Abstract
Objective
Epidemiological studies suggested that n-6 fatty acids, especially linoleic acid (LA), have beneficial effects on coronary heart disease (CHD), whereas some in vitro studies suggested that n-6 fatty acids, specifically arachidonic acid (AA), may have harmful effects. We examined the association of serum n-6 fatty acids with plasminogen activator inhibitor-1 (PAI-1).
Methods and Results
A population-based cross-sectional study recruited 926 randomly selected men aged 40–49 without cardiovascular disease during 2002 to 2006 (310 Caucasian, 313 Japanese, and 303 Japanese-American men). Plasma PAI-1 was analyzed in free form, both active and latent. Serum fatty acids were measured with gas-capillary-liquid-chromatography. To examine the association between total n-6 fatty acids (including LA and AA, respectively) and PAI-1, multivariate regression models were used. After adjusting for confounders, total n-6 fatty acids, LA, and AA were inversely and significantly associated with PAI-1 levels. These associations were consistent across three populations.
Conclusions
Among 915 middle-aged men, serum n-6 fatty acids had significant inverse associations with PAI-1.
Keywords: plasminogen activator inhibitor-1, linoleic acid, fatty acids
Introduction
Plasminogen activator inhibitor-1 (PAI-1), a primary inhibitor of plasminogen activators, has an anti-fibrinolytic function.(1) High levels of PAI-1 are associated with an increased risk for developing coronary heart disease (CHD) or stroke.(2–4) This increased risk of CHD may be due to promoting platelet adhesion and acute thrombus formation.(4)
Epidemiological studies suggest that linoleic acid (LA), a major component of n-6 fatty acid, has beneficial effects on both CHD and its risk factors, whereas some in vitro studies suggest that another n-6 fatty acid, arachidonic acid (AA), may have adverse effects. The Nurses’ Health Study showed that a high dietary intake of LA has a strong inverse association with CHD.(5) Additionally, a recent meta-analysis found that dietary intake of LA had a strong inverse association with non-fatal cardiovascular events.(6) The cardioprotective benefits(7) of n-6 fatty acids may be due to decreasing blood pressure,(8) reducing thrombosis,(9) and improving insulin sensitivity.(10) In contrast, several eicosanoids derived from AA are pro-inflammatory and pro-thrombotic, promoting vasoconstriction and enhance platelet aggregation. Thus, AA has been postulated to adversely affect CHD. However, recent studies identified AA-derived eicosanoids to have several beneficial attributes including vasodilation, platelet aggregation inhibition, and anti-inflammatory effects.(11) Interestingly, a recent meta-analysis showed that AA was not associated with fatal or non-fatal cardiovascular events.(6) These contradictory findings suggest that dietary or serum levels of AA have little association with CHD risk, possibly because AA levels are tightly regulated in the human body.(12–14) Although PAI-1 is known to be involved in developing atherothrombosis,(15, 16) very few studies reported their associations with n-6 fatty acids.
The purpose of this study was to test whether higher levels of serum n-6 fatty acids are associated with lower levels of PAI-1 in men aged 40–49. Additionally, we investigated whether higher levels of specific n-6 fatty acids, i.e., LA and AA, are associated with lower levels of PAI-1. To test these hypotheses, we examined data from a population-based cross-sectional study of 926 Caucasian, Japanese, and Japanese-American men aged 40–49 in the Electron-Beam Tomography, Risk Factor Assessment among Japanese and U.S. Men in the Post-World War II Birth Cohort (ERA-JUMP) study.(17)
Methods
Study population
Participants were a randomly selected population-based sample of 926 men aged 40–49 between 2002 and 2006 from three centers: 310 Caucasian men from Allegheny County, Pennsylvania, 313 Japanese men from Kusatsu, Shiga, Japan, and 303 Japanese-American men from Honolulu, Hawaii. Those with clinical cardiovascular or other severe diseases were excluded. Detailed descriptions of the study population were previously published.(17–19) Our final sample was 915 men (304 Caucasian, 313 Japanese, and 298 Japanese-American men) due to 11 missing data. Informed consent from each participant was obtained. The protocol for the study was approved by the Institutional Review Boards of the University of Pittsburgh (Pennsylvania), Shiga University of Medical Science (Otsu, Japan), and the Kuakini Medical Center (Honolulu, Hawaii).
