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European Journal of Clinical Nutrition (2012) 66, 932–941 & 2012 Macmillan Publishers Limited All rights reserved 0954-3007/12 www.nature.com/ejcn

ORIGINAL ARTICLE

Dietary intakes and food sources of phytoestrogens in the European Prospective Investigation into Cancer and Nutrition (EPIC) 24-hour dietary recall cohort R Zamora-Ros1, V Knaze1, L Luja´n-Barroso1, GGC Kuhnle2,3, AA Mulligan2, M Touillaud4, N Slimani4, I Romieu4, N Powell2, R Tumino5, PHM Peeters6,7, MS de Magistris8, F Ricceri9, E Sonestedt10, I Drake10, A Hjarta˚ker11, G Skie12, T Mouw7, PA Wark7, D Romaguera7, HB Bueno-de-Mesquita13,14, M Ros13,15, E Molina16,17, S Sieri18, JR Quiro´s19, JM Huerta17,20, A Tjønneland21, J Halkjær21, G Masala22, B Teucher23, R Kaas23, RC Travis24, V Dilis25,26, V Benetou25, A Trichopoulou25,26, P Amiano17,27, E Ardanaz17,28, H Boeing29, J Fo¨rster29, F Clavel-Chapelon30,31, G Fagherazzi 30,31, F Perquier30,31, G Johansson32, I Johansson33, A Cassidy34, K Overvad35 and CA Gonza´lez1 BACKGROUND/OBJECTIVES: Phytoestrogens are estradiol-like natural compounds found in plants that have been associated with protective effects against chronic diseases, including some cancers, cardiovascular diseases and osteoporosis. The purpose of this study was to estimate the dietary intake of phytoestrogens, identify their food sources and their association with lifestyle factors in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. SUBJECTS/METHODS: Single 24-hour dietary recalls were collected from 36 037 individuals from 10 European countries, aged 35–74 years using a standardized computerized interview programe (EPIC-Soft). An ad hoc food composition database on phytoestrogens (isoflavones, lignans, coumestans, enterolignans and equol) was compiled using data from available databases, in order to obtain and describe phytoestrogen intakes and their food sources across 27 redefined EPIC centres. RESULTS: Mean total phytoestrogen intake was the highest in the UK health-conscious group (24.9 mg/day in men and 21.1 mg/day in women) whereas lowest in Greece (1.3 mg/day) in men and Spain-Granada (1.0 mg/day) in women. Northern European countries had higher intakes than southern countries. The main phytoestrogen contributors were isoflavones in both UK centres and lignans in the other EPIC cohorts. Age, body mass index, educational level, smoking status and physical activity were related to increased intakes of lignans, enterolignans and equol, but not to total phytoestrogen, isoflavone or coumestan intakes. In the UK cohorts, the major food sources of phytoestrogens were soy products. In the other EPIC cohorts the dietary sources were more distributed, among fruits, vegetables, soy products, cereal products, non-alcoholic and alcoholic beverages. CONCLUSIONS: There was a high variability in the dietary intake of total and phytoestrogen subclasses and their food sources across European regions. European Journal of Clinical Nutrition (2012) 66, 932–941; doi:10.1038/ejcn.2012.36; published online 18 April 2012 Keywords: phytoestrogens; intake; food sources; EPIC-Europe

1

Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Programme, Catalan Institute of Oncology (ICO-IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain; MRC Centre for Nutritional Epidemiology in Cancer Prevention and Survival, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK; 3 Department of Food and Nutritional Sciences, University of Reading, Reading, UK; 4Dietary Exposure Assessment Group, International Agency for Research on Cancer (IARC), Lyon, France; 5Cancer Registry and Histopathology Unit, ‘Civile M.P. Arezzo’ Hospital, Ragusa, Italy; 6Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, The Netherlands; 7Department of Epidemiology and Biostatistics, Imperial College School of Public Health, London, UK; 8Department of Clinical and Experimental medicine, Federico II University, Naples, Italy; 9Human Genetics Foundation (HUGEF), Turin, Italy; 10Department of Clinical Sciences, Research group in Nutrition Epidemiology, Lund University, Malmo¨, Sweden; 11Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; 12Department of Community Medicine, University of Tromsø, Tromsø, Norway; 13National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; 14Department of Gastroenterology and Hepatology, University Medical Centre Utrecht (UMCU), Utrecht, The Netherlands; 15Department of Epidemiology, Biostatistics and HTA, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 16 Andalusian School of Public Health, Granada, Spain; 17CIBER Epidemiologı´a y Salud Pu´blica (CIBERESP), Spain; 18Nutritional Epidemiology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy; 19Public Health and Health Planning Directorate, Asturias, Spain; 20Department of Epidemiology, Murcia Regional Health Authority, Murcia, Spain; 21 Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark; 22Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute – ISPO, Florence, Italy; 23Department of Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany; 24Cancer Epidemiology Unit, University of Oxford, Oxford, UK; 25 WHO Collaborating Center for Food and Nutrition Policies, Department of Hygiene, Epidemiology and Medical Statistics, University of Athens Medical School, Athens, Greece; 26Hellenic Health Foundation, Greece; 27Public Health Division of Gipuzkoa, Institute Investigation BioDonostia, Basque Country, Spain; 28Navarre Public Health Institute, Pamplona, Spain; 29Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbru¨cke, Nuthetal, Germany; 30Inserm, Centre for Research in Epidemiology and Population Health, Institut Gustave Roussy, Villejuif, France; 31Paris South University, Villejuif, France; 32Department of Clinical Medicine and Public Health/Nutritional Research, Umeå University, Umea, Sweden; 33Department of Odontology/Cariology, Umeå University, Umea, Sweden; 34Norwich Medical School, University of East Anglia, Norwich, UK and 35Department of Epidemiology, School of Public Health, Aarhus University, Aarhus, Denmark. Correspondence: Dr R Zamora-Ros, Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Programme, Catalan Institute of Oncology (ICO-IDIBELL); Avda Gran Via 199-203, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. E-mail: [email protected] Contributors: RZ-R and CAG designed the research; RZ-R. and VK conducted the research; RZ-R and LL-B performed the statistical analysis; RZ-R wrote the manuscript; all authors read, critically reviewed and approved the final manuscript. Received 30 June 2011; revised 26 January 2012; accepted 26 January 2012; published online 18 April 2012 2

