CHD is a leading cause of disability and mortality, especially among the elderly(Reference Mattiuzzi and Lippi1). It has been shown that myocardial ischaemia is a valuable predictor of CHD and cardiac mortality(Reference Tanai and Frantz2,Reference Fallavollita, Heavey and Luisi3) . Myocardial ischaemia is defined as an imbalance between myocardial oxygen supply and demand due to blood flow reduction through coronary arteries, leading to cardiac and microvascular dysfunction, myocardial infarction and sudden death(Reference Shimokawa and Yasuda4). The optimal clinical method to assess early plasma changes related to myocardial ischaemia is the cardiac stress test(Reference Limkakeng, Henao and Voora5), as indicated by transient ST-segment depression in electrocardiogram (ECG) during a stress or exercise test(Reference Laukkanen, Mäkikallio and Rauramaa6).
Previously in the Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD), higher exercise-induced myocardial ischaemia has been related to an increased risk of CHD and sudden death(Reference Laukkanen, Mäkikallio and Rauramaa6,Reference Hagnäs, Lakka and Kurl7) . Moreover, it has been shown that exercise-induced myocardial ischaemia can predict the risk of atherosclerosis and the prognosis of future cardiac events by influencing the sub-endocardial blood flow and myocardial oxygen consumption(Reference Lalonde, Poirier and Sylvestre8,Reference Matsuzaki, Patritti and Tajimi9) .
Among dietary factors, the cardiovascular benefits of the n-3 PUFA, especially the long-chain n-3 PUFA from fish, have been quite well established(Reference Djuricic and Calder10). However, the possible benefits of the n-6 PUFA on CVD risk have been more debated, although the n-6 PUFA, especially the main n-6 PUFA linoleic acid (LA, 18:2n-6), has been associated with lower risk of CHD(Reference Farvid, Ding and Pan11) and CVD(Reference Marklund, Wu and Imamura12). This also seems to be independent of the exposure to the n-3 PUFA(Reference Marklund, Wu and Imamura12).
LA and the n-3 PUFA α-linolenic acid (ALA; 18:3n-3) are the two essential fatty acids for humans because they cannot be synthesised in the body. They can be used as the precursors for synthesis of longer-chain n-6 and n-3 PUFA, respectively. LA, which in diet is found mainly in vegetable oils, nuts and seeds(Reference Abedi and Sahari13), can be endogenously converted to other n-6 PUFA including γ-linolenic acid (GLA, 18:3n-6), dihomo-γ-linolenic acid (DGLA; 20:3n-6) and arachidonic acid (AA, 20:4n-6). AA is also found in some foods such as eggs and meat(Reference Abedi and Sahari13), and it has been associated with lower risk of CHD and CVD in some studies(Reference Marklund, Wu and Imamura12,Reference Chowdhury, Warnakula and Kunutsor14) . There are very few dietary sources of GLA and DGLA, so their concentrations reflect endogenous processes. In addition to diet, also other factors affect the concentrations and the synthesis of the longer-chain n-3 and n-6 PUFA in the body, including nutritional status, certain diseases, genetics, ageing and lifestyle factors(Reference Sasaki, Sueyasu and Tokuda15–Reference Videla, Hernandez-Rodas and Metherel17).
Although there are potential mechanisms that have been proposed to explain the beneficial associations of LA with CVD risk(Reference Farvid, Ding and Pan11,Reference Virtanen, Wu and Voutilainen18) , especially the beneficial impact on serum LDL-cholesterol reduction, little is known about the associations of LA and the other n-6 PUFA with myocardial ischaemia during exercise. Therefore, the aim of our study was to elucidate the associations of the serum n-6 PUFA concentrations with exercise-induced myocardial ischaemia among middle-aged and older men from the KIHD cohort.