Venipuncture was performed in all participants in the early morning after a 12-hour fast, as previously described.(17) Fasting serum and plasma samples were stored at −80°C, and shipped on dry ice to the Heinz Laboratory at the University of Pittsburgh to examine levels of low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc), total cholesterol, triglycerides, insulin, and glucose, as published elsewhere.(17) A calorimetric-competitive-enzyme-linked immunosorbent assay was utilized to assess CRP (C-reactive protein). Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, or anti-hypertensive medication usage. Diabetes mellitus was defined as fasting glucose level ≥7mmol/L (126mg/dL) or anti-diabetic medication usage. ‘Alcohol drinker’ was defined as a man who drinks alcohol two days or more per week. ‘Current smoking’ was defined as a man who smoked during the prior month.
Measurement of PAI-1
As previously reported,(20, 21) plasma PAI-1 was measured at the clinical biochemistry research laboratory of the University of Vermont. Briefly, plasma PAI-1 levels were analyzed in citrated plasma(20) by a two-site ELISA, which was sensitive to free PAI-1 form but not to complexes between t-PA and PAI-1(21), which was developed by Dr. Collen and colleagues.(22) Inter-assay coefficient of variations (CVs) for PAI-1 was 7.7%.
Measurements of serum fatty acids
To measure serum fatty acids in a percentage of total fatty acid amounts, gas-capillary-liquid chromatography (PerkinElmer Clarus 500; PerkinElmer, Waltham, MA) was performed.(23) The intra-assay CVs of LA (18:2n-6) and AA (20:4n-6) in serum n-6 fatty acids were 1.6% and 2.8%, respectively.(23) The CVs for other fatty acids ranged from 2.5% to 9.8%.(23)
Statistical analyses
Log-transformed PAI-1 was used for the analyses, because the distribution of PAI-1 was skewed. To compare descriptive distributions across centers, an analysis of variance (ANOVA) for a continuous variable and a Mantel-Haenszel test for a categorical variable were performed. When a significant difference among the three groups exists, we examined multiple comparison tests using a Bonferroni test. To pool the data, we tested interactions according to centers on associations of PAI-1 with LA as well as AA. We also assessed the center-specific associations of n-6 fatty acids on PAI-1 according to the three study centers (Table 3). After confirming no interaction and the same direction of the associations by centers, we pooled the data. To estimate the association between serum n-6 fatty acids and PAI-1 levels, we performed multivariate linear regression analyses, adjusting for covariates as followings: in the model I, we adjusted for age and center; in the model II, we further adjusted for body mass index (BMI), current smoking, alcohol drinker, hypertension, and diabetes; in the model III, we further adjusted for LDLc, HDLc, triglycerides, and CRP; in the model IV, we further adjusted for marine n-3 and trans fatty acids. Because some eicosanoids of n-3 fatty acids were reported to inhibit the production of AA derived eicosanoids,(11) we tested marine n-3 as well as trans fatty acids as covariates. The level of significance was considered to be p<0.05. All reported p-values were based on two-sided tests. All statistical analyses were performed using SAS 9.2 for Windows (SAS Institute, Inc., Cary, North Carolina, U.S.).
Table 3.
