Coronary artery calcification (CAC) can reliably predict cardiovascular events. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are thought to inhibit vascular calcification on a cellular level and in animal models, however, the correlation in humans is controversial.
In symptomatic patients, CAC was quantified according to Agatstons’ method using non-contrast coronary CT. We assessed the association of EPA and DHA with early-onset coronary atherosclerosis, defined as presence of CAC above the 75th Agatston-Score (AS) percentile in sex adjusted age categories. Erythrocyte fatty acid composition was analyzed with a standardized methodology. The percentage of EPA and DHA in relation to all fatty acids present in the erythrocyte membrane is regarded the Omega-3 Index®.
Among 71 patients, 51 were below and 20 were above the 75th AS-percentile. No differences were seen in age, gender, cardiovascular risk factors, and relevant medication. In univariable analysis, significantly lower values for EPA (0.77%[0.63; 0.97] vs. 0.93%[0.72; 1.21]; p = 0.045), DHA (4.90%[4.12; 5.57] vs. 5.50%[4.58; 6.52]; p = 0.038) and the Omega-3 Index (5.73%[4.75; 6.35] vs. 6.22%[5.46; 7.71]; p = 0.034) were seen in patients above the 75th AS-percentile. All other fatty acids showed no significant differences. In multivariable analysis, the Omega-3 Index showed a significant inverse association with early onset of CAC (OR: 0.533 (95%CI: 0.303–0.938; p = 0.029)), independent of age, gender, statin use, and creatinine level (all p > 0.05).
Low levels of EPA and DHA (Omega-3 Index) are associated with early-onset coronary atherosclerosis. This finding needs to be validated in larger cohorts and might help understand the beneficial cardiovascular effects of omega-3 fatty acids.
Epidemiological and interventional studies have demonstrated beneficial effects of poly-unsaturated fatty acids especially omega-3 (n-3) fatty acids on cardiovascular disease and mortality [1,2,3,4,5]. Most recently, the Reduction of Cardiovascular Events with EPA—Intervention Trial (REDUCE-IT) has confirmed the prognostic value of high-dose eicosapentaenoic acid (EPA) intake in patients with established cardiovascular disease or with diabetes and other risk factors . Pathophysiological mechanisms however are not fully understood. Whether n-3 fatty acids in humans can inhibit vascular calcification as shown on a cellular level  and in animal models  or act through other pathways is unclear.
Coronary artery calcification (CAC), reflecting coronary atherosclerosis, can be detected and quantified non-invasively in a standardized way using coronary computed tomography (CT), and is a robust marker of cardiovascular mortality. Absence of CAC has consistently been reported to have high negative predictive value for the prediction of cardiovascular events and to be associated with good prognosis in different cohorts [9,10,11,12]. In contrast, an increasing burden of CAC has been shown to be associated with a substantial 10-year risk for major adverse cardiovascular events . An established method to quantify CAC by CT is the Agatston Score (AS), initially described by Arthur Agatston and colleagues. The AS-percentiles reflect the burden of CAC in relation to age-groups and gender, allowing for risk classification in respect to cardiovascular events .
The aim of this study was to determine whether the level of n-3 fatty acids in red blood cell membranes, especially EPA and docosahexaenoic acid (DHA), is associated with early-onset coronary atherosclerosis in symptomatic patients.
Study design and population
In this prospective analysis, we included 71 consecutive symptomatic patients with low- to intermediate pretest-likelihood for coronary artery disease, who presented with atypical angina. All patients were referred for clinically indicated coronary CT. Exclusion criteria were non-sinus rhythm, known coronary artery disease or pregnancy/nursing. Also, patients who were or had been taking nutritional supplements containing fish-oil or other polyunsaturated fatty acids were not eligible for this study. For fatty acid analysis, blood was drawn at the time of presentation before the CT scan was performed. Patients provided written informed consent and the local institutional review board approved the study.
Coronary artery calcium analysis
Coronary calcification was detected using non-contrast enhanced coronary CT (prospectively triggered at 60% of RR-interval, tube voltage 120 kV, tube current-time product 80 mAs, 2 × 128 × 0.6 mm collimation). The burden of CAC was quantified according to Agatstons’ method  on a dedicated workstation using semiautomated software (MMWP, Siemens Healthcare, Forchheim, Germany). Coronary artery calcium volume (mm³), mass (g) and the Agatston Score were determined on a patient-level. Early-onset coronary atherosclerosis was defined as presence of CAC above the 75th AS percentile in age and sex adjusted categories.
