Conditions: Disorder of Vitamin C Atrial Fibrillation Complications Due to Coronary Artery Bypass Graft Heart Valve Disease
Keywords: Vitamin C Coronary Artery Bypass Grafting Atrial Fibrillation Cardiac Valve Repair
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 6
Healthy volunteers: no

Brief summary:
Coronary artery bypass grafting (CABG) is the most common procedure performed by cardiac surgeons. Post-operative atrial fibrillation (AF) is the most common adverse event following CABG, experienced in 20-50% of patients; the highest incidence of AF occurs by the third post-operative day. Reduction of AF by various drugs is moderately effective, but involves either rate control with beta blockers or rate conversion with amiodarone after the myocardial damage processes initiating AF have already occurred. Decreasing the incidence of post-operative AF, and hence the morbidity and mortality of high-risk CABG patients, could be more fruitfully approached by targeting the upstream combined processes of inflammation and coagulation activation induced by the surgical insult and associated ischemia-reperfusion (I/R). We propose that cell damage induced by oxidative stress and I/R injury could be prevented and/or inhibited by antioxidant supplementation. Specifically the investigators hypothesize that high-dose intravenous (IV) vitamin C supplementation will ameliorate ROS and therefore damp down upstream inflammatory processes, leading to a reduction of downstream adverse events with demonstrable links to inflammation processes, such as AF.

Detailed description:
Coronary artery bypass grafting (CABG) is the most common procedure performed by cardiac surgeons. More complicated surgeries were enabled by the development of cardiopulmonary bypass (CPB) in the 1930s; CABG procedures increased to peak at over 500,000 in 2000, although numbers have decreased somewhat in the last decade. In 2010 over 150,000 major cardiac procedures involved CABG; 18,008 involved both aortic valve replacement and CABG; 2,378 involved mitral valve replacement and CABG; and 4,635 involved mitral valve repair and CABG. This procedure is costly; in 2010 mean hospital charges for CABG and valve procedures were $124,404 and $171,270 with a mean length of hospital stay of 9 and 11 days and an in-hospital death rate of 1.8% and 3.9%, respectively.

Post-operative atrial fibrillation (AF) is the most common adverse event following CABG, experienced in 20-50% of patients; the highest incidence of AF occurs by the third post-operative day. Post-operative AF is associated with overall poorer prognosis resulting 4-fold increase in disabling stroke and cognitive impairment, 3-fold risk of cardiac-related death, and increased length of hospital stay. These risks are increased by the changing composition of the patient population over the last 10 years: patients are older and sicker, and are presenting with more complex disease states and co-morbidities, such as severe coronary artery disease (CAD), congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), diabetes, previous cardiac surgeries with mechanical support device placements, and complex pharmacological histories. A higher prevalence of AF with increased transfusions among CPB patients has also been reported, suggesting a link with increased plasma load of inflammatory markers and mediators from transfused red cells. Reduction of AF by various drugs is moderately effective, but involves either rate control with beta blockers or rate conversion with amiodarone after the myocardial damage processes initiating AF have already occurred.

Decreasing the incidence of post-operative AF, and hence the morbidity and mortality of high-risk CABG patients, could be more fruitfully approached by targeting the upstream combined processes of inflammation and coagulation activation induced by the surgical insult and associated ischemia-reperfusion (I/R). During CPB, the heart is subjected to ischemic periods of varying duration. The cardiac tissue at that point is electrically resting and its metabolic demands are accordingly low. However, there is also a severely diminished supply of oxygen and nutrients. This results in slow but direct myocardium cellular damage, which increases as ischemia duration increases. However, after reperfusion with oxygenated blood, oxidative stress actually peaks because of accumulated oxidative substrates and cellular depletion of reductive compounds. Cardiac damage related to ischemia-reperfusion will be exacerbated in the presence of co-morbidities, such as diabetes and CAD, which are pathologies with a significant reactive oxygen species (ROS) injury component. High levels of ROS in the myocardium contribute to both electrical and structural remodeling of the cardiac muscle, resulting in the development of AF.

I/R-related oxidative stress and ROS activation contribute to cell damage by modifying protein DNA and phospholipids, resulting in lipid peroxidation and thiol-group oxidation, which in turn are linked to the induction of inflammatory cascades. Lipids are important small-molecule metabolites that have roles in a wide variety of physiological processes. The lipidome of eukaryotic cells contains thousands of lipid entities that structurally and chemically regulate cell membranes, store energy, or are precursors of bioactive metabolites. Defects in lipid regulation and metabolism are therefore significant contributors to disease pathophysiology. In particular, eicosanoids are lipid mediators linking coagulation and inflammatory pathways; intracellular oxidant species are essential mediators in eicosanoids synthesis pathways. Thus lipidomic analyses are a potential novel tool for the identification of drivers of underlying pathogenesis and new diagnostic biomarkers.

During CABG, total peroxide (TP) and oxidative stress index approximately double Increased post-bypass plasma levels of inflammatory markers such as C-reactive protein (CRP) and platelets and leukocyte activation have been observed during CPB. Increases in oxidative stress associated with CPB are correlated with a reduction in total plasma antioxidant capacity. These observations are of interest because an antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by auto-oxidation; hence antioxidants are often reducing agents. The investigators propose that cell damage induced by oxidative stress and I/R injury could be prevented and/or inhibited by antioxidant supplementation. Specifically the investigators hypothesize that high-dose intravenous (IV) vitamin C supplementation will ameliorate ROS and therefore damp down upstream inflammatory processes, leading to a reduction of downstream adverse events with demonstrable links to inflammation processes, such as AF. Vitamin C is an viable therapeutic candidate because it is a powerful antioxidant with an excellent safety profile, and an emerging history of reducing inflammatory markers in both critically ill and traumatically injured patients. Scattered trials have indicated possible benefits of vitamin C in reducing incidence of post-CABG AF (see Literature search). An antioxidant such as vitamin C acts as a scavenger of intracellular oxidant species; as these are essential mediators in eicosanoids synthesis pathways, it is expected that vitamin C supplementation would decrease the thrombotic potential of eicosanoid-mediated pathways, such as the thromboxane (TBX) production pathway. Impaired cardiac blood flow and clot formation associated with AF contributes to congestive heart failure and stroke; therefore reduction of thrombotic potential is another essential feature for the proposed therapeutic agent.

