Published ahead of print on October 6, 2005, doi:10.1164/rccm.200503-372OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200503-372OC
Omega-3 Fatty Acid Prevents Heart Rate Variability Reductions Associated with Particulate MatterInstituto Nacional de Salud Pública, Cuernavaca, Mexico; Division of Pulmonary Allergy and Critical Care, Emory University School of Medicine, Atlanta, Georgia; and Laval University, Lipid Research Center, Quebec City, Canada Correspondence and requests for reprints should be addressed to Fernando Holguin, M.D., 1600 Clifton Road, NE, MS E-17, Atlanta GA 30333. E-mail: fch5{at}cdc.gov
Context: Environmental exposure to particulate matter of 2.5 µm or less (PM2.5) has been associated with changes in heart rate variability (HRV). Objective: To evaluate the effect of supplementation with omega-3 polyunsaturated fatty acids on the reduction of HRV associated with PM2.5 exposure. Design: Randomized double-blind trial. Setting: Mexico City, Mexico. Participants: 50 nursing home residents older than 60 yr. Intervention: Randomization to either 2 g/d of fish oil versus 2 g/d of soy oil as the control, with 6 mo follow-up (1-mo presupplementation and 5-mo supplementation) or repeated HRV measurements. PM2.5 was monitored indoors and outdoors. Main Outcome Measure: The association between HRV and 1 SD change in PM2.5 (8 µg/m3). Results: In the group receiving fish oil, the reduction in HRV–high-frequency log10-transformed associated with a 1-SD change in PM2.5 was –54% (95% confidence interval, –72, –24) in the presupplementation phase, and only –7% (95% confidence interval, –20,+7) in the supplementation phase (p < 0.01 for the effect of supplementation), with changes in other HRV parameters also being significantly less pronounced during supplementation. Small decreases in PM2.5-associated reductions in HRV parameters also occurred in the group receiving soy oil, but these were not significant. Fish oil supplementation was significantly better in preventing the reduction in percentage of successive normal RR intervals differing by more than 50 ms (p = 0.03) and the root square of the mean of the sum of the squares of differences between adjacent intervals (p = 0.05) than soy oil supplementation. Interpretation: Supplementation with 2 g/d of fish oil prevented HRV decline related to PM2.5 exposure in the study population.
Key Words: elderly • heart rate variability • Mexico • omega-3 polyunsaturated fatty acids • PM2.5 Exposure to airborne particulate matter (PM) has been associated with increased cardiovascular mortality in the elderly (1, 2) and reductions in heart rate variability (HRV), a measure of cardiac autonomic regulation (3–6). The effect of PM on HRV, an independent risk factor for cardiac arrhythmias, sudden death, and myocardial infarction (MI) (7, 8), may provide a mechanism for the PM-associated increases cardiovascular mortality (9, 10). Increased intake of n-3 polyunsaturated fatty acids (n-3 PUFA) appears to decrease the risk of sudden death, nonsudden death from MI and nonfatal MI (11–14). The protective effect of n-3 PUFA may be linked in part to its cardiac and arrhythmic effects, including increasing HRV (15–16). We have previously shown that supplementation with omega-3 fatty acids (fish oil) leads to a rapid onset and sustained increase in HRV among elderly subjects (17). Therefore, on the basis of our previous findings and the plausible mechanism for a preventive effect of n-3 PUFA on cardiovascular events, we conducted a randomized double-blind trial among elderly nursing home residents of Mexico City to evaluate the effect of n-3 PUFA supplementation either as marine (fish oil) or plant-derived (soy oil) sources of n-3 PUFA on the reduction of HRV resulting from increases in exposure to ambient PM of 2.5 µm or less (PM2.5).
