Ageing improves the antioxidant response against postoperative atrial fibrillation: a randomized controlled trial

Abstract

OBJECTIVES

Extracorporeal circulation is associated with ischaemia–reperfusion-induced oxidative stress and atrial fibrillation (AF). Ageing is the major risk factor for this arrhythmia. Reinforcing the antioxidant defence system would be expected to protect the myocardium against ischaemia–reperfusion damage. We studied the effect of antioxidant reinforcement and ageing on the occurrence of postoperative AF.

METHODS

A randomized, double-blind, placebo-controlled trial was performed in patients scheduled for cardiac surgery with extracorporeal circulation. Supplementation with omega-3 polyunsaturated fatty acid (PUFA) and vitamins C and E, or placebo was administered to 152 patients divided by age: young patients (30–59 years) and old patients (60–80 years). For the detection of AF, we used 12-lead and Holter EKGs. Antioxidant enzyme activity was determined in atrial tissue. Oxidative stress was assessed through plasma malondialdehyde levels.

RESULTS

Supplemented patients over 60 years old showed higher glutathione peroxidase activity than control patients in the same age range. There was no difference between the supplemented and placebo groups in patients under 60 years old. The decrease in the incidence of postoperative AF in supplemented patients was more marked in the older patients than in the younger ones.

CONCLUSIONS

Since the antioxidant supplementation with n-3 PUFAs and vitamins C and E resulted in a more marked reduction of postoperative AF in older patients, it could be suggested that the efficacy of this therapy improves with ageing.

INTRODUCTION

Postoperative atrial fibrillation (AF) is one of the most common occurrences during follow-up after coronary artery bypass graft surgery and other surgical procedures performed with extracorporeal circulation. The incidence of postoperative AF in patients undergoing cardiac surgery is between 20 and 60% [1], and advanced age seems to be the most powerful risk factor for its development. Furthermore, older age has consistently predicted a higher incidence of postoperative AF, being increased by at least 50% per decade of older age [2]. On the other hand, postoperative AF involves an increased risk of other major complications after cardiac surgery, while also prolonging the length of stay in the hospital and increasing costs.

A role of oxidative stress in the pathogenesis and perpetuation of AF [3] has been suggested. The reintroduction of molecular oxygen into the ischaemic tissue upon reperfusion has been proved to lead to the excessive formation of reactive oxygen species (ROS). Increased ROS may overwhelm the antioxidant defence capacity of tissues, leading to cell damage. This myocardial oxidative injury can finally lead to an increased susceptibility to postoperative AF [3].

The currently available pharmacological preventive therapy of postoperative AF has demonstrated suboptimal results and novel therapies have arisen. Thus, antioxidant intervention has emerged as a preventive strategy against postoperative AF [4]. The n-3 polyunsaturated fatty acids (PUFAs) have been shown to protect against reperfusion arrhythmias [5]. The mechanism by which this protection occurs has not been fully elucidated; however, it could be related to their effects on membrane fluidity and the ion transport through myocardial cell membranes [6]. This probably results from non-hypoxic preconditioning. Nevertheless, different clinical trials have reported controversial results in the use of n-3 PUFAs [7–9]. The administration of antioxidants, such as vitamins E and C, known to potentiate each other in order to counteract the effect of ROS has been used. Thus, the biological effects of these vitamins could contribute not solely to the scavenging of ROS, but also to diminish their formation.

In older people, oxidative stress has greater importance in the genesis of pathological events, such as AF, as it has a direct relationship with ageing [10]. Moreover, it has been reported that patients over 60 years old present higher levels of lipid peroxidation, a lower enzymatic activity of glutathione peroxidase (GSH-Px) and a decreasing in the total antioxidant capacity of plasma [11]. As the preconditioning effect of n-3 PUFAs is based on the induction of sub-lethal ROS concentrations [12], enough to enhance the enzymatic antioxidant defences, it is reasonable to assume that older patients would present a better antioxidant response than younger ones. Paradoxically, the increased vulnerability of older patients to an oxidative challenge could be expected to favour the antioxidant response through the generation of sub-lethal oxidative damage. This study was designed to test the hypothesis that antioxidant reinforcement through n-3 PUFAs and antioxidant vitamins reduces the postoperative AF incidence, and this response would be more marked in patients older than 60 years old.