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

933 INTRODUCTION Phytoestrogens are polyphenolic secondary plant metabolites that induce biological responses in animals and can mimic or modulate the action of endogenous estrogens, usually by binding to estrogen receptors.1,2 The principal phytoestrogens in foods are isoflavones, lignans, coumestans, enterolignans and equol. Isoflavones are a subclass of flavonoids that occur mainly in soy products. They are also found in much lower concentrations in other plant products3,4 and even in animal products.5 Classically, the most studied isoflavones were daidzein, genistein and glycitein. In the last two decades, biochanin A and formononetin have also been considered, because they are converted by the intestinal microbiota into genistein and daidzein, respectively.6 Lignans are a diverse group of nonflavonoid compounds widely distributed in the plant kingdom, especially in seeds, such as flax and sesame.7 A large variety of plant lignans exists, but only a few of them are converted into enterolignans by the intestinal microbiota, and therefore able to be absorbed by the human body.8 Highlighted in this group of bioavailable enterolignans are secoisolariciresinol and matairesinol. More recently, lariciresinol and pinoresinol, have also shown a high degree of conversion into enterolignans (around 55%).9 Along with lignans, isoflavone daidzein is also metabolized by intestinal microbiota and transformed into equol.8 As a result of such isoflavone and lignan microbial metabolism in animal gut, these metabolites may also be present in foods of animal origin.5 However, because their occurrence and biosynthesis are different, some authors classify these enterolignans and equol in separate groups, particularly in epidemiological studies.4 The bioactivity of phytoestrogens is based mostly on their weak affinity to the estrogen receptor b and to a lesser extent estrogen receptor a. Therefore, potential effects on hormone-related diseases such as breast, prostate, colorectal and endometrial cancers; and also on lung cancer, CVD, osteoporosis and menopausal symptoms, have been suggested.4,8,10 Furthermore, phytoestrogens may reduce oxidative stress by activating intracellular kinase cascades, leading to acute activation of endothelial nitric oxide synthase and modulating redox-sensitive gene transcription via NF-kB (nuclear factor-kB) and Nrf2 (nuclear factor erythroid 2-related factor 2).11,12 A meta-analysis investigating the relationship between soy foods, isoflavone intakes and breast cancer showed an inverse association in Asian populations, but not in Western populations (Europe and US),13 which tend to have low phytoestrogen intake. Several studies have published intake estimations of dietary isoflavones.14–18 Few descriptive articles exist on lignans,19,20 and even fewer are available to date on coumestans, enterolignans and equol intakes, especially in Europe.4 Moreover, the assessment methods used in these studies have been quite heterogeneous and some used old food composition databases (FCDB), so it is straightforward comparing phytoestrogen intakes. Therefore, the aim of this study was to estimate the intake of total, subgroups and individual phytoestrogens and to present their main food sources in 10 European countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC). Intakes were also assessed across geographical regions, sociodemographic, lifestyle and anthropometric strata to determine the factors associated with phytoestrogen intake.

MATERIALS AND METHODS Study population EPIC is a large multicentre prospective cohort study designed to investigate the role of dietary, environmental and lifestyle factors in the etiology of cancer in 23 centres of 10 European countries: Denmark, France, Germany, Greece, Italy, Norway, Spain, Sweden, The Netherlands and the United Kingdom.21,22 The initial 23 EPIC administrative centres were redefined into 27 centres for the analysis of dietary patterns & 2012 Macmillan Publishers Limited

according to geographical south–north gradient.23 Participants were mostly recruited from the general population residing within defined geographical areas, with some exception: female members of a health insurance company for teachers and school workers (France), women attending mammography screening (Utrecht-The Netherlands; and Florence-Italy), and mostly blood donors (centres in Italy and Spain). In the United Kingdom, a ‘health-conscious’ group involving mainly vegetarians, essentially from Oxford, was considered separately from a ‘general population’ group recruited by general practitioners in Cambridge and Oxford.22 The cohorts in France, Norway, Utrecht (The Netherlands) and Naples (Italy) recruited only women. All participants provided written informed consent, and the EPIC study was approved by the ethical review boards of all local recruiting institutions. The EPIC calibration study designed to correct measurement error on baseline dietary intake measurements was carried out between 1995 and 2000, and has been described elsewhere.23 Briefly, a stratified random sample (n ¼ 36 994) by age, sex and centre, and weighted for expected cancer cases in each stratum of the whole cohort was selected for a single 24-hour dietary recall (24-HDR) interview. We excluded 941 participants younger than 35 or older than 74 years of age as participation in these age groups was too low to describe intakes across centres meaningfully. After further exclusion of 16 participants who did not complete food frequency questionnaires, a total of 36 037 participants were finally included in this analysis.

Dietary and lifestyle information Dietary data of the 24-HDR was administered by trained dieticians using EPIC-Soft, a standardized computerized interview programe developed specifically for the EPIC calibration study.24,25 The 24-HDR was administered in a face-to-face interview, except in Norway, where it was obtained by telephone interview. Data on other lifestyle factors, including educational level, total physical activity and smoking history, were collected at baseline through standardized questionnaires, and have been described elsewhere.22,23,26 Data on age, body weight and height were self-reported by the participants during the 24-HDR interview. The mean time interval between these baseline questionnaire measures and the 24-HDR interview varied by country, from 1 day to 3 years later.23

FCDB We developed an ad hoc phytoestrogen FCDB based on data from the US Department of Agriculture database on isoflavones updated in 20083 and expanded with the Phenol-Explorer database on polyphenols released in 2009,27 which provide exhaustive and worldwide food composition data published to date on flavonoids/polyphenols. Moreover, our FCDB was also expanded with phytoestrogen data from specific countries such as the UK,4 The Netherlands7 and Canada.28 Approximately, 8.6%, 0.1% and 23.1% of our database come from USDA, Phenol-Explorer and the other publications, respectively. Unavailable values were estimated, as far as possible, using recipes (15.7%), estimations by similar food (25.3%) or food group items (2.5%) and logical zeros (16.5%), as in the previous studies.29,30 The variation in phytoestrogen content of foods after cooking is generally minimal;31 therefore we did not apply any retention factors. Finally, the FCDB created contained a total of 1877 food items and only 8.2% had unknown values, which are calculated as a zero by default. Dietary phytoestrogen intake was estimated for five major subclasses and their 13 components in each subclass: isoflavones (daidzein, genistein, glycitein, biochanin A and formononetin), lignans (secoisolariciresinol, matairesinol, lariciresinol and pinoresinol), coumestans (coumestrol), enterolignans (enterolactone and enterodiol) and equol. Phytoestrogens are expressed as phytoestrogen aglycones per 100 mg of fresh weight and are calculated as the sum of the available forms (glycosides and aglycones) in the literature.