Study Population and Design We recruited residents from a nursing home in Mexico City located in an area with elevated levels of PM2.5. Subjects were randomly assigned in a double-blind fashion to receive either fish oil (n-3 PUFA) or soy oil. The study was conducted in two phases: a presupplementation phase of 1 mo (May 20 to June 19) and a supplementation phase of 5 mo (June 20 to November 12). During the presupplementation phase, participants performed HRV studies once a week. During the supplementation phase, subjects received either daily fish oil or soy oil supplements and underwent HRV studies on alternate days (except weekends and holidays). In addition, ambient PM2.5 levels were measured indoors (within the nursing home) and outdoors (at the nursing home site). This design allowed the comparison of the changes in HRV as related to ambient PM2.5 levels before and during the supplementation phase for each supplementation group and the comparison of the potential benefits due to fish oil and soy oil supplements. The eligibility criteria were as follows: age older than 60 yr, absence of cardiac arrhythmias, no cardiac pacemaker, no allergies to omega-3 fatty acids or fish, no treatment with oral anticoagulants, no history of bleeding diathesis, and being able to undergo HRV measurements in the supine position. Among 58 residents invited to the study, 55 agreed to participate and 52 were eligible. These provided written, informed consent for both randomization and supplementation. Two subjects left the study early because of the constraint of the HRV measurements, and 50 completed the entire follow-up. At baseline, all participants responded to a general-purpose questionnaire and a validated food frequency questionnaire applied by a nutritionist (18). Medical histories were abstracted from the medical files. The trial protocol was approved by the Institutional Research Board and Ethical Committee of the National Institute of Public Health in Mexico.
Randomization to n-3 Fish Oil or Soy Oil
Fatty Acids in Erythrocyte Membranes Blood samples were immediately centrifuged and frozen in liquid nitrogen. The omega-3 concentration in red cell membranes was measured at Laval University, "Centre de Recherche sur les Maladies Lipidiques," Quebec City, Canada, using a gas chromatograph (HP 5890 gas chromatograph; Hewlett Packard, Toronto, ON, Canada) and a capillary column DB-23 (30 m x 0.25-mm diameter x 0.25-µm film thickness; Agilent Technologies, Palo Atto, CA) with nitrogen as the carrier gas (19, 20). Erythrocyte fatty acid profiles were expressed as the relative percentage areas of total fatty acids. Laboratory technicians were blinded to the treatment group assignment (fish oil or soy oil).
HRV Analysis
Pollutants and Temperature Measurements
Ambient levels of PM10 (PM
Statistical Analysis
All regression models were adjusted for age, sex, heart rate, body mass index (weight/height2), and hypertension because hypertensive patients are more likely to have an abnormal cardiac autonomic nervous system, manifested by reduced HRV (8, 24). In subsequent models, we also adjusted for systolic or diastolic blood pressure, as continuous variables, and day of the week. We also conducted stratified analyses by hypertension diagnosis to assess its potential modifying effect. The response variables were transformed using a log10. For HRV-HF and HRV-LF (ms2), the transformation was log10 (HF/100,000) and log10 (LF/100,000), respectively. The same-day indoor PM2.5 level, measured in the room where HRV measurements were conducted, was assigned as PM2.5 exposure for subjects tested on a given day (12 subjects/d). We estimated the percentage of change in each HRV parameter for a 1-SD increase for PM2.5 and O3 as (10[
Table 1 presents the general characteristics of participants according to their assignment to either the fish oil or soy oil supplementation groups. Few participants reported current smoking, 40% reported ever smoking, and the amount of cigarettes smoked for current smokers was low (mean, 5; range, 1–15/d). Hypertension and chronic obstructive lung disease were more frequent in the fish oil group (80.8 vs. 58.3% and 34.6 vs. 16.7%, respectively), although these differences were not statistically significant. Only two participants in the fish oil and the soy oil groups were treated with  -blockers. The average presupplementation HRV for the time domain components (r-MSSD and pNN50) was significantly higher in the soy oil group. This difference was likely due to the slightly larger number of subjects with hypertension in the fish oil group. No significant difference was observed with regard to heart rate and blood pressure between the groups. Subjects assigned to the soy oil group were slightly thinner than those assigned to the fish oil group. During the presupplementation phase, participants completed an average of 4 (SD = 2; range, 1–7) HRV, heart rate, and blood pressure measurements and, during the supplementation phase, an average of 30 (SD = 10; range, 8–47) measurements.
