrogenase deficiency (G6PD) and (d) cognitive impairment that 
precludes giving informed consent or ability to follow protocol instructions, (e) originally taking 
vitamin C 500mg/day or more, (f) women who are pregnant.  
Female patients with HF are considered past child-bearing potential: 1) if they are ≥60yrs of age 
or 2) if they are age 50 to 59 years and have not menstruated for 12 months (will confirm by 
self-report). For women < 50 years old (with child-bearing potential), we will confirm by self-
report that they are not pregnant and confirm this by completing a urine pregnancy test prior to 
enrollment and assess for likelihood of pregnancy on a monthly basis. 
 
4. INTERVENTION  
The intervention is designed to complement medical management. Therefore, all 
patients will continue to be medically managed by their health care provider as usual. 

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Participants will have a visit at baseline and Month 3 (with some flexibility to do the final visit at 3 
months +/- 1 month) coordinated with their regular clinic visits that are typically scheduled every 
3 months. 
Vitamin C supplementation. Throughout the study, patients will continue with their routine 
diets.  Patients randomized to the intervention groups will be provided with 4-month supply of 
either Vitamin C 500mg or Vitamin C tablets (1000mg) in a bottle and the control group will be 
provided placebo tablets in a bottle. All participants in the intervention group will be instructed to 
take one vitamin C tablet (500mg/ day or 1000mg/day) with one meal daily from baseline for 3 
months and all control group participants will be instructed to take one placebo tablet with one 
meal daily from baseline to 3 months. Participants will return their un-used investigational drugs 
on the day of their 3-month follow-up. We will count remaining pills to determine participant’s 
treatment adherence with both the vitamin C supplementation (500mg or 1000mg) and control 
(placebo) groups. After counting the pills, the un-used pills will be delivered to the 
Investigational Drug Services (IDS) pharmacy to dispose. We will draw blood to measure serum 
vitamin C level to determine drug accountability.  
Role of the Investigational Drug Services (IDS). The IDS will provide oversight for the 
storage, dispensing, labeling, distribution and disposal of all investigational medications (vitamin 
C tablets and placebo) for the study.  
5. PROCEDURES AND RECRUITMENT STRATEGY 
 Permission for this study will be obtained from the University of North Carolina at Chapel 
Hill Institutional Review Board. A dedicated and trained research assistant will recruit HF 
patients from the UNCH clinics. A list of HF patients who will come to the UNCH clinics will be 
generated and obtained from the staff in charge for the scheduling information in the clinics one 
week before patients’ visits. On the day that the potential participants come to the clinic, the 
research assistant will screen the potential participant’s eligibility based on inclusion and 
exclusion criteria. The research assistant also will consult with the potential participant’s treating 
healthcare provider to make sure the potential participant is appropriate for the study and 
patient’s projected lifespan is greater than or equal to 6 months. Once the research assistant 
confirms the potential participants are eligible and gets approval from the provider, the research 
assistant will approach and briefly explain the study to recruit potential participants while they 
wait to see their physicians. If potential participants are interested in participation, the research 
assistant will then confirm eligibility. For eligible patients, study requirements will be explained in 
detail. If the patient agrees to participate, a signed informed consent will be obtained based on 
consent process (see below) and we will administer the survey, measure cardiac impedance 
and obtain the blood samples.  All eligible patients will be randomized to either the control group 
or to the intervention group using the Medsharing application for clinical trials.  
 
Baseline visit. Data on socio-demographics, clinical characteristics medications, symptom 
burden, PROMIS depression, fatigue, and sleep disturbance, HRQOL, will be collected at the 
beginning of the visit and blood will be drawn to measure serum vitamin C level and oxidative 
stress (8-iso-PGF2a isoprostane). All participants will be asked to return their respective 
supplementation bottles to the research assistant at Month 3. We will count pills to determine 
patient adherence to taking the placebo or vitamin C supplementation. 
Follow up phone call (Month 1 and 2). Follow up phone calls will occur within 1 week after 
enrollment to follow-up whether they are taking the investigational pills (vitamin C or placebo) 
daily and whether they have any adverse events from taking the investigational pills, as well as 
at Month 1 and 2. The research assistant will inquire about general welfare and collect data on 
HF symptom burden and HRQOL (for both groups).  