The proposed pilot study described below represents a marked advance on previously-reported trials in that (a) study design is directed towards minimization of systemic bias, and (b) the investigators will perform simultaneous comparisons of multivariate inflammatory and coagulation profile changes over the immediate post-operative time period when inflammatory changes are expected to be at a maximum. The novelty of this approach is the systems-level analysis of lipids and their interacting moieties; new technology allows for rapid quantitative analysis of over 150 lipid mediators in a sample, time and cost effective manner.

Arms:
arm 1: Intravenous ascorbic acid 50 mg/kg in 50 mL normal saline every six hours for 48 hours. arm 2: Intravenous normal saline 50 mL every six hours for 48 hours

Interventions: intervention 1: Ascorbic Acid intervention 2: Placebo
Intervention descriptions: intervention 1: Comparing safety and efficacy of IV Vitamin C in CABG patients intervention 2: Placebo intervention

Protocol document:
Prot_SAP_000.pdf: 
November 16, 2016 
1 
 
 
 
 
 
 Title: High Dose Vitamin C in Cardiac Surgery Patients   
PI: Donald Brophy, PharmD, MS 
 
NCT #: NCT02762331 
 
Unique Protocol ID: HM20000539 
 
Document approval date: November 16, 2016 
 
Document Type: Protocol and SAP 
 
 


November 16, 2016 
2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PILOT STUDY TO ASSESS EFFECT OF HIGH DOSE ASCORBIC ACID (VITAMIN C) ON 
INFLAMMATION REDUCTION IN CARDIAC SURGERY PATIENTS 
 
PI: Donald F. Brophy, PharmD, MS 
 
 
 
 
 
 
 
 
 
Virginia Commonwealth University Medical Center Departments of 
Cardiothoracic Surgery, Pharmacy, and Anesthesiology Investigational 
Protocol 


November 16, 2016 
3 
 
 
 
 
BACKGROUND AND SIGNIFICANCE 
 
Coronary artery bypass grafting (CABG) is the most common procedure performed by 
cardiac surgeons. More complicated surgeries were enabled by the development of 
cardiopulmonary bypass (CPB) in the 1930s; CABG procedures increased to peak at over 
500,000 in 2000, although numbers have decreased somewhat in the last decade. In 2010 
over 150,000 major cardiac procedures involved CABG; 18,008 involved both aortic valve 
replacement and CABG; 2,378 involved mitral valve replacement and CABG; and 4,635 
involved mitral valve repair and CABG.1 This procedure is costly; in 2010 mean hospital 
charges for CABG and valve procedures were $124,404 and $171,270 with a mean length of 
hospital stay of 9 and 11 days and an in-hospital death rate of 1.8% and 3.9%, 
respectively.1-3 
 
Post-operative atrial fibrillation (AF) is the most common adverse event following CABG, 
experienced in 20-50% of patients; the highest incidence of AF occurs by the third post- 
operative day. Post-operative AF is associated with overall poorer prognosis 4,5 , resulting 
4-fold increase in disabling stroke and cognitive impairment, 3-fold risk of cardiac-related 
death6, and increased length of hospital stay.7  These risks are increased by the changing 
composition of the patient population over the last 10 years: patients are older and sicker, 
and are presenting with more complex disease states and co-morbidities, such as severe 
coronary artery disease (CAD), congestive heart failure (CHF), chronic obstructive 
pulmonary disease (COPD), diabetes, previous cardiac surgeries with mechanical support 
device placements, and complex pharmacological histories. A higher prevalence of AF with 
increased transfusions among CPB patients has also been reported, suggesting a link with 
increased plasma load of inflammatory markers and mediators from transfused red cells.7 
Reduction of AF by various drugs is moderately effective, but involves either rate control 
with beta blockers or rate conversion with amiodarone8,9, after the myocardial damage 
processes initiating AF have already occurred. 
 
Decreasing the incidence of post-operative AF, and hence the morbidity and mortality of 
high-risk CABG patients, could be more fruitfully approached by targeting the upstream 
combined processes of inflammation and coagulation activation induced by the surgical 
insult and associated ischemia-reperfusion (I/R). During CPB, the heart is subjected to 
ischemic periods of varying duration. The cardiac tissue at that point is electrically resting 
and its metabolic demands are accordingly low. However, there is also a severely 
diminished supply of oxygen and nutrients. This results in slow but direct myocardium 
cellular damage, which increases as ischemia duration increases. 7,9 However, after 
reperfusion with oxygenated blood, oxidative stress actually peaks because of accumulated 
oxidative substrates and cellular depletion of reductive compounds.9 Cardiac damage 
related to ischemia-reperfusion will be exacerbated in the presence of co-morbidities, such 
as diabetes and CAD, which are pathologies with a significant reactive oxygen species 
(ROS) injury component. High levels of ROS in the myocardium contribute to both electrical 
and structural remodeling of the cardiac muscle, resulting in the development of AF.5 
 
I/R-related oxidative stress and ROS activation contribute to cell damage by modifying 
protein DNA and phospholipids, resulting in lipid peroxidation and thiol-group oxidation, 