MATERIALS AND METHODS

Study design, subjects and methodology

The primary endpoint of the study was the occurrence of AF at any time after surgery, until hospital discharge. The secondary endpoints included biochemical evidence of oxidative stress in plasma and atrial tissue samples.

This study is a randomized, double-blind, placebo-controlled trial conducted in 248 patients admitted to cardiac surgery with extracorporeal circulation, between July 2007 and February 2009 in the Cardiovascular Department of the University of Chile Clinical Hospital (Clinical Trial Registration Information: http://www.controlled-trials.com/Unique Identifier: ISRCTN45347268). Valve, coronary artery bypass graft and mixed surgeries were included. The enrolled patients were between 30 and 80 years of age with sinus rhythm. The exclusion criteria included previous cardiac surgery, chronic or paroxysmal AF, other preoperative arrhythmias, advanced pulmonary and hepatic disease or chronic renal failure (serum creatinine >2.0 mg/dl). According to these criteria, 171 patients were selected, and 152 patients were analysed (Figs 1 and 2). The participants were grouped into those older than 60 years or younger. Both age groups were randomized 7 days before surgery to a placebo or supplemented group. Randomization was done centrally and was unstratified, block-based and computer-generated. Treatment was initiated immediately after randomization and consisted of daily doses of n-3 PUFAs (2 g/d) with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as ethyl esters in an average 1:2 EPA/DHA ratio, as previously described [9]. Two days before surgery, vitamin C (1 g/d) plus vitamin E (400 IU/d) were added. The whole supplementation regime continued until the discharge from hospital. The placebo group received an equal number of capsules of identical size and colour, containing caprylic/capric triglyceride (825 mg per capsule), inert microgranules (500 mg) and vegetable oil (400 mg) replacing n-3 PUFAs, vitamin C and vitamin E, respectively. The products were provided by Procaps (Colombia) and Gynopharm CRF (Chile) Laboratories. Patients enrolled in the study were hospitalized 2 days before the surgery, following 5 days of n-3 PUFA supplementation through an ambulatory administration.

Figure 1:

Flow chart of the patients under 60 years of age screened and enrolled in the study protocol.

Figure 2:

Flow chart of the patients over 60 years of age screened and enrolled in the study protocol.

Blood samples and atrial tissue

Five blood samples were obtained throughout the protocol: just before the beginning of the supplementation (Day −7), just before the administration of antioxidant vitamins (Day −2), before the surgery (Day 0), 6–8 h after surgery (Day +1) and 5 days after surgery (Day +5). Samples were stored in containers cooled with disodium EDTA (4 mmol/l) and centrifuged at 3000 g for 10 min. Subsequently, blood cells underwent haemolysis with distilled water, and the red blood lysates and plasma supernatants were stored at −70°C. Right atrial appendage samples (∼400 mg) were obtained immediately before starting extracorporeal circulation, and frozen in liquid nitrogen and stored at −70°C. Biochemical determinations in atrial tissue from 29 patients (15 supplemented and 14 placebos) were not possible due to medical reasons or insufficient samples.

Oxidative stress-related parameters

1. Malondialdehyde (MDA): The determination of MDA was performed in plasma and atrial tissue. It was assayed by a high-performance liquid chromatographic method for measuring the MDA-thiobarbituric acid adducts using a fluorometric detection. The compound 1, 1′,3,3′- tetramethoxy-propane was used as the external standard and the results were expressed as μmol/l or μmol/mg protein, for plasma and atrial tissue, respectively.

2. Activity of antioxidant enzymes (measured on red blood cells and atrial tissue): Determinations of Cu-Zn superoxide dismutase, catalase and GSH-Px activities were performed as published elsewhere.