Materials and methods
Study population
We performed a cross-sectional analysis among the participants from the KIHD cohort, using data from the baseline analyses from 1984 to 1989. The KIHD is a prospective population-based study designed to investigate risk factors for CVD, carotid atherosclerosis and related outcomes. The participants are an age-stratified sample of men from eastern Finland with a total of 2682 men (82·9 % of the eligible population) aged 42, 48, 54 or 60 years at the baseline examinations(Reference Salonen19). We excluded participants with missing data on serum n-6 PUFA (n 202) and with the history of CHD (n 609) from the analysis. Hence, the final analysis was conducted with 1871 men.
Measurements
At the baseline examinations, fasting venous blood samples were collected between 08.00 and 10.00. The subjects were instructed to abstain from ingesting alcohol for 3 d and from smoking and eating for 12 h before giving the sample. Details of the other determinants, including medical history, current medications, smoking status, alcohol intake, serum lipids and resting blood pressure measurements, have been reported previously(Reference Salonen, Nyyssönen and Korpela20). Physical activity was determined based on the 12-month leisure-time physical activity questionnaire and expressed as kcal/d(Reference Lakka, Venalainen and Rauramaa21). BMI was computed as the ratio of weight in kilograms to the square of height in metres. Height and weight were measured by a study nurse during the study visit. Hypertension was defined as systolic/diastolic blood pressure > 140/90 mmHg or use of antihypertensive medication. Dietary intakes were assessed at the time of blood sampling in 1984–1989 with an instructed and interviewer-checked 4-d food record (three weekdays and one weekend day) by household measures(Reference Virtanen, Mursu and Tuomainen22). The information of the years of education and annual income of study participants were obtained by using self-administered questionnaires.
Serum fatty acid measurements
Serum 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(Reference Laaksonen, Lakka and Lakka23). Serum fatty acids were extracted with chloroform-methanol. The chloroform phase was evaporated and treated with sodium methoxide, which methylated esterified fatty acids. Quantification was conducted with reference standards (Check Prep Inc., Elysian, MN). Each analyte had an individual reference standard, and an internal standard was eicosan. Fatty acids were chromatographed in an NB-351 capillary column (HNU-Nordion) by a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard Company, since 1999 Agilent Technologies Inc.) with a flame ionisation detector. Results were presented as a proportion of total serum fatty acids. For repeated serum fatty acid measurements, the CV was 8·7 % for LA (18:2n-6), 11·6 % for GLA (18:3n-6), 8·3 % for DGLA (20:3n-6) and 9·9 % for AA (20:4n-6). For the serum total n-6 PUFA concentration, we used the sum of LA, GLA, DGLA and AA.
Exercise test and exercise electrocardiography
A maximal symptom-limited exercise stress test was conducted at the baseline examinations in 1984–1989. It was performed between 08.00 and 10.00 using an electrically braked bicycle ergometer (Medical Fitness Equipment 400 L bicycle Ergometer)(Reference Lakka, Laukkanen and Rauramaa24). ECG was recorded continuously with the Kone 620 electrocardiograph (Kone)(Reference Laukkanen, Mäkikallio and Rauramaa25). The Mason-Likar lead system including V1, V5 and a VF lead connection was used. An ECG was recorded in 30 s intervals during exercise and at least 5 min during recovery while the subject was sitting on the bicycle. The criteria for ischaemia were horizontal ECG during exercise or down-sloping ST depression ≥ 1·0 mm at 80 ms after J point or any ST depression of > 1·0 mm at 80 ms after J point(Reference Laukkanen, Mäkikallio and Rauramaa25).
Statistical analysis
The univariate associations of the participants’ serum total n-6 PUFA concentration with demographic, lifestyle and clinical characteristics at baseline were assessed by means and linear regression for continuous variables and χ 2 test and Mantel-Haenszel test for categorical variables. We applied Spearman’s correlation coefficients (r) to estimate the correlations between the fatty acids.
