Omega-3 Fatty Acids and Pollution-Induced Cardiac Effects
Omega-3 Fatty Acids and Pollution-Induced Cardiac Effects
Twenty-nine participants ranging from 50 to 72 years of age (mean 58 ± 1 years) were enrolled in the study. They were nonsmokers for at least 1 year, with no history of heart disease, uncontrolled hypertension [mean blood pressure (BP): 123 ± 3/77 ± 2 mmHg], pulmonary disease, diabetes mellitus, hypercholesterolemia, or active allergy. Participants were not taking β-adrenergic receptor blockers, n-3 FA, anti-inflammatory drugs, or antioxidant supplements (such as beta-carotene, selenium, vitamin C, or vitamin E). All participants were instructed to refrain from using any pain medications for 2 weeks before each exposure. They were also asked to abstain from alcohol and caffeine and to adhere to a low-fat diet for 24 hr before exposures. The Biomedical Institutional Review Board at the University of North Carolina–Chapel Hill and the U.S. EPA approved the study protocol, recruitment materials, and consent forms. All study participants gave informed consent and received monetary compensation for their participation.
This study was conducted from July 2009 to August 2010. All exposures were conducted at the U.S. EPA Human Studies Facility on the University of North Carolina–Chapel Hill campus. A diagram of the study design is shown in Figure 1. Sixteen participants were assigned to receive 3 g/day (three 1-g capsules daily) of marine-derived n-3 FA (fish oil; FO), and 13 participants received 3 g/day (three 1-g capsules daily) of olive oil (OO) for 28 days before the filtered air exposure day. FO and OO assignments were made using a randomized, double-blinded study design. Each participant was exposed first to filtered air and then to CAP on the next day. The exposures were conducted at the same time of the day and same day of the week. Participants were exposed for 2 hr through a face mask in an exposure chamber in which temperature and humidity were controlled. Participants remained at rest in a seated position throughout the exposure.
(Enlarge Image)
Figure 1.
Schematic representation of the study design.
The following tests were done on each participant beginning at approximately 0800 hours (2 hr before exposure to filtered air): Venous blood was collected (120 min before exposure); Holter electrodes were applied and HRV and repolarization data obtained (105 min before exposure); and brachial artery diameter was measured by ultrasound (60 min before exposure). The ultrasound measurements will be reported elsewhere. The same tests were done immediately after the 2-hr air exposure (Post), and again the next morning at approximately 0800 hours (Follow-up). These latter measurements also served as the preexposure values for the CAP exposure. At approximately 1000 hours on the second day, participants were exposed to CAP for 2 hr. Post and Follow-up measurements were obtained immediately after exposure and again beginning at 0800 hours the next morning. The participants wore a portable ambulatory Holter device for the entire 48-hr period and time domain HRV variables were calculated from the two 24-hr periods.
All participants were asked to refrain from food containing n-3 FA for 2 weeks before and 4 weeks during the dietary supplementation period. Participants were also asked to keep 3-day food records during the 2nd and 4th weeks of the supplementation period to assess compliance with the dietary restrictions. Nutrition Data System for Research software (version 2011; Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN) was used to analyze the food records and estimate intakes of nutrients that may confound n-3 FA measurements. Each 1,000-mg FO capsule contained approximately 65% n-3 FA (410 mg eicosapentaenoic acid and 274 mg DHA). Each 1,000-mg OO capsule contained < 1% n-3 FA (73% oleic acid and 12% palmitic acid). Pharmavite, LLC (Mission Hills, CA) provided the FO and OO supplements. The ratios of the major plasma fatty acids were measured at the end of the supplementation period to determine whether ratios were consistent with expectations for the FO and OO groups.
CAP was generated as described previously (Samet et al. 2009) by drawing ambient air from above the roof of the Human Studies Facility and passing the air through a 2-stage aerosol Harvard concentrator which produces up to a 30-fold increase in particle number and mass. Air temperature and humidity were controlled inside the chamber. The concentration of particles delivered to the chamber varied with the level of naturally occurring ambient particles in Chapel Hill air. However, a particle dilution system was used to limit the maximal particle concentration and prevent it from exceeding 600 μg/m for > 6 min at any time during exposure. A face mask was used to maximize the PM concentration inhaled by the participants. Particle mass and number concentrations at the chamber inlet were monitored continuously as described previously (Samet et al. 2009). Filter samples were also obtained and analyzed for particle mass.