Statistical analyses Dietary intake data are shown as means (least square means) and s.e. stratified by sex and by 27 redefined EPIC centres ordered geographically from south to north. The mean intake data were adjusted for age and weighted by season and day of the recall using generalized linear modelling. The contribution of the individual phytoestrogen to the subgroup and total intake, as well as the contribution of the subgroup to total intake, was calculated as a percentage. The contribution of each food group to the total intake of phytoestrogens was also computed as a European Journal of Clinical Nutrition (2012) 932 – 941

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

934 percentage. Differences in phytoestrogen intake stratified by sex were also compared by the categories of age (35–44, 45–54, 55–64, 65–74), educational level (none, primary completed, technical/professional, secondary school and university degree), smoking status (never smoker, current smoker and former smoker), level of physical activity (inactive, moderately inactive, moderately active and active), body mass index (BMI; o25, 25 to o30, X30 kg/m2) and European region (Mediterranean (MED) countries: all centres in Greece, Spain, Italy and the South of France; non-MED countries: all centres in the North-East and North-West of France, Germany, The Netherlands, Denmark, Sweden and Norway; United Kingdom general population; and finally United Kingdom health-conscious group). We used these four European regions because in the UK, regular flour is supplemented with soy flour (one of the richest sources of isoflavones) in the elaboration of breads, cakes and biscuits.32 Moreover, the UK healthconscious group form a distinct subgroup with higher consumptions of fruit, vegetables, legumes, nuts and seeds, cereals and soy products compared with the other EPIC centres.33 These models were adjusted for sex, age, centre, BMI and energy intake. All models were weighted by season and day of the week of the 24-HDR using generalized linear models to control for different distributions of 24-HDR interviews across seasons and days of the week. P-values o0.05 (two-tailed) were considered statistically significant. All analyses were conducted using the SPSS Statistics software (version 17.0, SPSS Inc., Chicago, IL, USA).

RESULTS The mean daily intake of total phytoestrogens ranged from 1.29 mg/day (Greece) to 24.90 mg/day (UK health-conscious cohort) in men (Table 1a) and from 0.99 mg/d (Granada, Spain) to 21.05 mg/d (UK health-conscious cohort) in women (Table 1b). Table 1a.

For both sexes, the UK health-conscious group had the highest intake of isoflavones, lignans and coumestans. The highest enterolignan and equol intake was reported in Umea (Sweden) in men. For women, the highest enterolignan and equol intakes were reported in Asturias (Spain) and in North-East of France, respectively. Whereas for both sexes, the UK health-conscious group and Ragussa (Italy) had the lowest intakes of enterolignans and equol. Greek men had the lowest intakes of total isoflavones and coumestans; Spanish (Asturias) of lignans. For women, the lowest intakes of isoflavones, lignans and coumestans were observed in San Sebastian and Asturias of Spain and in Greece, respectively. Annexes 1 and 2 show the mean intakes and s.e. for individual phytoestrogens stratified by centre, adjusted for age and sex and weighted for season and day of the week. In MED and non-MED countries, the main contributing phytoestrogens were the group of lignans (58.1–67.3%) and isoflavones (30.4–37.9%) then followed by coumestans (1.5–3.3%), enterolignans (0.7–0.8%) and equol (0.2–0.3%) (Table 2). However, in both UK centres, the highest contributors were isoflavones (58.4–86.9%) followed by lignans (11.1–39.0%), coumestans (2.0–2.2%), enterolignans (o0.4%) and equol (o0.12%). Table 3 shows the evaluation of the effect of sociodemographic, lifestyle and anthropometric characteristics on phytoestrogen intake adjusted for age, sex, centre, energy and BMI and weighted for season and day of the week. Men had a statistically significant higher intake of lignans than women (1.22 vs 1.18 mg/day), but less of enterolignans (0.013 vs 0.014 mg/day) and equol (0.004 vs 0.005 mg/day). There were no statistically significant differences in

Adjusteda mean daily intakes of total and subgroups of phytoestrogens by centre ordered from south to north in men

Country and centre

N

Phytoestrogens (mg/day) Mean

s.e.

Isoflavones (mg/day) Mean

Lignans (mg/day)

Coumestans (mg/day)

Enterolignans (mg/day)

Equol (mg/day)

s.e.

Mean

s.e.

Mean

s.e.

Mean

s.e.

Mean

s.e.

1314

1.294

0.144

0.236

0.137

1.037

0.034

0.011

0.003

0.010

0.000

0.003

0.000

214 243 444 490 386

1.412 1.725 1.444 1.582 1.339

0.356 0.334 0.247 0.235 0.265

0.298 0.314 0.363 0.395 0.302

0.339 0.318 0.235 0.225 0.252

1.083 1.388 1.045 1.154 1.003

0.083 0.078 0.058 0.055 0.062

0.015 0.012 0.023 0.019 0.015

0.008 0.007 0.005 0.005 0.006

0.017 0.011 0.012 0.015 0.018

0.001 0.001 0.000 0.000 0.001

0.004 0.003 0.004 0.005 0.005

0.000 0.000 0.000 0.000 0.000

168

1.368

0.401

0.237

0.383

1.100

0.094

0.022

0.009

0.009

0.001

0.003

0.000

271 676 327

1.837 1.658 1.542

0.316 0.200 0.288

0.599 0.382 0.346

0.301 0.191 0.274

1.196 1.240 1.149

0.074 0.047 0.067

0.031 0.026 0.034

0.007 0.004 0.006

0.011 0.010 0.013

0.001 0.000 0.001

0.004 0.004 0.004

0.000 0.000 0.000

1034 1233

2.492 2.659

0.162 0.148

0.695 0.813

0.154 0.141

1.722 1.772

0.038 0.035

0.065 0.063

0.003 0.003

0.011 0.011

0.000 0.000

0.004 0.004

0.000 0.000

1024

2.217

0.164

0.734

0.156

1.391

0.038

0.078

0.004

0.014

0.000

0.004

0.000

403 113

4.240 24.897

0.259 0.489

2.462 21.804

0.247 0.466

1.663 2.626

0.061 0.115

0.098 0.458

0.006 0.011

0.017 0.009

0.001 0.001

0.005 0.003

0.000 0.000

Denmark Copenhagen Aarhus

1356 567

2.305 2.414

0.141 0.218

0.561 0.647

0.135 0.208

1.631 1.641

0.033 0.051

0.098 0.111

0.003 0.005

0.015 0.016

0.000 0.000

0.005 0.005

0.000 0.000

Sweden Malmo¨ Umea

1421 1344

2.145 1.965

0.141 0.142

0.978 0.721

0.135 0.136

1.070 1.150

0.033 0.033

0.081 0.072

0.003 0.003

0.016 0.021

0.000 0.000

0.006 0.007

0.000 0.000

Greece Spain Granada Murcia Navarra San Sebastian Asturias Italy Ragusa Naples Florence Turin Varese Germany Heidelberg Potsdam The Netherlands Bilthoven Utrecht United Kingdom General population Health-conscious

a

Adjusted for age and weighted by season and day of recall.

