(PDF) A study protocol for a randomized controlled trial on the prevention of atrial fibrillation after coronary artery bypass g

F1000Research 2018, 7:215 Last updated: 08 MAY 2018 STUDY PROTOCOL A study protocol for a randomized controlled trial on the prevention of atrial fibrillation after coronary artery bypass grafting surgery using Tocotrienol, an isomer of Vitamin E derived from palm oil [version 1; referees: 1 approved, 1 approved with reservations] Ahmad Farouk Musa 1, Jeswant Dillon2, Mohamed Ezani Md Taib2,  Alwi Mohamed Yunus2, Rusli Bin Nordin 1, Yuen Kah Hay3 1Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia 2Department of Cardiothoracic Surgery, National Heart Institute, Kuala Lumpur, Malaysia 3School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia First published: 22 Feb 2018, 7:215 (doi: 10.12688/f1000research.12912.1) Open Peer Review v1 Latest published: 08 May 2018, 7:215 (doi: 10.12688/f1000research.12912.2) Referee Status:     Abstract Background: One of the most common complications following coronary artery bypass grafting (CABG) surgery is atrial fibrillation (AF), which   Invited Referees contributes towards increasing morbidity and mortality, length of hospital stay 1   2 (LoHS) and reduced quality of life (QoL) of patients. Objectives: To determine whether the intake of Tocotrienol, a Vitamin E    isomer derived from palm oil, before and immediately following CABG prevents version 2 AF, reduces LoHS, and improves the QoL of patients. published Protocol: The study is registered with the National Medical Research Register 08 May 2018   with a trial number NMRR-17-1994-34963 and designed as a prospective, randomized controlled trial (RCT) with parallel groups. The experimental group version 1 will receive two 200mg Tocotrienol capsules each day, while the control group published report report 22 Feb 2018 will receive two identical placebo (palm Super Olein) capsules per day. ECG readings will be used to detect AF post operatively, LoHS will be measured by checking the records from the National Heart Institute Hospital register, and the 1 Whady Armino Hueb, University of São health-related Quality of Life (HRQoL) analysis (the Malay version of the Short Paulo, Brazil Form 36 Questionnaire) will be used to analyse QoL. The sample size was Paulo Cury Rezende, University of São calculated to be 140 in each arm of the RCT for a power of 0.8 and a significance level of 0.05. Paulo, Brazil Funding: HOVID Berhad funds this research project. Expected outcomes: The primary endpoint is the development of 2 Olaf Stanger, University Hospital of Bern, postoperative AF, whilst the secondary endpoints are the LoHS and HRQoL of Switzerland patients post CABG. Future implications: Prevention of AF and its complications such as Discuss this article cardiovascular or cerebrovascular events, especially stroke, is an important Comments (0) output. Malaysia is one of the biggest producers and exporters of palm oil and palm oil products. Thus, the possibility of marketing Tocotrienol, in reducing AF post CABG surgery, is a very important proposition indeed. Trial number: NMRR-17-1994-34963 Keywords   Page 1 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Keywords Coronary Artery Bypass, Atrial Fibrillation Corresponding author: Ahmad Farouk Musa (

) Author roles: Musa AF: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Dillon J: Project Administration, Resources, Supervision, Validation, Visualization; Md Taib ME: Project Administration, Resources, Validation, Visualization; Mohamed Yunus A: Project Administration, Resources, Validation, Visualization; Nordin RB: Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Kah Hay Y: Funding Acquisition, Project Administration, Resources, Supervision, Visualization Competing interests: No competing interests were disclosed. How to cite this article: Musa AF, Dillon J, Md Taib ME et al. A study protocol for a randomized controlled trial on the prevention of atrial fibrillation after coronary artery bypass grafting surgery using Tocotrienol, an isomer of Vitamin E derived from palm oil [version 1; referees: 1 approved, 1 approved with reservations] F1000Research 2018, 7:215 (doi: 10.12688/f1000research.12912.1) Copyright: © 2018 Musa AF et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Grant information: HOVID Berhad (contact information: 121, Jalan Tunku Abdul Rahman, 30010 Ipon, Perak, Malaysia) sponsored this study (grant number, MMRD-MS-1801, awarded to the principle investigator, Ahmad Farouk Musa). The study design is solely developed by the main investigator. The sponsor, HOVID Berhad, supplies the materials for the research and the financial support.  The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. First published: 22 Feb 2018, 7:215 (doi: 10.12688/f1000research.12912.1)    Page 2 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Introduction Previous data have shown that both Vitamin E and C are known Atrial fibrillation (AF) after coronary artery bypass graft- to have antioxidant properties, and have the ability to coun- ing (CABG) surgery is common in clinical practice, with an ter the action of free radicals11. Vitamin E is able to maintain incidence of up to 70%. It has the potential to double the risk membrane stability. It could also prevent the process of lipid of mortality and is associated with a 6-fold increase in the peroxidation. Vitamin C removes water-soluble free radicals risk of stroke1. and acts synergistically with Vitamin E. In a recent local study by Musa et al. on post-CABG patients, There have been numerous mechanistic studies on the role of it was demonstrated that patients who developed postopera- antioxidant vitamins in the prevention of postoperative AF. How- tive AF had a prolonged Intensive Care Unit (ICU) stay and ever, the therapeutic potential is uncertain and there is no uni- High Dependency Unit (HDU) stay, and a prolonged total hos- versally accepted protocol. It is interesting to note that Carnes pital stay. This leads to an increase in resource utilization. et al.12 reported a 16.3% incidence of postoperative AF in With an increased rate of morbidity and mortality, preoperative Vitamin C-treated patients compared to 34.9% in controls in prophylactic strategies are necessary to reduce the incidence of their prospective study. In their meta-analysis, Harling et al.13 AF post-operatively and improve the Quality of Life (QoL) of demonstrated a reduction in the incidence of postoperative AF patients2. and other forms of arrhythmia following antioxidant vitamin therapy, which remained significant when only randomized The financial burden in managing AF is huge, and many new controlled trials (RCTs) were analyzed. They also showed that innovative ideas and materials have been tested in order to this reduction in incidence was not related to the bypass or cross- reduce the occurrence of post-operative AF. One of the very clamp time, where there was no significant difference between promising but not widely studied treatment alternatives is the the two-groups. Lastly, the results of the meta-analysis compar- use of Tocotrienol, an isomer of vitamin E. ing the groups of anti-oxidants and control also demonstrated a reduced stay at the ICU and at the hospital. While this finding AF is thought to be initiated by rapid electrical activity aris- cannot be attributed solely to a reduction in postoperative AF, it ing from the muscular sleeves of pulmonary veins1,3. Multi- is possible that this reduction in both ICU and hospital stay was a ple re-entrant wavelets, shorter refractory periods, conduction result of the reduced incidence of AF in the treatment group. delays and fragmented electrograms maintain the arrhythmia1. Rasoli et al.14 came to a similar conclusion that the use of If this arrhythmia continues, electrical remodeling occurs, antioxidant vitamins plays a role in the reduction of post- which will further facilitate AF maintenance. The pathogen- operative AF. However, they argued that the effect was somewhat esis of post-operative AF is believed to be multifactorial. variable, because both vitamins, C and E, were weak anti-oxidants. Previously it was thought to be caused by inflammatory pathways, They were limited in their capacity to cross the cell membrane in but now it is known that oxidative stress plays a significant role4. order to counter the superoxides. Reactive oxygen species (ROS) are the cause of oxidative stress, and generate numerous pathological processes such as DNA We are proposing a more potent isomer of vitamin E, namely damage, apoptosis, and cellular hypertrophy, as well as signal Tocotrienol, which is derived from palm oil, instead of the pathway modulation5. usual isomer of vitamin E, namely tocopherol, which was being used in all previous research. Tocotrienol has been proven to Cardiac surgery is characterized by ischaemia and