November 16, 2016 
4 
 
 
 
 
which in turn are linked to the induction of inflammatory cascades. Lipids are important 
small-molecule metabolites that have roles in a wide variety of physiological processes. The 
lipidome of eukaryotic cells contains thousands of lipid entities that structurally and 
chemically regulate cell membranes, store energy, or are precursors of bioactive 
metabolites. 1, 2 Defects in lipid regulation and metabolism are therefore significant 
contributors to disease pathophysiology. In particular, eicosanoids are lipid mediators 
linking coagulation and inflammatory pathways; intracellular oxidant species are essential 
mediators in eicosanoids synthesis pathways. Thus lipidomic analyses are a potential novel 
tool for the identification of drivers of underlying pathogenesis and new diagnostic 
biomarkers.10 
 
During CABG, total peroxide (TP) and oxidative stress index approximately double11 
Increased post-bypass plasma levels of inflammatory markers such as C-reactive protein 
(CRP)6-10, and platelets and leukocyte activation have been observed during CPB.12 
Increases in oxidative stress associated with CPB are correlated with a reduction in total 
plasma antioxidant capacity.11 These observations are of interest because an antioxidant is 
a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction 
that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation 
reactions can produce free radicals. In turn, these radicals can start chain reactions. When 
the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants 
terminate these chain reactions by removing free radical intermediates, and inhibit other 
oxidation reactions by auto-oxidation; hence antioxidants are often reducing agents. 
 
We propose that cell damage induced by oxidative stress and I/R injury could be prevented 
and/or inhibited by antioxidant supplementation. Specifically, we hypothesize that high- 
dose intravenous (IV) vitamin C supplementation will ameliorate ROS and therefore damp 
down upstream inflammatory processes, leading to a reduction of downstream adverse 
events with demonstrable links to inflammation processes, such as AF. Vitamin C is a viable 
therapeutic candidate because it is a powerful antioxidant4, 12 with an excellent safety 
profile, and an emerging history of reducing inflammatory markers in both critically ill6, 13, 
14 and traumatically injured13 patients. Scattered trials have indicated possible benefits of 
vitamin C in reducing incidence of post-CABG AF (see Literature search). An antioxidant 
such as vitamin C acts as a scavenger of intracellular oxidant species; as these are essential 
mediators in eicosanoids synthesis pathways, it is expected that vitamin C supplementation 
would decrease the thrombotic potential of eicosanoid-mediated pathways, such as the 
thromboxane (TBX) production pathway. Impaired cardiac blood flow and clot formation 
associated with AF contributes to congestive heart failure and stroke; therefore, reduction of 
thrombotic potential is another essential feature for the proposed therapeutic agent. 
 
The proposed pilot study described below represents a marked advance on previously- 
reported trials in that (a) study design is directed towards minimization of systemic bias, 
and (b) we will perform simultaneous comparisons of multivariate inflammatory and 
coagulation profile changes over the immediate post-operative time period when 
inflammatory changes are expected to be at a maximum. The novelty of our approach is our 
systems-level analysis of lipids and their interacting moieties; new technology in our labs 
allows for rapid quantitative analysis of over 150 lipid mediators in a sample, time and cost 


November 16, 2016 
5 
 
 
 
 
effective manner. 
 
METHODS/DESIGN 
 
LITERATURE SEARCH 
 
A rapid systematic evidence assessment was conducted on three occasions in PUBMED. 
Search terms, filtered by identifier clinical trials, were: 
 
((high dose vitamin C) OR (ascorbic acid)) AND surgery; 
((vitamin C) OR (ascorbic acid)) AND (cardiac AND surgery); 
(ascorbate OR vitamin C OR ascorbic acid) AND Superoxide AND (Nitric Oxide); 
 
From a total of 285 studies, 20 were retained; the remainder was excluded for lack of 
relevance or duplication. Studies were archived and analyzed in Review Manager 5.2.13 
 
A search of ClinicalTrials.gov using the search terms (ascorbate OR vitamin C OR ascorbic 
acid) AND (cardiac surgery OR cardiovascular) AND/OR (IV OR intravenous) resulted in 9 
relevant trials (total 90). As of December 17, 2014, five were completed, one terminated, 
two of unknown status, and one not yet recruiting; no data from these studies were 
available. 
 
With very few exceptions, most studies were characterized by high risk of bias in six bias 
domains (selection, performance, detection, attrition, reporting, other).14 Lack of 
randomization and blinding, and failure to explicitly define clinical outcomes contributed to 
these problems. The majority of studies used oral vitamin C supplementation. Oral doses, 
even those in excess of 200 mg, are associated with little or no clinically significant increases 
in circulating Vitamin C levels15, and pharmacokinetics of oral ascorbic acid 
dosing differs considerably from intravenous dosing.16 However most assessed studies 
reported positive effects of vitamin C supplementation. Meta-analysis of these papers 
indicated a significant overall reduction in both post-operative AF (9 studies; n= 1039 
patients; odds ratio 0.37 [95% CI 0.28, 0.50]) and a small reduction in length of hospital 
stay (6 studies; n= 672 patients; mean difference -1.3 days [95% CI -1.6, -1.0]). A few 
papers reported measurements of isolated inflammatory and coagulation markers; 
however, results were equivocal. For example, vitamin C supplementation of CABG 
patients (usually in combination with various antioxidant cocktails) seemed to attenuate 
post-operative oxidative stress,17 myocardial “injury” (defined as a five-fold elevation of 
cardiac biomarker values and cardiac dysrhythmia),18 and endothelial dysfunction.19 In 
contrast, a study of cardiac surgery patients on or off CPB did not detect statistically 
significant differences between oral vitamin C and control groups in C-reactive protein 
(CRP) concentration, white blood cell (WBC) count, or fibrinogen concentration, markers of 
inflammation and coagulopathy.20 However the study was underpowered because of early 
study termination, and data were uncorrected for non-normality, invalidating subsequent 
statistical analyses. In summary, although suggestive of benefit, current evidence of efficacy 
of high-dose vitamin C was of low quality with limited and/or irrelevant outcomes, 
necessitating development of more rigorous clinical trials. 