European Journal of Clinical Nutrition (2012) 932 – 941

& 2012 Macmillan Publishers Limited

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

935 Table 1b.

Adjusteda mean daily intakes of total and subgroups of phytoestrogens by centre ordered from south to north in women

Country and centre

N

Phytoestrogens (mg/day)

Isoflavones (mg/day)

Lignans (mg/day)

Coumestans (mg/day)

Enterolignans (mg/day)

Equol (mg/day)

Mean

s.e.

Mean

s.e.

Mean

s.e.

Mean

s.e.

Mean

s.e.

Mean

s.e.

1373

1.059

0.140

0.220

0.134

0.822

0.033

0.008

0.003

0.009

0.000

0.003

0.000

Spain Granada Murcia Navarra San Sebastian Asturias

300 304 271 244 324

0.992 1.196 1.066 1.079 1.083

0.300 0.299 0.316 0.333 0.289

0.147 0.151 0.186 0.107 0.277

0.286 0.285 0.301 0.318 0.276

0.818 1.021 0.842 0.939 0.765

0.070 0.070 0.074 0.078 0.068

0.011 0.009 0.022 0.017 0.021

0.006 0.006 0.007 0.007 0.006

0.016 0.014 0.016 0.017 0.020

0.001 0.001 0.001 0.001 0.001

0.004 0.004 0.004 0.005 0.006

0.000 0.000 0.000 0.000 0.000

Italy Ragusa Naples Florence Turin Varese

138 403 784 392 794

1.314 1.319 1.484 1.597 1.227

0.443 0.259 0.186 0.263 0.185

0.409 0.271 0.548 0.502 0.337

0.423 0.247 0.177 0.251 0.176

0.878 1.001 0.891 1.056 0.848

0.104 0.061 0.044 0.062 0.043

0.019 0.037 0.035 0.029 0.030

0.010 0.006 0.004 0.006 0.004

0.008 0.010 0.010 0.010 0.011

0.001 0.001 0.000 0.001 0.000

0.003 0.004 0.004 0.004 0.004

0.000 0.000 0.000 0.000 0.000

France South coast South North-East North-West

620 1425 2059 631

2.416 2.508 2.177 1.844

0.209 0.138 0.115 0.207

1.300 1.387 1.058 0.769

0.199 0.131 0.109 0.197

1.065 1.069 1.064 1.021

0.049 0.032 0.027 0.049

0.034 0.036 0.039 0.038

0.005 0.003 0.002 0.004

0.016 0.016 0.016 0.015

0.000 0.000 0.000 0.000

0.006 0.006 0.007 0.006

0.000 0.000 0.000 0.000

Germany Heidelberg Potsdam

1087 1061

2.136 1.982

0.159 0.160

0.623 0.504

0.152 0.153

1.446 1.413

0.037 0.038

0.055 0.054

0.003 0.003

0.012 0.011

0.000 0.000

0.004 0.004

0.000 0.000

The Netherlands Bilthoven Utrecht

1086 1870

1.988 2.077

0.160 0.121

0.788 0.747

0.152 0.115

1.117 1.246

0.037 0.028

0.070 0.066

0.003 0.003

0.014 0.018

0.000 0.000

0.004 0.006

0.000 0.000

571 196

3.831 21.053

0.218 0.371

2.257 18.201

0.208 0.354

1.479 2.415

0.051 0.087

0.080 0.428

0.005 0.008

0.015 0.009

0.000 0.001

0.005 0.003

0.000 0.000

Denmark Copenhagen Aarhus

1484 510

1.928 1.972

0.135 0.230

0.460 0.461

0.129 0.220

1.384 1.411

0.032 0.054

0.072 0.086

0.003 0.005

0.013 0.014

0.000 0.000

0.005 0.005

0.000 0.000

Sweden Malmo¨ Umea

1711 1574

2.089 1.737

0.127 0.131

1.085 0.701

0.122 0.125

0.913 0.955

0.030 0.031

0.076 0.064

0.003 0.003

0.015 0.017

0.000 0.000

0.005 0.006

0.000 0.000

Norway South and East North and West

1004 793

2.677 2.201

0.166 0.186

1.474 1.096

0.158 0.178

1.062 0.959

0.039 0.044

0.126 0.132

0.004 0.004

0.015 0.015

0.000 0.000

0.005 0.004

0.000 0.000

Greece

United Kingdom General population Health-conscious

a

Adjusted for age and weighted by season and day of recall.

total phytoestrogen, isoflavone and coumestan intakes between the sexes. Participants who were between 45 and 64 years had the highest intakes of total phytoestrogens, isoflavones, lignans and coumestans. Northern European countries (non-MED countries) had higher intakes of total phytoestrogens, isoflavones, lignans and coumestans than southern countries (MED countries), with a maximum intake in the UK, especially in the UK health-conscious group, was observed. Decreasing intake gradients when assessing total phytoestrogens and some phytoestrogen subgroups in relation to increasing categories of BMI, were observed. Levels of intake of phytoestrogens overall and of isoflavones did not vary significantly by educational level, smoking status and physical activity and phytoestrogen and isoflavone intakes were observed. Physically active individuals, non-smokers and participants with a higher level of education had higher intakes of lignans, enterolignans and equol. However, for coumestans the opposite occurred. Soy products were the most abundant food sources of phytoestrogens overall (79.6%), isoflavones (89.9%) and coumestans (82.2%) in the UK health-conscious group. In the MED and & 2012 Macmillan Publishers Limited

non-MED countries and UK general population the sources were more varied, with vegetables, fruits, cereals, non-alcoholic (coffee and tea) and alcoholic beverages (wines, beers and ciders), and soy products being important contributors (up to 30% each) (Table 4). For MED countries, the major food sources of lignans were fruits, vegetables, beverages and cereal products, whereas for non-MED countries, they were beverages, vegetables, cereals products and fruits. For both UK centres, they were beverages, vegetables, fruits, nuts and seeds, and cereals products. For MED and non-MED countries, coffee was the most important contributors of coumestans, whereas for the UK general population, the highest food source was the cereal and cereals products. Dairy products were by far the most important contributor to enterolignans and equol intakes in all regions (82.3–92.1%), although intakes of both were low across nearly all centres. DISCUSSION To our knowledge, this is the first study to describe the total, subgroup and individual phytoestrogen intakes and their food European Journal of Clinical Nutrition (2012) 932 – 941