Postoperative atrial fibrillation detection

Continuous electrocardiogram (EKG) monitoring after surgery was maintained in the cardiovascular intensive care unit 24–48 h after finalizing surgery, to detect any new onset AF. A Holter device was used until 4 days post-operation, while a 12-lead EKG was employed in patients with arrhythmia symptoms between the removal of the Holter monitoring device and discharge. Postoperative AF lasted at least 1 min, and was confirmed by a monitoring system, EKG-Holter device or a 12-lead EKG if any symptomatic episode that required intervention occurred.

Ethics

The study followed the Helsinki Declaration of the World Medical Association (2000) and, at the same time, the research protocol was approved by the ethics committees of the participating institutions (the University of Chile’s Clinical Hospital and Faculty of Medicine). All the patients gave their written informed consent before any procedure was applied.

Statistical analysis

Results were expressed as means ± standard deviation (SD) for continuous variables. On the one hand, normally distributed variables were compared by a t-test. On the other hand, non-normally distributed variables were expressed by the median and interquartile ranges and compared with the two-sample Wilcoxon rank-sum test. Differences in continuous variables between and within groups were assessed through the analysis of variance (ANOVA), followed by Bonferroni's or Tukey's post hoc test as appropriate. For the frequency comparison, Fisher's exact test was used. A time-to-first-event analysis was calculated through the Kaplan–Meier method and compared by a log-rank test. Cox proportional hazards models with the Breslow method for ties were used to calculate the hazard ratio in the survival curves between the groups. P < 0.05 was considered statistically significant. Statistical analysis was performed using Microsoft Excel, Stata 10.0 for Windows and Graphpad Prism 5.0.

Sample size and power calculation

The sample size calculation was performed on the basis of the primary endpoint of the trial. The assumptions used for this purpose included an expected 35 and 10% occurrence of AF in the placebo and supplemented groups, respectively. The sample size calculation was based on the differences in the mean between two groups with an equal sample size, prespecified 5% alpha error and 80% power. The resulting sample size was 71 patients in each treatment group (Win Episcope 2.0 for Windows). The power estimate to detect the differences of the primary endpoint between the groups, as well as the time-related evolution of the secondary endpoints within a group and the treatment effect were determined by the CHANGE method of Stata software.

RESULTS

Clinical features

No significant differences were found between both groups regarding demographic characteristics, comorbidities, pharmacological treatment and surgery data (Table 1).

Table 1:

The demographic characteristics, electrocardiographic parameters, comorbidities, clinical and pharmacological treatments in 144 patients subjected to cardiac surgery with extracorporeal circulation