Center-specific and pooled associations between n-6 fatty acids and log(PAI-1)
| Standardized parameter estimates | ||||
|---|---|---|---|---|
| Total (n=915) |
Caucasian men (n=304) |
Japanese men (n=313) |
Japanese- American men (n=298) |
|
| Total n-6 Fatty acids | ||||
| Univariate | −0.27* | −0.32* | −0.25* | −0.27* |
| Model I | −0.32* | −0.32* | −0.24* | −0.27* |
| Model II | −0.24* | −0.18* | −0.20* | −0.21* |
| Model III | −0.13* | −0.06* | −0.12* | −0.10* |
| Model IV | −0.14* | −0.09* | −0.12* | −0.09* |
| Linoleic acid | ||||
| Univariate | −0.23* | −0.29* | −0.20** | −0.23* |
| Model I | −0.24* | −0.29* | −0.19** | −0.23* |
| Model II | −0.17* | −0.15* | −0.15* | −0.18* |
| Model III | −0.10* | −0.07* | −0.07* | −0.12* |
| Model IV | −0.10* | −0.08* | −0.04* | −0.11* |
| Arachidonic acid | ||||
| Univariate | −0.16* | −0.13*** | −0.19** | −0.11 |
| Model I | −0.14* | −0.13 | −0.18** | −0.11 |
| Model II | −0.10* | −0.08* | −0.17* | −0.07* |
| Model III | −0.01* | −0.003* | −0.10* | 0.04* |
| Model IV | −0.01* | −0.003* | −0.10* | 0.04* |
p<0.001;
p<0.01;
p<0.05
Total n-6 fatty acids indicate the sum of linoleic acid (18:2n-6), gamma-linoleic acid (18:3n-6), dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6).
Marine-derived n-3 fatty acids were defined as eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and docosahexaenoic acid (22:6n-3).
Total n-3 fatty acids indicate marine-derived n-3 fatty acids, eicosatetraenoic acid (20:4n-3) and α-linolenic acid (22:18n-3).
Trans fatty acids indicate the sum of palmitelaidic acid (16n-7:1t), trans 9-octadecanoic acid (18n-9:1t) and linolelaidic acid (18n-6:2tt).
Model I: adjusted for age;
Model II: additionally adjusted for BMI, current smoking, alcohol drinker, hypertension, and diabetes;
Model III: further adjusted for LDL cholesterol, HDL cholesterol, triglycerides, and CRP (C-reactive protein);
Model IV: continuously adjusted for marine n-3 fatty acids, and trans fatty acids.
Results
General characteristics of the 915 study participants are shown in Table 1. The average age was 45 years old. In the total study population, participants with hypertension and diabetes were 24.9% and 7.5%, respectively. Median level (interquartile range) of PAI-1 was 37.4 ng/mL (23.3 to 58.4).
Table 1.
General characteristics of the study participants
| Total (n=915) |
Caucasian men (n=304) |
Japanese men (n=313) |
Japanese- American men (n=298) |
|
|---|---|---|---|---|
| Age (year) | 45.4 | 45.0 | 45.1† | 46.1‡ |
| BMI (kg/m2) | 26.5 | 27.9* | 23.7† | 28.0 |
| Waist circumference (cm) | 92.6 | 98.7* | 85.2† | 94.0‡ |
| Systolic blood pressure (mmHg) | 125.1 | 122.6 | 125.0 | 127.6‡ |
| Diastolic blood pressure (mmHg) | 75.8 | 73.1* | 76.5 | 77.7‡ |
| Hypertension (%) | 24.9 | 15.1* | 26.5 | 33.2‡ |
| LDL cholesterol (mg/dL) | 129.5 | 134.7 | 132.2† | 121.4‡ |
| Triglyceride (mg/dL)§ | 140.5 (96.0–224.0) | 128.0(92.0–185.5) | 137(103.0–182.0) | 140.5(96.0–224.0) |
| HDL cholesterol (mg/dL) | 50.9 | 47.8* | 54.1† | 50.7 |
| Total cholesterol (mg/dL) | 212.0 | 212.1 | 217.2† | 206.5 |
| Glucose (mg/dL) | 106.7 | 101.3* | 106.8† | 112.0‡ |
| Insulin (µIU/mL) | 13.5 | 15.2* | 10.3† | 15.1 |
| Diabetes (%) | 7.5 | 3.3 | 6.1† | 13.4‡ |
| Current smoker (%) | 23.5 | 7.2* | 49.2† | 13.1 |
| Alcohol drinker (%) | 49.6 | 44.1* | 67.1† | 36.9 |
| C-reactive protein (mg/l)§ | 0.6 (0.3–1.3) | 1.0 (0.5–1.8) | 0.3 (0.2–0.7) | 0.7 (0.3–1.3) |
| PAI-1 (ng/mL)§ | 37.4 (23.3–58.4) | 27.4 (15.7–41.3)* | 41.2 (24.3–67.3) | 45.9 (31.6–61.7)‡ |
BMI=body mass index; LDL=low density lipoprotein; HDL=high density lipoprotein;
Significance test was based on ANOVA, followed by Bonferroni test if the overall ANOVA was significant.
