Discussion

In this prospective cohort study of well-characterized young and middle-aged women with 18 years of follow-up and repeated measures of dietary intake, we observed that a higher intake of anthocyanins was associated with a 32% reduction in risk of MI and that this inverse association was independent of established dietary and nondietary CVD risk factors. These compounds are present in red/blue fruits and vegetables and are readily incorporated into the habitual diet, and simple dietary change could have an impact on prevention efforts.

Our current knowledge of modifiable risk factors to prevent MI in young and middle-aged women is limited. Over the last few decades, several small case-control studies have reported on the increased risk of MI associated with use of oral contraceptives, heavy smoking, history of diabetes mellitus, and hypertension in younger women.^2,3,23–25 From a dietary perspective, the only available case-control data suggest a reduction in risk with moderate alcohol consumption and a modest increase in risk with very high coffee intake (≥10 cups per day).^26,27 Our data suggest a potential role for flavonoids, specifically anthocyanins, in reducing risk.

To date, limited randomized, controlled trials have examined the impact of anthocyanins on blood pressure and endothelial function relative to other subclasses.⁸ However, a recent 3-month randomized, controlled trial in dyslipidemic patients showed that anthocyanin intake improved the lipoprotein profile, resulting in an increase in high-density lipoprotein cholesterol levels and a decrease in low-density lipoprotein cholesterol levels, effects that were thought to be mediated via cholesteryl ester transfer protein inhibition.²⁸ A growing body of evidence from animal models and in vitro experiments supports a cardioprotective role for anthocyanins and their degradation products or metabolites; however, the concentrations used in many cell culture experiments are frequently higher than are physiologically achievable through dietary intake. In addition, many in vitro studies focus on the parent anthocyanin glycosides, and their biological effects may not reflect the effects of in vivo degradation or the colonic metabolites that may be responsible for at least part of the cardiovascular bioactivity of anthocyanins.⁷ In vitro, anthocyanins inhibit angiotensin-converting enzyme activity,²⁹ exert antiinflammatory effects,^30,31 and inhibit inducible nitric oxide synthase protein and mRNA expression and the activation of nuclear factor-κB and nitric oxide production in a dose-dependent manner.^32–34 Some of these biological effects were also observed after exposure to their metabolites, including protocatechuic acid.^30,31 In a rat heart model, anthocyanins increased cardiac glutathione concentrations and reduced infarct size after coronary occlusion and perfusion, which resulted in the myocardium being less susceptible to ischemia and reperfusion injury ex vivo.³⁵ In an apolipoprotein E–deficient mouse model, anthocyanins enhanced atherosclerotic plaque stabilization, suppressed the development of atherosclerotic lesions and the metabolite protocatechuic acid, and directly inhibited atherosclerosis development.^36–38 Anthocyanins have also been shown to act on a range of cells involved in atherosclerosis development, including suppressed tumor necrosis factor-α–induced monocyte chemoattractant protein-1 secretion in primary endothelial cells³⁹ and reduced expression of vascular endothelial growth factor–stimulated platelet-derived growth factor in vascular smooth muscle cells via deactivation of p38 mitogen-activated protein kinases and c-Jun N-terminal kinase.⁴⁰

These mechanistic insights into anthocyanins are important because the pathogenetic mechanisms underlying MI in young and middle-aged women may differ from those in older women and men. For example, risk in younger women may particularly reflect atherosclerosis with plaque disruption and ulceration leading to MI with nonobstructive coronary artery disease.⁴ Coronary artery spasm, a consequence of endothelial dysfunction, also may underlie CHD in younger women. Available data suggest that it is a frequent cause of acute coronary syndrome, with coronary spasm identified in 50% of patients.⁵

In an attempt to explore the biological pathways underlying the associations we observed, we added intermediate conditions, including history of hypertension, hypercholesterolemia, diabetes mellitus, and angina, to the multivariate models, but the risk estimates were not substantially attenuated. This suggests that other mechanisms beyond these may be involved. There was a suggestion of a stronger association in those with prevalent hypertension, although the interaction was not significant (Table 3), potentially because of a lack of statistical power. The addition of other plant-based food constituents, including potassium or folate, to our model also did not substantially attenuate the relationship, nor did adjusting for total intake of fruits and vegetables. These models suggest that the benefits are specific to a food constituent in anthocyanin-rich foods (including blueberries, strawberries, eggplants, blackberries, blackcurrants) and not necessarily to nonspecific benefits among participants who consume high intakes of fruits and vegetables.

We did not observe a substantial change in risk in the middle 3 quintiles of anthocyanin intake, but the difference in intake in these 3 quintiles was only 8 mg. With such small differences in intakes, measurement error or misclassification is likely to be greatest. However, when we examined the possible nonlinear relationship across the entire distribution of anthocyanin intake, we did not detect any significant deviations from linearity. To date, limited dose-response trials of anthocyanins and biomarkers of MI risk have been conducted, but given our knowledge of other subclasses, including the isoflavones and flavan-3-ols, there is likely a threshold of intake for a biological effect, and very low levels of intake are unlikely to be bioactive. When we compared extreme deciles of intake, those in the top decile had a 47% reduction in risk of MI, suggesting a continual dose-response at higher levels of habitual intake.

In food-based analyses, we similarly observed a trend toward a reduction in risk of MI with increasing intake of the 2 main sources of anthocyanins, strawberries and blueberries (which equated to almost 60% of total anthocyanin intake), with a 34% decrease in risk for those who consumed >3 portions per week compared with those who ate these fruits ≤1 time per month. These data are important from a public health perspective because these fruits can be readily incorporated into the habitual diet.