	Over 60 years			Under 60 years
	Placebo	Supplemented	P-value	Placebo	Supplemented	P-value
	(n = 34)	(n = 35)		(n = 38)	(n = 37)
Characteristics
Age, year	65 (7)	66 (6)	0.74	52 (29)	53 (29)	0.31
Sex (males/females)	28/6	28/7	1.0	33/5	31/6	0.75
Body mass index (kg/m2)	25.1 (0.3)	25.2 (4.4)	0.66	24.6 (3.2)	24.5 (4)	0.43
Heart rate (beats/min)	69 (12)	73 (12)	0.45	74 (10)	77 (9)	0.08
Left ventricular ejection fraction, %	66 (10.8)	67.3 (7.4)	0.07	66.4 (8.1)	67.1 (7.5)	0.42
Left atrial AP dimension, mm	49.3 ± 3.79	47.8 ± 5.7	0.20	50.9 ± 4.32	49.2 ± 5.1	0.12
Comorbidities
Systemic hypertension, n (%)	10 (28.6)	12 (32.4)	0.8	11 (27.5)	12 (30.0)	1.00
Diabetes mellitus, n (%)	19 (54.2)	23 (62.2)	0.63	16 (40.0)	20 (50.0)	0.5
Hypercholesterolaemia, n (%)	19 (54.2)	18 (48.6)	0.65	16 (40.0)	16 (40.0)	1.00
Smoking history, n (%)	11 (31.4)	13 (35.1)	0.81	12 (30.0)	13 (32.5)	1.00
Perioperative features
Revascularization, n (%)	17 (48.5)	16 (43.2)	1.00	20 (50.0)	20 (50.0)	1.00
Valve replacement, n (%)	10 (28.5)	11 (29.7)	0.9	12 (30.0)	9 (22.5)	0.62
Mixed aetiology, n (%)	8 (22.8)	10 (27.0)	0.89	8 (20.0)	11 (27.5)	0.83
Cross-clamp time, min	74 (33.0)	82 (29.0)	0.99	84 (42.0)	82 (54.0)	0.13
CPB time, min	95 (19.0)	91 (16.5)	0.66	82 (20.0)	87 (17.0)	0.71
Pharmacological treatments
Aspirin, n (%)	18 (51.4)	27 (72.9)	0.09	27 (67.5)	26 (65.0)	1.00
Atorvastatin, n (%)	16 (45.7)	15 (40.5)	0.81	14 (35.0)	12 (30.0)	0.81
Other statins, n (%)	6 (17.1)	1 (2.7)	0.06	3 (7.5)	1 (2.5)	0.62
ACE inhibitors, n (%)	20 (57.1)	22 (59.5)	1.00	22 (55.9)	29 (72.5)	0.16
ARBs, n (%)	10 (28.6)	10 (27.0)	1.00	9 (22.5)	8 (20.0)	1.00
Diuretics, n (%)	12 (34.3)	11 (29.7)	0.8	10 (25.0)	12 (30.0)	0.8
β-Blockers, n (%)	20 (57.1)	20 (54.1)	0.82	20 (50.0)	19 (47.5)	1.00
Calcium channel blockers, n (%)	7 (20.0)	5 (13.5)	0.54	1 (2.5)	2 (5.0)	1.00
Nitrates, n (%)	3 (8.6)	5 (13.5)	0.71	1 (2.5)	2 (5.0)	1.00
Insulin, n (%)	2 (5.7)	2 (5.4)	1.00	1 (2.5)	0 (0.0)	1.00
Sulfanilylurea, n (%)	5 (14.3)	6 (16.2)	1.00	2 (5.0)	4 (10.0)	0.68
Biguanides, n (%)	8 (22.9)	10 (27.0)	0.79	10 (25.0)	11 (27.5)	1.00

The results are expressed as means + SEM for continuous variables and percent for categorical parameters. The quantitative continuous variables are expressed as mean ± SD if they present a normal distribution, or median (interquartile range) if they do not present a normal distribution. The categorical variables are expressed in frequency (%).

AP: antero-posterior; CPB: cardiopulmonary bypass; ACE: angiotensin I converting enzyme; ARBs: angiotensin II receptor blockers.

Postoperative atrial fibrillation

Patients under 60 years of age

Twelve out of 40 (30.0%) patients who did not receive supplementation suffered from postoperative AF, whereas five out of 40 (12.5%) patients who received n-3 and antioxidant vitamin supplementation experienced an event. Nevertheless, the difference in the survival curve was not statistically significant (Fig. 3).

Figure 3:

The Kaplan–Meier graph estimating the occurrence of postoperative AF following an anastomosis by cardiac surgery with extracorporeal circulation until hospital discharge. The patients over 60 years of age receiving the supplementation show a low incidence of postoperative AF. A log rank-test was used.

Patients over 60 years of age

Ten out of 35 patients (28.5%) who did not receive supplementation experienced postoperative AF, whereas only two out of 37 (5.4%) patients who received supplementation experienced an event. In contrast to the patients under 60 years of age, there are significant differences (log-rank P = 0.0076) in the incidence of postoperative AF between both the groups, represented in the Kaplan–Meier plots, which illustrate the occurrence of postoperative AF in the study groups during hospitalization (Fig. 3).

Glutathione peroxidase activity

At the time of the surgery, GSH-Px activity in atrial tissue was higher in patients over 60 years who received the supplementation compare with the other groups (P < 0.05) (Fig. 4).

Figure 4:

The enzyme activity of GSH-Px in the supplemented or placebo groups on the day of surgery (Day 0). The results are expressed as means ± SEM. For the analysis ANOVA and Bonferroni's post test were used.

Oxidative stress-related biomarkers and post-operative atrial fibrillation

Patients with postoperative AF showed higher plasma MDA levels after surgery and lower atrial GSH-px activity compared with those who did not present postoperative AF (P < 0.01) (Figs 5 and 6).

