Serum n-6 polyunsaturated fatty acids and risk of atrial fibrillation: the Kuopio Ischaemic Heart Disease Risk Factor Study

The KIHD is a population-based study designed to investigate risk factors for CVD, atherosclerosis, and related outcomes in men from eastern Finland [16], a total of 2682 men (82.9% of those eligible) who were 42, 48, 54 or 60 years old and living in the city of Kuopio or its surrounding areas participated in the baseline examinations in 1984–1989. The baseline characteristics of the entire study population have been described previously [17]. The KIHD protocol was approved by the Research Ethics Committee of the University of Kuopio and complies with Declaration of Helsinki (ClinicalTrials.gov Identifier: NCT03221127). All the subjects signed a written informed consent. Study participants were not involved in the design, or conduct, or reporting, or dissemination plans of the current study. The manuscript complies with the STROBE checklist. Subjects with a history of AF at baseline (n = 32) were excluded from the analyses. We also excluded men with missing data on serum n-6 PUFAs (n = 200), leaving 2450 men for the analyses. The stratified analyses based on disease history included 1795 men without history of CHD or congestive heart failure and 655 men with history of these diseases. For the dietary analyses we excluded men with missing data on dietary intakes (n = 72), leaving 2610 men for the analyses, 1902 men without history of CHD or congestive heart failure and 708 men with history of these diseases.

Subjects gave venous blood samples between 8 A.M. and 10 A.M. at the baseline examinations. The subjects were instructed to abstain from ingesting alcohol for 3 days and from smoking and eating for 12 h before giving the sample. Comprehensive description of the determination of serum lipids and lipoproteins, assessment of medical history and medications, smoking, and alcohol consumption have been reported previously [18].

Physical activity was evaluated based on the 12-month leisure-time physical activity questionnaire and expressed as kcal/day [19]. The most common leisure-time physical activities were recorded, including the average duration, intensity, and frequency of each activity. BMI was computed as the ratio of weight in kilograms to the square of height in meters. Education and annual income were assessed using self-administered questionnaires. Hypertension diagnosis was defined as systolic/diastolic blood pressure > 140/90 mmHg at study visit or use of hypertension medication [18]. Dietary intakes were assessed using 4-day food recording at the time of blood sampling [13]. Nutrient intakes were estimated using the NUTRICA® 2.5 software (Social Insurance Institution, Turku, Finland). The databank of the software is mainly based on Finnish values of nutrient composition of foods. For the total n-6 PUFA intake, we used the sum of LA and AA intakes. The database does not have information on GLA or DGLA content in foods. Healthy Nordic diet score used in the analyses is based on the Baltic Sea diet score [20] but modified slightly to comply with the availability of the dietary data in the KIHD cohort [21]. The score ranges from 0 to 25. The higher the score, the higher the adherence to a healthy Nordic diet.

Serum esterified and nonesterified fatty acids were measured in 1991 from samples that had been stored at − 80 °C in one gas chromatographic run without preseparation, as described previously [22]. Serum fatty acids were extracted with chloroform–methanol. Chloroform phase was evaporated and treated with sodium methoxide, which methylated esterified fatty acids. Quantification was carried out with reference standards (Check Prep Inc., Elysian, MN). Each analyte had individual reference standard, and an internal standard was eicosan. Fatty acids were chromatographed in an NB-351 capillary column (HNU-Nordion, Helsinki, Finland) by a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard Company, Avondale, PA, since 1999 Agilent Technologies Inc.) with a flame ionization detector. Results were obtained in micromoles per litter and in the data analyses proportion of the fatty acid of the total serum fatty acids was used. The interassay coefficient of variation (CVs) for repeated measurements were 8.7% for LA, 11.6% for GLA, 8.3% for DGLA, and 9.9% for AA. For the serum total n-6 PUFA concentration, we used the sum of LA, GLA, DGLA and AA.

All AF events that occurred between study entry and December 31, 2014, were included. Data on events were obtained by record linkage from the national computerized hospitalization registry, which covers every hospitalization in Finland. Subjects were hospitalized because of AF or had AF when they were hospitalized for other reasons. Data on vital status were obtained from Statistics Finland. Cardiovascular causes of AF were coded according to International Classification of Diseases codes (8th revision code 427.4, 9th revision code 427.3, and 10th revision code I48) and the accuracy was verified by a physician [23].

The univariate associations of the serum n-6 PUFA with demographic, lifestyle and clinical characteristics at baseline were assessed by means and linear regression for continuous variables and Chi²-test for categorical variables. Correlations between the individual n-6 PUFAs were evaluated by Spearman correlation. Cox proportional hazards regression models adjusted for relevant covariates were used to estimate hazard ratios (HRs) of incident events. The validity of the proportional hazard assumption was evaluated using Schoenfeld residuals, and the assumptions were met. The analyses were controlled for possible confounders, which were selected based on established risk factors for AF [15], or on associations with exposures or outcomes in the present analysis.