reperfusion possess more potent antioxidant activity than tocopherols15. Lab- injury, which leads to the release of ROS. This causes oxida- oratory studies have proven that Tocotrienol has the ability to tive stress and initiates a systemic inflammatory response6. The scavenge peroxyl radicals in liposomes16. Tocotrienol is seen to be extracorporeal circulation during on-pump surgery also activates more evenly distributed in the phospholipid bilayer of the plasma inflammatory cells, increasing the oxidative stress7,8. Therefore it membrane. It is also shown to have more efficient collision was always thought that an off-pump surgery would reduce the with radicals. These are some of the reasons why Tocotrienol is incidence of AF. thought to have a more potent antioxidant properties15. Oxidative stress causes the depletion of endogenous antioxidants, All these recent discoveries suggest the promising antioxi- and this results in oxidative damage. This oxidative stress is mainly dant properties for Tocotrienol. We hypothesize that it would the result of on-pump surgery that requires aortic cross-clamping be able to reduce the incidence of postoperative AF and and the bypass machine9. subsequently reduce the complications associated with post- operative AF, along with the economic burden of prolonged Almost all cardiac centers have guidelines on the pharmaco- intensive care stay and prolonged hospital stay. Overall, we also logical management of postoperative AF10. But the challenge has hypothesized that Tocotrienol will increase the health related always been how to prevent AF from happening. Efforts have quality of life (HRQoL) of patients. The potential value of mar- been made to develop alternative preventive strategies. With the keting a national product that can be produced locally from palm recent understanding of the pathogenesis of AF via the oxidative oil and its potential uses, not only for patients, who are undergo- pathway, perhaps antioxidant vitamins would be a promising ing CABG, but also for patients with standalone AF, makes this treatment. an even more attractive product. Page 3 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Protocol Study design Ethical statement Study site: National Heart Institute, Kuala Lumpur, Malaysia The National Heart Institute Ethics Committee has approved the study protocol (IJNREC/20112017). The ethics com- The study is designed as a prospective, randomized controlled mittee will also serve as the data safety committee. Simi- trial, with two parallel groups of the same demographics and co- lar approval has been obtained from MUHREC - Monash morbidities. The main aim is to look at the effect of Tocotrienol in University Human Research Ethics Committee – (9277) and from reducing AF post CABG. MREC – the Malaysian Research Ethics Committee. The study is registered with the National Medical Research Registry (NMRR), Patients who are scheduled for CABG under the co-researchers Ministry of Health, Malaysia, (NMRR-17-1994-34963) and will be at the National Heart Institute will be approached for their con- carried out in accordance with the ethical principles outlined in the sent to be enrolled in this study once they are admitted to the Malaysian Good Clinical Practice Guidelines. wards. We will assign the patients enrolled in the study to one of two groups: Written informed consent for participation in the trial will be 1. The control group: Routine CABG surgery procedure obtained from each participant on recruitment (Supplementary File 1). plus placebo containing palm Super Olein capsules (supplier: HOVID Berhad, Malaysia). The placebo is an identical-looking capsule that contains tocotrienols- The clinical data of patients enrolled in this study will be de- stripped palm oil (termed palm Super Olein), which is identified. No personal data will be disclosed to anyone outside also the non-active excipient in the treatment capsule. of the National Heart Institute Ethics Committee. The choice of control is to mimic the treatment capsule This is Protocol Version 2.0 dated on 15 June 2017. as much as possible, without the active component of tocotrienols. Trial registration: NMRR-17-1994-34963 he study group: Routine CABG surgery procedure plus 2. T Trial registry: National Medical Research Register, Malaysia Tocotrienol capsules (supplier: HOVID Berhad, Malaysia) Trial registration date: 13th March 2017 For patients in the study group, Tocotrienol will be administered as two capsules of 200mg per day in two divided doses, and Study status: The study has not yet started at the time of administration will start immediately after randomization and will submission of this manuscript since we are still waiting for the continue until the first follow-up, which is normally six weeks Clinical Trial Exemption (CTX) from the National Pharmaceutical after discharge. Regulatory Agency (NPRA). Since there is no available data for preferred dose of treat- Hypothesis ment, we decided on two divided doses of Tocotrienol of Tocotrienol is a commercial product produced by Hovid Berhad, one capsule twice daily of 200mg Tocovid. This dosage was and is marketed as Tocovid Suprabio, containing 61.52mg alpha- estimated based on the regime used by Olaf Stanger et al.17 at the Tocotrienol, 112.80mg gamma-Tocotrienol, 25.68mg delta- Department of Cardiac Surgery, Paracelcus Medical University Tocotrienol and 91.60IU alpha-tocopherol. Its availability in Salzburg, Austria, where three ampoules of 45IE Vitamin E were Malaysia has allowed this innovative and promising study to be used, with 30mg per ampoule or 90mg in total. Because in our conducted. We hypothesize that: study we would be using an oral preparation instead of an IV • Intake of Tocotrienol capsules before and immediately preparation as in the Austrian study, and absorption of Tocotrienol following CABG prevents AF post CABG; has been shown to be low and incomplete via the oral route, we estimated a higher dosage18. Considering the bioavailability of • I ntake of Tocotrienol capsules before and immediately Tocotrienol can be as low 10–30% if administered orally18; and following CABG improves the QoL of patients post CABG; taking into consideration that this is a pivotal study, it is reason- • I ntake of Tocotrienol capsules before and immediately able to use the highest dose possible that is safe without any following CABG shortens the length of hospital stay (LoHS) of adverse effects. Many clinical studies with Tocotrienol have used patients post CABG. 400mg daily in two divided doses and have been proven to be safe19,20. Objectives • To determine whether the intake of Tocotrienol capsules before Tocovid will be administered right after randomization, and will and immediately following CABG prevents AF post CABG. continue after the patient is discharged until the first follow- up, which is normally about six-weeks later. If the patient is on • o determine whether the intake of Tocotrienol capsules T prolonged ventilation in the ICU, Tocotrienol will be adminis- before and immediately following CABG improves the QoL of tered through a nasogastric tube. The intensive care nurses and patients post CABG. cardiothoracic ward nurses will monitor compliance. • To determine whether the intake of Tocotrienol capsules before and immediately following CABG shortens the LoHS of All patients undergoing CABG surgery, either or with valve patients post CABG. surgery, will be included. Similarly, this study will include Page 4 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 participants undergoing both on-pump surgery using cold potassium randomly to either the study arm or the control arm based on the cardioplegia, and off-pump beating heart surgery. sequence assigned to them. All patients will be admitted to the intensive care unit (ICU) Inclusion/exclusion criteria after CABG surgery, with close monitoring on one-patient- Inclusion criteria: one-nurse basis. They will then be transferred to a monitored 1. Males or females high dependency unit (HDU) if their condition is stable, while some might be transferred straight back to the ward. Continuous 2. More than 18 years of age rhythm monitoring will be performed using the 12-lead lective, on-pump or off-pump CABG surgery of coronary 3. E ECG for all patients in ICU and HDU. The monitoring will be artery revascularization, single or double procedure continued for the first four to five postoperative days on the normal cardiothoracic wards using Holter monitors until the patients are Exclusion criteria: discharged. 