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Month 3 visit. Data on socio-demographics, clinical characteristics medications, symptom 
burden, PROMIS depression, fatigue, and sleep disturbance, HRQOL, will be collected at the 
beginning of the visit and blood will be drawn to measure serum vitamin C level and oxidative 
stress (8-iso-PGF2a isoprostane). All participants will be asked to return their vitamin C (or 
placebo) bottle to the research assistant at Month 3. We will count pills to determine their 
adherence to taking the placebo or vitamin C supplementation.   
 
6. CONSENT PROCESS  
The consent process includes: 1. Obtain written informed consent from the study subject; 2. 
Ensure that the most recent version of the IRB-approved consent/HIPAA authorization form is 
used; 3. Review the informed consent/HIPAA authorization form with the subject in a location 
that allows privacy; 4. Allow the subject time to read the document and ask questions. 
Encourage input from family members, if appropriate; 5. Ensure that the subject signs and dates 
the document in the required sections of the consent and HIPAA authorization form; 6. Ensure 
the consenter signs and dates (same date) the document in the required section of the consent 
form; 7. Provide a singed copy of the informed consent/HIPAA form document to the subject; 
and 8. Document and date consent procedure and file the signed document and checklist back 
to locked cabinet. 
The data (from self-report, impedance cardiograph, and blood drawn) will be collected at 
baseline clinic appointment and Month 3 in person for all patients. HF symptom burden and 
HRQOL will be collected at baseline and Month 3, in person and also Month 1 and 2, by phone. 
All patients will be compensated up to $50 for participating in this study: $25 at baseline visit 
and $25 at Month 3 visit. 
Under special circumstances that we are not allowed to collect in-person data at Month 3 
(such as Covid-19 pandemic stay-at-home order that prevents our in-person data collection), we 
will not collect data from impedance cardiograph or blood drawn. We will only collect self-report 
data using one of the following 2 ways based on subjects’ preference: 1) by phone; 2) by mail--
we will send subjects the questionnaires ahead of time and ask them to complete and send the 
questionnaire back to us. We also will ask them to send their un-used vitamin C pills back to us 
by mail.  
 
7. MEASUREMENTS AND STUDY EVALUATIONS 
Serum vitamin C level. We will measure participant’s serum vitamin C level at baseline and 
Month 3 to measure the effect of vitamin C supplementation. Sample vitamin C will be 
measured in serum using MyBioScouce ELISA kits.  
Symptom burden. Physical symptom burden will measured using items from the Memorial 
Symptom Assessment Scale—Heart Failure (MSAS-HF).8,23  The MSAS-HF is an 8-item 
questionnaire adapted from Portenoy’s Memorial Symptom Assessment Scale that is designed 
to provide multidimensional information about HF symptoms experienced by patients with HF. 
Patients will be first asked if the symptom was present during the previous 7 days. If present, 
three characteristics of each symptom will be rated: frequency of symptom, severity of 
symptom, and degree of symptom distress. Frequency is rated on a scale from 1 to 4 (rarely to 
almost constantly), severity rated on a scale from 1 to 4 (mild to very severe) and distress rated 
on a scale from 0 to 4 (not at all to very severe). Burden score for each symptom can range 
from 0 (no burden) to 12 (greatest symptom frequency, severity, and distress). The scale has 
good reliability, ranges of 0.73-0.92 in patients with HF.8,23 
Health-related quality of life (HRQOL). The Minnesota Living with Heart Failure 
questionnaire (MLHFQ) is a measure of HRQOL that assesses the patient’s perceptions of the 

