IJC International Journal of Cancer
Total dietary antioxidant capacity, individual antioxidant intake and breast cancer risk: The Rotterdam study Athanasios Pantavos1, Rikje Ruiter1,2, Edith F. Feskens3, Catherine E. de Keyser1,4, Albert Hofman1, Bruno H. Stricker1,4,5, Oscar H. Franco1 and Jessica C. Kiefte-de Jong1 1
Department of Epidemiology, Erasmus MC, Rotterdam, The Netherlands Department of Internal Medicine, Groene Hart Hospital, Gouda, The Netherlands 3 Department of Human Nutrition, Wageningen University, Wageningen, The Netherlands 4 Health Care Expectorate, The Hague, The Netherlands 5 Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands 2
The incidence of breast cancer cases is increasing worldwide; predominantly in high-income countries.1,2 Worldwide, it has been estimated that 1.38 million new cases were diagnosed in Key words: FRAP, breast cancer, antioxidants, nutrition Additional Supporting Information may be found in the online version of this article. Grant sponsors: Erasmus MC University Medical Center and Erasmus University Rotterdam; The Netherlands Organisation for Scientific Research (NWO); The Netherlands Organisation for Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); The Netherlands Genomics Initiative (NGI); The Ministry of Education, Culture and Science; The Ministry of Health, Welfare and Sports; the European Commission (DG XII) and the Municipality of Rotterdam; Grant sponsors: A.P., O.H.F. and J.C.K. work in ErasmusAGE, a center for aging research across the life course funded by Nestle Nutrition (Nestec Ltd.), Metagenics Inc. and AXA DOI: 10.1002/ijc.29249 History: Received 6 May 2014; Accepted 12 Sep 2014; Online 4 Oct 2014 Correspondence to: J.C. Kiefte-de Jong, RD, PhD, Department of Epidemiology, Room NA2903, PO Box 2040, 3000 CA, Rotterdam, The Netherlands, Tel.: 131 10 7043536, Fax: 131 10 7044657, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
2008 which accounts for 23% of all diagnosed cancers among women.3 Several risk factors for breast cancer have been identified such as family history of breast cancer, reproductive history, the absence of breastfeeding, weight gain, moderate alcohol consumption and postmenopausal hormone therapy.1,2,4 Dietary factors might also contribute to breast cancer prevention. For instance, experimental studies in vitro suggest that high antioxidant intake may reduce the risk of developing cancer.5 However, observational studies on antioxidant status and the risk of breast cancer show inconsistent results.6,7 In addition, case control studies have found that fruits and vegetable intake are associated with a reduced breast cancer risk which may be due to high intake of antioxidants.8,9 However, the European Prospective Investigation on Cancer (EPIC cohort) did not find any prospective association between fruit and vegetable intake and breast cancer risk across Europe.10 In addition, the World Cancer Research Fund/American Institute for Cancer Research has concluded that the overall evidence for fruit, vegetables and antioxidants and breast cancer risk is still too limited.1,2 So far, studies mainly focused on evaluating the contribution of individual factors to breast cancer risk and did not take into account the overall diet or the contribution of
Epidemiology
Some studies suggest a favorable role of antioxidants on breast cancer risk but this is still inconclusive. The aim of this study was to assess whether overall dietary antioxidant capacity, as assessed by dietary ferric reducing antioxidant potential (FRAP), and individual dietary antioxidant intake were associated with breast cancer risk. Data was used from women participating in the Rotterdam Study, a prospective cohort study among subjects aged 55 years and older (N 5 3,209). FRAP scores and antioxidant intake (i.e., vitamin A, C, E, selenium, flavonoids and carotenoids) was assessed at baseline by a food frequency questionnaire. Incident cases of breast cancer were confirmed through medical reports. During a median follow-up of 17 years, 199 cases with breast cancer were identified. High dietary FRAP score was associated with a lower risk of breast cancer [hazard ratio (HR): 0.68; 95% confidence intervals (CI): 0.49, 0.96]. No overall association between individual antioxidant intake and breast cancer risk was found. However, low intake of alpha carotene and beta carotene was associated with a higher risk of breast cancer among smokers (HR: 2.48; 95% CI: 1.21, 5.12 and HR: 2.31; 95% CI: 1.12, 4.76 for alpha and beta carotene, respectively) and low intake of flavonoids was associated with breast cancer risk in women over the age of 70 (HR: 1.80; 95% CI: 1.09, 2.99). These results suggest that high overall dietary antioxidant capacity is associated with a lower risk of breast cancer. Individual effects of dietary carotenoids and dietary flavonoids may be restricted to subgroups such as smokers and elderly.
