The efficacy of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in preventing recurrence of atrial fibrillation (AF) is controversial and their effects on inflammation and oxidative stress in this population are not known. This study examined the effects of high-dose marine n-3 PUFAs added to conventional therapy on the recurrence of AF and on markers of inflammation and oxidative stress. Patients with paroxysmal or persistent AF were randomized to n-3 PUFAs (4 g/day; n = 126) or placebo (n = 64) in a 2:1 ratio in a prospective, double-blind, placebo-controlled, parallel group study. The primary outcome was time to recurrence of AF. Secondary outcomes were changes in biomarkers of inflammation (serum interleukin [IL]-6, IL-8, IL-10, tissue necrosis factor alpha, monocyte chemoattractant protein-1, and vascular endothelial growth factor), N-terminal-pro-brain-type natriuretic peptide, and oxidative stress (urinary F 2 -isoprostanes). AF recurred in 74 patients (58.7%) randomized to n-3 PUFAs and in 30 patients (46.9%) who received placebo; time to recurrence of AF did not differ significantly in the 2 groups (hazard ratio 1.20; 95% confidence interval 0.76 to 1.90, adjusted p = 0.438). Compared with placebo, n-3 PUFAs did not result in clinically meaningful changes in concentrations of inflammatory markers, N-terminal-pro-brain-type natriuretic peptide or F 2 -isoprostanes. In conclusion, in patients with paroxysmal or persistent AF, treatment with n-3 PUFAs 4 g/day did not reduce the recurrence of AF, nor was it associated with clinically important effects on concentrations of markers of inflammation and oxidative stress. (Clinical trial registration number, NCT 00552084 .)
Few studies have specifically addressed the hypothesis that inflammation and oxidative stress play a direct role in atrial fibrillation (AF) and that treatment targeted at these mechanisms may be beneficial. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid and have antiarrhythmic, anti-inflammatory, and antioxidant effects. Thus, n-3 PUFAs are attractive as a potential therapy for AF. In vitro studies, as well as some epidemiological and interventional studies, support a role for n-3 PUFAs in the prevention of AF, but others do not. There is little information about the efficacy and effects on inflammation and oxidative stress of high doses of n-3 PUFAs added to conventional AF therapy. Accordingly, we designed this randomized, placebo-controlled trial to examine the effects of high-dose n-3 PUFAs added to conventional AF therapy on the recurrence of AF and on markers of inflammation and oxidative stress.
Methods
This was a prospective, double-blind, placebo-controlled, parallel group, 6-month study with randomization to n-3 PUFAs or placebo in a 2:1 ratio. The study was conducted at 4 centers in Nashville, Tennessee and at the Marshfield Clinic in Madison, Wisconsin and was approved by the institutional review boards of all participating organizations. All patients provided written informed consent.
We studied patients ≥21 years of age with a history of at least 2 occurrences of AF or atrial flutter, at least 1 of which was AF, and an electrocardiogram within the previous 12 months showing AF or atrial flutter. Patients were in sinus rhythm at randomization. Those patients taking antiarrhythmic drugs continued to do so. Key exclusion criteria included permanent AF, New York Heart Association class III or IV heart failure or Canadian Cardiovascular Society class III or IV angina pectoris, cardiothoracic surgery, myocardial infarction or stroke within the previous 3 months, reversible causes of AF, cancer, currently taking fish oil, allergy to fish, serious bleeding in the previous year, and dialysis or renal transplantation.
Participants received 4 g/day of n-3 PUFAs (Lovaza, GlaxoSmithKline, Research Triangle Park, North Carolina) or an identical corn oil placebo. Each 1 g n-3 PUFA capsule contained approximately 465 mg of EPA and 375 mg of docosahexaenoic acid. Randomization was performed according to a computer-generated permuted block scheme with 4 strata according to baseline antiarrhythmic therapy: (1) no antiarrhythmic therapy, (2) amiodarone, (3) class I antiarrhythmic drugs, and (4) sotalol or dofetilide.
Subjects took the first dose of study medication under direct observation and this was considered the time of randomization. Patients were seen at baseline and then at weeks 2, 4, 8, 12, 18, and 24. At each visit, adverse events were recorded and adherence monitored by capsule count and heart rhythm was monitored. Patients were provided with eCardio Post-Event Recorder which is a transtelephonic electrocardiographic monitoring device (eCardio Diagnostics, Houston, Texas) and made routine transmissions every 2 weeks, and in addition, if they had symptoms suggestive of arrhythmia. Electrocardiograms were coded, de-identified, and evaluated blindly by 2 cardiac electrophysiologists (DD and KM). Patients were followed for 6 months or until AF recurred. Patients who changed antiarrhythmic drug or dose during the study were withdrawn.