November 16, 2016 
6 
 
 
 
 
SPECIFIC AIMS 
 
Specific Aim 1: We will test the prediction that patients administered high-dose vitamin C 
will show clinically-significant reductions in pro-inflammatory and coagulation markers at 
peak response and at 48 hours relative to normal saline controls. Specifically, we will 
measure 
 
a)  Levels of circulating pro-inflammatory biomarkers profiles (CRP, IL-6, TNF-α), 
NGAL (in urine), and lipid pro- and anti-inflammatory mediators (in plasma); 
 
b)  Levels of coagulation biomarker and platelet function (e.g. fibrinogen, platelet 
counts, thrombomodulin, R, K, MA, G, angle, LYO) 
 
Specific Aim 2: We will test the prediction that reduced post-surgical inflammatory 
burden of patients administered high-dose vitamin C will result in clinically significant 
reductions in hospital-based outcomes (such as length of hospital stay, length of ICU stay.) 
 
STUDY DESIGN 
 
This is a prospective, double-blind, placebo-controlled, randomized pilot trial comparing 
high-dose intravenous vitamin C to placebo (normal saline) infusions in elective non- 
emergent cardiac bypass patients. Double blinding will be used at enrollment, 
randomization, and throughout treatment, sampling, analysis, and data interpretation 
phases. 
 
The purpose of this pilot study is to demonstrate performance feasibility and proof of 
concept (PoC) before initiation of a full-fledged clinical trial. Both financial and personnel 
constraints limit the total patient enrollment to 24 (12 per arm). Because sample sizes are 
so small, hypothesis testing cannot be a central feature of this proposal. Instead, success 
criteria will consist of tangible and measurable performance metrics relating to trial 
performance and PoC. Performance success criteria relate to the logistics and processes 
involved in the execution of the study itself (e.g. recruitment, retention, study flow, 
operations, management, obtaining samples, sample processing, documentation, 
compliance oversight). Proof of concept will be determined by large effect size differences 
(20%) between test and experimental intervention groups in one or more of the key 
inflammation and coagulation markers (see OUTCOMES). 
 
It is unethical to consider running a phase III trial without sufficient data or feasibility 
information. This pilot trial will be extremely useful to inform the planning and execution 
of the future large-scale trial, especially with respect to feasibility and sample size 
calculations. Proof of concept information will be essential for writing major grants, which 
usually require such preliminary information for assessing scientific validity21-23 
 
Dose/route. Previous studies describing use of IV vitamin C24-26 suggests that a total 
dose of 200mg/kg/day administered as 4 intravenous doses in 50mL D5W every 6 hours 
per day for 4 days will be sufficient to permit biologically-effective plasma concentrations, 
and thus are hypothesized to reduce deleterious effects associated with CPB-induced 


November 16, 2016 
7 
 
 
 
 
inflammation. The intervention schedule in our study was based on a general knowledge of 
inflammation trajectory; inflammation typically peaks during CPB itself then stabilizes 
within a 24-hour period27.  Accordingly, in this study intravenous Vitamin C will be given 
as 200mg/kg/day divided over 4 intravenous doses every 6 hours per day for only 2 days 
perioperative. 
 
Completion of study drug administration: Study drug administration will be 
stopped when one of the following conditions is met, whichever comes first: 
1. 
Final drug dose at 48 hours 
3. 
Inability to obtain peripheral access after removal of central line. 
4. 
Discharge from the hospital 
5. 
Withdrawal from study 
6. 
Death 
 
ETHICAL OVERSIGHT 
 
Prior to study initiation, this study will be approved by the Virginia Commonwealth 
University (VCU) Institutional Review Board, and an IND requested from the Food and Drug 
Administration (FDA). The trial will be registered with ClinicalTrials.gov, and the definitive 
protocol submitted for publication in one of the open-access clinical trial specific journals 
(e.g. Trials). This is an investigator-initiated study, falling under an existing IND held by 
another investigator who is not acting as PI.  In accordance with GCP, monitoring will occur 
through the use of a monitor not affiliated with this study. This monitor, while employed at 
the institution conducting the study, has not been affiliated with the development or 
execution of this study. 
 
STUDY POPULATION 
 
The study population will consist of adult patients scheduled for elective non-emergent 
cardiac surgery at Virginia Commonwealth University Medical Center, and who have 
consented to participation in the study. 
 
Inclusion criteria are: (1) adult (≥18 years) patients (2) scheduled for elective non- 
emergent cardiac valve replacement or repair with cardiopulmonary bypass, and/or multi- 
vessel CABG surgery. 
 
Exclusion criteria are: (1) known allergy to Vitamin C; (2) pregnant or breastfeeding; (3) 
prisoner; (4) emergent cardiac surgery patients; (5) scheduled for single CABG procedure; 
(6) low ejection fraction (< 35%); (7) current autoimmune disease or on 
immunosuppressant therapy, including prednisone, prednisolone, dexamethasone, 
mycophenolate, tacrolimus, cyclosporine, or azathioprine; (8) presence of renal calculi, or 
renal dysfunction (pre-operative creatinine clearance < 40, or serum creatinine greater 
than 1.8 mg/dl); (9) known bleeding diathesis such as hemophilia, Von Willebrand's 
disease, or liver failure, or receiving concurrent anticoagulant therapy (such as warfarin, 
dabigatran or apixaban) for venous or arterial thrombosis; (10) active infection or tumor; 
(11) prior history of atrial fibrillation (AF); (12) known diagnosis of diabetes mellitus, or 
preoperative (this surgery) A1C test result ≥ 6.5; (13) diagnosed with diabetic ketoacidosis
 


November 16, 2016 
8 
 
 
 
 
(DKA); (14) known coagulopathy prior to surgery (INR > 1.5). 
 