2
Dietary antioxidant capacity and breast cancer
What’s new? A diet rich in anti-oxidants may protect from breast cancer but studies focusing on individual antioxidant intake have been notoriously inconsistent. Here the authors assessed the overall dietary antioxidant capacity in more than 3000 postmenopausal Dutch women by calculating the so-called ferric acid reducing antioxidant potential (FRAP). They show that high dietary FRAP, but not individual anti-oxidant intake, is associated with an overall lower risk of breast cancer, a result that may help design more consistent studies of anti-oxidant intake and cancer risk in the future.
multiple elements to an overall antioxidant effect which may have contributed to the contradictory results. Measuring the “total antioxidant capacity of the diet” is a challenge and different alternatives have been explored which show similar ranking of foods according to their antioxidant capacity.11 The ferric reducing antioxidant potential (FRAP) measures the antioxidant capacity of the diet by measuring the reduction of ferric ion to ferrous ion which can be a useful marker to capture overall effects of antioxidants according to dietary components12 instead of individual effects of foods or antioxidants. In addition, previous studies have suggested that iron can induce oxidative stress that may influence mammary carcinogenesis13 but whether a more complete measurement of the antioxidant capacity of the diet, as assessed by FRAP, would provide a more relevant effect in terms of prevention of breast cancer remains unclear. Therefore, the primary objective of our study was to assess whether FRAP was associated with the risk of breast cancer in postmenopausal women. A second aim was to assess whether individual dietary antioxidant intake was associated with breast cancer risk.
Methods
Epidemiology
Study population
The present analyses were performed in the Rotterdam Study, a population-based prospective cohort study which has been described in detail previously.14 We used data from the first cohort of the Rotterdam Study which consists of 7,983 persons living in the Ommoord district in Rotterdam, The Netherlands. All subjects were above the age of 55 years and have been recruited between July 1989 and September 1993.14 The participants first underwent an interview at home and an extensive physical examination in the research center. These examinations were repeated every 3–4 years. Assessment of diet
Dietary intake was evaluated at baseline examination by using a two step-protocol, in order to minimize the loss of information in dietary reporting in an older population. During the home interview, subjects were provided with a meal-based checklist to indicate the foods they had consumed at least twice per month in the previous year. Subsequently, a dietary interview was performed at the research center by using a validated semiquantitative food-frequency questionnaire (SFFQ).15 The SFFQ was designed to measure
‘typical’ diet by asking questions about the frequency and amount of food consumption. It consisted of 170 food items. Frequency of food intake was recorded in times per day, week or month, and serving sizes were specified in standardized units, household measures, or grams. Data from the SFFQ were then analysed and transformed into macronutrient and antioxidant intake using the Dutch Food Composition Table.16 Nutrient intake from the SFFQ was validated against a 15 days food diary of food recording over a 1-year period.15 Pearson correlation after adjustment for age, sex, energy and within-person variation were between 0.44 and 0.85 for macro and micronutrients as described in detail previously.15 Assessment of antioxidant capacity and dietary antioxidant intake
To assess dietary antioxidant capacity from the SFFQ, each food’s contribution to FRAP was calculated on the basis of the Antioxidant Food Table published by the Institute of Nutrition Research, University of Oslo, which includes measurements of >3,000 foods.17 Based on a modified version of the FRAP assay to assess the antioxidant capacity of individual food items by measuring the ability of antioxidants in food items to reduce ferric iron (Fe31) to ferrous iron (Fe21).12 Since this food table consisted of foods from several manufacturers, we consulted nutritional experts at Wageningen University (the Netherlands) to determine linkage of foods from several manufacturers that were the closest to the Dutch food products. For each participant, we multiplied the consumption frequency of each food by the corresponding FRAP value (in mmol/100g), and summed these values across all dietary sources. Detailed data on vitamin supplementation was not available, and, therefore, not included in the FRAP assessment. Most variation in dietary FRAP scores was explained by intakes of coffee (65%) and tea (21%). As it had been suggested that this FRAP table is best suited for ranking individuals than using the exact values,17 FRAP was analyzed after stratification into tertiles of the total population of analysis (Tertile 1: 18.00 mmol/day, tertile 2: 18.01–22.25 mmol/day and tertile 3: 22.26 mmol/day). Additionally, we assessed the dietary intake of the following individual antioxidants: vitamin A, C, E, selenium, flavonoids, alpha and beta carotene, lutein, zeaxanthine, beta cryptoxanthin and lycopene. These have been suggested to be potentially related to cancer risk.1 C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
3
Pantavos et al.