Previous prospective studies on flavonoids and CVD risk have been mixed,^41,42 in part because, until recently, databases did not contain the comprehensive range of flavonoids present in the diet. Two recent studies suggest inverse associations between increased flavanone and anthocyanin intake and fatal CVD risk in older women,^9,10 although 1 study was based on the earlier 2003 US Department of Agriculture database. Our data included both fatal CHD and nonfatal MI, and we had insufficient power to examine the association between anthocyanin intake and fatal CHD risk (n=36 cases). In relation to age, in these middle-aged women (median age, 48.9 years at diagnosis; age at baseline, 25–42 years), we observed a 32% reduction in risk in a comparison of extreme intake quintiles, a finding that supports our previous study in which we observed the greatest magnitude of effect on blood pressure in younger and middle-aged women.¹¹ This compares with the 2 previous studies of older women (mean age, 69 years; age range, 55–69 years at baseline, respectively) in which the magnitude of the association was lower: 18% and 9% reduction in risk, respectively.^9,10

We hypothesized that increased flavan-3-ol intake would also be associated with a reduction in risk, given the wealth of mechanistic support for this subclass.^6,8,12 However, to the extent that we could assess intake, no relationship was apparent for habitual intake in this population. Our recent meta-analysis of flavan-3-ol randomized, controlled trials and CVD risk biomarkers suggested that >50 mg/d epicatechin, one of the main flavan-3-ol compounds, is required for beneficial effects on systolic and diastolic blood pressures.¹² By their very nature, FFQs cannot capture all sources of flavonoids, and some sources of flavan-3-ols may not have been accurately captured. Specifically, dark chocolate is one of the main sources of flavan-3-ols, but its overall consumption in the 1990s was quite low, and most FFQs of that era did not assess different chocolate types.

There was a trend toward a reduction in risk of MI with increasing intake of flavonoid polymers and flavonols, although this did not reach statistical significance. The association with flavonoid polymer intake is intriguing because, to date, many of these compounds remain poorly defined, and we currently know little about their biological activity and bioavailability. This subclass includes proanthocyanidins, theaflavins, and thearubigins, found predominantly in the habitual diet in tea and apples, and the relative impact of these different constituents on biomarkers of CVD risk merits further investigation in future randomized, controlled trials.

The strengths of this study include the prospective design, focus on middle-aged and not older women, large sample size with long-term follow-up, repeat measures of dietary intake, detailed data on important risk factors and confounders for CVD risk, and comprehensive assessment of the range of flavonoid subclasses present in the habitual diet. The limitations of our study also warrant discussion. We adjudicated cases of acute MI using standard criteria, but we did not collect catheterization results. Therefore, although we speculated that coronary spasm may underlie CHD in younger women, we were unable to delineate which women had evidence of coronary spasm during their infarction. Although we adjusted for possible confounders that are strongly associated with MI risk (including body mass index, smoking, family history), there is still the possibility of residual or unmeasured confounding from additional unmeasured factors, which may be greatest when those consuming the highest and the lowest anthocyanin intakes are compared. However, given our detailed and updated adjustment for potential confounders, it is unlikely that these would account fully for the observed results, and in our stratified analyses, we showed that even in nonsmokers and in normal-weight, physically active participants, the point estimates remained similar (Table 3). Of all the flavonoid subclasses assessed, only anthocyanin intake was associated with a reduction in MI risk, suggesting something specific about this subclass. Our FFQ assesses intake of each food up to 6 times per day; therefore, even among the few participants who consume that many servings of blueberries or strawberries, we would be able to accurately assess their intakes. Our data may underestimate the true benefits of lowering blood pressure because we have only a dichotomous variable. It is likely that anthocyanin intake exerts benefits across the entire range of blood pressure, not just at an artificial threshold set for hypertension diagnosis. We used repeated measurements of diet to obtain a more accurate assessment of long-term flavonoid intake and to reduce measurement error. Mean cumulative dietary flavonoid intakes were calculated from a database developed from the most recent US Department of Agriculture databases¹¹ with additional input from other sources. These data sets allowed us to quantify a broad range of flavonoid subclass intakes more robustly than previous analyses. The flavonoid content of foods varies, depending on growing conditions and manufacturing processes, but despite this variation, these data allow us to rank order intakes and to compare high and low intakes in large population groups. Although correlations between the major dietary sources of flavonoids (fruits, vegetables, tea, wine) have been determined for our FFQ,^16,17 our FFQ has not been validated specifically for the intake of flavonoid subclasses. However, in a recent study, the sum of 7 flavonoid biomarkers measured in 24-hour urine samples was correlated with intakes of fruits and vegetables (0.43–0.66),⁴³ correlations similar to our validation studies. There are currently no specific biomarkers for anthocyanins because there is currently a limited understanding of their degradation and metabolism after ingestion. It is possible that our findings for anthocyanins might be due to other constituents found in the foods that contribute most to this subclass; however, the addition of other potentially beneficial constituents of fruits, including potassium, folate, and total fruit and vegetable intake, to our multivariate model did not substantially attenuate the relationship between anthocyanins and MI risk, suggesting that anthocyanins may be another important cardioprotective constituent. However, in a population-based study like ours, it is impossible to disentangle the relative influence of all the constituents of fruits and vegetables.

Our findings suggest that bioactive compounds present in red and blue fruits and vegetables commonly consumed in the habitual diet may be associated with a reduced risk of MI in young and middle-aged women. Further prospective studies, including studies with biomarkers of CHD risk to elucidate mechanisms, are needed to confirm these associations. Randomized trials focusing on commonly consumed anthocyanin-rich foods are also needed to examine dose-response effects and to be of long-enough duration to assess clinically relevant end points.