Under Bonferroni test, significant difference between Caucasian and Japanese men, p<0.01
Under Bonferroni test, significant difference between Japanese and Japanese-American men, p<0.01
Under Bonferroni test, significant difference between Caucasian and Japanese-American men, p<0.01;
Median (interquartile range)
Serum proportions of fatty acids are listed in Table 2. Total n-6, total n-3, saturated, and monounsaturated fatty acids made up 39.0%, 6.5%, 31.2%, and 20.4%, respectively. LA and AA were 28.8% and 8.1%, respectively. A correlation coefficient between LA and AA was 0.06 (p=0.059).
Table 2.
Distribution of serum fatty acids (%)
| Total (n=915) |
Caucasian men (n=304) |
Japanese men (n=313) |
Japanese- American men (n=298) |
|
|---|---|---|---|---|
| Polyunsaturated fatty acids | 45.4 | 45.6* | 44.3† | 46.5 |
| Total n-6 fatty acids | 39.0 | 41.3* | 34.7† | 41.1 |
| Linoleic acid | 28.8 | 29.9* | 26.5† | 30.0 |
| Arachidonic acid | 8.1 | 9.0* | 6.6† | 8.9 |
| Total n-3 fatty acids | 6.5 | 4.2* | 9.6† | 5.4‡ |
| Marine n-3 fatty acids | 6.0 | 3.8* | 9.3† | 4.9‡ |
| α-linolenic fatty acids | 0.3 | 0.3* | 0.2† | 0.4‡ |
| Monounsaturated fatty acids | 20.4 | 20.3* | 21.2† | 19.6 |
| Saturated fatty acids | 31.2 | 30.9* | 31.7† | 30.9 |
| Trans fatty acids | 0.8 | 1.0* | 0.6† | 0.9‡ |
Total n-6 fatty acids indicate the sum of linoleic acid (18:2n-6), gamma-linoleic acid (18:3n-6), dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6).
Marine-derived n-3 fatty acids were defined as eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and docosahexaenoic acid (22:6n-3).
Total n-3 fatty acids indicate marine-derived n-3 fatty acids, eicosatetraenoic acid (20:4n-3) and α-linolenic acid (22:18n-3).
Saturated fatty acids indicate the sum of myristic aicd (14:0), palmitic acid (16:0) and stearic acid (18:0).
Monounsaturated fatty acids indicate the sum of palmitoleic acid (16:1n-7), oleic acid (18:1n-9), and cis-vaccenic acid (18:1n-7).
Trans fatty acids indicate the sum of palmitelaidic acid (16n-7:1t), trans 9-octadecanoic acid (18n-9:1t) and linolelaidic acid (18n-6:2tt).
Significance test was based on ANOVA followed by Bonferroni test if the overall ANOVA was significant.