Patient numbers/sample size: This is a feasibility/ proof of concept pilot trial; therefore 
power calculations are not appropriate. Patient enrollment numbers are limited a priori by 
the funding available for this project and costs inherent to enrollment, randomization and 
blinding procedures, sampling, and sample processing. 
 
Consent Procedures. All eligible patients will be approached prior to their scheduled 
surgery by the study coordinator or attending surgeon. A formal Institutional Review 
Board (IRB)-approved Informed Consent form will be discussed with the patient, and all 
questions answered prior to obtaining informed patient consent in writing. Informed 
consent will be documented in the patient medical record, and the clinical research office 
and each enrolled patient will receive a copy of his/her signed consent form. 
 
INTERVENTION AND CONTROLS 
 
This is a two-arm trial comparing high-dose IV vitamin C to normal saline placebo controls 
(Fig. 1). Once informed consent is obtained, the time of surgery will be communicated to 
the VCU investigational pharmacy so that the medication can be appropriately prepared 
and delivered to the operating room. Prior to preparation, patients will be randomized to 
receive either control or Vitamin C solution. Randomization will be performed by a 
computer-generated permuted block design conducted by the investigational pharmacy. 
Both test and placebo preparations will be prepared by pharmaceutical staff in identical de- 
identified 50-mL medication bags so as to be indistinguishable by OR/ICU staff and 
investigators. 
 
Vitamin C will be administered in doses of 200 mg/kg/day group divided into four doses 
per day at six hour intervals 48 hours. Patients randomized to the control (normal saline) 
group will receive equivalent fluid volume in identical dispensing bags; total study fluid 
volumes will be approximately 400 mL over the two-day study period. Blinded 
interventions will be administered in the OR by the anesthesiologist, and post-operatively 
by Cardiac Surgery ICU nursing staff and/or Cardiac Stepdown Units. All participating 
hospital units will be provided with thorough in-servicing. Nurses will be prompted to 
administer the medication via the patient-specific electronic medical record (EMR). 
 
OUTCOMES 
 
Enrollment Goals: (1) Up to 40 may be enrolled in order to reach completion of 24 out of 
24 subjects to allow for withdrawals; (2) complete follow-up and follow-up data for 24/24 
(100%) of enrolled subjects; (3) all patients (24/24) to receive study drug according to 
randomization designation; (4) all patients (24/24) to receive every scheduled dose of the 
study drug in a blinded manner; (5) all patients (24/24) to have coagulation and biolipid 
determinations performed for all. 
 
 
 
 
Biomarker outcomes: 


November 16, 2016 
9 
 
 
 
 
Coagulation and inflammation markers: We hypothesize that oxidative stress acquired 
during CPB and surgery will upregulate coagulation pathways and vitamin C 
administration will reduce overall expression and/or reduce the duration of the response. 
Coagulation testing will include RoTEM, fibrinogen, thrombomodulin, and platelet count. 
Fibrinogen is a liver-produced acute-phase reactant that increases as a result of 
inflammation. The release of soluble thrombomodulin is associated with increased pro- 
coagulant response. Thrombomodulin must be expressed on the endothelium to convert 
thrombin to an anti-coagulant. The RoTEM measures the viscoelastic properties of clot 
formation and fibrinolysis. Cytokines are soluble proteins acting as cell signalling and 
regulator molecules to coordinate generalized inflammatory responses. We will measure a 
few selected pro-inflammatory cytokines (e.g. IL -6, TNF-) and non-cytokine markers such 
as C-reactive protein (CRP) and white blood cell counts. NGAL (neutrophil gelatinase- 
associated lipocalin) is a marker protein that starts to increase early (earlier than 
creatinine) in acute kidney injury (AKI). AKI is a very common clinical problem in cardiac 
surgery ICU (~40%). Both NGAL and creatinine will be followed. 
 
Lipidomics: We hypothesize that oxidative stress acquired during CPB and surgery leads to 
oxidation of both substrate phospholipids and already-generated precursor molecules, 
such as arachidonic acid (AA). The oxidation of these lipids would deprive the eicosanoid 
biosynthetic enzymes of necessary precursors and effectively dysregulate the production of 
lipid mediators important in maintaining platelet function as well as modulating 
platelet/leukocyte/endothelial cell interplay. We further hypothesize that administration 
of high dose vitamin C will attenuate or correct these processes. We have preliminary data 
suggesting a time-dependent conversion of 2 AA phosphatidylcholine (2-AA-PC) to 
oxidized 2-AA-PC without a significant effect on total 2-AA-PC levels. This effect correlated 
with a dramatic reduction in the production of TXB2. We also have data to show that co- 
treatment of blood with vitamin C and AA attenuates the loss of TXB2 levels caused by 
oxidative stress. Thus, we are confident about the relationship between loss of lipid 
mediators and previously-demonstrated loss of platelet function induced by oxidative 
stress. 
 