1. Less than 18 years of age We will review the electrocardiographic (ECG) data on a daily 2. Refusal to have surgery basis. We will also review all the printouts of all abnormal 3. Urgent or emergency surgery rhythms, which will then be included in the clinical records. Additionally, an ECG will also be recorded when patients are 4. Inability to give informed consent symptomatic or when there is a suspicion of arrhythmia clini- 5. Documented allergy to palm oil cally. We will treat all AF episodes, in both the study arm and control arm, according to the protocols of the Cardiothoracic Patients will be recruited from those who are scheduled elec- Department. The patients will be managed by the cardiothoracic tively for surgery under the Consultant Surgeons, JD, MEMT, surgeon and their team. The first-line drug used for treatment of and AMY, who are co-researchers in this project. All public and new onset AF post-operatively by the Cardiothoracic team in the private patients are eligible to be enrolled in this study. Consent National Heart Institute (IJN) Hospital is Amiodarone, unless will be taken upon admission to the wards. contraindicated, as a 300mg infusion over one hour to be followed by 900mg over the next 23 hours. Patients can choose to withdraw from the study at any time. Patients of both the study and control arms will come back Subjects may be withdrawn if the investigator deems that it is for follow-up at the Cardiothoracic Clinic at the IJN usually detrimental or risky for the subject to continue. The patients who six weeks after discharge. They will also be advised to report withdraw will not be replaced. to the Outpatient Department at the IJN if they develop any symptoms. During follow-up, all patients will be assessed routinely Study endpoints with blood work and a 12-lead ECG. As the primary study endpoint, we will look at the develop- ment of AF post-surgery. This will be documented with ECG. We take a 30 second duration as a cut-off point and define AF as For the study flowchart, see Figure 1. when there is a loss of p-waves and irregular ventricular rate or a confirmed atrial flutter21. Hovid Berhad follow strict GLP Guidelines in the manufactur- ing process of Tocotrienols and matching placebo capsules. The secondary endpoint would include the LoHS after surgery, Each 200mg capsule of Tocotrienol will contain 61.52mg alpha- which will be obtained from the IJN registry, and the HRQoL. Tocotrienol, 112.80mg gamma-Tocotrienol, 25.68mg delta- Three measurements will be used to determine the LoHS: Tocotrienol and 91.60IU alpha-tocopherol. The placebo capsules will contain palm Super Olein oil. (1) Total number of days that the patients stay in the Intensive care unit (ICU) Randomization The study is designed as a randomized controlled trial (RCT), (2) Total number of days in the high-dependency unit (HDU); and where patients with coronary artery disease requiring CABG (3) Total number of days in the hospital, overall21. surgery at the IJN will be prospectively and randomly divided into two parallel groups by means of computer generated numbers We will measure the HRQoL of patients using the validated using Excel 2016 (Microsof, Redmont, WA, USA) in a block of 10 Malay and English versions of Short-Form 36 Questionnaires experimental to 10 matching controls at enrollment. The experi- (SF-36)22–24. mental group will receive Tocovid capsules whilst the control group will receive identical placebo capsules containing palm Super Since the researchers are blinded to the trial, we will be evaluat- Olein oil. ing all endpoints, primary and secondary, independently. We will also review the patients’ clinical records and all ECG tracings. The unblinded pharmacist will generate the allocation sequence to In fact, all the trial participants, care-givers, outcome assessors assign the participants to the interventions. Surgeon investigators and data analysts will be blinded to the study treatment alloca- will enroll the participants. The participants will then be assign tion. An assigned pharmacist maintaining the randomisation list Page 5 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Figure 1. Study flowchart. Page 6 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 will be unblinded to the study treatment allocation. In the event heart. The anastomosis is done by sewing the open end of of serious adverse events or emergency clinical treatment requir- the internal mammary to the coronary artery, namely the left ing knowledge of the study treatment, a request for unblinding anterior descending artery. Chest is closed in the usual manner shall be submitted to the pharmacist with reasonable justifi- after placement of the drains and the temporary pacing wire. cation for unblinding. The unblinding details and justifica- tion will be documented in the case record form. If unblinded, Post-operative the participant will be withdrawn from the study in the event of Both groups will continue taking either two capsules of Tocot- unblinding, and post-treatment follow-up conducted as per rienol or two capsules of placebo (palm Super Olein) daily for protocol or as applicable. Where possible, the outcome assessor the entire hospitalization period until follow-up at six weeks will remain blinded to all treatment allocation until end of study, after discharge. The capsules will be taken on a bd – twice- even after participants are withdrawn from the study. daily dosing. If the patient is still ventilated in the cardiac ICU, the capsules will be broken and the content administered via Study procedures a Ryle’s tube. Preoperative The two randomized groups, based in the IJN, will be matched After surgery, patients will be admitted to the ICU with according to sex, age, NYHA criteria, ejection fraction, and close monitoring on one-patient-one-nurse basis and will be diabetic status. Operative and peri-operative conditions will subsequently transferred to a monitored high dependency unit also be similar for both groups. All subjects will receive an if their condition is stable. Continuous heart rhythm monitor- identical prophylactic antibiotic regime consisting of Cefazolin ing will be carried out using the 12-lead ECG. The monitoring 2gm at induction and 1gm 12 hourly for 48 hours. Gentamicin will be continued for the first four to five postoperative days on 2mg/kg will also be given at induction. the normal cardiothoracic wards using Holter monitors until the patients are discharged. Intra-operative The electrocardiographic data will be stored for 24 h and • On-pump CABG reviewed on a daily basis by the cardiothoracic team involved A standard procedure of performing CABG will be done under in the research. The printouts of all abnormal rhythms will also general anaesthesia. Veins will be harvested from the legs and be reviewed for any episodes of arrhythmia. All printouts will the left internal mammary will be taken down once the chest is be included in the clinical records. Additionally, an ECG will opened. Titanium clips will be used to secure all side branches also be recorded in case of symptoms or when arrhythmia is of saphenous veins and also the branches of the internal mam- suspected on clinical grounds; AF episodes will always be treated mary artery. The patient will then be cannulated and placed on a under the direction of the attending cardiothoracic surgeon. heart-lung machine. Once the ascending aorta is cross-clamped, cold cardioplegic solution will be perfused in an ante-grade After discharge, all patients will be asked to report to the manner through the aortic root into the heart and the pericardial outpatient department of our institution in case of any rel- sac will be buried with ice sludge to create myocardial evant symptom. Additionally, all patients will be followed up hypothermia. six weeks after discharge; this will include physical examinaton and a 12-lead ECG measurement. The diseased coronary arteries will then be identified and arteri- otomies will be performed beyond the level of blockages. The Measurements open ends of the saphenous veins and the internal mammary ECG readings to detect AF post operatively artery are sewn to the openings artertomies using Prolene 7/0 Diagnostic criteria: sutures. Once the distal anastomoses are constructed, proximal 1. Absent P waves anastomoses will commence. vidence of fibrillation (f) waves instead of P waves. 