Flavonols and flavones are antioxidant phenolic compounds found in vegetables, fruits, tea, and red wine. They are known to be effective scavengers of free radicals 1 and chelators of transitional metals (Fe, Cu) that catalyze free radical production. 2 They also reduce macrophage-mediated low-density lipoprotein oxidation. 3,4 Quercetin and apigenin have inhibited hemostasis in vitro but not in concentrations found in human plasma. 5,6 Under certain reaction conditions, flavonoids can also have pro-oxidant activity. 7,8
Epidemiologic studies have not shown a consistent association between flavonoid intake and the risk of coronary heart disease. In three studies, flavonoids had an inverse association with coronary heart disease, 9–11 but the results of two other studies did not support these findings. 12,13 We investigated the associations between flavonol and flavone intake and the risk of nonfatal myocardial infarction and coronary death in a cohort of Finnish male smokers.
Subjects and Methods
Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study
The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) was a double-blind, placebo-controlled primary prevention trial undertaken to determine whether supplementation with alpha-tocopherol, beta-carotene, or both would reduce the incidence of lung cancer in male smokers. The rationale, design, and methods of the study as well as the characteristics of the participants have been described in detail. 14
The participants of the ATBC Study were male smokers recruited from the total male population 50–69 years of age in southwestern Finland (N = 290,406). To be eligible, they had to smoke at least five cigarettes per day at entry and to give written informed consent. The exclusion criteria included a history of cancer or other serious disease limiting long-term participation; use of vitamin E, vitamin A, or beta-carotene supplements in excess of predefined doses [vitamin E >20 mg per day, vitamin A >20,000 international units (4,000 retinol equivalents) per day, or beta-carotene >6 mg per day] and treatment with anticoagulant agents.
After these exclusions, 29,133 men were randomized into one of the four supplementation regimens: alpha-tocopherol alone (daily dose 50 mg), beta-carotene alone (20 mg), alpha-tocopherol and beta-carotene, or placebo. Follow-up continued for 5–8 years (median 6.1 years).
Diet was assessed at baseline using a self-administered, modified diet history questionnaire. 15 This questionnaire was satisfactorily completed by 27,111 participants (93%). In addition, 1,739 men reported a previous myocardial infarction diagnosed by a physician. Thus, 25,372 men were included in the present analyses.
At baseline, the men completed a questionnaire on general background characteristics and medical and smoking histories. Height, weight, and blood pressure were measured, and serum samples were stored at −70°C. Serum total cholesterol and high-density lipoprotein cholesterol levels were determined enzymatically cholesterol oxidase-4-aminophenazone (CHOD-PAP) method, Boehringer Mannheim, Mannheim, Germany.
The diet questionnaire included 276 food items and mixed dishes. It was used with a portion size picture booklet of 122 photographs of foods, each with three to five different portion sizes. The subject was asked to report the usual frequency of consumption and the usual portion size of foods during the previous 12 months. The frequencies were reported as the number of times per day, week, or month. At the first baseline visit, the questionnaire and the picture booklet were given to the subject to be completed at home. At the second baseline visit 2 weeks later, the questionnaire was returned and reviewed and completed with the help of a nurse.
The food consumption data were used to compute daily nutrient intake values based on the food composition database and related software at the National Public Health Institute. These data are based on chemical analyses of Finnish foods, with the exception of flavonol and flavone content, which is mainly based on composition analyses done by Hertog and colleagues. 16,17 The flavonol content of berries is, however, based on Finnish analyses. 18 Total flavonol and flavone intake was calculated as a sum of intakes of quercetin, kaempherol, myricetin, luteolin, and apigenin.
The dietary method was validated in a pilot study carried out among 190 men before the ATBC Study. 15 The men completed the questionnaire first and then kept 24 days of food records, spread over 6 months, as the reference method. They filled in the questionnaire again at the end. The energy-adjusted correlations between the first or the second dietary questionnaire and the food records were 0.59/0.66 for flavonols and flavones.