Under Bonferroni test, significant difference between Caucasian and Japanese men, p<0.01
Under Bonferroni test, significant difference between Japanese and Japanese-American men, p<0.01
Under Bonferroni test, significant difference between Caucasian and Japanese-American men, p<0.01
Pooled and center-specific analyses reveal that serum n-6 fatty acids were inversely associated with PAI-1 in the total population as well as in each of three different populations (Table 3). Pooled analyses showed that serum total n-6 fatty acids, LA, and AA had significant inverse associations with PAI-1 levels, even after adjusting for covariates. In center-specific analyses, PAI-1 had significant inverse associations with total n-6 fatty acids and LA over three study populations. These significant associations remained after multivariate adjustments. PAI-1 was inversely associated with AA. No significant interaction existed in the associations of serum n-6 fatty acids, LA, and AA with PAI-1 according to the study centers (p=0.09, 0.08, and 0.24, respectively). Standard parameter estimates indicate a standard deviation (SD) unit change in log-transformed PAI-1 per a 1 SD unit increase in serum n-6 fatty acids.
Discussion
This population-based cross-sectional study found that total serum n-6 fatty acids were inversely and significantly associated with PAI-1 among 915 men, aged 40–49. Additionally, both LA and AA showed significant inverse associations with PAI-1 levels (both, p<0.0001).
Our present study may provide a novel mechanism on the cardioprotective benefits of n-6 fatty acids by improving the fibrinolytic response, such as reducing PAI-1. Our finding of an inverse association between serum n-6 fatty acids and PAI-1 is consistent with the results of several previous studies,(24, 25) but not all.(26) These findings of n-6 fatty acids suggest a favorable fibrinolytic response on vascular thrombosis, including a decrease in platelet aggregation. Fleischman et al. found an increased platelet aggregation time (p<0.05) and a decreased disaggregation time (p<0.01) on a dietary LA in each for two weeks from about 2.9% to about 5.0% of energy among 66 subjects.(24) A crossover study by Thijssen et al. also demonstrated an increased platelet aggregation time while on a LA diet in comparison to on a saturated fatty acid diet in 18 men (p=0.04).(27) O’Brien et al. conducted a clinical trial in 39 healthy men for six weeks with either a PUFA diet (sunflower oil based foods, 65% LA) replaced for saturated fat or a normal diet.(25) They found the decreased platelet count (p=0.01), and the increased bleed time (p=0.05).(25) Further, previous studies, including the Nurses’ Health Study,(5) have suggested that n-6 fatty acids lower the CHD risk, through a decrease in blood pressure,(8) a reduction of thrombosis,(9) and an improvement in insulin sensitivity.(10)
Our results of the inverse association between serum n-6 fatty acids and PAI-1 are partially inconsistent with the results of a previous study. Byberg et al. showed that PAI-1 activity has a significant inverse association with serum LA but a significant positive association with serum AA in their sub-analysis with 381 men from a population-based cross-sectional sample of 871 men aged 70 years.(26) The discrepancy in the association of PAI-1 with AA may be attributed to different measurements of PAI-1 and fatty acids or to different ages of participants. In measurements of PAI-1 and fatty acids, Byberg et al. measured PAI-1 activity (i.e., a free active form) and serum cholesterol ester for fatty acid measurements in older participants (mean average 70 years), whereas we measured total plasma PAI-1 levels (i.e., free active, free latent, and complex with t-PA forms) and fatty acids in serum cholesteryl ester, phospholipids, and triglycerides, in middle-aged men (ages 40–49). Although the previous study demonstarted a linear association between PAI-1 activity and PAI-1 antigen (r=0.80 in platelet-poor plasma; r=0.88 in platelet-rich plasma), about 66.7% of PAI-1 antigen in plasma was active.(22) Considering very short half-life of PAI-1 levels, and various processes (e.g., temperature, time, or pH) for handling the blood samples, as well as significant diurnal change of PAI-1, the PAI-1 antigen measurement as in our study may have the advantage of detecting comprehensive forms of relatively unstable total plasma PAI-1, rather than measuring only an active form.