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

936 Table 2.

Contributiona of individual and subgroups of phytoestrogen intake in the EPIC cohort

Subgroup

Compound

Mediterranean countries % of subgroup

Isoflavones Daidzein Genistein Glycitein Biochanin A Formononetin

32.95 44.14 5.03 12.64 5.24

Secoisolariciresinol Matairesinol Lariciresinol Pinoresinol

23.59 3.07 37.86 35.48

Lignans

Coumestans Coumestrol

100.00

Enterolignans Enterolactone Enterodiol

99.86 0.14

Equol Equol

100.00

% of total 30.39 10.02 13.42 1.53 3.84 1.59 67.27 15.87 2.07 25.47 23.87 1.50 1.50 0.83 0.83 0.00 0.27 0.27

Non-mediterranean countries % of subgroup 38.82 42.94 4.42 6.74 7.09 25.17 3.17 37.20 34.45 100.00 99.74 0.26 100.00

% of total 37.87 14.70 16.26 1.67 2.55 2.69 58.09 14.62 1.84 21.61 20.01 3.34 3.34 0.69 0.69 0.00 0.24 0.24

UK general population

UK health-conscious

% of subgroup

% of subgroup

41.22 49.36 6.46 2.27 0.70 22.60 3.50 40.95 32.94 100.00 99.95 0.05 100.00

% of total 58.43 24.09 28.84 3.77 1.32 0.41 39.00 8.82 1.36 15.97 12.85 2.18 2.18 0.39 0.39 0.00 0.12 0.12

39.52 51.61 8.14 0.64 0.11 15.49 2.26 36.65 45.60 100.00 99.92 0.08 100.00

% of total 86.91 34.34 44.85 7.07 0.55 0.09 11.09 1.72 0.25 4.06 5.06 1.96 1.96 0.04 0.04 0.00 0.01 0.01

a

Adjusted for age and sex and weighted by season and day of dietary recall.

sources in a large adult European cohort, comparing intakes within 10 European countries and by individual and lifestyle characteristics. The use of a unique and updated FCDB and a standardized 24-HDR for the entire study facilitated the comparison across countries and thus reduces the error in testing for the potential relationships between phytoestrogen intake and diseases. The highest intake of phytoestrogen was in the UK healthconscious group (22.41 mg/day). MED countries had the lowest phytoestrogen intake (1.52 mg/day), followed by non-MED countries (2.11 mg/day) and UK general population (4.04 mg/day), showing similar results as those published in individual European countries, such as The Netherlands,34 Sweden35,36 and UK4 (0.58–3.17 mg/day), and in the US (1.11–2.02 mg/day)37 or Canada (1.17 mg/day).20 The UK health-conscious cohort is made up predominantly of lacto-ovo vegetarians and vegans; therefore they consume more plant foods, especially soy products that are the most abundant food sources of phytoestrogens.33 Despite this, phytoestrogen intake was not statistically associated with healthy lifestyle characteristics, such as be active physical activity or never smoker, in the whole cohort. Our results are in concordance with previous results using plasma phytoestrogen concentrations in a sub-sample within the EPIC-Europe cohort.38 Many publications have estimated isoflavone intake, although most of them have only assessed the main isoflavones (daidzein and genistein). In our study, these contributed between 77 and 91% to the total isoflavone intake. MED countries had the lowest isoflavone intake (0.47 mg/day), but it was higher than previously reported (0.03–0.29 mg/day).39–42 The lower values obtained in both Greek42 and Spanish39 EPIC studies were probably because of the fact that neither the Phenol-Explorer database27 nor the UK composition data4 were accounted for. Moreover, the present study was based on a single 24 h recall, which may be more efficient in capturing foods rich in major phytoestrogens that are rarely consumed, notably soy-rich products, but only at population level. Non-MED countries had slightly higher, though still relatively low levels of estimated isoflavone intake (0.76 mg/day), which is comparable to other reported data (from 0.23 to 0.96 mg/day).4,34,35,43 These low intakes are consistent with estimates for other Western countries such as Canada (0.31 mg/day)20 and US (around 1.1 mg/day).37,44 In the UK general population, European Journal of Clinical Nutrition (2012) 932 – 941

the isoflavone intake was also higher (2.34 mg/day) compared with other European countries because of the addition of soy flour to regular flour used in baking.32 Therefore, cereals and cereals products, sugar and confectionary, cakes and biscuits contributed 69.1% of the total isoflavone intake. Isoflavone intake in vegetarians is higher than in non-vegetarians, as was observed in the health-conscious cohort in the present study (19.4 mg/day) and in a former UK study (10.1 mg/day), which includes a subset of participants from the current study.43 Nevertheless, Chinese and Japanese populations still have the highest isoflavone intakes worldwide (ranging from 27.1 to 68.6 mg/day).45–48 Asian populations consume more soy products, which are by far the most important food source of isoflavones in all the studies. However, the dietary habits of Asian immigrant populations living in America and Europe are becomimg more westernized and this may explain a twofold decrease in isoflavone intake observed in Japanese Brazilians46 and a fourfold decrease in intake observed in Japanese women living in the US.49 Lignans were the most abundant contributor of phytoestrogens in those with low phytoestrogen intakes, and the second highest contributor in those with a high intake, such as the UK centres. MED countries had a low lignan intake (1.02 mg/day), but higher than that reported in an Italian study (0.67 mg/day).50 Non-MED countries and the UK general population had slightly higher intakes of lignans (1.26–1.60 mg/day) than MED countries (1.02 mg/day). This is similar to the intake previously reported in The Netherlands (1.24 mg/day),19 Sweden (0.50–2.81 mg/day)35,36 and Finland (1.22 mg/day).51 In other Western countries such as Mexico, US and Canada, lignan intakes were much lower than our results (from 0.35 to 0.86 mg/day).20,37,52 To our knowledge, there are not descriptive data on dietary lignan intake in Asian populations, but probably their contribution to the total phytoestrogen intake would be low like in our UK health-conscious group (o16%). Several papers have reported on intakes of secoisolariciresinol and matairesinol,34,60 but most of them did not consider lariciresinol and pinoresinol, which contributed 470% to the total lignans in our cohort. Other lignans, such as syringaresinol and medioresinol have also been described, but to date, there is little food composition data for them. These lignans have a low conversion degree into enterolignans (o15%) compared with the other precursors described previously.9 Food & 2012 Macmillan Publishers Limited

& 2012 Macmillan Publishers Limited s.e.