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influence of HF on physical and emotional aspects of life.24,25 The 21 items are summed with 
higher scores indicating worse HRQOL. The questionnaire has been widely used to measure 
quality of life in this population. Researchers have demonstrated evidence for validity and 
reliability (Cronbach’s alphas ranging from .88 to .93).24-26  The MLHFQ was used in our study 
that demonstrated a significant relationship between vitamin C deficiency and HRQOL, as well 
as between HRQOL and re-hospitalization or mortality.26 
Cardiac function. Impedance Cardiography is a measurement method that is primarily 
used to determine aspects of Cardiac Output (CO) that we will use the MindWare Mobile 
impedance cardiograph (model number 50-2303-00) in this study. CO is the total amount of 
blood, pumped from both the left and right ventricles of the heart for each of its successive 
systolic phases, over the course of one minute. Left ventricular cardiac output is the CO from 
the left ventricle only and can be estimated via a non-invasive technique called Impedance 
Cardiography (ICG), which provides an approximation of Stroke Volume (SV) 
(CO=SV*HR).  We will measure patient’s cardiac output and heart rate variability to reflect the 
participant’s cardiac function using impedance cardiograph that is non-invasive, reproducible, 
valid and widely used in patients with cardiovascular disease.27 The MindWare Mobile 
impedance cardiograph is a commercially available impedance cardiograph that has been 
widely used in research studies.28 The MindWare Mobile impedance cardiograph is a small, 
portable device with a high precision frequency constant current of 400 micro amps which 
allows measurement of the impedance changes across the thorax to monitor cardiac function.28  
Oxidative stress biomarker. We will measure 8-iso-PGF2a isoprostane as a biomarker of 
oxidative stress. Elevated level of 8-iso-PGF2a isoprostane will serve as a biomarker of 
increased lipid peroxidation due to oxygen free radical production exceeding antioxidant 
capacity and will be measured in our Biobehavioral Lab. The level of the biomarker is expected 
to decrease in response to the vitamin C supplementation. Venous blood will be drawn into 
vacutainers containing trisodium citrate, EDTA and/or BHT depending on the analysis, 
centrifuged in a refrigerated centrifuge and serum separated and allotted to micro-tubes and 
kept at -70oC prior to analysis. 8-iso-PGF2a isoprostane will be measured in serum using Cell 
Biolabs ELISA kits. For all assays, samples and standards will be run in duplicate and inter- and 
intra-assay determined to assure accuracy and reproducibility.  
Covariates. We will collect participants’ demographic and clinical data to assess sample 
characteristics, characterize the sample, and obtain data on potential confounding variables.  
8. SAFETY EVALUATIONS 
All participants will receive a phone call within 1 week after enrollment, and also at Month 1 
and Month 2 to follow-up whether they are taking the investigational pills (vitamin C or placebo) 
daily and whether they have any adverse events from taking the investigational pills (see 
checklist in Appendix). If any adverse events were found/reported, we will follow them per 
recommendations of the medical monitor until the event resolves. Sarah Waters, a nurse 
practitioner with specialty in heart failure management at UNC Meadowmont Cardiology clinic 
will serve as a medical monitor on this study. Any adverse event will be reported to Sarah 
Waters in 72 hours or less. Sarah Waters will determine the severity of the adverse events and 
advise whether the participant needs to stop taking the investigational pills, withdraw from the 
study, or any medical treatment is needed. We will follow Sarah Waters’ advice immediately. 
When subjects have AE (define as new signs and symptoms after taking investigational 
drugs, such as digestive distress--diarrhea, nausea, abdominal bloating and any other 
unanticipated adverse events that could possibly be related to the intervention). We will report to 

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Sarah Waters to determine the severity of the AEs (see below). When mild AE is determined, 
subjects will continue to take their investigational drugs without further treatment and the RA will 
keep following up subject’s AE until AE resolves. When moderate AE is determined, we will ask 
subjects to stop taking their investigational drug, advise them to seek medical treatment, and 
continue to follow up until the AE resolves. If severity of any AE is more than moderate, we will 
ask subjects to seek for emergent medical treatment.  
• 
Mild Adverse Event – Event results in mild or transient discomfort, not requiring 
intervention or treatment; does not limit or interfere with daily activities (e.g., insomnia, 
mild headache).  
• 
Moderate Adverse Event – Event is sufficiently discomforting so as to limit or interfere 
with daily activities; may require interventional treatment (e.g., fever requiring antipyretic 
medication). 
• 
Severe and undesirable Adverse Event – Event results in significant symptoms that 
prevents normal daily activities; may require hospitalization or invasive intervention (e.g., 
anemia resulting in blood transfusion). 
The adverse event information will be reviewed in our monthly team meeting to monitor subject 
safety. The PI will also monitor the rate of AEs occurring in this study and decide if it is 
reasonable in light of the rate of AEs in the current literature to continue the study with the 
medical monitor’s input. 
The research assistant is responsible to report any AE related to the study to Sarah Waters and 
the PI. The PI will work with the medical monitor to evaluate the event and make a 
determination of relatedness if the event is related to the study, as it occurs and for notifying the 
Associate Dean for Research of the UNC School of Nursing and UNC IRB of the occurrence of 
any adverse event. A scale on relatedness ranging from “Not related to” to “Definitely related to” 
(Not related to, probably not related to, possibly related to, definitely related to) will be used to 
evaluate relatedness. Any “definitely related” and “possibly related” events will be reported to 
the UNC IRB. 
 