Figure 5:

Lipid peroxidation assessed by the MDA concentration in the plasma of patients with (white bars), or without postoperative AF (grey bars) 1 day after surgery. The results are expressed as means ± SEM. For the analysis a Wilcoxon rank-sum test was used.

Figure 6:

An antioxidant defence system represented by the enzyme activity of GSH-Px in the patients with (white bars), or without postoperative AF (grey bars) 1 day after surgery. The results are expressed as means ± SEM. For the analysis a Wilcoxon rank-sum test was used.

DISCUSSION

The present study shows, for the first time, that older patients have a more marked antioxidant response to omega-3 plus vitamins C and E supplementation as a preventive treatment against postoperative AF.

It is of interest that despite the documented clinical impact of postoperative AF, the lack of mechanistic understanding of its occurrence has contributed in part to explain why there are no uniformly accepted treatments for its prevention. The pathophysiological mechanisms of postoperative AF are influenced by preoperative, intraoperative and postoperative factors. In on-pump cardiac surgery, the unavoidable occurrence of an ischaemia–reperfusion event leads to the formation of ROS, causing oxidative stress and a systemic inflammatory response [13]. Moreover, some studies have demonstrated the implication of oxidative stress within the atrial tissue during AF, suggesting a potential role in the remodelling phenomenon [14].

The current finding, showing decreased postoperative AF incidence, is in agreement with previous data found using the antioxidant protocol [7–9, 15]. Interestingly, we found that the supplementation caused a more marked decrease in the relative risk of experiencing postoperative AF in patients over 60 years than in younger patients. Following supplementation, older patients had a five times lower risk of developing AF after cardiac surgery, thus accounting for the greater effectiveness of the intervention in this group. This result could be explained on the basis of a different baseline redox status and antioxidant defence profile. In fact, it has been observed that patients over 60 years of age present higher levels of lipid peroxidation, lower enzymatic activity of GSH-Px and decreased total antioxidant plasma capacity [11]. Consequently, ageing would allow more easily the generation of a sub-lethal oxidative challenge, which can induce the enzymatic antioxidant machinery. In contrast, this response would be attenuated in younger patients due to their higher activity of basal antioxidant defenses, consistent with the observed lower effectiveness of the supplementation treatment.

Indeed, in the present study, we observed on Day 0 an increased GSH-Px activity only in patients older than 60 years of age who received the supplementation. This result could be explained taking into account that patients under 60 years of age can handle n-3 PUFAs-induced oxidative stress more efficiently. Consequently, this effect decreases the extent of the stimulus able to induce an adaptive response aimed at preventing ischaemia/reperfusion injury secondary to extracorporeal circulation. In contrast, older patients who did show this adaptive response are expected to have a lesser vulnerability to the oxidative damage caused by surgery.

Atrial GSH-Px activity seems to be an important predisposing factor for the development of postoperative AF, as patients with higher activity showed lower incidence of this arrhythmia. Moreover, those patients who presented with postoperative AF also showed higher plasma MDA levels, a lipid peroxidation by-product.

The limitations of this study could be the low doses of vitamin C, not enough for ROS scavenging, but only for decreasing its production. In addition, these data do not allow us to know whether a longer time of n-3 exposure would result in further improvement of the anti-arrhythmic response.

In summary, while patients under 60 years of age seem to be capable of controlling the effects of ROS excess, older patients paradoxically undergo an enhanced antioxidant response despite their lower baseline antioxidant defences. In fact, higher enzymatic antioxidant actitvity and lower lipid peroxidation biomarkers turned out to be correlated with lower postoperative AF occurrence. It is suggested that this design could be actively tested in further large-scale trials for its application as a suitable way of improving the outcome of patients subjected to cardiac surgery, thus reducing the associated comorbidities, the length of stay in the hospital and overall cost.

FUNDING

We thank the Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT; grant number 1070948), Procaps Laboratory (Colombia) and Gynopharm CFR Laboratory (Chile) for their financial support of this study.

Conflict of interest: none declared.