16854 13766 5417

1709 10469 8038 7152 8155

17483 10288 7726

7463 11969 8400 6380

Body mass index (kg/m2) o25 25 to o30 X30

Level of schooling None Primary completed Technical/professional Secondary school University degree

Smoking status Never smoker Former smoker Current smoker

Physical activity Inactive Moderately inactive Moderately active Active

0.148 0.059 0.068 0.069 0.065

2.609 2.722 2.780 2.711

0.096 0.088 0.094 0.101

2.689 0.048 2.703 0.056 2.613 0.064

2.532 2.595 2.621 2.793 2.765

2.755 0.050 2.637 0.050 2.527 0.077

0.383

0.646

0.111

0.020

0.092 0.049 0.047 0.080

0.142 0.056 0.065 0.066 0.062

1.386 1.460 1.480 1.436

0.073 0.053 0.050 0.095

1.417 0.046 1.401 0.054 1.369 0.061

1.388 1.397 1.360 1.477 1.393

1.445 0.047 1.385 0.047 1.296 0.072

2.338 0.159 19.443 0.283

1.333 1.522 1.389 1.161

974 4.039 0.166 309 22.405 0.296

o0.001

o0.001

s.e.

1.370 0.055 1.434 0.040

Mean

0.468 0.048 0.762 0.034

0.096 0.052 0.049 0.083

0.705

P-value

0.762

0.914

0.734

0.184

o0.001

o0.001

0.337

P-value

Isoflavones (mg/day)

1.520 0.050 2.106 0.035

2.521 2.796 2.692 2.410

2.664 0.057 2.691 0.042

Mean

Phytoestrogens (mg/day) s.e.

0.022 0.012 0.011 0.019

0.034 0.014 0.016 0.016 0.015

1.143 1.182 1.220 1.196

0.022 0.020 0.022 0.023

1.198 0.011 1.225 0.013 1.152 0.015

1.066 1.116 1.183 1.237 1.294

1.231 0.011 1.172 0.011 1.152 0.018

1.596 0.039 2.514 0.069

1.016 0.012 1.258 0.008

1.108 1.188 1.226 1.180

1.215 0.013 1.178 0.010

Mean

0.003

0.001

o0.001

o0.001

o0.001

o0.001

0.023

P-value

Lignans (mg/day)

GLM models adjusted for sex, age, centre, energy intake, and body mass index and weighted by season and day of recall.

a

3335 12595 14940 5167

Age 35–44 45–54 55–64 65–74

11285 23469

13028 23009

Gender Men Women

European region Mediterranean countries Non-mediterranean countries UK general population UK health-conscious

N

Adjusteda mean daily intakes of total and subgroups of phytoestrogens by sex and selected characteristics

Stratification variable

Table 3.

s.e.

0.002 0.001 0.001 0.002

0.003 0.001 0.001 0.001 0.001

0.067 0.067 0.065 0.065

0.002 0.002 0.002 0.002

0.061 0.001 0.064 0.001 0.079 0.001

0.066 0.068 0.064 0.066 0.063

0.065 0.001 0.067 0.001 0.066 0.002

0.089 0.004 0.439 0.006

0.023 0.001 0.073 0.001

0.065 0.072 0.063 0.057

0.067 0.001 0.064 0.001

Mean

0.787

o0.001

0.013

0.362

o0.001

o0.001

0.077

P-value

Coumestans (mg/day) s.e.

0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000

0.013 0.013 0.014 0.014

0.000 0.000 0.000 0.000

0.014 0.000 0.013 0.000 0.013 0.000

0.012 0.013 0.013 0.014 0.014

0.014 0.000 0.013 0.000 0.013 0.000

0.016 0.000 0.009 0.001

0.012 0.000 0.014 0.000

0.014 0.013 0.014 0.013

0.013 0.000 0.014 0.000

Mean

o0.001

o0.001

o0.001

o0.001

o0.001

0.004

o0.001

P-value

Enterolignans (mg/day)

s.e.

0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000

0.004 0.005 0.005 0.005

0.000 0.000 0.000 0.000

0.005 0.000 0.005 0.000 0.004 0.000

0.004 0.004 0.005 0.005 0.005

0.005 0.000 0.005 0.000 0.004 0.000

0.005 0.000 0.003 0.000

0.004 0.000 0.005 0.000

0.005 0.005 0.005 0.004

0.004 0.000 0.005 0.000

Mean

o0.001

o0.001

o0.001

0.029

o0.001

0.294

o0.001

P-value

Equol (mg/day)

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

937

European Journal of Clinical Nutrition (2012) 932 – 941

European Journal of Clinical Nutrition (2012) 932 – 941

7.0 1.6 1.4 0.3 1.9 0.9 0.9

1.6 2.7 15.5

2.2

0.1 0.1

0.2 7.1

3.8

22.3

1.9 1.1 12.6 6.7

7.1 2.5 4.5 0.1

1.1

1.5 12.6

20.9 4.0 3.8 0.7 8.3 1.2 2.9

2.4 2.8 7.0

0.9

0.1 0.1

4.2 2.2

2.2

8.2

0.9 0.2 4.6 2.5

7.8 5.8 1.9 0.0

1.5

0.5 16.0

Fruits Citrus fruit Apple and pear Grape Stone fruits Berries Other fruits

Nuts and seeds Dairy products Cereal and cereal products Meat and meat products Fish and shellfish Egg and egg products Fat Sugar and confectionery Cakes and biscuits