At baseline, participants from the fish oil and soy oil groups reported low dietary intakes of foods rich in n-3 PUFA: 88% of the participants ate fresh fish less than once a week, 73% ate canned fish (tuna) less than once a week, and 92% ate green leafy vegetables once a week or less.
Fatty Acids in Erythrocytes
Environmental Exposure Data Participants in both groups spent, on average, 92.8% of their time indoors. Table 3 presents air pollutant levels during the study. The mean ambient PM2.5 level in the room where HRV studies were conducted was 18.6 µg/m3 (SD = 8.0), ranging from 5.1 to 49 µg/m3. These levels were highly correlated with PM2.5 levels measured in the living room of the nursing home (r = 0.84) and outdoors (r = 0.71). During the study phase, O3 1-h maximum ranged from 12 to 165 ppb (mean = 110 ppb, SD = 4) and PM10 24-h averaged from 6.4 to 47.7 µg/m3 (mean = 23.8 µg/m3, SD = 8.0). Air pollutant levels were similar during the two study phases (presupplementation and supplementation phases).
HRV and PM2.5 Exposure Table 4 presents the association of indoor PM2.5 levels and HRV components in the presupplementation and the supplementation phases for the fish and the soy oil groups.
In the fish oil group, after adjusting for age, sex, heart rate, hypertension, body mass index, and time, indoor PM2.5 levels were significantly associated with a substantial reduction in time and frequency domain components of HRV during the presupplementation phase, but not during the supplementation phase. There was a 54% reduction in HRV-HF (95% CI, –72, –24) per SD of PM2.5 (8.0 µg/m3) in the presupplementation phase compared with a –7% (95% CI, –7, 20) per SD of PM2.5 during the supplementation phase. Figure 2 presents the change in HRV-HF related to an increase in 1 SD (8 µg/m3) of PM2.5 during the presupplementation and the supplementation phases for both the fish and soy oil groups.
For SDNN, the decrease associated with PM2.5 in the presupplementation phase was –27% (95% CI, –37, –16) per SD. However, during the supplementation phase, PM2.5 levels were not significantly associated with SDNN (0.5%; 95% CI, –4, 3). The interaction between study phases with PM2.5 was significant for all HRV parameters (Table 4). In the soy oil group, after adjusting for the variables previously mentioned, HRV (HRV-HF, HRV-LFT, SDNN, and r-MSSD) was inversely associated with the SD of indoor PM2.5 levels. During the supplementation phase, the magnitude of the negative association decreased; however, the interaction between supplementation phases and PM2.5 was only marginally significant for the HRV-LFT parameter (Table 4). Further adjustment by systolic blood pressure and day of the week did not modify these results. When data were stratified by hypertension diagnosis, we observed that the adverse effect of indoor PM2.5 levels was larger among subjects with hypertension and that fish oil supplementation was beneficial; however, our capacity to draw conclusions from this interaction was limited by the small sample size in subjects without hypertension (n = 5; data shown in the online supplement). To determine which form of supplementation was more effective in preventing the PM2.5-associated reductions in HRV, we compared the coefficient of the interaction between the supplementation phase and PM2.5 exposure on HRV parameters in the fish and soy oil groups. There were no significant differences in the interaction for HFT and LFT components (p = 0.49 and p = 0.68, respectively) and SDNN (p = 0.18); however, the differences in p-NN50 and r-MSSD were statistically significant (p = 0.03 and p = 0.05, respectively).
HRV and O3 Exposure
In this randomized controlled trial, fish oil supplementation prevented the reduction in HRV associated with same-day exposure to indoor PM2.5. In contrast, soy oil, our comparison supplementation of plant-derived n-3 PUFA, was associated with a marginal, nonsignificant protection from the effects of PM2.5 on HRV. This is the first study to evaluate the supplementation of n-3 PUFA as a protective measure against the adverse effect of air pollution on cardiac autonomic function in the elderly.