Figure 1. Flow chart of the participants included in the study.
The characteristics of the population within the Rotterdam Study have been described previously.14 For our analysis, we used a population of 7,983 participants (n 5 7,983) and excluded men (n 5 3,105, 38.9%) and women with a history of breast cancer (n 5 1). The remaining population were 4,877 women over the age of 55 years old. In addition to this, we excluded 1,668 (38.1%) women without dietary data. Dietary data was missing when: (i) Individuals were included during the pilot phase of the study (between 1989 and 1990), (ii) Individuals were institutionalized and (iii) The research dietician considered the dietary data unreliable when subjects had difficulties with recall of their food intake or when dementia was suspected. The final population that was analyzed consisted of 3,209 women of whom 199 (6.3%) developed breast cancer during follow-up (Fig. 1).
Assessment of breast cancer
Statistical analysis
The diagnoses of breast cancer were obtained through the general practitioners and by linkage with a nationwide registry of histo and cytopathology in the Netherlands (PALGA). Two research physicians independently assessed the first date and diagnosis of breast cancer. All events were coded according to the International Classification of Diseases (ICD-10 code C50). In case of discrepancy, consensus was sought, or a cancer epidemiologist decided. Data on breast cancer incidence was available until December 2010.
First, we used Chi-square-test for the categorical variables to study whether there were any differences in characteristics between subjects with different FRAP intake. Continuous variables were analyzed by using one-way ANOVA test. Subsequently, crude and multivariate Cox proportional hazard models were performed to evaluate the association between dietary FRAP score, dietary antioxidant intake (per individual item) and breast cancer risk. Dietary FRAP score was analyzed as continuous variable and as categorical variable after stratification into tertiles (low: 18.00 mmol/day, medium: 18.01–22.25 mmol/day and high: 22.26 mmol/day). Dietary FRAP and dietary antioxidant intake were adjusted for total energy intake by using the residual method.18 The crude model was adjusted for age. Subsequently, in the multivariate model we adjusted for the following potential confounders: BMI, educational level, household income, family history of breast cancer, smoking status, alcohol consumption, dietary fat intake, dietary fiber intake, reproductive history, comorbidities to baseline and hormone and multivitamin use. To evaluate potential confounding, these variables were stepwise added to the crude model. When the effect estimate changed by more than 10%, the variable was kept in the final multivariate model.19 To evaluate potential effect modification, interaction was tested by smoking status, alcohol intake, BMI and age in the final multivariate model. Stratified analyses by median values were performed when p-value for interaction was less than 0.10. To reduce potential bias associated with missing data, a multiple imputation procedure of missing data was performed20 (N 5 5 imputations; Supporting Information Tables 1 and 2). To prevent potential reverse causality, sensitivity analysis was performed by excluding all breast cancer cases that occurred in the first 24 months of the follow up time. Also, sensitivity analysis was performed after excluding subjects with any use of supplementation. Results are presented as hazard ratios (HRs) and 95% confidence intervals (95% CI). A p-value of 0.05 was considered as
Assessment of covariates
At baseline of the Rotterdam Study, during the home interview the participants provided information about socioeconomic background, demographics and lifestyle habits. Weight and height were measured at first visit at the study center. Household income was categorized into two categories: low to intermediate (net income less than 1,090 euro per month) and intermediate to high income (net income more than 1,090 euro per month). Educational level was categorized into two categories: lower education (primary school) and higher education (secondary education and higher). Smoking status was categorized into three categories: (i) never smoked, (ii) former smoker, (iii) current smoker. Alcohol consumption was categorized into four categories: (i) less than one drink per week, (ii) one drink per week, (iii) two drinks per week and (iv) three or more drinks per week. Family history of breast cancer, menopausal hormone therapy and multivitamin use were categorized into dichotomous variables (yes vs. no). Reproductive history was categorized into three categories as having no children, having one to four children and having more than four children. Body mass index (BMI) (kg/m2) was categorized into four categories: underweight (