2. E These are irregular undulations of the base line in ECG. Once all the anastomoses are completed, the cross-clamp will be taken off and the heart-lung machine will then be gradu- 3. Irregular R-R intervals ally weaned off. Subsequently, the patient will be decannulated. Drainage catheters will be placed around the heart and temporary Length of hospital stay (LoHS) pacing will be sewn to the surface of the heart. Sternum will then This information will be obtained from the IJN registry of patients. be closed with steel wire and subcutaneous tissue and skin in Three measurements will be used to determine the LoHS: the usual manner. (1) Total Cardiac ICU length of stay; • Off-pump CABG (2) Total HDU length of stay; and In this case, the bypass surgery will be done without the use of (3) Total hospital length of stay. heart-lung machine. Surgery will be done on a beating heart. The procedure is similar to the on-pump surgery. Chest is Health related quality of life (HRQoL) opened and the left internal mammary artery is taken down. The analysis of HRQoL will be performed using the A stabilizer is placed on the heart to limit the motion of the validated Short-Form 36 Questionnaire (SF-36). SF-36 has Page 7 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 demonstrated its efficiency in clinical studies and has been ‘lost to follow up’ is as high as 20%, then the two proportions proven to be generally acceptable to patients, consistent and a will be 140 (Intervention Group) and 140 (Control Group) valid measure of health outcome. We will be using the validated with a total number of 280 patients. Malay version of SF-36, which is a thirty-six items questionnaire, which measures QoL across eight domains that encompass both Statistical analysis physical and mental. IBM SPSS Statistics version 25.0 will be used to analyze the results. The ITT analysis will be used to analyze all outcome. The questionnaire will be distributed to both groups of patients The RR of the two-binomial proportion analysis will be used to before CABG and at discharge and six weeks during clinic visit test the occurrence of postoperative AF in the two treatment postoperatively. The questionnaires are going to be administered groups. The Kaplan-Meier method will be used to look at the using the questionnaire-interview approach. cumulative risk. Log-rank test will compare the survival curves of the two treatment groups. Continuous variables will use mean Sample size calculations (±SD) while categorical variables will use frequencies (percent- We will use the PS Power and Sample Size Calculation ages). We will use unpaired Student-t test for continuous variables Software version 3.1.2 for sample size calculation. We plan to to look for differences between groups. And group differences will study a continuous response variable from independent control be examined by the chi-square or Fisher exact tests as appropri- and experimental subjects with 1 control per experimental subject. ate for categorical variables. In case of an expected frequency of less than 5 in any cell in a 2×2 table, a Fischer Exact test will Calculation of sample size in the present study (randomized be applied. A p value of less than 0.05 will be considered as controlled trial: RCT to rule out selection bias) requires precise statistically significant. specification of: In order to find the predictors of AF after surgery, a stepwise • The primary hypothesis of the study (Tocotrienol consumption multiple logistic regression analysis will be done. To examine reduces the incidence of post-operative atrial fibrillation in the mean (±SD) LoHS (number of days) differences between the subjects that had undergone CABG) two groups, we will use an unpaired Student-t test. • The method of analysis (using Relative Risk: RR). To examine the mean (+SD) QoL score differences between the We will also take into account the possibility of ‘loss to two groups (pre-operative, six weeks, and three months), the follow-up’ or attrition bias of subjects by analysing all subjects one-way mixed-mode repeated measure ANOVA with post- from the start to the completion of the study according to the hoc multiple comparison test (between and within subject) of groups that they were originally randomized. This is called an the two groups will be performed. Intention-To-Treat (ITT) analysis25. To calculate the desired sam- ple size based on the above consideration, we use the PS Power In order to look for the factors influencing the change in the and Sample Size Calculation Software for sample size quality of life after CABG, we will perform the univariate calculation26,27. simple logistic regression (SLogReg) initially, and examin- ing the statistical significance of each independent variable such In the present RCT, estimated sample size for the primary end- as the number of revascularization and duration of surgery on point (incidence of POAF) was computed on the basis of findings the outcome. Then, we will perform a multiple logistic regression from Calo et al.4 In that study, the incidence of post-operative AF (MLogReg) including variables with a level of significance less was 15.2% in the treatment group. Whereas in the placebo group, or equal to 0.25 in the univariate logistic regression and control- the incidence was 33.3% (RR=15.2/33.3 = 0.46). ling for the effects of possible confounding variables (sex, age, NYHA Criteria, ejection fraction, and diabetic status). A p value To account for possible subject withdrawal/non-compliance of less than 0.05 will be taken as significant. (attrition bias), we will adopt the ITT analysis so that subjects who are ‘lost to follow-up’/non-compliant will be analyzed accord- Monitoring ing to the groups that they were randomized at the beginning of The Principal Investigator, AFM, will be responsible for ensur- the study. The formula suggested by Wittes25 is “1/1-c” where ing participants’ safety on a daily basis and for reporting Seri- c is the proportion that is lost to follow-up/did not comply. When ous Adverse Events and Unanticipated Problems to his IJN c= 12%, for a total enrollment of 260 patients. However, when Review Board (IJNRB) as required. The study statistician pre- c=20%, the estimated sample size is increased to 278 (rounded pares reports that list adverse events, serious adverse events, to 280). deaths, and disease-or treatment-specific events required for monitoring body review in order to ensure good clinical care and Using the PS Power and Sample Size Calculator26,27 with α identify any emerging trends. The IJNRB will act in an advisory equivalent to 0.05 and power (1-β) is 0.8, the estimated sample capacity to the IJN Ethics Committee to monitor participants’ size based on the two proportions above is 130 intervention sub- safety, evaluate the progress of the study, to review procedures jects and 130 control subjects (Fisher’s exact test) if we assume for maintaining the confidentiality of data, the quality of data the “lost to follow up” is 12%. However, if we assume that the collection, management, and analyses. Page 8 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Data dessemination Competing interests Data dissemination will be done via three ways. Firstly, via No competing interests were disclosed. presentation at local and international conferences either in the form of posters and/or oral presentation. Secondly via writing Grant information for publication in scientific journals. Thirdly, via participation HOVID Berhad (contact information: 121, Jalan Tunku Abdul at international exhibitions and conventions on research and Rahman, 30010 Ipon, Perak, Malaysia) sponsored this study innovation. (grant number, MMRD-MS-1801, awarded to the principle investigator, Ahmad Farouk Musa). Study termination The sponsor has the full discretion to decide on the termination The study design is solely developed by the main investigator. The of the study at any time. Patients will be informed if the study is sponsor, HOVID Berhad, supplies the materials for the research terminated and follow-up visits will be arranged if needed. and the financial support. Data Availability The funders had no role in study design, data collection and No data are associated with this article. analysis, decision to publish, or preparation of the manuscript. Supplementary material Supplementary File 1: Informed consent form and participant information sheet. Click here to access the data. References 1. Nattel S, Allessie M, Haissaguerre M: Spotlight on atrial fibrillation-the ‘complete the incidence of postoperative atrial fibrillation. Circ Res. 2001; 89(6): E32–8. arrhythmia’. Cardiovasc Res. 2002; 54(2): 197–203. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 13. Harling L, Rasoli S, Vecht JA, et al.: Do antioxidant vitamins have an anti- 2. Musa AF, Dillon J, Omar R: Atrial Fibrillation after Coronary Artery Bypass arrhythmic effect following cardiac surgery? A meta-analysis of randomised Grafting: A Restrospective Review of a Single Centre Experience. Heart Surg controlled trials. Heart. 2011; 97(20): 1636–1642. Forum. 2008; 2: 23. PubMed Abstract | Publisher Full Text 3. Markides V, Schilling RJ: Atrial fibrillation: classification, pathophysiology, 14. Rasoli S, Kakouros N, Harling L, et al.: Antioxidant vitamins in the prevention of mechanisms and drug treatment. Heart. 2003; 89(8): 939–943. atrial fibrillation: What is the evidence? Cardiol Res Prac. 2011; 2011: 164078. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 4. Banach M, Kourliouros A, Reinhart KM, et al.: Postoperative atrial fibrillation 15. Packer L, Weber S, Rimbach G: Molecular aspects of alpha-tocotrienol - what do we really know? Curr Vasc Pharmacol. 2010; 8(4): 553–572. antioxidant action and cell signalling. J Nutr. 2001; 131(2): S369–S373. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 5. Huang CX, Liu Y, Xia WF, et al.: Oxidative stress: a possible pathogenesis of 16. Serbinova E, Kagan V, Had D, et al.: Free radical recycling and intramembrane atrial fibrillation. Med Hypotheses. 2009; 72(4): 466–467. mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. PubMed Abstract | Publisher Full Text Free Radic Biol Med. 1991; 10(5): 263–275. 6. Kim H, Kim KH: Role of nitric oxide in oxidative damage in isolated rabbit gastric PubMed Abstract | Publisher Full Text cells exposed to hypoxia-reoxygenation. Dig Dis Sci. 1998; 43(5): 1042–1049. 17. Stanger O, Aigner I, Schimetta W, et al.: Antioxidant Supplementation Attenuates PubMed Abstract | Publisher Full Text Oxidative Stress In Patients Undergoing Coronary Artery Bypass Graft 7. Fontaine D, Pradier O, Hacquebard M, et al.: Oxidative stress produced by Surgery. Tohoku J Exp Med. 2014; 232(2): 145–154. circulating microparticles in on-pump but not in off-pump coronary surgery. PubMed Abstract | Publisher Full Text Acta Cardiol. 2009; 64(6): 715–722. 18. Yap SP, Yuen KH, Lim AB: Influence of route of administration on the PubMed Abstract | Publisher Full Text absorption and disposition of alpha-, gamma- and delta-tocotrienols in rats. 8. Matata BM, Sosnowski AW, Galiñanes M: Off-pump bypass graft operation J Pharm Pharmacol. 2003; 55(1): 53–58. significantly reduces oxidative stress and inflammation. Ann Thorac Surg. PubMed Abstract | Publisher Full Text 2000; 69(3): 785–791. 19. Gopalan Y, Shuaib IL, Magosso E, et al.: Clinical investigation of the protective PubMed Abstract | Publisher Full Text effects of palm vitamin E tocotrienols on brain white matter. Stroke. 2014; 9. De Vicchi E, Pala MG, Di Credico G, et al.: Relation between left ventricular 45(5): 1422–1428. function and oxidative stress in patients undergoing bypass surgery. Heart. PubMed Abstract | Publisher Full Text 1998; 79(3): 242–247. 20. Magosso E, Ansari MA, Gopalan Y, et al.: Tocotrienols for normalisation of PubMed Abstract | Publisher Full Text | Free Full Text hepatic echogenic response in nonalcoholic fatty liver: a randomised placebo- 10. Dunning J, Treasure T, Versteegh M, et al.: Guidelines on the prevention and controlled clinical trial. Nutr J. 2013; 12(1): 166–174. management of de novo atrial fibrillation after cardiac and thoracic surgery. PubMed Abstract | Publisher Full Text | Free Full Text Eur J Cardiothorac Surg. 2006; 30(6): 852–872. 21. Mozaffarian D, Marchioli R, Gardner T, et al.: The ω-3 Fatty Acids for Prevention PubMed Abstract | Publisher Full Text of Post-Operative Atrial Fibrillation trial--rationale and design. Am Heart J. 11. Niki E: Interaction of ascorbate and alpha-tocopherol. Ann N Y Acad Sci. 1987; 2011; 162(1): 56–63.e3. 498: 186–199. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 22. Jenkinson C: The SF-36 physical and mental health summary measures: an 12. Carnes CA, Chung MK, Nakayama T, et al.: Ascorbate attenuates atrial pacing- example of how to interpret scores. J Health Serv Res Policy. 1998; 3(2): 92–96. induced peroxynitrite formation and electrical remodeling and decreases PubMed Abstract | Publisher Full Text Page 9 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 23. Savelieva I, Camm AJ: Clinical relevance of silent atrial fibrillation: prevalence, Rev. 2002; 24(1): 39–56. prognosis, quality of life, and management. J Interv Card Electrophysiol. 2000; PubMed Abstract | Publisher Full Text 4(2): 369–382. 26. Dupont WD, Plummer WD Jr: Power and sample size calculations: a review and PubMed Abstract | Publisher Full Text computer program. Control Clinical Trials. 1990; 11(2): 116–28. 24. Sararaks S, Azman AB, Low LL, et al.: Validity and reliability of the SF-36: the PubMed Abstract | Publisher Full Text Malaysian context. Med J Malaysia. 2005; 60(2): 163–179. 27. Dupont WD, Plummer WD Jr: Power and sample size calculations for studies PubMed Abstract involving linear regression. Control Clinical Trials. 1998; 19(6): 589–601. 25. Wittes J: Sample size calculations for randomized controlled trials. Epidemiol PubMed Abstract | Publisher Full Text Page 10 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Open Peer Review Current Referee Status: Version 1 Referee Report 19 April 2018 doi:10.5256/f1000research.13999.r32234 Olaf Stanger  Clinic for Cardiovascular Surgery, University Hospital of Bern, Bern, Switzerland The authors present the study protocol of a randomized controlled study to investigate the effect of a commercially available product (Tocovid Suprabio) that includes Tocotrienol, an Vitamin E isomer, against a placebo group, on the prevention of postoperative atrial fibrillation (AFib) in patients undergoing cardiac surgery.  The topic is of general interest. Postoperative AFib is common, is associated with complications and morbidity, thus consumes many resources and is, in part, potentially preventable. Because oxidative stress has been established as key mechanism, relatively cheap antioxidant treatment could be effective. The sample size under the current plan was calculated as 140 patients in each arm for a power of 0.8. The ambitious study plan has some important shortcomings that must be addressed. 1.  As the authors correctly note, postoperative AFib is assumed to be a multifactorial event and may be triggered by mechanisms other than oxidative stress. That has severe impact on exclusion criteria and statistical power calculations, that is difficult to assess for me without further information.   2.  The authors correctly note that the use of cardiopulmonary bypass is involved in ischemia-reperfusion injury and radical oxygen species (ROS) generation. On page 3 it its stated that it is thought in consequence that off-pump surgery would reduce the incidence of AFib. I miss references, a clear statement whether or not this is thought to be a fact. In any case I would strongly suggest not to include off-pump patients and mix up the cohorts with on-pump cases. It is difficult to precisely quantify the difference from a metabolic point, and it appears impossible to draw a conclusion on the effect of a given dose with unknown blood and tissue concentrations on different levels and sources of ROS.   3.  What is known about the pharmacokinetics of the product? How quick and (in)complete must absorption be expected? What levels in blood and tissue can be achieved? Are interactions and competing drugs known?   4.  The authors "propose using a more potent isomer of vitamin E, namely Tocotrienol, instead of the usual Tocopherol" (page 3). However in describing the commercially available drug on page 4 (Tocovid Suprabio by Hocid Berhad) it is clearly expressed, that the capsule contains Tocopherol (!) besides Tocopherol. So how will the investigators ever know which component, if any, was   Page 11 of 21 4.   F1000Research 2018, 7:215 Last updated: 08 MAY 2018 (!) besides Tocopherol. So how will the investigators ever know which component, if any, was effective?   5.  The authors aim at assessing the health-related quality of life in patients undergoing cardiac surgery; by definition a very sick group of elderly patients with usually many co-morbidities and with much drug intake. This aim is an extremely difficult matter involving many critical sources of definition, assessment tools (including SF-36), error and bias and I doubt that a meaningful result can be obtained.    6.  According to the study protocol patients are required to take the capsules "before" and immediately following CABG. Before is defined as "immediately after randomization" on page 4. It is quite normal that even elective cardiac surgery can not always be planned precisely on the exact day and hour, must be delayed for medical or organizational reasons. Therefore there will be patients with various time intervals of capsule intake and supposedly different blood and tissue concentrations of the study medication! Capsules intake should last for approx 6 weeks until the first follow-up. Nearly all cases of AFib occur within the first 4 days postoperative. It is extremely rare to observe first-time AFib later than that. Although the long intake is ambitious how do the investigators control full compliance?    7.  Again, bioavailibility is an issue. The authors state on page 4 that bioavailibility of oral Tocotrienol is as low as 10-30%; how long before surgery must the drug be given to guarantee an effective availability in all patients of the non-placebo group? Is the trial controlled for interference with other drugs, malabsorption, possibly even gastric resections or concomitant intestinal disease etc that may   prevent the study drug to be taken up effectively?   8.  