The PI will report to the UNC IRB any adverse events that are (1) unexpected, (2) related or 
possibly related to participation in the research, and (3) serious or suggest that there are new or 
increased risk(s) to subjects. 
 
Patient Safety and Confidentiality. All private health information (PHI) will be kept strictly 
confidential and subject to the Health Insurance Portability and Accountability Act (HIPAA).  All 
study data in transit will be stored on an encrypted lap top computer stored and locked within 
the office of the study PI.  All transit study data will be back up to the secure SON server that is 
password protected and maintained by information technology support and then be deleted from 
the encrypted laptop computer within 72 hours after data are collected. The secure SON server 
can only be accessed by study personnel on this study. Any new signs or symptoms reported by 
the patient to the research team will be promptly communicated to the patient’s health care 
team.  At the baseline visit, patients will be assessed for any vitamin supplementation, serum 
creatinine and history of kidney stones.  If subjects are currently taking a vit C supplement, we 
will advise them to stop taking any vitamin C supplement while they are on the study. We also 
will carefully review with the subjects if they are taking any multivitamin that contains vitamin C 
and advise them to stop taking any multivitamin that contains vitamin C. Although vitamin C is a 
water-soluble vitamin, we have implemented these steps to insure safety before randomization. 

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9. DATA MANAGEMENT AND STATISTICAL ANALYSIS  
Each participant will be assigned an anonymous ID. Data will be collected using touchscreen 
laptop computer, and the REDCap (Research Electronic Data Capture) system,29,30 a secure 
web-based application that is designed to support data capture for research studies.30  
All data analyses will be performed using SPSS 24.0. All estimates will be obtained along with 
95% confidence intervals. 
Descriptive statistics: All data with approximately symmetric distributions will be described by 
their mean and standard deviation, as well as number/frequency and percentage (for Aim 3) and 
variables with skew will be described by the median and interquartile range.  Categorical 
variables will be shown as total number and proportion and will include: race, sex, NYHA 
functional class, and comorbidity.  Associations between categorical variables will be tested by 
the Chi Square test (X²) or McNemar’s test.  Continuous variables will include symptom burden, 
PROMIS depression, fatigue, and sleep disturbance, HRQOL, cardiac function, serum vitamin c 
levels and biomarker for oxidative stress. Associations between categorical and continuous 
variables will be tested by t-test and analysis of variance (ANOVA).  Associations between 
continuous variables will be tested using Pearson’s correlation coefficient (r) will be used to 
establish if the correlation coefficient is statistically different.   
Efficacy analysis for the specific aims is described below: 
The primary endpoint is serum vitamin C level. For Aim 1 to test the effect of a vitamin C 
supplementation intervention on the primary outcome (serum vitamin C level) from baseline to 
3-month follow-up among 3 groups, we will use repeated measure of ANOVA to detect 
differences in the serum vitamin C level from baseline to 3-month follow-up in the intervention 
group and compared to the control group from baseline to 3-month follow-up.   
The secondary endpoints are symptom burden, HRQOL, cardiac function, and oxidative stress. 
For Aim 2 to test the effect of a vitamin C supplementation intervention on the secondary 
endpoint of HF symptom burden from baseline to 3-month follow-up among 3 groups, we will 
use repeated measure of ANOVA to detect differences in symptom burden from baseline to 3-
month follow-up in the intervention group and compared to the control group from baseline to 3-
month follow-up. Similarly, to test the effect of a vitamin C supplementation intervention on the 
secondary endpoint of HRQOL (cardiac function, and oxidative stress) compared to the control 
group, we will use repeated measure of ANOVA to detect differences in HRQOL (cardiac 
function, and oxidative stress) from baseline to 3-month follow-up in the intervention group and 
compared to the control group from baseline to 3-month follow-up.  
Similar analysis will be done for adverse events. to test the effect of a vitamin C 
supplementation intervention on the frequencies of advance events compared to the control 
group, we will use ANOVA to compare frequencies of adverse events during the 3-month period 
among 3 groups. 
Sample Size and Power: The total sample size of 42 participants will be selected as a pilot 
feasibility study.  With 42 patients (14 in each group) and an alpha level of .05, the power of the 
ANOVA F test to detect a significant group difference will be 80% if the effect size is 0.5.  