Non alcoholic beverages Juices Carbonated drinks Coffee Tea

Alcoholic beverages Wine Beer and ciders Spirits

Condiments and sauces Soups, bouillons Soy products

0.8

0.4 0.2 0.2 0.0

0.2 0.0 1.0 2.1

3.3

1.8

0.0 0.8

0.0 0.0

0.0

2.6 0.2 5.9

1.3 0.2 0.3 0.1 0.3 0.2 0.3

0.4

2.8 0.0 0.3 0.2 1.7 0.0 0.1 0.4

0.1

0.1 0.0 12.7 79.6

0.6

2.4 1.4 1.1 0.0

0.4 0.1 6.2 13.3

20.1

7.4

0.1 7.7

0.1 0.0

1.6

0.3 1.5 31.4

3.3 0.7 0.9 0.1 0.6 0.4 0.5

0.8

9.9 0.0 1.3 0.9 6.7 0.1 0.2 0.6

0.3

0.9 49.2

0.7

2.5 2.0 0.5 0.0

0.5 0.0 5.4 0.4

6.3

4.2

1.9 5.4

0.3 0.2

1.8

2.1 3.4 3.6

1.5 1.0 0.2 0.0 0.1 0.0 0.2

8.9

7.1 0.0 2.6 0.0 0.0 0.1 2.6 1.8

0.0

MED countries

3.1 31.9

0.8

1.7 0.7 0.9 0.0

1.6 0.0 11.0 0.5

13.2

7.9

0.1 16.7

0.2 0.1

5.1

0.9 2.9 10.5

0.8 0.4 0.1 0.0 0.0 0.0 0.2

0.3

4.0 0.0 0.7 0.0 0.0 0.0 3.1 0.1

0.0

0.8

0.0 0.0 0.0 0.0

0.0 0.0 0.4 0.1

0.5

1.9

0.0 0.8

0.0 0.0

0.0

0.1 0.1 5.5

0.1 0.0 0.0 0.0 0.0 0.0 0.0

0.4

0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.1 0.0 20.8 89.9

0.2

0.3 0.2 0.1 0.0

0.1 0.0 3.7 0.2

3.9

11.2

0.0 12.3

0.1 0.0

2.5

0.1 0.8 45.6

0.3 0.2 0.0 0.0 0.0 0.0 0.0

1.2

0.5 0.0 0.2 0.0 0.0 0.0 0.1 0.2

0.0

NonUK UK MED Gen health countries pop

Isoflavones (%)

0.3 0.2

1.8

10.6 7.9 2.7 0.0

1.2 0.2 3.0 3.6

8.0

1.3

5.5 0.6

0.1 0.0

0.4

2.7 1.4 8.8

30.8 5.5 5.6 1.0 12.6 1.9 4.3

0.4

26.4 1.5 9.7 1.5 10.0 1.1 0.8 1.7

0.6

MED countries

0.5 0.2

1.4

11.3 3.9 7.2 0.2

2.1 1.9 9.9 11.2

25.1

1.3

0.2 1.0

0.0 0.0

0.4

2.1 1.6 19.9

11.6 2.5 2.4 0.5 3.3 1.6 1.4

0.1

22.5 0.4 4.1 2.2 11.0 0.3 0.5 3.9

0.8

0.5

0.2

1.2

0.3 1.2

0.0 0.0

0.0

0.1 0.4

1.1

5.8 3.2 2.6 0.0

0.1 4.0

0.8

3.2 1.3 1.8 0.1

0.9 1.2 0.3 0.3 9.5 4.9 34.3 17.2

45.1 23.5

1.8

0.2 0.8

0.0 0.0

0.2

0.6 21.5 1.7 0.6 8.7 9.1

8.0 10.5 1.6 1.7 2.3 2.3 0.4 0.5 1.6 2.5 0.9 1.4 1.1 2.1

0.1

24.6 23.1 0.1 0.3 3.1 2.6 2.3 1.7 17.4 14.8 0.3 0.3 0.2 0.2 1.2 3.1

0.7

NonUK UK MED Gen health countries pop

Lignans (%)

0.0 17.2

0.1

0.5 0.5 0.0 0.0

0.2 0.0 63.0 0.5

63.8

0.3

0.1 1.1

0.1 0.2

1.0

0.4 2.3 1.0

2.3 1.1 0.5 0.1 0.4 0.1 0.1

2.8

6.6 0.1 0.0 0.0 0.0 0.0 5.9 0.6

0.1

MED countries

0.2 3.8

0.1

0.2 0.1 0.1 0.0

3.6 0.0 82.0 0.6

86.2

0.2

0.0 1.6

0.0 0.0

0.3

0.1 0.6 0.5

0.3 0.1 0.1 0.0 0.1 0.0 0.0

0.8

5.0 0.0 0.0 0.0 0.0 0.0 4.9 0.1

0.0

0.5

0.0 0.0 0.0 0.0

0.0 0.0 3.9 0.3

4.2

1.4

0.0 0.5

0.0 0.0

0.0

0.1 0.0 7.9

0.1 0.0 0.0 0.0 0.0 0.0 0.0

0.8

2.2 0.0 0.0 0.0 0.0 0.0 2.2 0.0

0.0

0.0 0.0 12.9 82.2

0.1

0.1 0.1 0.0 0.0

0.1 0.0 18.1 1.7

19.9

2.9

0.0 4.8

0.0 0.0

0.2

0.0 0.3 52.5

0.2 0.1 0.1 0.0 0.0 0.0 0.0

0.0

5.9 0.0 0.0 0.0 0.0 0.0 5.9 0.0

0.0

NonUK UK MED Gen health countries pop

Coumestans (%)

0.0 0.2

2.1

0.0 0.0 0.0 0.0

0.0 0.0 0.4 0.0

0.4

0.9

0.0 2.3

0.1 0.7

1.2

0.0 92.1 0.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.2 0.2

3.1

0.0 0.0 0.0 0.0

0.0 0.0 0.8 0.0

0.8

1.4

0.0 2.9

0.1 0.5

0.9

0.0 89.8 0.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.1 0.2

3.6

0.0 0.0 0.0 0.0

0.0 0.0 0.6 0.0

0.6

1.2

0.0 4.9

0.0 0.4

0.6

0.1 2.5

4.8

0.0 0.0 0.0 0.0

0.0 0.0 1.1 0.0

1.1

1.3

0.0 3.8

0.0 0.4

0.2

0.0 0.0 88.2 84.8 0.3 0.9

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

NonUK UK MED Gen health countries pop

Enterolignans (%) MED countries

Abbreviation: MED, Mediterranean. Values are percentages derived from models adjusted for age and sex and weighted by season and day of recall.

0.2

3.1

14.6 0.3 2.6 1.3 6.3 0.2 1.6 2.3

19.8 1.0 7.3 1.0 6.6 0.8 1.4 1.7

Vegetables Leafy vegetables Fruiting vegetables Root vegetables Cabbages Onions Sprout vegetables Other vegetables

Legumes

0.5

0.4

NonUK UK MED Gen health countries pop

Potatoes and other tubers

MED countries

Phytoestrogens (%)

Percent contribution of food groups and some main foods to the intake of total and subgroups of phytoestrogens by European region

Food groups and foods

Table 4.