Definitely exclude patients undergoing concomitant valve surgery (page 4)! Operating times are longer, the heart will be OPEN with further exposure to ROS-producing surfaces and valve pathologies, stretch and surgical maneuvers are all known to cause and influence postoperative AFib. Up to 7% of patients following aortic valve replacement require permanent pacemakers anyway! Again, I would also suggest not to mix on- and off-pump patients.   9.  Holter ECG, very essential, will be monitored for the first 5 days, which makes sense. The study drug is given for approx 6 weeks. There will be no continuous ECG monitoring between discharge and follow-up. Unless the patient would be symptomatic and decides to see a physician who may be able to make a documentation, investigators are likely never to know whether there was an episode of AFib at any time between 1 and 6 weeks after surgery.   10.  I miss any information on potential PREoperative AFib assessment; or at least the exclusion of patients with AFib or other arrhythmias before surgery!   11.  And authors should exclude patients with vitamin or antioxidant intake before randomization! Check for all medications as many are known to exert antioxidant side effects.    12.  Finally I would consider it extremely valuable to obtain blood samples and analyse various ROS and vitamin concentrations (or at least the antioxidant capacity) to proof that any clinical outcome is directly linked to a mechanism as hypothesized. Otherwise, any outcome can be spurious and by chance, in principle and by definition. Is the rationale for, and objectives of, the study clearly described?   Page 12 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Is the rationale for, and objectives of, the study clearly described? Yes Is the study design appropriate for the research question? Partly Are sufficient details of the methods provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Not applicable Competing Interests: No competing interests were disclosed. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 02 May 2018 Ahmad Farouk Musa, Monash University Malaysia, Malaysia The authors present the study protocol of a randomized controlled study to investigate the effect of a commercially available product (Tocovid Suprabio) that includes Tocotrienol, an Vitamin E isomer, against a placebo group, on the prevention of postoperative atrial fibrillation (AFib) in patients undergoing cardiac surgery.  The topic is of general interest. Postoperative AFib is common, is associated with complications and morbidity, thus consumes many resources and is, in part, potentially preventable. Because oxidative stress has been established as key mechanism, relatively cheap antioxidant treatment could be effective. The sample size under the current plan was calculated as 140 patients in each arm for a power of 0.8. The ambitious study plan has some important shortcomings that must be addressed. 1.  As the authors correctly note, postoperative AFib is assumed to be a multifactorial event and may be triggered by mechanisms other than oxidative stress. That has severe impact on exclusion criteria and statistical power calculations, that is difficult to assess for me without further information. Thank you Prof for your comments and suggestions. In the revised version of this study, we will include a few other exclusion criteria namely poor LV, off-pump surgery, combined valve surgery and currently on or indicated for long-term corticosteroid treatment. 1.  The authors correctly note that the use of cardiopulmonary bypass is involved in ischemia-reperfusion injury and radical oxygen species (ROS) generation. On page 3 it its stated that it is thought in consequence that off-pump surgery would reduce the incidence of AFib. I miss references, a clear statement whether or not this is thought to be a fact. In any case I would strongly suggest not to include off-pump patients and mix up the cohorts with on-pump cases. It is difficult to precisely quantify the difference from a metabolic point, and it appears impossible to draw a conclusion on the effect of a given dose with unknown blood and tissue concentrations on different levels and sources of ROS. Our assertion that off-pump surgery would reduce the incidence of AF post-CABG was   Page 13 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Our assertion that off-pump surgery would reduce the incidence of AF post-CABG was due to the fact that the extracorporeal circulation during on-pump surgery would activate the inflammatory cells thus increasing the oxidative stress that would later lead to AF. This was also acknowledged by Hashemzadeh K et al. in their paper published by the Journal of Cardiovascular and Thoracic Research 2013;5(2),45-49, with the title “Does off-pump coronary artery bypass reduce the prevalence of atrial fibrillation?, though we did not cite it. However, in the revised version of this Study Protocol, we have omitted Off-pump surgery. 1.  What is known about the pharmacokinetics of the product? How quick and (in)complete must absorption be expected? What levels in blood and tissue can be achieved? Are interactions and competing drugs known? Gan et al. published in Scientific Reports 2017; 7(1): p11542 on “Effect of palm-based tocotrienols and tocopherol mixture supplementation on platelet aggregation in subjects with metabolic syndrome: a randomised controlled trial” showed that a two-week supplementation of the same product and same dosing schedule resulted in plasma levels of approximately 0.58 ug/mL. And Patel et al. published in the Journal of Nutrition 2012; 142(3):513-519 on “Oral Tocotrienols are transported to human tissues and delay the progression of the model for end-stage liver disease score in patients” demonstrated that oral supplementation of tocotrienols in surgical patients up to four weeks before surgery significantly increased the tissue concentrations in blood, skin, adipose, brain, cardiac muscle, and liver. In our study, plasma tocotrienols levels will be measured to indicate the levels in the blood pre and post-surgery, and also on follow-up to determine if the blood levels are sufficient to exert clinical effects at the end of the study. And Tocotrienols have not been noted to have any interference with other drugs so far. 1.  The authors "propose using a more potent isomer of vitamin E, namely Tocotrienol, instead of the usual Tocopherol" (page 3). However in describing the commercially available drug on page 4 (Tocovid Suprabio by Hocid Berhad) it is clearly expressed, that the capsule contains Tocopherol (!) besides Tocopherol. So how will the investigators ever know which component, if any, was effective? The proposed investigation uses a commercially available product of natural mixed tocotrienols. Almost all natural sourced and marketed tocotrienols products contain a small fraction of tocopherols. In palm oil-derived tocotrienols, the composition been established as approximately 80% tocotrienols, 20% tocopherols. The investigation aims to evaluate the commercially available product (mixed tocotrienols, tocopherols and phytosterols) in the proposed effect on AF, and rather than isolated tocotrienols effect. We believe, the overall anti-oxidative effect may in part be contributed by the action of tocopherols with enhanced bioactivity from the much higher fraction of tocotrienols. The effects could potentially be synergistic to result in a clinically significant outcome. 1.  The authors aim at assessing the health-related quality of life in patients undergoing cardiac surgery; by definition a very sick group of elderly patients with usually many co-morbidities and with much drug intake. This aim is an extremely difficult matter involving many critical sources of definition, assessment tools (including SF-36), error and bias and I doubt that a meaningful result can be obtained.  We acknowledged the concerns regarding the use of health-related quality of life (HRQOL) instruments, such as the SF-36, to document the QOL of principally sick, elderly patients undergoing CABG surgery. In order to improve the validity and reliability of the SF-36, a Malay version of the SF-36 has been developed and tested. Results suggest that the Malay-SF-36 is a valid and reliable tool to measure the HRQOL of patients undergoing CABG surgery at the National Heart Institute (IJN) Kuala Lumpur, Malaysia. Both English and Malay validated versions of SF-36 will be administered to the patients before surgery,   Page 14 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 and Malay validated versions of SF-36 will be administered to the patients before surgery, at discharge, and at 6-weeks follow-up visit. A one-way repeated measure ANOVA analysis will be undertaken to assess changes in HRQOL across the three time points. 1.  