Erythrocyte fatty acid analysis
For the assessment of the individual fatty acid profile, erythrocyte fatty acid composition of each patient was analyzed with a standardized analytical methodology (“HS-Omega-3 Index®”) as described previously . By acid transesterification, fatty acid methylesters were generated from erythrocytes and analysed using gas chromatography as shown in Fig. 1. A variety of fatty acids including EPA (C20:5n3) and DHA (C22:6n3) were identified by comparison with a standard mixture of fatty acids characteristic of erythrocytes and expressed as a percentage of total identified fatty acids. The amount of EPA and DHA in relation to all fatty acids present in the red blood cell membrane is regarded the Omega-3 Index.
Continuous variables are presented as mean ± standard deviation and categorical variables are presented as frequencies and percentages. We used the chi-squared test for categorical variables, the student’s t-test for comparison of parametric and the Mann–Whitney U test for comparison of nonparametric data. For multivariable analysis, logistic regression was used. In the multivariable model, we included age, gender, statin use, and creatinine level as covariates to account for potential effects. A two-sided p value of <0.05 was considered to indicate statistical significance. All analyses were performed using SPSS (Version SE 14.2, StataCorp LP, College Station, TX).
Overall patient characteristics
The mean age of all patients (49 men and 22 women) was 62 ± 8 years. The mean BMI was 28 ± 4 kg/m2 and the mean number of cardiovascular risk factors was 2.4 ± 1. While 26 (37%) patients had no coronary calcification, 45 patients (63%) had detectable CAC (median [1.,3. quartile]): 29 mm³ [0;265], 5.5 g [0;50], Agatston Score of 30 [0;284]). The Agatston Score was between 1 and 10 in 4 (5.6%) patients, between 11 and 100 in 15 (21%) patients, between 101 and 400 in 11 (16%) patients, and over 400 in 15 (21%) patients. The mean erythrocyte levels of EPA and DHA were 0.89% [0.70; 1.10] and 5.27% [4.51; 5.97], respectively. The mean Omega-3 Index was 6.04% [5.19; 7.12].
Sex- and age-specific Agatston Score groups (above and below the 75th percentile)
Cardiovascular risk profile and burden of CAC
Among 71 patients, 51 were below and 20 were above the age and sex adjusted 75th Agatston-Score percentile. The mean age did not differ between groups (61 ± 7 vs. 63 ± 8 years, p = 0.27), and no differences were seen according to gender (female 15/51 (29%) vs. 7/20 (35%), p = 0.65). Also no differences existed in regard to BMI, traditional cardiovascular risk factors, the number of cardiovascular risk factors, and relevant medication between both groups (all p > 0.05). As expected, CAC measurements (CAC volume, mass, and Agatston Score) did differ significantly (all p < 0.001) as listed in Table 1.
Erythrocyte fatty acid composition and association with CAC
In patients above the 75th AS-percentile, significantly lower values for EPA (0.77% [0.63; 0.97] vs. 0.93% [0.72; 1.21]; p = 0.045) and DHA (4.90% [4.12; 5.57] vs. 5.50% [4.58; 6.52]; p = 0.038) were seen. The same was true for the Omega-3 Index (5.73% [4.75; 6.35] vs. 6.22% [5.46; 7.71]; p = 0.034). All other fatty acids showed no significant differences as listed in Table 2 (all p > 0.05). Although in univariable analysis no differences were found for other variables beyond EPA and DHA, we performed multivariable logistic regression analysis to account for potentially influencing factors like statin use and creatinine level. Even though Agatston Score percentiles already respect age and gender, we nevertheless also included these as covariates in the regression model to ensure accounting for potentially occult effects . In this logistic regression model the Omega-3 Index showed a significant inverse association to early onset of CAC with an odds ratio of 0.533 (95%CI: 0.303–0.938; p = 0.029). This significant association was independent of age (p = 0.319), gender (p = 0.634), statin use (p = 0.107) and creatinine level (p = 0.061)
In this prospective analysis of symptomatic patients undergoing cardiac CT, EPA and DHA showed significant inverse association to early onset of coronary atherosclerosis according to age and sex adjusted calcium score percentiles.
