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Original Research Communications |
r Christensen1 From the Department of Nephrology, Aalborg Hospital, Aalborg, Denmark; the Department of Medicine, Hjørring/Brønderslev Hospital, Hjørring, Denmark; and Medi-Lab, Copenhagen, Denmark.
2 Supported by The Medical Research Foundation of the County of Northern Jutland, and Aalborg Stifts Julelotteri. LUBE A/S of Hadsund, Denmark, provided the Pikasol capsules.
3 Address reprint requests to J Dyerberg, Medi-Lab, 5–7 Adelgade, PO Box 2, 1001 Copenhagen K, Denmark.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: The objectives of this study were to 1) examine the correlations between the content of fatty acids in blood cell membranes (platelets and granulocytes) and HRV in healthy subjects, and 2) assess the effect on HRV of dietary intervention with n-3 PUFA in different doses.
Design: Sixty healthy volunteers (25 women and 35 men) were randomly assigned to 3 treatment groups in a double-blind design. Subjects received a daily supplement of either 6.6 g n-3 PUFA, 2.0 g n-3 PUFA, or placebo (olive oil). A 24-h Holter recording was obtained for each subject before supplementation and after 12 wk of supplementation; the 24-h HRV was then related to the content of fatty acids in granulocytes and platelets.
Results: Before supplementation, positive correlations were observed in men between the content of docosahexaenoic acid in cell membranes and HRV indexes (r = 0.50, P < 0.01), whereas such correlations were not found in women. Dietary intervention revealed a dose-dependent effect of n-3 PUFA on HRV in men, whereas no effect was found in women.
Conclusion: The study showed a beneficial effect of n-3 PUFA on HRV in healthy men, suggesting an antiarrhythmic effect of n-3 PUFA. No such effect was observed in healthy women.
Key Words: n-3 fatty acids • omega-3 fatty acids • fish oil • sudden cardiac death • cardiac arrest • ventricular arrhythmias • heart rate variability • granulocytes • platelets
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We (11) reported in 1996 that dietary n-3 PUFA significantly increased heart rate variability (HRV) in human survivors of myocardial infarction (MI). This points to an antiarrhythmic effect of n-3 PUFA, because low HRV is strongly associated with increased mortality and arrhythmic events in post-MI patients (12–15). Furthermore, we found the content of n-3 PUFA in cell membranes to be positively correlated with HRV in these patients (16). This finding was reproduced in another group of patients with increased risk of SCD, namely patients with chronic renal failure treated with maintenance hemodialysis (17).
The aim of the present study was to examine the possible correlation between fatty acids in blood cell membranes and HRV in healthy subjects. Furthermore, the subjects were randomly assigned to dietary supplementation with either n-3 PUFA in 2 different doses or an olive oil placebo to investigate the possible effect of dietary supplementation with n-3 PUFA on HRV.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dietary supplements
In a double-blind design, the subjects were randomly assigned to 3 groups. Group 1 received daily supplements of 6.6 g n-3 PUFA as 3.0 g eicosapentaenoic acid (EPA) and 2.9 g docosahexaenoic acid (DHA) [10 capsules of Pikasol; LUBE A/S, Hadsund, Denmark, a reesterified triacylglycerol (EPAX 5500) Pronova Biocare A/S, Sandefjord, Norway]. Group 2 received supplementation with 2.0 g n-3 PUFA daily (0.9 g EPA and 0.8 g DHA, in 3 capsules of Pikasol) and 7 capsules of placebo (olive oil). Group 3 received 10 capsules of placebo daily. The subjects took 3 capsules in the morning and 7 capsules with the evening meal, taking the capsules from 2 different boxes to maintain the blinding. The supplements were given for 12 wk.
Heart rate variability analyses
A 24-h Holter recording was obtained for each subject on a flash card by using a 3-channel digital monitor (Diagnostic Monitoring, Santa Ana, CA). This was done before and after 12 wk of dietary supplementation. The recordings were analyzed by using a commercially available software program, DIAGNOSTIC MONITORING (Diagnostic Monitoring). The following time-domain HRV variables were analyzed: 1) RR, the mean of all normal R-R intervals during the 24-h recording; 2) SDNN, the SD of all normal R-R intervals during the 24-h recording; 3) SDNNindex, the mean of the SDs of all normal R-R intervals for all 5-min segments of the 24-h recording; 4) SDANNindex, the SD of the mean of R-R intervals measured in successive 5-min periods; 5) pNN50, the percentage of successive R-R interval differences 
50 ms; 6) RMSSD, the square root of the mean of the sum of the squares of differences between adjacent intervals. QRS complexes with abnormal morphology were excluded from HRV analysis and the recordings were processed without knowledge of other subject variables.