Venous blood was collected, separated immediately, and stored in aliquots frozen at −70°C until assayed. Serum interleukin (IL)-6, IL-8, IL-10, tissue necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1, vascular endothelial growth factor, and N-terminal-pro-brain-type natriuretic peptide (NTpBNP) were measured by multiplex enzyme-linked immunosorbent assay (Linco Research and/or Millipore Corp, St. Louis, Missouri) as previously described. Urine samples for F 2 – and F 3 -isoprostanes (IsoPs), markers of oxidative stress, were quantified using gas chromatography and mass spectroscopy and expressed as ng/mg creatinine.
The primary outcome was the time to documented recurrence of AF (symptomatic or asymptomatic). Secondary outcomes included changes in measures of inflammation and oxidative stress and their relation with recurrence of AF. Initial sample size estimations (n = 450) sought to provide not only excellent power for the primary and secondary outcomes but also for determining whether efficacy was related to changes in secondary outcomes. In 2010, because of slow enrollment, the sample size was recalculated to focus on the primary outcome and indicated that a sample size of n = 180 would provide 94% power to detect a difference of 30% in the recurrence rate of AF between groups. We estimated that 10% of patients would drop out and thus sought to enroll approximately 198 subjects.
Baseline demographic and clinical characteristics were examined using median and interquartile ranges or frequencies and proportions and compared using the Wilcoxon rank-sum test for continuous variables or chi-square test for categorical variables. The cumulative probability of not having a recurrence of AF was estimated using the Kaplan-Meier product limit method for n-3 PUFAs and placebo groups. Comparisons between the 2 survival curves were made using the log-rank test. For the primary outcome, time to recurrence of AF, we used a Cox proportional hazard regression model to evaluate the effect of treatment after adjusting for covariates. Clinically relevant covariates were selected a priori as potential risk factors for AF and included age, race, gender, duration of AF, coronary heart disease, congestive heart failure, and randomization stratification factor. Duration of AF was included as a flexible smooth parameter using splines. A prespecified analysis was performed in the subpopulation of patients who remained in the study 30 days after randomization. Subgroup analyses were adjusted for only age, gender, and race due to smaller sample sizes. Proportional hazard assumptions were checked using the Schoenfeld residuals method. Hazard ratios (HRs) and their 95% confidence intervals (CIs) are reported. For biomarkers, we calculated HRs per increase in interquartile range. We used intent-to-treat as the primary analysis.
To assess differences between n-3 PUFAs and placebo groups for the secondary outcomes (biomarkers), we performed analysis of covariance multiple linear regression analysis using the end-of-study value of the response variable with the baseline value as a covariate; additional covariates were as described for the primary outcome.
Additionally, we evaluated whether baseline or end-of-study concentration of biomarkers were differentially associated with recurrence of AF by treatment status. We used Cox proportional hazard models and a cross-product term between biomarker (baseline or end-of-study) and treatment status. In adjusted analyses, covariates included were limited to age, race, and gender. Biomarkers were natural log transformed in regression analyses. All statistical analyses were performed using open source R statistical software version 3.0.2 ( http://www.r-project.org ).
Results
From December 2007 to December 2012, 241 patients were enrolled and 190 (178 at Vanderbilt and 12 at other sites) ultimately randomized to n-3 PUFAs (n = 126) or placebo (n = 64; Figure 1 ). The 2 groups were well matched with regard to age, race, gender, and baseline antiarrhythmic therapy, but coronary heart disease was more frequent in the control group ( Table 1 ). Of the 190 patients randomized, 173 (91%) completed the study.
| Baseline Characteristics | n-3-PUFA (N=126) | Placebo (N=64) | P value |
|---|---|---|---|
| Age (years) | 62 (12) | 61 (11) | 0.57 |
| Women | 59 (47%) | 22 (34%) | 0.10 |
| White | 119 (94%) | 61 (95%) | 0.29 |
| Body mass index (kg/m 2 ) | 30.1 (7.2) | 31.9 (7.3) | 0.04 |
| Hypertension | 78 (62%) | 44 (69%) | 0.35 |
| Diabetes mellitus | 23 (18%) | 13 (20%) | 0.73 |
| Coronary heart disease | 9 (7%) | 14 (22%) | 0.003 |
| Prior myocardial infarction | 7 (6%) | 8 (12%) | 0.09 |
| Heart failure | 14 (11%) | 13 (20%) | 0.09 |
| Lone atrial fibrillation | 20 (16%) | 7 (11%) | 0.36 |
| Duration of atrial fibrillation (months) | 50 [13,111] | 54 [22,112] | 0.32 |
| Ejection fraction (% ∗ ) | 58 ± 7 | 57 ± 6 | 0.31 |
| Left atrial size (mm ∗ ) | 41 ± 7 | 43 ± 7 | 0.13 |
| Therapy | |||
| No anti-arrhythmic therapy | 46 (37%) | 21 (33%) | 0.91 † |
| Amiodarone | 12 (10%) | 8 (12%) | |
| Class I agent | 30 (24%) | 15 (23%) | |
| Sotalol or dofetilide | 38 (30%) | 20 (31%) | |
| Beta blocker | 71 (56%) | 41 (64%) | 0.31 |
| Statin | 45 (36%) | 28 (44%) | 0.28 |
| ACE inhibitor | 33 (26%) | 16 (25%) | 0.86 |
| Angiotensin receptor blocker | 22 (17%) | 6 (9%) | 0.14 |
| Warfarin | 63 (50%) | 28 (44%) | 0.41 |
∗ Measurements for left atrial size and ejection fraction were available in 152 and 169 patients, respectively.