Total dietary antioxidant capacity, individual antioxidant intake and breast cancer risk: The Rotterdam study Athanasios Pantavos1, Rikje Ruiter1,2, Edith F. Feskens3, Catherine E. de Keyser1,4, Albert Hofman1, Bruno H. Stricker1,4,5, Oscar H. Franco1 and Jessica C. Kiefte-de Jong1 1
Department of Epidemiology, Erasmus MC, Rotterdam, The Netherlands Department of Internal Medicine, Groene Hart Hospital, Gouda, The Netherlands 3 Department of Human Nutrition, Wageningen University, Wageningen, The Netherlands 4 Health Care Expectorate, The Hague, The Netherlands 5 Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands 2
The incidence of breast cancer cases is increasing worldwide; predominantly in high-income countries.1,2 Worldwide, it has been estimated that 1.38 million new cases were diagnosed in Key words: FRAP, breast cancer, antioxidants, nutrition Additional Supporting Information may be found in the online version of this article. Grant sponsors: Erasmus MC University Medical Center and Erasmus University Rotterdam; The Netherlands Organisation for Scientific Research (NWO); The Netherlands Organisation for Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); The Netherlands Genomics Initiative (NGI); The Ministry of Education, Culture and Science; The Ministry of Health, Welfare and Sports; the European Commission (DG XII) and the Municipality of Rotterdam; Grant sponsors: A.P., O.H.F. and J.C.K. work in ErasmusAGE, a center for aging research across the life course funded by Nestle Nutrition (Nestec Ltd.), Metagenics Inc. and AXA DOI: 10.1002/ijc.29249 History: Received 6 May 2014; Accepted 12 Sep 2014; Online 4 Oct 2014 Correspondence to: J.C. Kiefte-de Jong, RD, PhD, Department of Epidemiology, Room NA2903, PO Box 2040, 3000 CA, Rotterdam, The Netherlands, Tel.: 131 10 7043536, Fax: 131 10 7044657, E-mail: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
2008 which accounts for 23% of all diagnosed cancers among women.3 Several risk factors for breast cancer have been identified such as family history of breast cancer, reproductive history, the absence of breastfeeding, weight gain, moderate alcohol consumption and postmenopausal hormone therapy.1,2,4 Dietary factors might also contribute to breast cancer prevention. For instance, experimental studies in vitro suggest that high antioxidant intake may reduce the risk of developing cancer.5 However, observational studies on antioxidant status and the risk of breast cancer show inconsistent results.6,7 In addition, case control studies have found that fruits and vegetable intake are associated with a reduced breast cancer risk which may be due to high intake of antioxidants.8,9 However, the European Prospective Investigation on Cancer (EPIC cohort) did not find any prospective association between fruit and vegetable intake and breast cancer risk across Europe.10 In addition, the World Cancer Research Fund/American Institute for Cancer Research has concluded that the overall evidence for fruit, vegetables and antioxidants and breast cancer risk is still too limited.1,2 So far, studies mainly focused on evaluating the contribution of individual factors to breast cancer risk and did not take into account the overall diet or the contribution of
Epidemiology
Some studies suggest a favorable role of antioxidants on breast cancer risk but this is still inconclusive. The aim of this study was to assess whether overall dietary antioxidant capacity, as assessed by dietary ferric reducing antioxidant potential (FRAP), and individual dietary antioxidant intake were associated with breast cancer risk. Data was used from women participating in the Rotterdam Study, a prospective cohort study among subjects aged 55 years and older (N 5 3,209). FRAP scores and antioxidant intake (i.e., vitamin A, C, E, selenium, flavonoids and carotenoids) was assessed at baseline by a food frequency questionnaire. Incident cases of breast cancer were confirmed through medical reports. During a median follow-up of 17 years, 199 cases with breast cancer were identified. High dietary FRAP score was associated with a lower risk of breast cancer [hazard ratio (HR): 0.68; 95% confidence intervals (CI): 0.49, 0.96]. No overall association between individual antioxidant intake and breast cancer risk was found. However, low intake of alpha carotene and beta carotene was associated with a higher risk of breast cancer among smokers (HR: 2.48; 95% CI: 1.21, 5.12 and HR: 2.31; 95% CI: 1.12, 4.76 for alpha and beta carotene, respectively) and low intake of flavonoids was associated with breast cancer risk in women over the age of 70 (HR: 1.80; 95% CI: 1.09, 2.99). These results suggest that high overall dietary antioxidant capacity is associated with a lower risk of breast cancer. Individual effects of dietary carotenoids and dietary flavonoids may be restricted to subgroups such as smokers and elderly.