0.0 0.1

1.6

0.0 0.0 0.0 0.0

0.0 0.0 0.2 0.0

0.2

2.2

0.0 2.4

0.1 10.0

0.5

0.0 82.8 0.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

MED countries

0.3 0.1

2.7

0.0 0.0 0.0 0.0

0.0 0.0 0.5 0.0

0.5

3.0

0.0 2.0

0.2 6.8

0.8

0.0 83.3 0.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.1 0.3

2.3

0.0 0.0 0.0 0.0

0.0 0.0 0.3 0.0

0.3

3.8

0.0 2.8

0.2 5.9

0.2

0.1 2.2

2.8

0.0 0.0 0.0 0.0

0.0 0.0 0.6 0.0

0.6

4.0

0.0 2.2

0.0 5.4

0.1

0.0 0.0 84.0 82.3 0.1 0.2

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

NonUK UK MED Gen health countries pop

Equol (%)

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

938

& 2012 Macmillan Publishers Limited

Phytoestrogen intake in the EPIC study R Zamora-Ros et al

939 sources of lignans varied depending on the region. Fruits, vegetables and wine were the most abundant sources in MED countries. On the other hand, the highest contributors were vegetables, cereal and cereals products, tea coffee and fruits in non-MED countries. Likewise, in Northern European countries (UK, The Netherlands, Sweden and Canada) flaxseed, bread, tea, and some vegetables and fruits have also described in the literature as the main food sources.19,36,53,54 Lignans are the precursors of enterolignans, but enterolignans can also occur in animal-derived foods after their transformation by the intestinal microbiota. Therefore, their main food source was dairy products, so the intake differences across centres were equivalent to the consumption of dairy product previously observed in EPIC.55 Our results were higher than those reported in the literature (0.01–0.02 mg/day).4,35,36 Enterolactone is the main contributor in all the studies, whereas enterodiol was an extremely minor enterolignan contributor (o0.3%). Equol is also a metabolite formed by the intestinal microbiota, but from daidzein. Equol intake (ranged from 0.003 to 0.005 mg/day) in our study was similar to that reported for the EPIC-Norfolk cohort (0.004 mg/day).4 However, two Swedish studies have reported lower equol intakes (about 0.001 mg/day).35,36 Dairy products were by far the most food source of equol intake in all regions; therefore its intake variance was similar to the dairy product consumption in EPIC centres.55 Coumestrol is the most common coumestan. MED countries had the lowest intake of coumestrol (0.02 mg/day) preceded by non-MED countries (0.07 mg/day) and the UK general population group (0.09 mg/day). Finally, the UK health-conscious group (0.44 mg/day) had the highest coumestrol intake because soy products were the main food sources in their diets. According to our data, coffee is the main source of coumestrol in diets low in soy, which is in line with previously published data from the USDA.3 It is noteworthy that values reported by others are at least 10-fold lower compared with our data, ranging from 0.0005 to 0.006 mg/day,4,20,36,37,52 except for those reported in a US casecontrol study (0.19 mg/day).56 The incidence rates for hormone-related cancers, especially breast cancer, in Asian countries have been much lower (up to sixfold) than those in Western countries.57 Soy products and isoflavones/phytoestrogens, which are widely consumed in Asian countries, have been extensively assessed as possibly being responsible for these beneficial effects. Even if phytoestrogens are absorbed from the gut in tiny amounts,8,58 their effects on health may be significant.59 However, this is not always consistent, especially in populations with low phytoestrogen intakes.8,13,60,61 The possible mechanism of action through which phytoestrogens may act is often related with their weak estrogenic activity,4 but it has even been suggested that phytoestrogens might simply reflect a healthy lifestyle.8 In our study, healthy lifestyle and anthropometric characteristics, which have been associated with risk of hormone-related diseases, were statistically associated with lignans, enterolignans and equol, but no differences were shown for total phytoestrogens, isoflavones or coumestans. This is a large study estimating the phytoestrogen intake across European countries. However, as not all the EPIC cohorts are population based, the findings on intake cannot be extrapolated to the general population of each region.62 Dietary data was collected B10 years ago therefore the current phytoestrogen intake may be higher because soy products and soysupplemented foods are now more available in Europe. Another limitation of this study is that each person contributed only one 24-HDR, hence variation in intakes cannot be evaluated at the individual level, especially when less frequently consumed foods, such as soy-rich products, are concerned. However, 24-HDRs are considered an acceptable method for estimating population mean intakes.63 Furthermore, the sampling of the EPIC calibration study & 2012 Macmillan Publishers Limited

was designed to control for seasonal and day-of-the-week variations in dietary intake.23 Thus, it is reasonable to expect that the population mean will not be too much influenced by dayto-day or seasonal variability. Underestimation of total phytoestrogen intake may also occur because of the unknown composition data, especially for the newly available soysupplemented products. However, our database was compiled from the most updated polyphenol databases, with 1877 food items and only 8% of missing values. Indeed, the major strength of the present study is the use of a unique and specifically developed FCDB on phytoestrogens, for that allowed results to be compared across countries. Further underestimation may be due to the omission of herb/plant supplements in this analysis (up to 5% of the consumers in Denmark, the highest consumer country).64 In summary, the data presented in this study show the mean intake of phytoestrogens (isoflavones, lignans, coumestans, enterolignans and equol) and their food sources in 10 European countries. The used FCDB comprises the most updated and available worldwide data on phytoestrogen in foods. Lignan, enterolignan and equol intakes were related with healthy lifestyle habits (non-smoking status, BMI, educational level and physical activity), but not total phytoestrogens (except BMI), isoflavones or coumestans. These data provide a useful basis for the further exploration of associations between phytoestrogen intake and disease risk. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was carried out with the financial support of the European Commission: Public Health and Consumer Protection Directorate 1993 to 2004; Research Directorate-General 2005; Ligue contre le Cancer, Institut Gustave Roussy, Mutuelle Ge´ne´rale de l’Education Nationale, Institut National de la Sante´ et de la Recherche Me´dicale (INSERM; France); German Federal Ministry of Education and Research; Danish Cancer Society: Health Research Fund (FIS) of the Spanish Ministry of Health (RTICC DR06/0020); the participating regional governments and institutions of Spain; Cancer Research UK; Medical Research Council, UK; the Stroke Association, UK; British Heart Foundation; Department of Health, UK; Food Standards Agency, UK; the Wellcome Trust, UK; the Stavros Niarchos Foundation and the Hellenic Health Foundation; Italian Association for Research on Cancer; Compagnia San Paolo, Italy; Dutch Ministry of Public Health, Welfare and Sports; Dutch Ministry of Health; Dutch Prevention Funds; LK Research Funds; Dutch ZON (Zorg Onderzoek Nederland); World Cancer Research Fund (WCRF); Swedish Cancer Society; Swedish Scientific Council; Regional Government of Skane, Sweden; Nordforsk - Centre of Excellence programe HELGA; Some authors are partners of ECNIS, a network of excellence of the 6FP of the EC. RZR is thankful for a postdoctoral programe Fondo de Investigacio´n Sanitaria (FIS; no. CD09/00 133) from the Spanish Ministry of Science and Innovation. We thank Raul M. Garcı´a for developing an application to link the FCDB and the 24-HDR. We would also like to thank Marleen Lentjes, Veronica van Scheltinga, Alison McTaggart and Amit Bhaniani for their invaluable contributions to the creation of the EPIC-Norfolk phytoestrogen database.

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