Logistic regression models were used to estimate the OR for the occurrence of exercise-induced myocardial ischaemia in exposure quartiles, with the lowest category as the reference. Two different models were used to control for potential confounding factors. Model 1 was adjusted for age (years) and year of examination. Model 2 included the variables in model 1 plus BMI (kg/m2), smoking (pack-years), leisure-time physical activity (kcal/d), alcohol consumption (g/week), serum total long-chain n-3 PUFA concentration (DHA + docosapentaenoic acid (DPA) + EPA as a percentage of total serum fatty acids), hypertension, family history of ischaemic heart disease, intake of SFA (% of total energy intake), and vegetables, fruit and berries (g/d). All quantitative variables were entered in the models as continuous variables. The covariables in the analyses were selected based on the previous studies in the KIHD with the serum n-3 PUFA(Reference Tajik, Tuomainen and Kurl26) or on associations with exposures or outcomes in the present analysis. Missing values in covariates (40 in pack-years of smoking, 15 in dietary intakes, 10 in leisure-time physical activity, 8 in BMI, 3 in alcohol consumption and 1 in education) were replaced with the cohort means. For assessing the linear trends across the n-6 PUFA quartiles, the median value of each fatty acid quartile was used as a continuous variable. Statistical significance of the potential interactions by age, BMI, leisure-time physical activity and alcohol consumption (using the median values as the cut-off) was assessed by likelihood ratio test using a multiplicative interaction term. All P-values were two-tailed (α = 0·05). Data were analysed using the SPSS software version 27 (IBM Corp).
Results
Baseline characteristics
The mean (s d) age of the participants was 52·4 (5·3) years. The mean (sd) serum total n-6 PUFA concentration, as a percentage of all serum fatty acids, was 33·04 % (4·61). Table 1 shows the mean concentrations and correlations between individual n-6 and n-3 PUFA. The correlations were mostly weak, except for the positive correlations between GLA and DGLA and between AA and the long-chain n-3 PUFA. Characteristics of the participants according to quartiles of the serum total n-6 PUFA concentration are presented in Table 2. Men with higher serum total n-6 PUFA concentration were younger, more educated, more physically active and less likely to have hypertension or diabetes, or to smoke. They also had higher ALA and fruit, berry, and vegetable consumption, lower alcohol and SFA intake, lower BMI and lower serum TAG and higher HDL-cholesterol concentrations.
Table 1. Mean values of serum n-6 and n-3 PUFA and Spearman’s correlation coefficients
(Mean values and standard deviations)
LA, linoleic acid; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; ALA, α-linolenic acid; DPA, docosapentaenoic acid.
* P-value < 0·05.
Table 2. Baseline characteristics according to the quartiles of total serum n-6 PUFA concentrations*
(Mean values and standard deviations; percentages)
Q; quartile; E%, percent of energy intake; ALA, α-linolenic acid; LA, linoleic acid; AA, arachidonic acid; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid.
* Values are mean (sd) or median (interquartile range, IQR) for continuous variables and n (%) for categorical data. Data were analysed with analysis of variance and linear regression for continuous variables and χ 2 test and Mantel-Haenszel test for categorical variables.
† Antihypertensive and anti-hyperlipidemic medication.
‡ Serum total long-chain n-3 PUFA was the sum of EPA, docosapentaenoic acid (DPA) and DHA.
The serum n-6 PUFA concentrations and odds of exercise-induced myocardial ischaemia
Exercise-induced myocardial ischaemia was found in 332 (17·7 %) of the 1871 men. Serum total n-6 PUFA concentration was not associated with myocardial ischaemia during exercise (Table 3). Among the individual fatty acids, only serum AA concentration was associated with the odds for exercise-induced myocardial ischaemia. After multivariable adjustments, those in the highest v. the lowest serum AA concentration quartile had 50 % lower odds for exercise-induced myocardial ischaemia (OR 0·50, 95 % CI 0·34, 0·76, P-trend across quartiles < 0·001) (model 2, Table 2). Each 1-sd increase in the serum AA concentration was associated with 17 % lower odds (OR 0·83, 95 % CI 0·72, 0·95) (Fig. 1). LA, GLA and DGLA were not associated with exercise-induced myocardial ischaemia (Table 3, Fig. 1). Age, physical activity, alcohol consumption or BMI did not modify the associations (P-values for interactions were > 0·73 for LA, > 0·13 for GLA, > 0·28 for DGLA and > 0·62 for AA).