According to the study protocol patients are required to take the capsules "before" and immediately following CABG. Before is defined as "immediately after randomization" on page 4. It is quite normal that even elective cardiac surgery can not always be planned precisely on the exact day and hour, must be delayed for medical or organizational reasons. Therefore there will be patients with various time intervals of capsule intake and supposedly different blood and tissue concentrations of the study medication!Capsules intake should last for approx 6 weeks until the first follow-up. Nearly all cases of AFib occur within the first 4 days postoperative. It is extremely rare to observe first-time AFib later than that. Although the long intake is ambitious how do the investigators control full compliance?  The National Heart Institute has a protocol that must be adhered known as CABG Clinical Pathway where patients should not stay longer than seven days before surgery. Though we initially planned that patients should at least be on study medication three days before surgery, after randomisation, we find it rather difficult to achieve since there will be patients who will be admitted just two days prior to surgery at our centre. And to ensure that all study patients have a rather stable and sustainable level of the study medication, we have made it compulsory that the study patients to take the study drugs a minimum of seven days prior to surgery. This could be achieved by randomising the patients upon referral to the Cardiothoracic Wards for surgery once they have consented to be enrolled. We would take their blood samples at the beginning of randomization, pre-op, on Day-4 post-op, at discharge and on the first follow-up at six-weeks. The variability in dosing duration will be noted and supported with pre-surgery blood levels of tocotrienols. We will control for the dosing duration during analysis, if necessary. However, due to the short half-life of tocotrienols, the small variation of dosing duration is not expected to have a significant impact in the blood levels of tocotrienols. And we also realised the fact that almost all AF occurred during the first four days post-operatively and our previous retrospective study at our centre showed that the median time for the development of AF was 45 hours post-CABG where the majority developed within three days post-surgery with the second day being most common. But we would like patients to consume the study medication until the first follow-up which is six-weeks after discharge and will try to ensure compliance by asking the research assistants to remind the patients intermittently at home and that their blood will be taken upon their follow-up visit and pill-counting will be done as well. The patients will be informed before discharge about the blood taking and pill counting to ensure that they have good compliance. Apart from first-time AF, we will continue to monitor the safety endpoints, including symptoms requiring medical treatment during the post-discharge supplementation of 6 weeks. This will allow us to evaluate the effects of continued supplementation in post-CABG recovery. 1.  Again, bioavailibility is an issue. The authors state on page 4 that bioavailibility of oral Tocotrienol is as low as 10-30%; how long before surgery must the drug be given to guarantee an effective availability in all patients of the non-placebo group? Is the trial controlled for interference with other drugs, malabsorption, possibly even gastric resections   Page 15 of 21 1.   F1000Research 2018, 7:215 Last updated: 08 MAY 2018 controlled for interference with other drugs, malabsorption, possibly even gastric resections or concomitant intestinal disease etc that may   prevent the study drug to be taken up effectively? The investigational product is formulated with a patented delivery system that provides 100-200% enhanced bioavailability, of mixed tocotrienols. If taken on fasted state, tocotrienols, as with all oil-soluble vitamins, have been shown to have poor and erratic absorption due to dependence on food status. However, this is resolved using the enhanced absorption delivery system. The subjects are proposed to initiate supplementation at least one week from surgery in order to ensure levels of tocotrienols in the blood and tissues. All concomitant medication and medical history will be noted upon enrolment and throughout the study to evaluate any possible interaction effects on the study outcomes. The medical history and plasma levels will help us determine if gastrointestinal issues or any drugs may cause significant impact on the absorption and final clinical outcome. 1.  Definitely exclude patients undergoing concomitant valve surgery (page 4)! Operating times are longer, the heart will be OPEN with further exposure to ROS-producing surfaces and valve pathologies, stretch and surgical maneuvers are all known to cause and influence postoperative AFib. Up to 7% of patients following aortic valve replacement require permanent pacemakers anyway! Again, I would also suggest not to mix on- and off-pump patients. Yes Prof, we will exclude the concomitant valve surgery patients and Off-pump CABG. This is reflected in the revised version of the Study Protocol. 1.  Holter ECG, very essential, will be monitored for the first 5 days, which makes sense. The study drug is given for approx 6 weeks. There will be no continuous ECG monitoring between discharge and follow-up. Unless the patient would be symptomatic and decides to see a physician who may be able to make a documentation, investigators are likely never to know whether there was an episode of AFib at any time between 1 and 6 weeks after surgery. Based on our previous retrospective study, only 15% of our patients developed AF after more than three days post-surgery. Yes, we will never know if they would develop AF at home unless they present themselves to our centre again. But considering all our patients were discharged in sinus rhythm, we do not anticipate that they would develop AF after being discharged from the hospital. 1.  I miss any information on potential PREoperative AFib assessment; or at least the exclusion of patients with AFib or other arrhythmias before surgery! Yes, all patients documented with AF pre-operatively will be excluded from the study. 1.  And authors should exclude patients with vitamin or antioxidant intake before randomization! Check for all medications as many are known to exert antioxidant side effects.  All pre-operative medications will be documented and patients on any form of anti-oxidant vitamins pre-operatively will be excluded. We will include this in our revised Study Protocol. 1.  Finally I would consider it extremely valuable to obtain blood samples and analyse various ROS and vitamin concentrations (or at least the antioxidant capacity) to proof that any clinical outcome is directly linked to a mechanism as hypothesized. Otherwise, any outcome can be spurious and by chance, in principle and by definition. Apart from anti-oxidative mechanisms, we postulate that tocotrienols may potentially inhibit the HMGCo-reductase to attenuate AF, similar to statins. We will consider including relevant blood biomarkers to support the findings of the study. However, due to transient   Page 16 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 relevant blood biomarkers to support the findings of the study. However, due to transient effect of oxidative stress and whether blood levels are representative of cardiac condition remains a concern. Competing Interests: No competing interests were disclosed. Referee Report 02 March 2018 doi:10.5256/f1000research.13999.r31123 Whady Armino Hueb 1, Paulo Cury Rezende 2  1 Heart Institute (InCor), University of São Paulo, São Paulo, Brazil 2 Heart Institute (InCor) Medical School, University of São Paulo, Sao Paulo, Brazil These reviewers suggest changing the title to: “Hypotheses, rationale, design, and methods for evaluation of a randomized controlled trial using Tocotrienol, an isomer of Vitamin E derived from palm oil, on the prevention of atrial fibrillation after coronary artery bypass grafting surgery”. We appreciate the opportunity to review the manuscript “A study protocol for a randomized controlled trial on the prevention of atrial fibrillation after coronary artery bypass grafting surgery using Tocotrienol, an isomer of Vitamin E derived from palm oil”. In this study, the authors aimed to test Tocotrienol, an isomer of Vitamin E derived from palm oil in the prevention of atrial fibrillation after myocardial revascularization surgery. The study is well designed, well written and well detailed in definitions and methods. However, some serious methodological failures should be avoided in order not to irreversibly compromise the study. It is known that myocardial revascularization surgery with the use of extracorporeal circulation adds non-physiological effects with direct damage to the myocardium. Such effects include: cardiac arrhythmias, low cardiac output, SIRS, thrombocytopenia, among others. The systemic inflammatory process is usually treated with corticosteroids. Thus, authors cannot ignore the differences between on-pump or off-pump. As suggestions, these reviewers recommend the following care in patient selection and data analysis. 