2
Dietary antioxidant capacity and breast cancer
What’s new? A diet rich in anti-oxidants may protect from breast cancer but studies focusing on individual antioxidant intake have been notoriously inconsistent. Here the authors assessed the overall dietary antioxidant capacity in more than 3000 postmenopausal Dutch women by calculating the so-called ferric acid reducing antioxidant potential (FRAP). They show that high dietary FRAP, but not individual anti-oxidant intake, is associated with an overall lower risk of breast cancer, a result that may help design more consistent studies of anti-oxidant intake and cancer risk in the future.
multiple elements to an overall antioxidant effect which may have contributed to the contradictory results. Measuring the “total antioxidant capacity of the diet” is a challenge and different alternatives have been explored which show similar ranking of foods according to their antioxidant capacity.11 The ferric reducing antioxidant potential (FRAP) measures the antioxidant capacity of the diet by measuring the reduction of ferric ion to ferrous ion which can be a useful marker to capture overall effects of antioxidants according to dietary components12 instead of individual effects of foods or antioxidants. In addition, previous studies have suggested that iron can induce oxidative stress that may influence mammary carcinogenesis13 but whether a more complete measurement of the antioxidant capacity of the diet, as assessed by FRAP, would provide a more relevant effect in terms of prevention of breast cancer remains unclear. Therefore, the primary objective of our study was to assess whether FRAP was associated with the risk of breast cancer in postmenopausal women. A second aim was to assess whether individual dietary antioxidant intake was associated with breast cancer risk.
Methods
Epidemiology
Study population
The present analyses were performed in the Rotterdam Study, a population-based prospective cohort study which has been described in detail previously.14 We used data from the first cohort of the Rotterdam Study which consists of 7,983 persons living in the Ommoord district in Rotterdam, The Netherlands. All subjects were above the age of 55 years and have been recruited between July 1989 and September 1993.14 The participants first underwent an interview at home and an extensive physical examination in the research center. These examinations were repeated every 3–4 years. Assessment of diet
Dietary intake was evaluated at baseline examination by using a two step-protocol, in order to minimize the loss of information in dietary reporting in an older population. During the home interview, subjects were provided with a meal-based checklist to indicate the foods they had consumed at least twice per month in the previous year. Subsequently, a dietary interview was performed at the research center by using a validated semiquantitative food-frequency questionnaire (SFFQ).15 The SFFQ was designed to measure
‘typical’ diet by asking questions about the frequency and amount of food consumption. It consisted of 170 food items. Frequency of food intake was recorded in times per day, week or month, and serving sizes were specified in standardized units, household measures, or grams. Data from the SFFQ were then analysed and transformed into macronutrient and antioxidant intake using the Dutch Food Composition Table.16 Nutrient intake from the SFFQ was validated against a 15 days food diary of food recording over a 1-year period.15 Pearson correlation after adjustment for age, sex, energy and within-person variation were between 0.44 and 0.85 for macro and micronutrients as described in detail previously.15 Assessment of antioxidant capacity and dietary antioxidant intake
To assess dietary antioxidant capacity from the SFFQ, each food’s contribution to FRAP was calculated on the basis of the Antioxidant Food Table published by the Institute of Nutrition Research, University of Oslo, which includes measurements of >3,000 foods.17 Based on a modified version of the FRAP assay to assess the antioxidant capacity of individual food items by measuring the ability of antioxidants in food items to reduce ferric iron (Fe31) to ferrous iron (Fe21).12 Since this food table consisted of foods from several manufacturers, we consulted nutritional experts at Wageningen University (the Netherlands) to determine linkage of foods from several manufacturers that were the closest to the Dutch food products. For each participant, we multiplied the consumption frequency of each food by the corresponding FRAP value (in mmol/100g), and summed these values across all dietary sources. Detailed data on vitamin supplementation was not available, and, therefore, not included in the FRAP assessment. Most variation in dietary FRAP scores was explained by intakes of coffee (65%) and tea (21%). As it had been suggested that this FRAP table is best suited for ranking individuals than using the exact values,17 FRAP was analyzed after stratification into tertiles of the total population of analysis (Tertile 1: 18.00 mmol/day, tertile 2: 18.01–22.25 mmol/day and tertile 3: 22.26 mmol/day). Additionally, we assessed the dietary intake of the following individual antioxidants: vitamin A, C, E, selenium, flavonoids, alpha and beta carotene, lutein, zeaxanthine, beta cryptoxanthin and lycopene. These have been suggested to be potentially related to cancer risk.1 C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
3
Pantavos et al.