Our targeted lipidomic analysis will examine a specific subset of lipids identified alterations 
in the arachidonic acid metabolite pathway associated with inflammation and 
coagulopathy. We will track patient condition before, during, and after CPB by mass 
spectrometric profiles of selected biolipids with the initial focus on free fatty acids, 
eicosanoids, 3-PUFA-derived lipid mediators, phospholipid substrates of cPLA2α (including 
oxidized forms), and isoprostanes, which are well-characterized markers of oxidative 
stress and redox status. Lipid entities will be quantitatively determined via LC tandem 
mass spectrometry.28-30 
 
Clinical outcomes: Clinical data will be obtained from the patient’s chart under direct 
supervision of the PI by a research coordinator and/or a graduate student listed in the 
study personnel. Data will include: (1) Demographics (age, race, ethnicity, gender, height, 
weight); (2) Pertinent medical history (hypertension, congestive heart failure); (3) Surgery 
data (cardiac surgeon, date and time of surgery, time and details of bypass, total heparin 
administered, total protamine administered, blood products administered (type, units), 


November 16, 2016 
10 
 
 
 
 
other concomitant medications [times, dosages/amounts]); (4) Surgical complications (e.g. 
fever, respiratory failure, intubation (Y/N, duration), chest tube drainage amount); (5) 
Incidence of AF; (6) Hospital outcomes: length of ICU stay, length of step-down and/or floor 
stay, and patient discharge disposition. Patients will be followed up through hospital 
discharge or in-hospital death. 
 
POST-RANDOMIZATION TREATMENT 
 
All clinically-indicated treatments (including blood product, medications, and fluid 
administration) will be given as appropriate per attending physician discretion. 
 
Study–specific blood samples will be obtained by the anesthesiologist or nursing staff, 
depending on the patient’s location at each time point, at four times: 
 
1st Draw: 
Baseline (after anesthesia induction but prior to drug administration) 
2nd Draw: 
Approx. 60 min. after protamine sulfate is administered (during CPB), 
3rd Draw: 
24 hours post-baseline (+/- 2 hours), 
4th Draw: 
48 hours post-baseline (+/- 2 hours). 
Approximately 20 mL of blood - 2 Na citrate tubes and 1 SST tube - will be needed to run 
assays described below. In addition, 3 urine samples will be collected, 5 mL each, at 
Baseline, 6hr, and 24 hrs. Blood and urine tubes will be transported to the analysis 
research lab by a study personnel member. 
 
DATA STORAGE AND MANAGEMENT 
 
Confidentiality will be maintained by storing paper-based records in a secure central 
location with access provided only to the study staff.  All records will be de-identified and 
data coded with the key stored in a separate secure location.  All personal identifying 
information will be kept in password protected files and these files will be deleted after 6 
years, per VCU requirements. Other de-identified records, such as lab results, will be kept 
in a locked file cabinet for 6 years after the study ends and will be destroyed at that time. 
No data will be stored indefinitely. 
 
Patients will be screened, recruited, and consented in a private exam room prior to their 
scheduled surgery. Collection of identifiable information will only be necessary to link the 
subject to medical records and/or subject data. Personal information about subjects may 
be shared with or copied by authorized officials of the Federal Food and Drug 
Administration and Department of Health and Human Services if required. Although results 
of this research may be presented at meetings or in publications, no identifiable personal 
information will be disclosed. 
 
 
 
 
 
 
DATA SAFETY MONITORING BOARD (DSMB) AND SAFETY OVERSIGHT 


November 16, 2016 
11 
 
 
 
 
An independent DSMB has been appointed to oversee safety monitoring and trial data 
comprised of physicians and a biostatistician who are not directly involved with the study 
and interventions. 
 
PROTOCOL ADHERENCE 
 
Deviations from study protocol will be reported by the Trial Coordinator to the PI and, as 
needed, to the VCU IRB. 
 
ADVERSE EVENTS AND/OR DRUG REACTIONS 
 
Very few side effects have been observed in clinical trials of intravenous high-dose vitamin 
C.14  The only known reported toxicity of Vitamin C originates after continuous (days to 
weeks) of infusions or intake,  resulting in the formation of oxalate renal calculi.12 Another 
potential concern is the hypothesized pro-oxidant property of high dose Vitamin C; 
however, evidence supporting this claim is weak; antioxidant properties at even higher 
doses were demonstrated to be predominant over pro-oxidant properties.32  The safety of 
the proposed dose has been verified in our preliminary human data, and the safety of 
higher doses has previously been verified in cancer trials.33 Overall, high-dose IV Vitamin C 
is regarded as having a good safety profile with a low risk of toxicity over a wide range of 
doses. 
 
High-dose vitamin C levels can lead to a false reading of blood glucose levels using point of 
care testing. Blood glucose levels are to be measured in the metabolic lab as detailed below 
in the Glucose Monitoring Plan section. 
 
GLUCOSE MONITORING PLAN: 
 
Ascorbic acid is known to artefactually raise POC blood glucose readings; however, it does 
not raise blood glucose readings from a basic metabolic panel (BMP). Thus, extreme care 
must be taken to assure an accurate blood glucose level from a metabolic laboratory before 
initiating any insulin therapy, including sliding scale or scheduled insulin. 
 