Ascertainment of Endpoints
The endpoints of this study were the first nonfatal myocardial infarction (alive on day 28 after the onset of the event) and death due to coronary heart disease (coronary death). Only the first of these events after randomization was registered as an endpoint. Endpoints were identified from national registers. In Finland, all hospitalizations are entered into the Hospital Discharge Register and all deaths into the Register of Causes of Death. Both registers use the codes of the International Classification of Diseases (the 8th revision was used through 1986 and the 9th revision thereafter).
Record linkage to the registers was done using the unique personal identification number. The first acute myocardial infarction (code 410) after randomization was searched for in the Hospital Discharge Register. When a case was found, survival beyond 28 days from the beginning of the attack was checked in the Register of Causes of Death, and the survivors were considered cases of nonfatal myocardial infarction. Coronary death cases were considered to be those who died within 28 days together with those fatal cases identified from the Register of Causes of Death with the underlying cause of death coded as 410–414. Register follow-up continued throughout the ATBC Study, and thus cases were also identified among dropouts. After 6.1 years of follow-up, there were 1,122 nonfatal myocardial infarctions and 815 coronary deaths.
Validity of the diagnoses of the coronary events in the registers has been evaluated; 94% of the cases of a random sample (N = 408) retained either definite or possible myocardial infarction in a review of clinical and autopsy data according to the FINMONICA criteria. 19
The participants contributed follow-up time from the date of randomization until first myocardial infarction, death, or end of trial (April 30, 1993). Men were grouped into quintiles of energy-adjusted intakes of flavonols and flavones and nutrients calculated from the food consumption data. Total flavonol and flavone intake and all nutrients were log-transformed before the energy adjustment, which was done by the regression residual method. 20 Alcohol intake was not energy adjusted.
Proportional hazards models were used to estimate the relative risks (RRs) and 95% confidence intervals (CIs) of coronary heart disease associated with intake of flavonols and flavones and selected foods, with simultaneous adjustment first for age and supplementation group and second for cardiovascular risk factors (systolic and diastolic blood pressure, serum total cholesterol, serum high-density lipoprotein cholesterol, body mass index, smoking years, number of cigarettes smoked daily, history of diabetes mellitus or coronary heart disease, marital status, education, and leisure-time physical activity).
The median intake of flavonols and flavones was 8.0 mg per day. There was more than a fourfold difference in the median flavonol and flavone intake between the highest and the lowest quintiles of energy adjusted intake (Table 1). Men who had the highest intake of flavonols and flavones were more educated; were physically more active; were more often wine or tea drinkers; and ate more fruits, berries, and vegetables than those with low intake of flavonols and flavones. In addition, men with high intake of flavonols and flavones had higher intake of polyunsaturated fatty acids, beta-carotene, vitamin C, and vitamin E and lower intake of saturated fatty acids than men with low intake of flavonols and flavones.
Correlations between intakes of flavonols and flavones and nutrients are shown in Table 2. The highest correlation was with vitamin C (r = 0.50). The highest correlations between flavonol and flavone intake and consumption of foods rich in flavonols and flavones were tea (r = 0.63) and vegetables (r = 0.44).
Flavonol and flavone intake was associated with the risk of nonfatal myocardial infarction (Table 3). After adjustment for age and supplementation group, the RR of nonfatal infarction among men in the highest compared with the lowest quintile of flavonol and flavone intake was 0.77 (95% CI = 0.64–0.93). In the multivariate model adjusting further for cardiovascular risk factors, the RR was unchanged (RR = 0.77, 95% CI = 0.64–0.93). When subjects reporting coronary heart disease or diabetes mellitus at baseline were excluded, the risk of nonfatal myocardial infarction was similar (RR = 0.80, 95% CI = 0.65–0.99).