Table 3. OR for exercise-induced myocardial ischaemia in quartiles of serum n-6 PUFA*
(Odds ratios and 95 % confidence intervals)
Q, quartile; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid.
* Values are means (95 % CI).
† Model 1: adjusted for age and examination year.
‡ Model 2: adjusted for model 1 + BMI, smoking status, leisure-time physical activity, alcohol intake, intake of SFA, fruits, berries and vegetables, serum total long-chain n-3 PUFA concentrations, family history of ischaemic heart disease and hypertension.
Fig. 1. Multivariable-adjusted odds of exercise-induced myocardial ischaemia for 1 sd change in the serum n-6 PUFA. LA, linoleic acid; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid
Discussion
In the present study among middle-aged and older men from eastern Finland, higher serum AA concentration was associated with lower odds of exercise-induced myocardial ischaemia, whereas the other n-6 PUFA, LA, GLA and DGLA had no association. To the best of our knowledge, the present study is the first one to evaluate the associations of the serum n-6 PUFA concentrations with exercise-induced myocardial ischaemia, so we are not able to compare our findings to other studies.
The findings in some dietary fat modification trials from the 1960s and 1970s suggested that very high LA intake could have unfavourable cardiovascular effects. However, these trials had many limitations, such as high dropout rate, short duration, low number of participants and possible high trans-fatty acid intake(Reference Virtanen27). Also, the LA intake in those trials was much higher than in typical Western diets or when compared with the dietary recommendations(Reference Virtanen27), and the fat sources with only n-6 PUFA used in some trials may have replaced fat sources with n-3 PUFA, thereby reducing n-3 PUFA intake(Reference Virtanen27). Indeed, there is some evidence that only the trials that used LA sources that included also n-3 PUFA reported some benefits, whereas trials that used fat sources with only n-6 PUFA did not(Reference Ramsden, Zamora and Leelarthaepin28). In contrast, for example, country-level comparisons have shown that LA content in the body was much lower in a region with high CHD mortality (eastern Finland) compared with a region with low CHD mortality (Italy)(Reference Riemersma, Wood and Butler29). In addition, cohort studies have consistently suggested an inverse association of higher exposure to LA with risk of CVD(Reference Farvid, Ding and Pan11,Reference Marklund, Wu and Imamura12) , and also short-term dietary trials have shown benefits on serum lipid profile(Reference Katan, Zock and Mensink30), without having harmful effects on, for example, inflammation(Reference Johnson and Fritsche31). Also in the current study population, higher serum LA concentrations have been associated with lower risk of CVD(Reference Virtanen, Wu and Voutilainen18,Reference Tajik, Tuomainen and Isanejad32) , although the findings from the current study suggest that the associations are not explained by effects on exercise-induced myocardial ischaemia.
Less is known of the cardiovascular effects of the minor n-6 PUFA GLA and DGLA. GLA and DGLA and its metabolites, including PGE1, have beneficial effects on reducing inflammation and improving cardiac function(Reference Awan, Needham and Evenson33,Reference Yamagata34) . In addition, in one study from the early 1980s, DGLA had an even stronger beneficial association with CHD than LA had(Reference Wood, Riemersma and Butler35). However, the findings in the current study are in line with our previous results in this same cohort, where serum GLA or DGLA concentrations have not been associated with risk of CVD outcomes(Reference Virtanen, Wu and Voutilainen18,Reference Tajik, Tuomainen and Isanejad32) .