Low HRV is an independent predictor of sudden cardiac death, myocardial ischemia, heart failure exacerbations, and cardiac arrhythmias in patients with coronary artery disease (25). Furthermore, a reduced HRV may also predict death and risk of cardiovascular mortality and arrhythmic events in apparently healthy middle-aged and elderly subjects (26); although the predictive value of transient changes in HRV is still not well understood, exposure to PM2.5 has been shown to significantly decrease HRV and may thus increase the risk of cardiovascular mortality in conjunction with other cardiovascular effects associated with particulate air pollution, such as an increase in systemic inflammation, deregulation of the coagulation system, and impaired endothelial-mediated vasodilation and microvascular inflammation (27). The PM-mediated inflammation may involve multiple pathways, including the following: (1) formation prostaglandins (28, 29), (2) intracellular downstream activation of inflammation via Toll-like receptor 2 (30), and (3) reactive oxygen species–mediated translocation of nuclear transcription factors (nuclear factor- During the supplementation with 2 g of fish oil, the n-3 PUFA content in the erythrocyte membrane increased significantly for both EPA and DHA and decreased for AA and LA, as observed in other supplementation studies (12). As we have previously reported (17), we observed an increase in HRV in the early weeks after fish oil supplementation, which also provided significant protection from PM2.5 -induced reductions in HRV. This was not observed in the soy oil group, where the content of n-3 PUFA in the erythrocyte membranes increased only in small amounts and therefore had a lesser effect on HRV. Because levels of n-3 PUFA in erythrocyte membranes were available for only 16 participants, we were not able to fully explore which component of n-3 fish oil EPA or DHA was responsible for the modulating effect on HRV and if the decrease in AA in membranes could also impact HRV.
Hypertension was more frequent in the fish oil group and may have been responsible for the lower HRV component observed in this group at baseline. Unfortunately, the small number of nonhypertensive participants in the study limited our capacity to adequately evaluate the effect of the supplementation on the basis of hypertension diagnosis. The other participants' characteristics, such as chronic obstructive pulmonary disease, diabetes, and use of A strength of the study is the comparability between supplementation groups that stems from exposure to similar air pollution levels and particulate composition and from sharing a common diet. In addition, there was no confounding from indoor passive tobacco exposure, in accordance with the nursing home regulations. Although our study involved subjects who smoked, only seven subjects of the whole group were active smokers. Excluding these subjects from the analysis did not modify our results. We used exposure to indoor levels of PM2.5 given that participants spent 92% of time indoors during the study and that the correlation with outdoor PM2.5 levels was high (r = 0.71). Information about other pollutants was available through a nearby automated monitoring station, situated 3 km upwind from the study site, allowing a reasonable estimation of the ambient environment outside the nursing home. Exposure to same-day levels of O3 was associated with reductions in HRV. Although the O3-associated reductions in HRV were significantly lessened in the supplementation phase, the interaction between O3 and supplementation phase was not significant. It is possible that our study may have been underpowered to detect the protective effect against O3-associated changes in HRV associated with either form of supplementation. PM10 levels were highly correlated with PM2.5 indoors (r = 0.69) and outdoors (r = 0.78), and therefore it is difficult to separate its effect from that of PM2.5. We used short-term HRV recordings, which could have hindered the possibility of performing an adequate assessment on very low frequencies; however, for HRV-HF, excellent correlation exists between short recordings using spectral analysis and longer time-domain analysis (7). In addition, the same trained technician performed the entire HRV recording, thus reducing intertest variability, and all recordings were reviewed by the same physician, blinded to the supplementation grouping. The presupplementation phase was limited to 1 mo and consequently the confidence limit around our point estimate for the effect of PM2.5 on HRV during this phase was wide. However, we