High intake of flavonol and flavones was associ- ated also with lowered risk of coronary death in the base model (RR in the highest vs the lowest quintile of intake = 0.79, 95% CI = 0.63–0.97). After adjusting further for cardiovascular risk factors, however, this association was attenuated (RR = 0.89, 95% CI = 0.71–1.11).
In the food group analyses, consumption of wine was inversely related to the risk of coronary heart disease in the multivariate model (Table 4). Those who drank at least one glass of wine per week had decreased RR of coronary death (RR = 0.71, 95% CI = 0.55–0.93) compared with those who drank less than one glass of wine per week. There was also an inverse association for nonfatal myocardial infarction (RR = 0.77, 95% CI = 0.62–0.95). After further adjustment for total alcohol intake, the association between wine consumption and the risk of nonfatal myocardial infarction was attenuated markedly (RR = 0.89, 95% CI = 0.72–1.11). The risk of coronary death was attenuated only marginally, however (RR = 0.73, 95% CI = 0.55–0.95).
In addition, those in the highest quintile of vegetable consumption had lower risk of nonfatal myocardial infarction and coronary death than those in the lowest quintile (RR = 0.77, 95% CI = 0.63–0.94, and RR = 0.68, 95% CI = 0.50–0.95, respectively). After adjusting further for flavonol and flavone intake, the association between vegetable consumption and nonfatal myocardial infarction was attenuated (RR = 0.84, 95% CI = 0.68–1.04); the same was true for coronary death (RR = 0.64, 95% CI = 0.46–0.91). Consumption of fruits, berries, or tea had little association with the risk of coronary heart disease. In a multivariate model that included simultaneously all foods (fruits, berries, vegetables, tea, and wine), RRs and their CIs were essentially the same as they were in the individual models of the foods.
In this large cohort of male smokers, we observed an inverse association between flavonol and flavone intake and the risk of nonfatal myocardial infarction, with a much more modest association with coronary death.
In a Dutch cohort of 805 older men with coronary deaths during 5-year follow-up, a strong inverse relation between the intake of flavonols and flavones and the risk of coronary death was found (RR in the highest vs lowest tertile of intake = 0.32, 95% CI = 0.15–0.71). 9 Among those 693 men with no history of myocardial infarction at baseline, the RR of nonfatal and fatal myocardial infarction (N = 38) was attenuated (RR = 0.52, 95% CI = 0.22–1.23), whereas the risk of coronary death (N = 20) remained similar (RR = 0.29, 95% CI = 0.09–0.93). In a Finnish cohort of 5,133 men and women followed for 25 years, an inverse association between intake of flavonols and flavones and the risk of coronary death was observed among men (N = 324; RR in the highest vs lowest quartile of intake = 0.67, 95% CI = 0.44–1.00) and women (N = 149; RR = 0.73, 95% CI = 0.41–1.32).10
The largest cohort (51,529 men) in which associations between the intake of flavonols and flavones and the risk of coronary events have been studied so far is The Health Professionals Follow-Up Study. 13 During 6 years of follow-up there were 496 first nonfatal myocardial infarctions, among which there was a small positive association with the intake of flavonols and flavones (RR in the highest vs lowest quintile of intake = 1.08, 95% CI = 0.81–1.43). Among 4,814 men with coronary heart disease at baseline, there was an inverse association between flavonol and flavone intake and the risk of coronary death (RR = 0.63, 95% CI = 0.33–1.20). In the Caerphilly study of 1,900 Welsh men without previous myocardial infarction who were followed up for 14 years, 186 men were diagnosed with ischemic heart disease during the follow-up and 131 died from ischemic heart disease. 12 The intake of flavonols was not related to ischemic heart disease incidence (RR in the highest vs with lowest quartile of intake = 1.0, 95% CI = 0.6–1.6) but was positively associated with coronary mortality (RR = 1.6, 95% CI = 0.9- 2.9). The authors suggested that this association could be explained by high tea consumption, which in turn is related to a less healthful life-style and lower social class in the United Kingdom, in contrast to other populations in which high tea consumption is associated with a more healthful life-style. In a recent cohort study 34,492 postmenopausal women were followed for 10 years during which 438 deaths from coronary heart disease occurred. 11 The risk of coronary death was inversely associated with flavonoid intake (RR in the highest vs lowest quintile of intake = 0.62, 95% CI = 0.44–0.87).