Various metabolites from multiple metabolic pathways of AA in the body, including PGE2, are related to the development of myocardial ischaemia and ischaemic reperfusion injury, but the relationships between these various pathways are unclear(Reference Zhang, He and Ni36). Moreover, AA is a ligand for both PPAR α and γ that have different characteristics. PPARα exhibits anti-inflammatory characteristics and preventive effects on vascular damage, while PPARγ has pro-inflammatory and atherosclerotic effects(Reference Kota, Huang and Roufogalis37,Reference Echeverría, Ortiz and Valenzuela38) . On the other hand, several AA-derived eicosanoids and metabolites may have an anti-inflammatory role(Reference Schmitz and Ecker39), vasodilatory effects and vascular healing role to prevent ischaemic heart disease and CVD(Reference Mitchell and Kirkby40). From different AA metabolites, PGD2(Reference Katsumata, Shinmura and Sugiura41), PGI2(Reference Xiao, Hara and Yuhki42) and epoxyeicosatrienoic acids (EET)(Reference Batchu, Lee and Qadhi43) have potentially protective effects on cardiac ischaemia/reperfusion injury. In addition, the potent anti-inflammatory metabolite lipoxin A4, derived from AA, increases in the body during exercise(Reference Gundala, Naidu and Das44), thereby exerting cardioprotective effects through the inhibition of PGE1 and leukotriene production, as well as by reducing other plasma inflammatory component concentrations, such as IL-6 and TNF-α (Reference Gundala, Naidu and Das44–Reference Das46). The role of AA as a precursor for multiple cardioprotective eicosanoids provides one plausible explanation for the specific protective of AA.
In addition, exercise can change the concentrations of n-6 PUFA metabolites and increase those with protective effects. Giordano et al. found an elevation of plasma EET and dihydroxyeicosatrienoic acid (DHET) during a bicycle ergometer exercise test on fourteen healthy participants(Reference Giordano, Newman and Pedersen47). Similarly, a study on six healthy subjects aged 23–53 years in Germany showed that during a maximal treadmill test, the plasma metabolites of AA in cytochromes P450 pathway, including EET and DHET, increased, while the concentration of lipoxygenase-dependent metabolites of AA with pro-inflammatory and vasoconstriction characteristics (thromboxane A, thromboxane B, etc.) were not changed(Reference Gollasch, Dogan and Rothe48). EET can affect the vasodilation and activation of Ca-activated K+ channels (BKca) in vascular cells, and DHET can affect the haemodynamics and responses of the cardiovascular system(Reference Gollasch, Dogan and Rothe48,Reference Hercule, Schunck and Gross49) . In addition, an experimental study found that EET to DHET ratio can modulate vascular tone due to inducing the release of endothelial nitric oxide; this ratio can increase during the exercise test(Reference Gollasch, Dogan and Rothe48,Reference Hercule, Schunck and Gross49) . Therefore, the exercise test, as a short-term exercise that increases haemodynamics like heart rate and blood pressure, might elevate the heart-protective AA metabolites(Reference Gollasch, Dogan and Rothe48), which may partially explain the lower probability of myocardial ischaemia during an exercise test among those with higher serum AA in our study.
The cardioprotective effects of the n-3 PUFA and their metabolites (three-series PG and five-series leukotrienes) have long been recognised(Reference Russo50). However, despite the concern about possible pro-inflammatory effects of the n-6 PUFA, especially AA, recent research has shown that some n-6 PUFA metabolites (like PGI2 and lipoxin A4 from AA) can have cardioprotective effects similar to the n-3 PUFA(Reference Farvid, Ding and Pan11). Furthermore, there is enzymatic competition between EPA and AA for synthesis of some metabolites(Reference Simopoulos51); however, sufficient amounts of AA and EPA are needed for their beneficial functions in cardiovascular health(Reference Das, Kumar and Mohan52). For instance, although higher EPA is associated with lower pro-inflammatory eicosanoids from AA(Reference Simopoulos51), AA provokes the metabolic pathway to increase PGI3 synthesis from EPA(Reference Bordet, Guichardant and Lagarde53). Therefore, adequate levels of these fatty acids may help prevent atherosclerosis and CHD(Reference Das, Kumar and Mohan52). In our study, there was a positive correlation between AA and the long-chain n-3 PUFA, which could indicate that the associations observed with AA could be due to the long-chain n-3 PUFA. However, adjustment for the long-chain n-3 PUFA concentrations did not attenuate the inverse association of AA with exercise-induced myocardial ischaemia.