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Interim/safety Analysis. We plan to conduct the interim/safety analysis when we have 50% of 
the intervention sample, and the findings (as well as adverse events) will be reported to the 
medical monitor. The medical monitor will make decisions about the safety of continuing (or 
stopping) the protocol, providing recommendations for recruitment, retention, safety, and other 
issues as appropriate. 
 
 
 

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10. APPENDIX: 
Checklist of adverse events for taking high dose of vitamin C supplement 
Study #: ___________________ 
 
Data collector: ________________________ 
Date of follow-up: ___________________(for Week 1, Month1, Month2, Month3) 
1. Diarrhea 
 
Yes  
No 
 
2. Nausea 
 
Yes  
No 
3. Abdominal bloating 
Yes  
No 
4. Others: any other unanticipated adverse events/problems that occurred  Yes    No 
____________________(described what it is) 
 
If any yes for item 1-4, describe in the box: 1) when did the event start, 2) how long did it last, 3) 
did the participant take any treatment/action, and 4) is it now resolved? 
 
 
 
 
 
 
 
 
 
 

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11. REFERENCES 
 
1. 
Mozaffarian D, Benjamin EJ, Go AS, et al. Heart Disease and Stroke Statistics-2016 Update: A 
Report From the American Heart Association. Circulation. 2016;133(4):e38-360. 
2. 
Mozaffarian D, Benjamin EJ, Go AS, et al. Heart disease and stroke statistics--2015 update: a 
report from the American Heart Association. Circulation. 2015;131(4):e29-322. 
3. 
Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart Disease and Stroke Statistics-2017 Update: A 
Report From the American Heart Association. Circulation. 2017;135(10):e146-e603. 
4. 
Ashor AW, Siervo M, van der Velde F, Willis ND, Mathers JC. Systematic review and meta-
analysis of randomised controlled trials testing the effects of vitamin C supplementation on 
blood lipids. Clinical nutrition (Edinburgh, Scotland). 2016;35(3):626-637. 
5. 
Hamner JB, Ellison KJ. Predictors of hospital readmission after discharge in patients with 
congestive heart failure. Heart Lung. 2005;34(4):231-239. 
6. 
Di Bari M, Pozzi C, Cavallini MC, et al. The diagnosis of heart failure in the community. 
Comparative validation of four sets of criteria in unselected older adults: the ICARe Dicomano 
Study. Journal of the American College of Cardiology. 2004;44(8):1601-1608. 
7. 
Heo S, Lennie TA, Okoli C, Moser DK. Quality of life in patients with heart failure: ask the 
patients. Heart & lung : the journal of critical care. 2009;38(2):100-108. 
8. 
Zambroski CH, Moser DK, Bhat G, Ziegler C. Impact of symptom prevalence and symptom 
burden on quality of life in patients with heart failure. European journal of cardiovascular 
nursing : journal of the Working Group on Cardiovascular Nursing of the European Society of 
Cardiology. 2005;4(3):198-206. 
9. 
Arcand J, Floras V, Ahmed M, et al. Nutritional inadequacies in patients with stable heart failure. 
Journal of the American Dietetic Association. 2009;109(11):1909-1913. 
10. 
Song EK, Kang SM. Vitamin C Deficiency, High-Sensitivity C-Reactive Protein, and Cardiac Event-
Free Survival in Patients With Heart Failure. The Journal of cardiovascular nursing. 2016. 
11. 
Gorelik O, Almoznino-Sarafian D, Feder I, et al. Dietary intake of various nutrients in older 
patients with congestive heart failure. Cardiology. 2003;99(4):177-181. 
12. 
Ashor AW, Siervo M, Lara J, Oggioni C, Afshar S, Mathers JC. Effect of vitamin C and vitamin E 
supplementation on endothelial function: a systematic review and meta-analysis of randomised 
controlled trials. The British journal of nutrition. 2015;113(8):1182-1194. 
13. 
Ellis GR, Anderson RA, Lang D, et al. Neutrophil superoxide anion--generating capacity, 
endothelial function and oxidative stress in chronic heart failure: effects of short- and long-term 
vitamin C therapy. Journal of the American College of Cardiology. 2000;36(5):1474-1482. 
14. 
Ellis GR, Anderson RA, Chirkov YY, et al. Acute effects of vitamin C on platelet responsiveness to 
nitric oxide donors and endothelial function in patients with chronic heart failure. Journal of 
cardiovascular pharmacology. 2001;37(5):564-570. 
15. 
Hornig B, Arakawa N, Drexler H. Effect of ACE inhibition on endothelial dysfunction in patients 
with chronic heart failure. Eur Heart J. 1998;19 Suppl G:G48-53. 
16. 
Hornig B, Arakawa N, Haussmann D, Drexler H. Differential effects of quinaprilat and enalaprilat 
on endothelial function of conduit arteries in patients with chronic heart failure. Circulation. 
1998;98(25):2842-2848. 
17. 
Hornig B, Arakawa N, Kohler C, Drexler H. Vitamin C improves endothelial function of conduit 
arteries in patients with chronic heart failure. Circulation. 1998;97(4):363-368. 
18. 
Moser MA, Chun OK. Vitamin C and Heart Health: A Review Based on Findings from 
Epidemiologic Studies. International journal of molecular sciences. 2016;17(8). 