Thus, most studies support a modest inverse association between the intake of flavonols and flavones and the risk of death from coronary heart disease, whereas no such association is evident for nonfatal myocardial infarction. We, however, found an inverse moderate association between flavonoid intake and risk of nonfatal myocardial infarction but only a weak association with coronary death.
The discrepant findings between our results and those of other studies may be due to different study populations. The intake of flavonols and flavones in our cohort was lower than in any of the previously reported cohorts, except for the other Finnish cohort. 10 The mean intake of flavonols and flavones was only 9.9 mg per day in our study, whereas in the previous studies the average intake has varied from 20 13 to 26 9 mg per day. The main reason for the low intake of flavonols and flavones is low consumption of tea and wine in Finland. On the other hand, all subjects in our study were male smokers, whereas other studies have also included women and the proportion of smokers has varied from 10% to 50%. It is possible that the effects of flavonoids are different between smokers and nonsmokers.
Flavonols and flavones have actions that could influence the risk of coronary heart disease. In in vitro studies they have inhibited the oxidation of low-density lipoprotein, which is considered an essential event in the development of atherosclerosis. Thus, flavonols and flavones could retard the progression of atherosclerosis. In vitro studies have indicated antithrombotic effects 5 that may be protective against thrombosis formation in the acute phase of myocardial infarction. Only a fraction of coronary deaths are caused by acute thrombosis in the coronary arteries, and so far there is no evidence that flavonols and flavones could influence other mechanisms of coronary death.
We found that men drinking on average one glass of wine weekly had lower risk of nonfatal myocardial infarction and coronary death than other men. This risk reduction was, however, unlikely to be due to flavonols and flavones, 21 because the difference in the total intake of flavonols and flavones was only 3 mg per day between the groups. The most likely explanation is differences in life-style factors. Wine drinkers are a very specific group of people in Finland; they are much more educated and urban than those who do not drink wine. Thus, although several background factors were taken into account in the effect estimates, some residual confounding probably remained. This interpretation is supported by our finding that the inverse association between wine consumption and the risk of nonfatal myocardial infarction was markedly attenuated after adjusting for alcohol intake.
Many studies have observed an inverse association between the consumption of vegetables and the risk of cardiovascular diseases. We also found lowered risk of nonfatal myocardial infarction and coronary death in the highest quintile of vegetable consumption. It is clear, however, that flavonols and flavones cannot explain our finding. First, the effect estimates were little changed when the intake of flavonols and flavones were simultaneously added to the model. Secondly, the difference in the median intake of flavonols and flavones was 6.7 mg per day between the highest and the lowest quintiles of vegetable consumption. A similar difference in flavonol and flavone intake was observed between the highest and the lowest quintiles of consumption of fruits and berries, but little difference in coronary risk was evident. Thus, other compounds in vegetables, such as carotenoids, vitamin C, potassium, magnesium, or dietary fiber, may be the true factors behind the inverse association with vegetable consumption. Similarly, men drinking on average one cup of tea daily had 10 mg per day higher intake of flavonols and flavones than those drinking less tea, but once again little difference was evident in coronary risk.
The largest decrease in coronary risk in our data was observed between the lowest and second-lowest quintiles of flavonol and flavone intake; differences among the four highest quintiles of intake were small. This pattern may indicate that only very low intake of flavonols and flavones increases the risk of coronary heart disease or that low intake is a surrogate measure for life-style risk factors related to the risk of coronary heart disease.
1. Husain SR, Cillard J, Cillard P. Hydroxy radical scavenging activity of flavonoids. Phytochemistry 1987; 26: 2489–2492.