Guidance for blood glucose monitoring in patients enrolled this study: 
 Critical care Nursing and Physician leadership at all study sites must be informed of 
vitamin C’s effect on point of care (glucometer) blood glucose and arterial blood gas 
glucose point of care values. 
 In-service training will be documented in the Study Training Log 
 Bold signage will be displayed on all study instructions, data collection forms, and at 
the patient’s head of bed, stating: 
   STOP! This patient is enrolled in a study with Vitamin C, which artefactually 
increases POC glucose testing 
 
   Do NOT use Accuchek or other Point of Care devices to measure glucose on 
this patient 


November 16, 2016 
12 
 
 
 
 
   Use ONLY metabolic glucose screening performed by central laboratory as 
ordered in Cerner 
 
   Do NOT initiate or utilize sliding scale, scheduled insulin, or continuous insulin 
infusion without laboratory confirmation of blood glucose level 
 
 
Those receiving insulin infusion or sliding scale insulin will have metabolic glu- 
cose screening on the schedule determined by the primary physician and paid for 
by the study 
 
Blood glucose monitoring for insulin administration guidance should  only be by a 
metabolic laboratory measured blood glucose results, whether or not the study 
patient is receiving insulin 
 
To accommodate the required glucose monitoring via the metabolic lab, each sub- 
ject will have a dedicated peripheral line 
 
Study personnel will follow each study patient closely to monitor insulin use to 
ensure that point of care glucose screening is suspended for the research subject. 
 
If subject loses central venous access (PICC line and arterial line acceptable), Vita- 
min C infusions are to stop but subject not withdrawn.  Data will be collected 
through end of study. 
 
Point of care glucose testing may resume 36 hours after the last infusion of study 
drug. 
 
 
 
ADVERSE EVENT REPORTING/SAFETY REPORTING 
 
Investigators will determine daily if any clinical adverse experiences occur during the pe- 
riod from informed consent through study hour 48 and will be followed up through reso- 
lution, resolved with sequelae, unresolvable or death. 
 
It is expected that diseases/illnesses/symptoms associated with the study population will 
occur in the study population, independent of investigation product exposure.  These asso- 
ciated diseases/illnesses/symptoms will be considered as part of the study inclusion pro- 
cesses and/or study assessments and as such will not be considered ‘reportable’ Adverse 
Events (AE)/Serious Adverse Events (SAE) unless the Investigator has a reasonable doubt 
regarding the relatedness of the event to the investigational product. 
 
The investigator will evaluate any changes in laboratory values and physical signs and will 
determine if the change is clinically important and different from what is expected in the 
course of treatment of the study patients.  If clinically important and unexpected adverse 
experiences occur, they will be recorded on the adverse event case report form. 
 
 
 
 
The following will be considered reportable adverse events: 
 
For this trial, a reportable adverse event is defined as: 


November 16, 2016 
13 
 
 
 
 
 
 
1. 
Any clinically important untoward medical occurrence in a patient receiving study 
drug or undergoing study procedures which is different from what is expected in 
the clinical course of a patient undergoing cardiac bypass surgery, or, 
2. 
Any clinically important, untoward medical occurrence that is thought to be associ- 
ated with the study drug or procedures, regardless of the “expectedness” of the event 
for the course of a patient undergoing cardiac bypass surgery. 
3. 
Investigators will report all serious, unexpected, AND study-related adverse events from 
the time of informed consent through study hour 48 that are considered to be harm- 
ful and unintended responses to the investigational product and/or study related 
procedures in the participants’ case report forms. ‘Responses to investigational prod- 
uct’ means that the causal relationship between an investigational product and an ad- 
verse event cannot be ruled out. 
 
The following will be reported as adverse events: 
 
Investigators will report all unanticipated problems that involve risk or harm to a re- 
search participant AND was not anticipated or foreseen (e.g., not described in the 
consent form) AND is probably or definitely related to or caused by the research to 
the sponsor/IND Holder by phone and email within 24 hours of becoming aware of event. 
The Institutional Review Board will be notified within 5 business days of receiving notice 
of the unanticipated problem. 
 
The study team will report all unanticipated problems, defined as problems that involve 
risk or harm to a research participant AND was not anticipated or foreseen (e.g., not 
described in the consent form) AND is probably or definitely related to or caused by 
the research, to the DSMB within 7 calendar days of the Sponsor/IND Holder being noti- 
fied of the event. 
 
The DSMB will also determine if the serious adverse event is unexpected for Vitamin C.  If 
the DSMB determines that any serious and study-related adverse event is unexpected for 
Vitamin C, the FDA will be notified within 7 calendar days. Such events may also meet the 
definition of Unanticipated Problems as described below. 
 
Investigators must report Unanticipated Problems, regardless of severity, associated with 
the study drug or study procedures within 24 hours.  An unanticipated problem is defined 
as follows: 
 
Any incident, experience, or outcome that meets all of the following criteria will be re- 
ported from the time of consent through study hour 48 until resolved, withdrawn from the 
study, death occurs or lost to follow up: 
•  Unexpected, in terms of nature, severity, or frequency, given the research procedures 
that are described in the protocol-related documents, such as the IRB-approved re- 
search protocol and informed consent document; and the characteristics of the sub- 
ject population being studied; 


November 16, 2016 
14 
 
 
 
 
•  Related or possibly related to participation in the research. In this guidance docu- 
ment, possibly related means there is a reasonable possibility that the incident, ex- 
perience, or outcome may have been caused by the procedures involved in the re- 
search; 
•  Suggests that the research places subjects or others at a greater risk of harm (in- 
cluding physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
WITHDRAWAL CRITERIA 
 
The Principal Investigator may withdraw the subject at any time due to subject failure to 
adhere to protocol, or for safety concerns. 
 
TRIAL MANAGEMENT 
 
It is expected that the trial will terminate when the intended sample size (24 patients) has 
been achieved. However, the trial will be stopped prior to completion if: (1) the 
intervention is associated with adverse effects that call into question the safety of the test 
intervention; (2) problems with study recruitment or retention will significantly impact the 
ability to evaluate the study endpoints; (3) any new information that becomes available 
during the trial necessitates discontinuation of the trial. 
 
The trial protocol and results will be published in peer-reviewed journals. All publications 
will follow the Consolidated Standards for Reporting Trials (CONSORT) statement [25]. 
Credit for the study will be assigned to collaborators who have actually performed 
designated work. The results of the trial will be reported first to trial collaborators. 
Summary outcome data and links to published data will be uploaded to the 
ClinicalTrials.gov website. 
 