1.  The surgery should be only on-pump or just off-pump.   2.  Patients with valvular heart disease cannot be included in patient selection. Aortic stenosis is often accompanied by myocardial hypertrophy, and Mitral insufficiency accompanies an increase in the left atrium. Both conditions facilitate the development of atrial fibrillation.   3.  The authors should include only patients with preserved left ventricular function. Ventricular   Page 17 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 3.  The authors should include only patients with preserved left ventricular function. Ventricular dysfunction is one of the leading causes of AF.   4.  The use of corticosteroids should be prespecified and, preferably, avoided because of its known anti-inflammatory actions.   5.  Timing from randomization and consequently beginning of Tocotrienol or placebo to surgery could be a relevant issue. A short time since medication start and surgery could compromise a possible significant effect of the drug on AF development.   6.  A Holter monitoring must be installed only in the first 72 hours. It is known that the most sensitive moment for the onset of AF is the immediate postoperative period. However, the use of the Holter monitoring for a longer period, even if uncomfortable, may be useful.   7.  In statistical analysis, the stepwise multiple logistic regression analysis should include atrium data such as size and/or volume, and procedure-related variables, such as use of inotropes and other vasoactive drugs, cross-clamp and cardiopulmonary bypass time. All of these variables might be associated with the occurrence of AF after revascularization procedures and should be included in the final model. On the other hand, overfitting should be avoided.   8.  In the multiple logistic regression model for the analysis of the factors influencing the change in quality of life after CABG, the authors should also include in the model angina data such as angina presence or frequency. In order to assess whether Tocotrienol is associated to better quality of life measures, it would be helpful if the authors control for angina relief after surgery. Is the rationale for, and objectives of, the study clearly described? Yes Is the study design appropriate for the research question? Yes Are sufficient details of the methods provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Author Response 30 Mar 2018 Ahmad Farouk Musa, Monash University Malaysia, Malaysia Thank you Prof Hueb and Prof Rezende. Your comments were highly beneficial for the improvement of this project. Since we have not started this project yet and waiting for CTX - Clinical Trial Exemption from NPRA - National Pharmaceutical Regulatory Agency, we would   Page 18 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 Clinical Trial Exemption from NPRA - National Pharmaceutical Regulatory Agency, we would include all the suggestions in the Study Protocol which will be resubmitted for Ethical Clearance. And to make reading easier, I'll copy the comments and reply immediately below. 1.  The surgery should be only on-pump or just off-pump. Since the National Heart Institute is an on-pump center, only a small minority of less than 5% were off-pump cases. This was based on the previous retrospective research that we have done on atrial fibrillation after CABG. Since we have not started this study yet and waiting for the CTX –Clinical Trial Exemption approval, we will include this in the Exclusion Criteria. Refer: A retrospective study on atrial fibrillation after coronary artery bypass grafting surgery at the National Heart Institute, Kuala Lumpur. F1000Research 2018; 7:164. 2.  Patients with valvular heart disease cannot be included in patient selection. Aortic stenosis is often accompanied by myocardial hypertrophy, and Mitral insufficiency accompanies an increase in the left atrium. Both conditions facilitate the development of atrial fibrillation. In our center, less than 10% of cases are combined surgery based on the same retrospective research cited above. And based on that the same paper we cited above, we noticed that there was a higher incidence of AF in patients with combined surgery although the association was not significant after adjustment. However since some paper noticed this correlation, we have no issue in classifying this under the Exclusion Criteria.   3.  The authors should include only patients with preserved left ventricular function. Ventricular dysfunction is one of the leading causes of AF. Similarly we also noticed a statistically significant difference in the previous study we cited above in between patients with poor left ventricular function and preserved left ventricular function. And since these patients only constitute less than 10% (for EF<30%) and less than 1% (for EF<20%) of our total patients load of about 1800 CABG patients per year, we will also exclude them from the study.   4.  The use of corticosteroids should be prespecified and, preferably, avoided because of its known anti-inflammatory actions. It is not customary for our surgeons especially the co-researchers to add corticosteroids in the pump. We will specify that all patients must not be given corticosteroids. Patients with a history of long-term corticosteroids treatment at screening will be excluded.   5.  Timing from randomization and consequently beginning of Tocotrienol or placebo to surgery could be a relevant issue. A short time since medication start and surgery could compromise a possible significant effect of the drug on AF development. This is an issue which we have thought and discussed for quite some time. We wanted to load the patient a minimum of two days prior to surgery or preferably three days. The Institute protocol of Clinical Pathway for CABG does not allow patients to stay longer than 72hours prior to surgery. So we will ensure that all patients will receive their tocotrienol right after randomization and not less than two   Page 19 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 patients will receive their tocotrienol right after randomization and not less than two days prior to surgery.   6.  A Holter monitoring must be installed only in the first 72 hours. It is known that the most sensitive moment for the onset of AF is the immediate postoperative period. However, the use of the Holter monitoring for a longer period, even if uncomfortable, may be useful. We do not normally use Holter for our patients post-operatively but we will monitor them via continuous ECG monitoring. Our previous study cited above showed that 95% of patients developed AF within the first 48hours. We will monitor them using continuous ECG monitoring for 72hours post-operatively. 7.  In statistical analysis, the stepwise multiple logistic regression analysis should include atrium data such as size and/or volume, and procedure-related variables, such as use of inotropes and other vasoactive drugs, cross-clamp and cardiopulmonary bypass time. All of these variables might be associated with the occurrence of AF after revascularization procedures and should be included in the final model. On the other hand, overfitting should be avoided. We will include data on atrium size and/or volume, use of inotropes and other vasoactive drugs, cross-clamp and cardiopulmonary bypass time in the study proforma and the final model. We will modify and strengthen the proforma we used when collecting the data for our retrospective research before. Using multiple logistic regressions, we will choose the most parsimonious model and avoid overfitting of the model. Calibration of the model will be evaluated with the Hosmer-Lemeshow goodness-of-fit test, and discrimination with the Areas Under the ROC Curve (AUC) will be used as a guide towards model fitting strategy.   8.  In the multiple logistic regression model for the analysis of the factors influencing the change in quality of life after CABG, the authors should also include in the model angina data such as angina presence or frequency. In order to assess whether Tocotrienol is associated to better quality of life measures, it would be helpful if the authors control for angina relief after surgery. Yes, the frequency of angina will be included and controlled in the multiple logistic regression models for factors influencing the change in the quality of life after CABG. Thank you, Professors. Your comments are highly appreciated.  Competing Interests: There is no competing interest. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias   Page 20 of 21 F1000Research 2018, 7:215 Last updated: 08 MAY 2018 You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact 

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