2. Korkina LG, Afanas’ev IB. Antioxidant and chelating properties of flavonoids. Adv Pharmacol 1997; 38: 151–163.
3. Aviram M, Fuhrman B. Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis. Atherosclerosis 1998; 137: S45–S50.
4. de Whalley CV, Rankin SM, Hoult JRS, Jessup W, Leake DS. Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem Pharmacol 1990; 39: 1743–1750.
5. Janssen KPLTM, Mensink RP, Cox FJJ, Harryvan JL, Hovenier R, Hollman PCH, Katan MB. Effects of the flavonoids quercetin and apigenin on hemostasis in healthy volunteers: results from an in vitro
and a dietary supplement study. Am J Clin Nutr 1998; 67: 255–262.
6. Conquer JA, Maiani G, Azzini E, Raguzzini A, Holub BJ. Supplementation with quercetin markedly increases plasma quercetin concentration without effect on selected risk factors for heart disease in healthy subjects. J Nutr 1998; 128: 593–597.
7. Laughton MJ, Halliwell B, Evans PJ, Hoult JR. Antioxidant and pro-oxidant actions of the phenolics quercetin, gossypol and myricetin: effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA. Biochem Pharmacol 1989; 38: 2859–2865.
8. Sahu SG, Gray GC. Pro-oxidant activity of flavonoids: effects on glutathione and glutathione S
-transferase in isolated rat liver nuclei. Cancer Lett 1996; 104: 193–196.
9. Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993; 342: 1007–1011.
10. Knekt P, Järvinen R, Reunanen A, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study. BMJ 1996; 312: 478–481.
11. Yochum L, Kushi LH, Meyer K, Folsom AR. Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am J Epidemiol 1999; 149: 943–949.
12. Hertog MG, Sweetnam PM, Fehily AM, Elwood PC, Kromhout D. Antioxidant flavonols and ischemic heart disease in a Welsh population of men: the Caerphilly Study. Am J Clin Nutr 1997; 65: 1489–1494.
13. Rimm EB, Katan MB, Ascherio A, Stampfer MJ, Willett WC. Relation between intake of flavonoids and risk for coronary heart disease in male health professionals. Ann Intern Med 1996; 125: 384–389.
14. The ATBC Cancer Prevention Study Group. The Alpha-Tocopherol, Beta-Carotene Lung Cancer Prevention Study: design, methods, participant characteristics, and compliance. Ann Epidemiol 1994; 4: 1–10.
15. Pietinen P, Hartman AM, Haapa E, Räsänen L, Haapakoski J, Palmgren J, Albanes D, Virtamo J, Huttunen JK. Reproducibility and validity of dietary assessment instruments. I. A self-administered food use questionnaire with a portion size picture booklet. Am J Epidemiol 1988; 128: 655–666.
16. Hertog MG, Hollman PC, Katan MB. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J Agric Food Chem 1992; 40: 2379–2383.
17. Hertog MG, Hollman PC, van de Putte P. Content of potentially anticarcinogenic flavonoids in tea infusions, wines, and fruit juices. J Agric Food Chem 1993; 41: 1242–1246.
18. Häkkinen SH, Kärenlampi SO, Heinonen IM, Mykkänen HM, Törrönen AR. Content of the flavonols quercetin, myricetin and kaempherol in 25 edible berries. J Agric Food Chem 1999; 47: 2274–2279.
19. Rapola JM, Virtamo J, Korhonen P, Haapakoski J, Hartman AM, Edwards BK, Heinonen OP. Validity of diagnoses of major coronary events in national registers of hospital diagnoses and deaths in Finland. Eur J Epidemiol 1997; 13: 133–138.
20. Willett WC. Nutritional Epidemiology. New York: Oxford University Press, 1990.
21. Ness AR, Powles JW. Fruit and vegetables, and cardiovascular disease: a review. Int J Epidemiol 1997; 26: 1–13.