ETHICAL CONSIDERATIONS 
 
This research will involve the use of fresh blood drawn from patients. The blood draws will 
be done, when possible, from in-dwelling arterial line and central line catheters. Additional 
venous blood draws from peripheral sites (e.g. antecubital, radial vein) may be required if 
the arterial and central line catheters have been removed before the conclusion of 48-hour 
study period, heparinized, or would be compromised by extra blood draws. Blood draw 
volumes will be approximately 20 mL. 
 
The toxicity of Vitamin C is very low; therefore, potential benefit far outweighs known 
risks. 
 
Patients will not be paid for participation and there will be no penalties imposed for either 
refusal to participate or withdrawal of consent after enrollment. 
 
Patient Drop-Out. Patients may refuse consent and leave the study at any time for any 
reason. This will be done by notifying the study physician or study coordinator.  Subjects 
can be withdrawn from the study emergently by the attending study physician if they 


November 16, 2016 
15 
 
 
 
 
develop signs and symptoms of adverse reactions. 
 
DATA ANALYSES 
 
Baseline demographic and clinical characteristics of study participants will be reported as 
descriptive summary statistics. Categorical variables will be reported as absolute and 
percent values. Continuous variables will be expressed as means and standard deviations, 
or median and interquartile ranges. 
 
For each primary and secondary outcome, we will report the point estimate of effect and its 
precision (e.g. 95% confidence interval). 
 
Biomarkers will be analyzed both individually and as clusters, or constellations, of 
variables. These clusters will be determined by exploratory factor analysis, discriminant 
analysis and/or vector analysis. Each cluster can be interpreted as a specific 
pathophysiological network process. The clusters can then be used as an empirical model 
to determine inter-dependencies between individual biomarkers. Discriminant function 
analysis is used to predict patient membership in either the Vitamin C treatment group or 
the control group by the entire suite of inflammation and coagulation variables (the 
predictor variables). Unlike cluster analysis, discriminant analysis is used when groups are 
known a priori. The measures of effect size are the canonical correlation (correlation 
between groups and the function), the proportion of variance for each function, calculated 
as (λx/Σλi) where λx is the eigenvalue for the function and Σλi is the sum of all eigenvalues, 
and percent correctly classified.34 Finally vector analysis35 can allow determination of the 
simultaneous directional change of all the measured inflammatory, biolipid and/or 
coagulation markers as a function of treatment group. These techniques allow a 
comparison of dynamic responses rather than a direct comparison of static levels which 
may not be particularly revealing of complex patterns of interactions between multiple 
variables. 
 
No interim analyses will be performed because this trial is a small pilot. 
 
LIMITATIONS OF PROPOSED RESEARCH 
 
Small sample size. This is a pilot study designed to assess feasibility and proof of concept; 
constraints due to restricted funding limited the scope of this initial trial. Because this is a 
pilot trial, statistical significance cannot and should not be emphasized in determining 
clinical relevance and significance of findings as this trial is not powered to detect minimal 
differences between groups.22 
 
Missing markers. Preliminary subsets of biomarkers were carefully chosen according to 
biological relevance, greatest relevance to cardiovascular conditions, and published strong 
associations with clinical outcomes. Nevertheless, relatively few biomarkers will be 
screened, so it is possible the relevant features of the inflammatory response may not be 
captured, and/or sufficient alteration in the inflammatory profile may not occur to allow 
discrimination between test interventions. However, the sensitivity of TA1 is optimized to 
the femto-molar range for most analytes. If this is not sufficient to detect much lower 


November 16, 2016 
16 
 
 
 
 
concentrations present at early stages of the post-operative inflammatory response (as 
indicated by sample analyses and mathematical modeling) we will employ a charge 
reversal derivatization for detecting sub-low limit of quality level of oxilipins and free fatty 
acids.36,37 
 
Misidentified markers. Possible sample contamination may result in some non-lipid 
metabolites being misidentified as important markers. Analytes will be checked for 
conformity to identification parameters of experimental or theoretical lipid databases. 
 
FUTURE RESEARCH 
 
Pilot study results will be used to determine if proceeding to a large, adequately-powered, 
main trial is feasible. There are four possible feasibility outcomes:22 
 
a)  Stop: Main study not feasible (because of poor recruitment retention, staffing, 
insufficient resources, or high probability of treatment futility); 
b)  Continue, but modify protocol: Feasible with modifications (most likely 
scenario; this could include modifications of operational logistics, dose loads, 
dosing schedules, data acquisition protocols, etc.); 
c)   Continue without modifications, but monitor closely (for example, if the 
major limitation is ensuring patients get the appropriate test intervention at the 
appropriate time, we could have in-place monitor); 
d)  Continue without modifications: feasible as is. 


November 16, 2016 
17 
 
 
t- 
 
 
Figure 1. Trial flow per CONSORT guidelines 
 
 
ELIGIBILITY SCREENING 
•  Adult,non-emergent, elective valve 
replacement or CABG surgery patients 
• EF > 35% 
•  No known coagulopathy 
•  No known relevant comorbidities 
 
 
 
 
 
 
 
INFORMED CONSENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
IV VITAMIN C 
PLACEBO 
 
Did not receive 
 
 
 
RECEIVED 
INTERVENTION 
 
 
 
 
ANALYZED 
t- 
RECEIVED 
INTERVENTION 
 
 
 
 
ANALYZED 
 
intervention; 
Discontinued 
intervention; 
Lost to follow- 
up; 


November 16, 2016 
18 
 
 
 
 
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