Vit C Study Protocol / 14 
Protocol 17-1809 –  
Protocol version: 4  
Version date: 04-21-2020 
19. 
Witte KK, Nikitin NP, Parker AC, et al. The effect of micronutrient supplementation on quality-of-
life and left ventricular function in elderly patients with chronic heart failure. European heart 
journal. 2005;26(21):2238-2244. 
20. 
Tomiyama H, Watanabe G, Yoshida H, Doba N, Yamashina A. Reduction of oxidative stress 
augments natriuretic effect of furosemide in moderate heart failure. American heart journal. 
2003;145(1):E2. 
21. 
Ho KK, Pinsky JL, Kannel WB, Levy D. The epidemiology of heart failure: the Framingham Study. 
Journal of the American College of Cardiology. 1993;22(4 Suppl A):6A-13A. 
22. 
Walsh CR, Larson MG, Evans JC, et al. Alcohol consumption and risk for congestive heart failure 
in the Framingham Heart Study. Annals of internal medicine. 2002;136(3):181-191. 
23. 
Son YJ, Lee Y, Song EK. Adherence to a sodium-restricted diet is associated with lower symptom 
burden and longer cardiac event-free survival in patients with heart failure. Journal of clinical 
nursing. 2011;20(21-22):3029-3038. 
24. 
Moser DK, Yamokoski L, Sun JL, et al. Improvement in health-related quality of life after 
hospitalization predicts event-free survival in patients with advanced heart failure. Journal of 
cardiac failure. 2009;15(9):763-769. 
25. 
Rector TS, Tschumperlin LK, Kubo SH, et al. Use of the Living With Heart Failure questionnaire to 
ascertain patients' perspectives on improvement in quality of life versus risk of drug-induced 
death. Journal of cardiac failure. 1995;1(3):201-206. 
26. 
Wu M, Brazier JE, Kearns B, Relton C, Smith C, Cooper CL. Examining the impact of 11 long-
standing health conditions on health-related quality of life using the EQ-5D in a general 
population sample. The European journal of health economics : HEPAC : health economics in 
prevention and care. 2015;16(2):141-151. 
27. 
Van De Water JM, Miller TW, Vogel RL, Mount BE, Dalton ML. Impedance cardiography: the next 
vital sign technology? Chest. 2003;123(6):2028-2033. 
28. 
Nakonezny PA, Kowalewski RB, Ernst JM, et al. New ambulatory impedance cardiograph 
validated against the Minnesota Impedance Cardiograph. Psychophysiology. 2001;38(3):465-
473. 
29. 
Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture 
(REDCap)--a metadata-driven methodology and workflow process for providing translational 
research informatics support. J Biomed Inform. 2009;42(2):377-381. 
30. 
Klipin M, Mare I, Hazelhurst S, Kramer B. The process of installing REDCap, a web based 
database supporting biomedical research: the first year. Appl Clin Inform. 2014;5(4):916-929.