Two different models were used to control for confounding factors. The model 1 was adjusted for age (years) and examination year. The multivariable model 2 included model 1 and BMI (kg/m²), smoking (pack/years), years of education, leisure-time physical activity (kilocalories/day), intake of alcohol (grams/week), serum triglycerides (mmol/L), serum long-chain n-3 PUFA concentration (% of all serum fatty acids), systolic and diastolic blood pressure (mm Hg), family history of ischemic heart disease, and use of hypercholesterolemia or hypertension medications at baseline or during follow-up (yes or no). In the analyses with dietary n-6 PUFA intakes, also total energy intake was used as a covariate. Additional adjustments for serum HDL cholesterol or LDL cholesterol concentrations, income, history of type 2 diabetes, or intakes of fruits, berries and vegetables, eggs, whole grains, meat, fish, dairy, butter, or a healthy Nordic diet score did not appreciably change the associations (< 5% change in estimates).”

Cohort means were used to replace missing values in covariates (< 0.5%). Statistical significance of the interactions on a multiplicative scale was assessed by likelihood ratio tests with a cross-product term. Tests of linear trend across categories were conducted by assigning the median values for each category of exposure variable and treating those as a single continuous variable. Potential nonlinear associations were assessed semi-parametrically using restricted cubic splines. All P values were two-sided (α = 0.05). Data were analyzed using the SPSS software version 27 for windows (Armonk, NY: IBM Corp.) and Stata version 14.1 for spline analysis (Stata Corp).

The mean (SD) age of the participants was 53.0 (5.2) years. The mean (SD) serum concentrations, as a percentage of all serum fatty acids, were 32.79% (4.85) for serum total n-6 PUFA concentration, 26.40% (4.65) for LA, 0.29% (0.11) for GLA, 1.34% (0.30) for DGLA, and 4.77% (1.01) for AA. The intercorrelations between the individual n-6 PUFA were weak, except for a moderate correlation between GLA and DGLA; (−0.19 for LA and GLA, P < 0.001), (− 0.09 for LA and DGLA, P < 0.001), (0.13 for LA and AA, P < 0.001), (0.54 for GLA and DGLA, P < 0.001), (0.19 for GLA and AA, P < 0.001), (0.09 for DGLA and AA, P < 0.001).

Baseline characteristics of the participants according to quartiles of the total n-6 PUFA concentration are presented in the Table 1. Men with higher concentration were more likely to have a higher annual income and education, leisure-time physical activity, and serum HDL and LDL cholesterol concentrations, higher intakes of LA and AA, fruits, berries and vegetables, eggs, total grains, whole grains, red meat and total meat products, and a higher adherence to a healthy Nordic diet, but lower serum triglyceride concentration and systolic and diastolic blood pressure, and lower intakes of fish, dairy products, and butter. They also had a lower BMI and alcohol intake and were less likely to have hypertension, congestive heart failure, ischemic heart disease, diabetes, and to smoke.

During the mean follow-up of 22.4 years, AF was diagnosed in 486 (19.8%) of the 2450 men. The risk for AF was 33% lower (HR 0.76, 95% CI 0.59–0.99, P trend across quartiles = 0.007) in the highest vs. the lowest serum total n-6 PUFA quartile after adjustment for age and examination year (Model 1, Table 2). Further adjustments for potential confounders slightly attenuated the association (extreme-quartile HR 0.79, 95% CI 0.58–1.08, P trend = 0.04) (Model 2, Table 2). Among the individual fatty acids, only LA was associated with reduced risk of AF (the multivariable-adjusted extreme-quartile HR 0.69, 95% CI 0.51–0.94, P trend = 0.02) (Model 2, Table 2). GLA, DGLA and AA were not associated with the risk of AF (Table 2). Similarly, when evaluated continuously, 1-SD increase in serum total n-6 PUFA and serum LA concentrations were associated with 15% lower risk of AF (HR 0.85, 95% CI 0.75–0.95 and HR 0.85, 95% CI 0.76–0.96, respectively) (Fig. 2). Restricted cubic splines analysis confirmed a relatively linear inverse association of serum total n-6 PUFA and LA with the risk of AF and showed little evidence for nonlinearity (Fig. 1). No associations were observed between GLA, DGLA, AA and risk of AF (Table 2, Supplemental Fig. 1).

The associations of the total n-6 PUFA and LA were slightly stronger among the subjects without history of CHD or congestive heart failure at baseline, whereas no statistically significant associations were observed among the men with history of these diseases (P-interaction = 0.05, Fig. 2). Among the 1795 men without disease history, 319 AF events occurred. Each 1-SD increase in serum total n-6 PUFA and LA concentrations were associated with a multivariable-adjusted 22% (95% CI 8–34%) and 21% (95% CI 7–33%) lower risk of AF, respectively (Fig. 2). There were no statistically significant associations between the serum GLA, DGLA and AA and risk of AF in these analyses, either (P for interactions ≥ 0.61) (Fig. 2).

During the follow-up, among the 1902 men without disease history, 335 AF events occurred, while AF was diagnosed in 176 of the 708 men with disease history. Similar associations with the risk of AF as with the serum n-6 PUFA concentrations were also observed with the intakes of total n-6 PUFA, LA and AA. Higher intakes of total n-6 PUFA and LA were associated with reduced risk of AF [the multivariable-adjusted extreme-quartile HR = 0.66 (95% CI 0.43–0.98, P trend = 0.04) for total n-6 PUFA, and HR = 0.64 (95% CI 0.43–0.95, P trend = 0.03)] for LA, only among those without history of CHD or congestive heart failure at baseline (Supplemental Table 1). There were no statistically significant associations between the dietary intake of AA and risk of AF among men with or without disease history (Supplemental Table 1).