Ambulatory ECG data were collected for HRV analysis as described previously (Samet et al. 2009). Briefly, 12-lead Holter ECG data were collected for approximately 48 hr using a Mortara H12+ Recorder (Mortara Instrument, Milwaukee, WI). HRV indices in both the time and frequency domains and ventricular repolarization were calculated from the raw Holter ECG data using SuperECG software (version 4.0; Mortara Instrument). HRV was measured to evaluate the influence of CAP exposure on the autonomic nervous system control on the heart. Approximately 90 min before both the filtered air and CAP exposures, the participants were asked to recline quietly in a darkened room for 30 min. During the final 10 min of the resting period, data were collected and used to calculate frequency domain parameters of HRV [normalized LF (nLF), normalized HF (nHF), and the high frequency/low frequency ratio (HF/LF ratio)] and repolarization parameters. This 30-min regimen was repeated 15 min after exposure to filtered air and CAP and again the next morning to obtain Post and Follow-up measurements, respectively. Time domain HRV parameters {SDNN [standard deviation of normal to normal (NN) intervals], PNN50, [fraction of consecutive NN intervals that differ by more than 50 msec], RMSDD [the square root of the mean of the sum of the squares of differences between adjacent NN intervals]} were calculated from the data collected over two 24-hr periods (from 2 hr before air exposure until 0800 hours the next morning, and from 2 hr before CAP exposure until 0800 hours the next morning). The HRV parameters were determined according to established guidelines (Task Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology Circulation 1996). The QT interval corrected for heart rate (QTc) was measured to determine the influence of exposure to CAP on ventricular repolarization. The interval from the onset of QRS to the peak of the T wave (QTp), the interval from the peak of the T-wave to the end of the T-wave (Tp-Te), the ratio of the transmural dispersion of repolarization relative to the total duration of repolarization (Tp-Te/QT) were measured to assess the effects of CAP exposure on spatial dispersion of repolarization.
Venous blood was collected 2 hr before both the filtered air and CAP exposures ("Pre"), immediately after each exposure, and again at 0800 hours the morning after CAP exposure. LabCorp (Burlington, NC) performed complete blood counts, including a differential count and a lipid panel.
To assess changes in biological end points between the two exposures and among the FO and OO groups, we used a two-factor (supplement and PM concentration) mixed effects model with a subject-specific random intercept. Changes in HRV, cardiac repolarization, and blood parameters were assessed at two time points: immediately and approximately 20 hr after exposure to CAP and filtered air, denoted "Post" and "Follow-up" respectively. To control for day-to-day variability, time domain measures of HRV, cardiac repolarization, blood counts, and lipids were normalized by dividing the Post and Follow-up values by the values measured before filtered air exposure (Post/Pre, Follow-up/Pre). Time domain HRV parameters were calculated once, over a 24-hr period. Changes are expressed as percent change after CAP exposure (per 100-μg/m increase in CAP) relative to change after air exposure. R statistical software (version 2.11.1; R Developement Core Team; http://www.r-project.org/) was used for analysis, and a p-value of < 0.05 was considered significant.
Methods
Study Participants
Twenty-nine participants ranging from 50 to 72 years of age (mean 58 ± 1 years) were enrolled in the study. They were nonsmokers for at least 1 year, with no history of heart disease, uncontrolled hypertension [mean blood pressure (BP): 123 ± 3/77 ± 2 mmHg], pulmonary disease, diabetes mellitus, hypercholesterolemia, or active allergy. Participants were not taking β-adrenergic receptor blockers, n-3 FA, anti-inflammatory drugs, or antioxidant supplements (such as beta-carotene, selenium, vitamin C, or vitamin E). All participants were instructed to refrain from using any pain medications for 2 weeks before each exposure. They were also asked to abstain from alcohol and caffeine and to adhere to a low-fat diet for 24 hr before exposures. The Biomedical Institutional Review Board at the University of North Carolina–Chapel Hill and the U.S. EPA approved the study protocol, recruitment materials, and consent forms. All study participants gave informed consent and received monetary compensation for their participation.
Study Design
This study was conducted from July 2009 to August 2010. All exposures were conducted at the U.S. EPA Human Studies Facility on the University of North Carolina–Chapel Hill campus. A diagram of the study design is shown in Figure 1. Sixteen participants were assigned to receive 3 g/day (three 1-g capsules daily) of marine-derived n-3 FA (fish oil; FO), and 13 participants received 3 g/day (three 1-g capsules daily) of olive oil (OO) for 28 days before the filtered air exposure day. FO and OO assignments were made using a randomized, double-blinded study design. Each participant was exposed first to filtered air and then to CAP on the next day. The exposures were conducted at the same time of the day and same day of the week. Participants were exposed for 2 hr through a face mask in an exposure chamber in which temperature and humidity were controlled. Participants remained at rest in a seated position throughout the exposure.
(Enlarge Image)
Figure 1.
Schematic representation of the study design.
The following tests were done on each participant beginning at approximately 0800 hours (2 hr before exposure to filtered air): Venous blood was collected (120 min before exposure); Holter electrodes were applied and HRV and repolarization data obtained (105 min before exposure); and brachial artery diameter was measured by ultrasound (60 min before exposure). The ultrasound measurements will be reported elsewhere. The same tests were done immediately after the 2-hr air exposure (Post), and again the next morning at approximately 0800 hours (Follow-up). These latter measurements also served as the preexposure values for the CAP exposure. At approximately 1000 hours on the second day, participants were exposed to CAP for 2 hr. Post and Follow-up measurements were obtained immediately after exposure and again beginning at 0800 hours the next morning. The participants wore a portable ambulatory Holter device for the entire 48-hr period and time domain HRV variables were calculated from the two 24-hr periods.