did not observe a significant effect of fish oil supplementation, which might have been larger with a more precise estimate of the effect of PM2.5 on HRV during the presupplementation phase. A major concern in a dietary supplementation study is related to defining effective dosage. In our study, soy oil was intended to act as placebo; however, soy oil contains a small amount of ALA—further elongated in EPA and DHA—and resulted in a small increase in n-3 PUFA in erythrocyte membranes (41). This increase was not sufficient to reverse the adverse effect of PM2.5. Our results suggest that 2 g/d of fish oil might be necessary to significantly decrease the adverse impact of PM2.5 on HRV in elderly subjects without prior cardiovascular disease other than hypertension. This dose is similar to the concentration of omega-3 PUFA provided in secondary prevention studies. (Evidence from prospective secondary prevention studies suggests that EPA + DHA supplementation ranging from 0.5 to 1.8 g/d significantly reduces subsequent cardiac and all-cause mortality [12]). However, for adults without diagnosed cardiovascular disease, the American Heart Association has advised that everyone should have at least two meals of oily fish per week (200–400 g fatty fish/wk provides an additional 0.5–0.8 g/d of n-3 PUFA). Because we do not have information regarding dose–response effect, we cannot exclude that doses lower than 2 g/d of fish oil supplementation could be equally effective to prevent PM2.5-induced changes in HRV. Our study population had a low baseline intake of fish and other sources of n-3 PUFA. Therefore, the effective doses in other populations might differ depending on the baseline nutrient status and state of health. The dose range of n-3 PUFA supplementation used in this study is considered safe (12) and was well tolerated by our participants. Therefore, fish oil supplement as a source of n-3 PUFA could be considered as a potential form of preventive measure to reduce the risk of arrhythmia and sudden death in elderly subjects exposed to ambient air pollution. However, larger intervention studies are needed to confirm the modulating effect of n-3 PUFA on the adverse cardiovascular outcomes related to air pollution exposure in susceptible populations and to clarify the dose–response relationship.
The authors thank the CENICA (Centro Nacional de Investigación y Capacitación Ambiental en la Ciudad de México) group and Victor Gutierrez for their contribution in performing the gravimetric analyses in our study; the Departamento de Integración Familiar, México, for allowing us to perform the study in their nursing home; the participants of the study; Isabel García and Rafael Santibáñez for their technical support; Josep Maria Anto and Stephanie London for their critical review of the manuscript and their thoughtful comments; and the Galcaps International Laboratory for providing the fish oil and soy oil supplements.
Supported by research grants from the Mexican "Consejo Nacional de Ciencia y Tecnologia" (no. 34483-M), the Mexican Ministry of Health, the National Center for Environmental Health from the Centers for Disease Control and Prevention, Atlanta, Georgia, and the Heart and Stroke Foundation of Canada. M.-C.B. was a recipient of a doctoral fellowship from the FRSQ Cardiovascular Health Network. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200503-372OC on October 6, 2005 Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 9, 2005; accepted in final form October 4, 2005
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ABSTRACT
METHODS
-linolenic acid, a plant-derived n-3 PUFA (C18:3 n3 ALA), 16.3% of saturated fat, 54.5% of linoleic acid (C18:2 n-6 LA), and 22.5% of oleic acid (C18:1 n-9) in each gram. Fish oil and soy oil capsules were provided at no cost by Gelcaps Mexico. Neither the participants nor the study personnel, such as the nurses providing the supplementation or the technician conducting HRV measurements, were aware of the randomization assignment (
10-µm diameter), ozone (O3), nitrogen dioxide (NO2), and sulfur dioxide (SO2), as well as climatic variables, were obtained from an automated monitoring station (Pedregal, Thermo Electron Corp.) located at 3 km (northeast) upwind from the study site. Given the low concentrations observed for SO2 and NO2, these pollutants were excluded from further analysis. 
2 or Fisher's exact test were used for discrete variables and frequencies. A p value of less than 0.05 was considered significant. All statistical analyses were conducted using Stata 7.0 (Stata Corp., College Station, TX). 
B) (