Figure 1. Flow chart of the participants included in the study.
The characteristics of the population within the Rotterdam Study have been described previously.14 For our analysis, we used a population of 7,983 participants (n 5 7,983) and excluded men (n 5 3,105, 38.9%) and women with a history of breast cancer (n 5 1). The remaining population were 4,877 women over the age of 55 years old. In addition to this, we excluded 1,668 (38.1%) women without dietary data. Dietary data was missing when: (i) Individuals were included during the pilot phase of the study (between 1989 and 1990), (ii) Individuals were institutionalized and (iii) The research dietician considered the dietary data unreliable when subjects had difficulties with recall of their food intake or when dementia was suspected. The final population that was analyzed consisted of 3,209 women of whom 199 (6.3%) developed breast cancer during follow-up (Fig. 1).
Assessment of breast cancer
Statistical analysis
The diagnoses of breast cancer were obtained through the general practitioners and by linkage with a nationwide registry of histo and cytopathology in the Netherlands (PALGA). Two research physicians independently assessed the first date and diagnosis of breast cancer. All events were coded according to the International Classification of Diseases (ICD-10 code C50). In case of discrepancy, consensus was sought, or a cancer epidemiologist decided. Data on breast cancer incidence was available until December 2010.
First, we used Chi-square-test for the categorical variables to study whether there were any differences in characteristics between subjects with different FRAP intake. Continuous variables were analyzed by using one-way ANOVA test. Subsequently, crude and multivariate Cox proportional hazard models were performed to evaluate the association between dietary FRAP score, dietary antioxidant intake (per individual item) and breast cancer risk. Dietary FRAP score was analyzed as continuous variable and as categorical variable after stratification into tertiles (low: 18.00 mmol/day, medium: 18.01–22.25 mmol/day and high: 22.26 mmol/day). Dietary FRAP and dietary antioxidant intake were adjusted for total energy intake by using the residual method.18 The crude model was adjusted for age. Subsequently, in the multivariate model we adjusted for the following potential confounders: BMI, educational level, household income, family history of breast cancer, smoking status, alcohol consumption, dietary fat intake, dietary fiber intake, reproductive history, comorbidities to baseline and hormone and multivitamin use. To evaluate potential confounding, these variables were stepwise added to the crude model. When the effect estimate changed by more than 10%, the variable was kept in the final multivariate model.19 To evaluate potential effect modification, interaction was tested by smoking status, alcohol intake, BMI and age in the final multivariate model. Stratified analyses by median values were performed when p-value for interaction was less than 0.10. To reduce potential bias associated with missing data, a multiple imputation procedure of missing data was performed20 (N 5 5 imputations; Supporting Information Tables 1 and 2). To prevent potential reverse causality, sensitivity analysis was performed by excluding all breast cancer cases that occurred in the first 24 months of the follow up time. Also, sensitivity analysis was performed after excluding subjects with any use of supplementation. Results are presented as hazard ratios (HRs) and 95% confidence intervals (95% CI). A p-value of 0.05 was considered as
Assessment of covariates
At baseline of the Rotterdam Study, during the home interview the participants provided information about socioeconomic background, demographics and lifestyle habits. Weight and height were measured at first visit at the study center. Household income was categorized into two categories: low to intermediate (net income less than 1,090 euro per month) and intermediate to high income (net income more than 1,090 euro per month). Educational level was categorized into two categories: lower education (primary school) and higher education (secondary education and higher). Smoking status was categorized into three categories: (i) never smoked, (ii) former smoker, (iii) current smoker. Alcohol consumption was categorized into four categories: (i) less than one drink per week, (ii) one drink per week, (iii) two drinks per week and (iv) three or more drinks per week. Family history of breast cancer, menopausal hormone therapy and multivitamin use were categorized into dichotomous variables (yes vs. no). Reproductive history was categorized into three categories as having no children, having one to four children and having more than four children. Body mass index (BMI) (kg/m2) was categorized into four categories: underweight (