Our current study had several strengths. The study included a large sample size with information from an exercise test with ECG measurements. The use of serum n-6 PUFA measurements instead of assessing dietary intakes reduces the bias of misreporting and misclassification that can introduce random error and attenuate associations. In this cohort, serum LA and AA concentrations both associate with their dietary intakes(Reference Virtanen, Wu and Voutilainen18). However, the concentrations are known to be influenced also by genetic factors that can also have an impact on the association of the n-6 PUFA with CVD risk(Reference Marklund, Wu and Imamura12). For instance, polymorphisms of the FADS1 gene do not only regulate the n-6 PUFA concentrations but are also linked to, for example, heart rate and some CVD outcomes(Reference Marklund, Wu and Imamura12,Reference Guan, Steffen and Lemaitre54,Reference Den Hoed, Eijgelsheim and Esko55) . Unfortunately, we do not have data on such genetic factors.
The extensive database from the KIHD enabled examination of potential confounders. One potential limitation of our study was that the participants were middle-aged and older men from eastern Finland, so the findings may not be generalisable to other populations, age groups or to women. Another limitation related to the cross-sectional study design is that we cannot derive information on causal relationships between the serum n-6 PUFA concentrations and exercise-induced myocardial ischaemia. Moreover, in contrast to the n-6 PUFA in adipose tissue and erythrocyte fatty acid measurements that reflect long-term intakes, our data were obtained from serum fatty acids, which reflect only more recent intake(Reference Hodson, Skeaff and Fielding56). However, all the baseline serum n-6 PUFA concentrations in this study cohort correlate reasonably well with repeated measurements several years apart (correlation coefficients ≥ 0·5)(Reference Virtanen, Wu and Voutilainen18), suggesting that they reflect typical concentrations. Finally, we did not have data on plasma levels of cytokines and prostanoids to investigate the associations of n-6 and n-3 PUFA with these particles to understand the full potential and beneficial/harmful actions of the PUFA.
In conclusion, higher serum AA concentration was associated with lower occurrence of exercise-induced myocardial ischaemia among middle-aged and older men. This finding may provide one explanation for the previously observed inverse association of AA with risk of CHD and CVD. However, no association was found with the other n-6 PUFA in the current study. Additional large-scale studies in different populations of different ages and sexes are needed to confirm our findings and to provide knowledge of the mechanisms of the n-6 PUFA on myocardial ischaemia.
Acknowledgements
This research received no specific grant from any funding agency, commercial or not-for-profit sectors. The KIHD project was mainly funded by research grants from the NIH and the Finnish Academy to Jukka T. Salonen and George A. Kaplan. The funding sources had no role in the design, analysis or writing of this article.
The authors’ responsibilities were as follows: H. E., B. T., T-P. T. and J. K. V. designed the research; T-P. T., J. T. S., S. K. and J. K. V. acquired data; H. E., B. T. and J. K. V. had full access to all the data and statistically analysed the data; J. K. V. takes responsibility for integrity of the data and the accuracy of the data analysis. H. E. drafted the manuscript; B. T., T-P. T., J. T. S., S. K. and J. K. V. critically revised the manuscript for important intellectual content; and all authors read and approved the final manuscript.
Jukka T. Salonen is the CEO of MAS-Metabolic Analytical Services Oy. The other authors report no conflicts of interest.
This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and the study protocol was approved by the Research Ethics Committee of the University of Kuopio (1.12.1983). Written informed consent was obtained from all participants.
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