Fatty acid analysis
Blood samples were drawn in the fasting state before the Holter recording, both before and after 12 wk of dietary supplementation. Granulocytes and platelets were isolated and their fatty acid composition was measured by gas chromatography and expressed as a percentage of total fatty acid content, as described previously (18).
Statistical analysis
Paired t tests were used to test for any differences within the groups before and after supplementation. Differences among the groups (placebo, low-dose n-3 PUFA, and high-dose n-3 PUFA) were tested by one-way analysis of variance. If significant differences were found when comparing the 3 groups, Tukey's test was applied. Correlations between the cellular concentrations of fatty acids and HRV variables were tested by simple linear regression analysis using the SPSS software package, version 6.0 (SPSS, Chicago). Bonferroni-corrected correlations were used if multiple correlations were made. Furthermore, we used Lowess' locally weighted regression smoothing, which is an iterative weighted least-squares method (19). A P value <0.05 (two-tailed) was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The 3 intervention groups were comparable before supplementation regarding HRV indexes, cellular content of fatty acids (Table 2), and plasma lipids and lipoproteins.
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). Although these differences were significant, they were rather small. There were distinct differences in fatty acid contents between platelets and granulocytes in both women and men, particularly for 18:1n-9 and 20:4n-6. However, significant correlation coefficients in the range of 0.25–0.85 were found between the contents of the fatty acids in granulocytes and in platelets, except for 18:1n-9, for which no correlation was found (r = 0.00). The highest correlation coefficient was found for EPA (r = 0.85, P < 0.001).
Fatty acids in granulocytes and HRV in women and men
In women, significant positive correlations were found between the content of 20:4n-6 in granulocytes and the 6 HRV indexes used. There was also a significant positive correlation between the EPA content of granulocytes and RR in women (r = 0.50, P < 0.01).
In men, a significant positive correlation was found between DHA in granulocytes and all the HRV indexes, with correlation coefficients ranging from 0.38 (P < 0.05) to 0.50 (P < 0.01). The correlation in men before supplementation between DHA in granulocytes and the clinically important parameter, SDNN, is shown in Figure 1. In this figure, it is apparent that Lowess regression smoothing (dotted line) showed a dose-response relation between 22:6n-3 and SDNN with a steep rise in SDNN when the content of 22:6n-3 in granulocytes was between 1.3% and 2.1%. The correlation coefficient in this interval was 0.68 (P < 0.001).
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In men, there were significant positive correlations between the content of DHA in platelets and RR, SDNN, SDNNindex, SDANNindex, and pNN50, with correlation coefficients ranging from 0.32 (P < 0.05) to 0.46 (P < 0.01).
DHA and HRV in men
The group of 35 men was dichotomized according to median content of DHA in platelets and in granulocytes and the HRV indexes were calculated for each subgroup. In Table 3 it is apparent that the men with the highest content of DHA in their cell membranes also had the highest HRV indexes.
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. It is apparent that when results for women and men were analyzed together, dietary n-3 PUFA supplementation had no effect on HRV. The 3 groups had normal serum concentrations of potassium, magnesium, and calcium before and after dietary supplementation (data not shown).
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); thus, the subjects (both women and men) were divided into 2 groups according to the median SDNN (150 ms) before supplementation. There was a significant increase in RR after supplementation with n-3 PUFA (both doses) and a nonsignificant increase in other HRV indexes in subjects with before-supplementation SDNN < 150 ms (Table 5). No such effect was found in the group with the highest SDNN before supplementation (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We found small differences between women and men in the content of certain fatty acids, especially in granulocytes. Such sex differences were described previously in a study that related dietary intake of fatty acids to the same fatty acids in plasma phospholipids (22). It seems likely that women and men consume fatty acids in slightly different proportions.
The dose-response relation found between the cellular content of DHA and SDNN before supplementation may suggest a threshold effect, although these findings should be viewed with caution because of the small number of men studied. However, a threshold effect was also suggested in a recently published, large prospective cohort study of US male physicians (10). Consumption of 1 fish meal/wk was associated with a 52% lower risk of SCD compared with consumption of fish less than monthly, but increasing the intake of fish further did not confer any additional benefit in the prevention of SCD (10). A smaller, population-based case-control study (80% men) with 334 case subjects with primary cardiac arrest and 493 control subjects concluded that dietary intake of n-3 PUFA equal to 1 fatty fish meal/wk was associated with a 50% reduction in the risk of primary cardiac arrest compared with no intake of n-3 PUFA (9). Data from epidemiologic studies (23) also show an apparent cardioprotective effect of n-3 PUFA in populations with a rather low intake of fish.