Dietary Supplementations
All participants were asked to refrain from food containing n-3 FA for 2 weeks before and 4 weeks during the dietary supplementation period. Participants were also asked to keep 3-day food records during the 2nd and 4th weeks of the supplementation period to assess compliance with the dietary restrictions. Nutrition Data System for Research software (version 2011; Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN) was used to analyze the food records and estimate intakes of nutrients that may confound n-3 FA measurements. Each 1,000-mg FO capsule contained approximately 65% n-3 FA (410 mg eicosapentaenoic acid and 274 mg DHA). Each 1,000-mg OO capsule contained < 1% n-3 FA (73% oleic acid and 12% palmitic acid). Pharmavite, LLC (Mission Hills, CA) provided the FO and OO supplements. The ratios of the major plasma fatty acids were measured at the end of the supplementation period to determine whether ratios were consistent with expectations for the FO and OO groups.
Controlled Exposure
CAP was generated as described previously (Samet et al. 2009) by drawing ambient air from above the roof of the Human Studies Facility and passing the air through a 2-stage aerosol Harvard concentrator which produces up to a 30-fold increase in particle number and mass. Air temperature and humidity were controlled inside the chamber. The concentration of particles delivered to the chamber varied with the level of naturally occurring ambient particles in Chapel Hill air. However, a particle dilution system was used to limit the maximal particle concentration and prevent it from exceeding 600 μg/m for > 6 min at any time during exposure. A face mask was used to maximize the PM concentration inhaled by the participants. Particle mass and number concentrations at the chamber inlet were monitored continuously as described previously (Samet et al. 2009). Filter samples were also obtained and analyzed for particle mass.
Ambulatory Electrocardiography (ECG) Measurements
Ambulatory ECG data were collected for HRV analysis as described previously (Samet et al. 2009). Briefly, 12-lead Holter ECG data were collected for approximately 48 hr using a Mortara H12+ Recorder (Mortara Instrument, Milwaukee, WI). HRV indices in both the time and frequency domains and ventricular repolarization were calculated from the raw Holter ECG data using SuperECG software (version 4.0; Mortara Instrument). HRV was measured to evaluate the influence of CAP exposure on the autonomic nervous system control on the heart. Approximately 90 min before both the filtered air and CAP exposures, the participants were asked to recline quietly in a darkened room for 30 min. During the final 10 min of the resting period, data were collected and used to calculate frequency domain parameters of HRV [normalized LF (nLF), normalized HF (nHF), and the high frequency/low frequency ratio (HF/LF ratio)] and repolarization parameters. This 30-min regimen was repeated 15 min after exposure to filtered air and CAP and again the next morning to obtain Post and Follow-up measurements, respectively. Time domain HRV parameters {SDNN [standard deviation of normal to normal (NN) intervals], PNN50, [fraction of consecutive NN intervals that differ by more than 50 msec], RMSDD [the square root of the mean of the sum of the squares of differences between adjacent NN intervals]} were calculated from the data collected over two 24-hr periods (from 2 hr before air exposure until 0800 hours the next morning, and from 2 hr before CAP exposure until 0800 hours the next morning). The HRV parameters were determined according to established guidelines (Task Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology Circulation 1996). The QT interval corrected for heart rate (QTc) was measured to determine the influence of exposure to CAP on ventricular repolarization. The interval from the onset of QRS to the peak of the T wave (QTp), the interval from the peak of the T-wave to the end of the T-wave (Tp-Te), the ratio of the transmural dispersion of repolarization relative to the total duration of repolarization (Tp-Te/QT) were measured to assess the effects of CAP exposure on spatial dispersion of repolarization.
Blood Chemistry and Lipids
Venous blood was collected 2 hr before both the filtered air and CAP exposures ("Pre"), immediately after each exposure, and again at 0800 hours the morning after CAP exposure. LabCorp (Burlington, NC) performed complete blood counts, including a differential count and a lipid panel.
Statistical Analysis
To assess changes in biological end points between the two exposures and among the FO and OO groups, we used a two-factor (supplement and PM concentration) mixed effects model with a subject-specific random intercept. Changes in HRV, cardiac repolarization, and blood parameters were assessed at two time points: immediately and approximately 20 hr after exposure to CAP and filtered air, denoted "Post" and "Follow-up" respectively. To control for day-to-day variability, time domain measures of HRV, cardiac repolarization, blood counts, and lipids were normalized by dividing the Post and Follow-up values by the values measured before filtered air exposure (Post/Pre, Follow-up/Pre). Time domain HRV parameters were calculated once, over a 24-hr period. Changes are expressed as percent change after CAP exposure (per 100-μg/m increase in CAP) relative to change after air exposure. R statistical software (version 2.11.1; R Developement Core Team; http://www.r-project.org/) was used for analysis, and a p-value of < 0.05 was considered significant.