The prognostic value of EPA (4 g icosapent ethyl per day), as compared with placebo, was assessed in the REDUCE-IT, that enrolled 8179 patients with established cardiovascular disease or with diabetes and other risk factors. The authors could demonstrate a significant reduction of the primary end point in the intervention group, which was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina over a follow-up of 4.9 years (17.2% vs. 22.0%; HR 0.75; 95% CI: 0.68–0.83; p < 0.001). Various beneficial effects of EPA have been described, among these alterations of plaque composition and plaque progression as demonstrated by intravascular ultrasound in humans [17, 18]. Whether this holds true for vascular calcification as shown on a cellular level  and in animal models  is unclear. In fact, our analysis demonstrated a significant association of low levels of EPA in the erythrocyte membrane to early onset of coronary atherosclerosis using cardiac CT. This finding is contrary to an analysis in a Japanese population, demonstrating no association between EPA and coronary calcification (OR: 0.99 (95%CI: 0.88–1.11; p = 0.87)) . Although the number of patients with any calcification is comparable to our study (66% vs. 63%), patients with high burden of CAC seem to be more frequent in our study (13% for CAC > 300 vs. 21% for CAC > 400). As associations to some risk factors may only become apparent in patients with more advanced disease, a higher burden of severe calcification in our patients, might explain why an inverse association of EPA was detectable in the underlying analysis. In line with our finding is a study looking at CAC progression, comparing 175 Japanese in Japan to 113 U.S. whites, who underwent a follow-up CT examination. . The authors could first of all demonstrate a significant higher level of EPA in Japanese (2,36% vs. 0,79%, p < 0.01), and second of all a lower rate of CAC progression in Japanese even after adjusting for various traditional cardiovascular risk factors and BMI (incidence rate ratio: 0.262 (95% CI: 0.094, 0.731; p = 0.01). In consequence, the influence of EPA on coronary plaque might be one potential mechanism translating into lower cardiovascular events, as shown in REDUCE-IT. A more detailed insight of the influence of EPA on coronary plaque is expected from the ongoing prospective EVAPORATE trial (Effect of Vascepa on Improving Coronary Atherosclerosis in People With High Triglycerides Taking Statin Therapy), which aims to include 80 patients undergoing coronary CTA pre- and post intervention with 4 g icosapent ethyl per day . Especially the effect of EPA over statin therapy will be of major interest, as it has been shown that statin therapy itself can promote coronary atheroma calcification in studies using serial intravascular ultrasound for plaque quantification . In our analysis, the significant inverse association of n-3 fatty acids to early onset of coronary calcification was independent of statin use.
Interestingly, we could also demonstrate a significant inverse association to DHA. This result is in line with a recently published study in 1074 Japanese men, demonstrating a significant inverse association of DHA with CAC in a multivariate analysis, adjusted for multiple covariates, among them age, BMI, C-reactive protein and traditional cardiovascular risk factors (OR: 0.87 (95%CI: 0.77–0.99; p = 0.03). However, according to outcome data, DHA do not seem to have the same beneficial health effects as compared with EPA [18, 23, 24]. Whether the methodology, how fatty acids are analyzed, might partly be responsible is unclear at the time being. In this regard, two important aspects need to be mentioned. First, fatty acid levels are frequently assessed in blood serum and not in red blood cell membranes. However, the latter was shown to have low biological variability, was shown to reflect tissue fatty acid composition and is therefore regarded a more robust marker to represent true omega-3 fatty acid levels . As EPA incorporation into plaque was shown to be rapid and to a greater extent than DHA , it is to assume that serum fatty acid levels might not necessarily reflect the true content in cellular membranes. Second, hardly any intervention trial assessed the effect of omega-3 fatty acid intake by measuring pre- and post-intervention levels of membrane fatty acid composition, as known from statin intervention trials, in which the determination of pre- and post-intervention LDL-cholesterol levels is a crucial aspect to show its effect. Especially as variability in response to fatty acid intake has been shown to vary significantly between individuals [27, 28], the intake per se does not allow to conclude its adequate effect, and thus emphasizes the pre- and post intervention measurements, that therefore seem to be also a crucial aspect for intervention trials with n-3 fatty acids.
Furthermore, in our analysis we assessed early onset of coronary atherosclerosis by measuring CAC in native CT datasets. Whether early onset of non-calcified atherosclerosis shows similar differences in regard to n-3 fatty acid levels remain unclear and need to be assessed in future analysis.
Some limitations need to be acknowledged. The number of patients included in this analysis is rather small and thus the statistical power to control for multiple covariates was limited. Intake of EPA- and DHA-rich sources of food were not evaluated, however, patients who were or had been taking nutritional supplements containing fish-oil or other polyunsaturated fatty acids were not eligible for this study. Although the results need to be validated in a bigger population, the significant associations observed in this small cohort can be hypothesis generating. Secondly, no follow up CT was performed, therefore comments on CAC progression in patients with different levels of omega-3 fatty acids are not possible.
Low levels of EPA and DHA (Omega-3 Index) are associated with early onset of coronary atherosclerosis according to age and sex adjusted calcium score percentiles. These findings need to be validated in a larger population, however our findings shed some light on the pathophysiology, potentially explaining the beneficial effects of omega-3 fatty acids.
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Conflict of interest
MM reports honoraria from Siemens HealthCare and Edwards Lifescience outside the submitted work, and YZ reports lecture fees from Baxter, MSD and Shire outside the submitted work. The other authors declare that they have no conflict of interest.
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Bittner, D.O., Goeller, M., Zopf, Y. et al. Early-onset coronary atherosclerosis in patients with low levels of omega-3 fatty acids. Eur J Clin Nutr 74, 651–656 (2020). https://doi.org/10.1038/s41430-019-0551-5
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