Two secondary prevention trials that studied dietary n-3 PUFA have been reported. In the Diet and Reinfarction Trial, 2033 men with a previous MI were randomly assigned to receive or not receive advice to eat fatty fish 2 times/wk (8). During a 2-y follow-up, the total mortality was reduced significantly, by 29%, in the intervention group, although no decrease in the number of reinfarctions was observed. The authors hypothesized that fish consumption might have reduced the risk of serious ventricular arrhythmias and SCD. In another randomized secondary prevention trial that included 605 post-MI patients (>90% men), the effect of a Mediterranean diet was compared with that of a control diet (24). The experimental diet involved increased intake of bread, fruit, vegetables, fish, and a margarine rich in 18:3n-3. After a mean follow-up of 27 mo there was a striking reduction in the number of cardiac deaths in the intervention group compared with the control group (3 and 16 cardiac deaths, respectively) and no cases of SCD were recorded in the intervention group. However, it is not known whether the high intake of 18:3n-3, the fish-derived n-3 PUFA, or other foods in the Mediterranean diet were responsible for the reduction in SCD (24).
Although there is increasing evidence of a protective effect of n-3 PUFA against SCD in men, such an effect in women has not yet been reported. In the present study, we did not find any correlation between n-3 PUFA content in cell membranes and HRV indexes other than RR in women, and in agreement with this, dietary intervention with n-3 PUFA did not affect HRV in women. The finding of a positive correlation between 20:4n-6 in granulocytes and HRV in women could not be reproduced in platelets. In general, it is believed that 20:4n-6 is arrhythmogenic, but in vitro studies have shown an antiarrhythmic potential of 20:4n-6 similar to that of EPA and DHA, if the activity of cyclooxygenase and lipoxygenase is inhibited (25). Thus, it may be that the cyclooxygenase and lipoxygenase metabolites of 20:4n-6 are arrhythmogenic whereas the free fatty acid of 20:4n-6 may possess antiarrhythmic properties.
In men we found a beneficial effect on HRV of dietary supplementation with n-3 PUFA, but only in those men with low HRV before supplementation. This is perhaps partly explained by the dose-response relation found between DHA and HRV before supplementation. According to Figure 1 it would be expected that only the men with low HRV would show an influence on HRV by increasing their DHA concentration in cell membranes, and this was in fact what was found in a dose-dependent manner. It should be emphasized that the granulocyte membrane concentrations and platelet concentrations of n-3 PUFA are also biomarkers of the composition of other cell membranes, including those of myocardial cells (26).
Our data may suggest that DHA is the principal active n-3 fatty acid that confers protection against arrhythmias, which is consistent with our previous observations in post-MI patients (16). Most animal studies that have shown an antiarrhythmic effect of n-3 PUFA have used mixtures of EPA and DHA, but a recent animal study using both EPA and DHA in separate preparations found DHA to be the active protector against arrhythmias (27). Another recent study suggests that the fluidity of the sarcolemmal membrane is a critical factor concerning asynchronous beating of the myocyte and only DHA, which has the longest chain of the n-3 PUFA, changes the membrane fluidity in a favorable way; this may underlie the antiarrhythmic effect of DHA (28). However, this specific effect may be obscured by the in vivo interconversion between EPA and DHA (26).
We used 24-h HRV as the endpoint because the available data suggest great stability of HRV measures derived from 24-h ambulatory monitoring in normal subjects (29, 30), post-MI patients (31), and patients with previous serious ventricular arrhythmias (32). The importance of measuring HRV in post-MI patients has recently been emphasized (33). Finally, a consensus report on HRV stated that because 24-h HRV indexes appear to be stable and free of placebo effects, HRV may be an ideal method of assessing intervention therapies (34).
Dietary supplementation with n-3 PUFA revealed a dose-dependent reduction in plasma triacylglycerol concentration. No effects on other plasma lipids or lipoproteins were observed, which is in agreement with results from previous studies (35).
With regard to the limitations of this study, the study groups were rather small, thereby increasing the risk of type 2 statistical errors. Also, the differences found between women and men made it necessary to examine the sexes separately, thereby decreasing the numbers of subjects under investigation. In addition, a direct method of evaluating the effects of an intervention therapy on ventricular arrhythmias in humans would be preferable. However, arrhythmias are seldom present, especially in healthy subjects. Therefore, it is necessary to rely on surrogate endpoints, such as HRV, for the development of serious arrhythmias.
This study found a beneficial effect of n-3 PUFA on HRV in healthy men, suggesting an antiarrhythmic effect of n-3 PUFA. Together with our previous observations of an increase in HRV in post-MI patients after dietary supplementation with n-3 PUFA, the results may help explain the reduction in SCD seen in men who regularly eat fish. However, larger studies are needed to clarify the dose-response relation observed in the present study. In addition, more attention should be paid to the effect of n-3 PUFA on arrhythmic tendency in women.
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REFERENCES |
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-linolenic acid-rich diet in secondary prevention of coronary heart disease. Lancet 1994;343:1454–9.[Medline]
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