Future studies are required in order to elucidate possible reasons of the discrepancy between in vitro and the population studies. Several in vitro studies have shown that LA increases the secretion of PAI-1 in HepG2 cells.(28, 29) In vitro studies reported that AA-produced eicosanoids promoted neutrophil adhesion(30) and IL-1β production by human monocytes(31). However, more recent studies demonstrated no effect or a beneficial effect. A double-blind placebo-controlled study with an AA supplementation of 840 mg/day for four weeks demonstrated no effect on platelet aggregation in 24 healthy Japanese men who had relatively high levels of fish oil consumption.(32) In another clinical trial of 10 healthy men taking a 200mg/day vs. 1,500mg/day AA regimen, Nelson et al. found a borderline significance between higher AA intake and prolonged bleeding time (p=0.06).(33) Although several AA-derived eicosanoids may indeed have a pro-inflammatory role, recent studies suggest that several AA-derived eicosanoids may play an anti-inflammatory role.(34)
Mechanisms responsible for the association of n-6 fatty acids with PAI-1 require future studies. However, two possibilities exist. First, n-6 fatty acids may delay platelet aggregation so that PAI-1, acting as an acute-phase reactant, is decreased within hemodynamic balance and thrombotic response, during vascular injury, in atherosclerosis and CHD. Several previous studies have shown that LA reduces platelet aggregation.(24, 25) Second, LA may reduce PAI-1 levels through its cholesterol lowering effect. A previous study from ERA-JUMP found that higher levels of serum LA and AA were associated with lower levels of LDL and VLDL.(35) An in vitro study showed that VLDL led to increased PAI-1.(36) These reduced cholesterol levels may improve or modulate the fibrinolytic response.
We additionally examined the associations of other fatty acids with PAI-1. Serum marine n-3 fatty acids over the three populations showed little overlapped distributions and the lack of consistent associations (supplemental table 1). Trans fatty acids of three populations were positively associated with PAI-1 (supplemental table 2).
The strengths of the present study include the following: a) the association was examined in a randomly selected population-based sample; and b) the sample size was relatively large. However, this study also has several limitations: a) the cross-sectional study design could not assess a causality; b) the study population included only men aged 40–49 years, which may limit the generalizability to other populations; and c) as an observational study, this present study may include residual confounding or potentially unmeasured factors, such as total energy intake.(37)
In conclusion, serum n-6 fatty acids were inversely and significantly associated with PAI-1 levels in a population-based cross-sectional study. Total n-6 fatty acids, especially LA and AA, were inversely and significantly associated with PAI-1 levels in both univariate and multivariate models. These findings suggest that n-6 fatty acids may have favorable effects on fibrinolysis. A future study to examine the causality between n-6 fatty acid and PAI-1 is warranted.
Supplementary Material
Acknowledgments
Funding Sources
This research was funded by the National Institutes of Health R01 HL68200 and HL071561 and from the Japanese Ministry of Education, Culture, Sports, Science and Technology, B 16790335 and A 13307016
Footnotes
Disclosures
Conflict of interests: none.
Sunghee Lee wrote the first manuscript.
J. David Curb, Takashi Kadowaki, Rbobert W. Evans, Tomoko Takamiya, Aiman El-Saed, Sayaka Kadowaki, Todd Seto, Daniel Edmundowicz, Hirotsugu Ueshima, Lewis Kuller, and Akira Sekikawa collected the data.
Sunghee Lee, Teruo Otake, and Akira Sekikawa analyzed the data.
Sunghee Lee, Rhobert W. Evans, Katsuyuki Miura, Chol Shin, Jina Choo, Akira Fujiyoshi, Teruo Otake, Hirotsugu Ueshima, Lewis H. Kuller, and Akira Sekikawa interpreted the data.
J. David Curb, Rhobert W. Evans, Katsuyuki Miura, Jina Choo, Todd Seto, Kamal Masaki, Daniel Edmundowicz, Lewis H. Kuller and Akira Sekikawa gave critical comments.
Sunghee Lee with Akira Sekikawa revised the paper.
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