† P-value represents comparison of the prevalence of 4 anti-arrhythmic therapies (no therapy, amiodarone, class I agent, and sotalol or dofetilide) in the two groups.
AF recurred in 74 (59%) of 126 patients randomized to n-3 PUFAs and in 30 (47%) of those who received placebo; the majority (81 of 104 recurrences, 78%) were symptomatic. There was no statistically significant difference in the primary outcome of time to recurrence of AF among patients randomized to receive n-3 PUFAs or placebo (HR 1.20, 95% CI 0.79 to 1.84, p = 0.39; Figure 2 ). Statistical adjustment for age, race, gender, randomization stratum, coronary artery disease, congestive heart failure, and duration of AF did not alter the findings materially (HR 1.20, 95% CI 0.76 to 1.90, p = 0.438). There was no statistically significant treatment effect in patients who remained in the study 30 days after randomization (HR 1.65, 95% CI 0.88 to 3.09, p = 0.119 age, race, and gender adjusted).
Concentrations of cytokines and NTpBNP ( Table 2 ) were similar to those we previously reported in a different group of patients with AF and higher than what would be expected in the general population ; however, n-3 PUFA therapy did not affect concentrations of IL-6, IL-8, IL-10, monocyte chemoattractant protein-1, or NTpBNP significantly ( Table 2 ). There were small differences in TNF-α and vascular endothelial growth factor concentrations of marginal significance that were not statistically significant after adjustment for covariates (p >0.05 for both). Concentrations of markers of inflammation and NTpBNP at baseline were not significantly associated with time to recurrence of AF (all p values >0.05), although there was a differential association between higher levels of IL-6 at baseline and time to recurrence of AF between subjects who received n-3 PUFAs and those that did not (p interaction = 0.019; n-3 PUFAs HR 1.23, 95% CI 0.94 to 1.59; placebo HR 0.61, 95% CI 0.35 to 1.08, adjusted for age, gender, and race).
| Biomarker | Baseline | End of Study | P value ∗ | P † value | ||
|---|---|---|---|---|---|---|
| n3-PUFAs | Placebo | n3-PUFAs | Placebo | |||
| IL-6 (pg/ml) | 2.8 (1.5-4.4) | 2.5 (1.3-4.5) | 2.2 (1.3-4.0) | 2.4 (1.5-4.7) | 0.212 | 0.77 |
| IL-8 (pg/ml) | 7.2 (5.5-10.0) | 5.6 (4.1-8.4) | 6.8 (5.0-9.2) | 5.8 (3.8-8.0) | 0.792 | 0.41 |
| IL-10 (pg/ml) | 2.0 (0-5.9) | 1.5 (0-8.4) | 1.4 (0-5.3) | 1.6 (0-6.3) | 0.634 | 0.92 |
| TNF-α (pg/ml) | 8.5 (6.0-11.2) | 7.8 (6.5-9.9) | 8.0 (5.5-10.9) | 7.6 (6.2-9.8) | 0.049 | 0.10 |
| MCP-1 (pg/ml) | 148 (97-210) | 142(110-176) | 131 (102-188) | 129 (104-172) | 0.941 | 0.62 |
| VEGF (pg/ml) | 95 (35-240) | 103(30-189) | 90 (33-228) | 69 (18-145) | 0.046 | 0.33 |
| NTpBNP (pg/ml) | 124.0 (0.35-386.0) | 157.5 (9.2-441.0) | 192 (11-723) | 288 (27-752) | 0.768 | 0.85 |
| Urinary F 2 -IsoPs (ng/mg Cr) | 1.41 (0.91-2.06) | 1.29 (1.05-1.83) | 1.25 (0.87-1.81) | 1.08 (0.83-1.98) | 0.991 | 0.95 |
| Urinary F 3 -IsoPs (ng/mg Cr) | 0.134 (0.089-0.23) | 0.141 (0.085-0.177) | 0.31 (0.19-0.53) | 0.12 (0.06-0.18) | <0.0001 | <0.0001 |
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